Future drug discovery

Drug repurposing to new medical uses and chiral switches are elements of secondary pharmaceuticals. This article focuses on drug repurposing/chiral switches of the diastereomeric quasi-enantiomeric antimalarial quinine and antiarrhythmic quinidine, based on the histories of these drugs (1638–2022), applying a widened scope. Quinine, an essential medicine, changed the world. Drug repurposing is a strategy for identifying new uses for approved or investigational drugs outside the scope of the original medical indications. Potential drugs are not included in the definition of drug repurposing. Drug repurposing may be within or outside the therapeutic group, e.g., quinidine to quinine repurposing, from treatment of arrhythmia or severe malaria to uncomplicated malaria. The scope of chiral switches included racemate to single enantiomer and other switches of the status of chirality, e.g., racemate and quasi-racemate to scalemic mixtures. There are 16 quinine/quinidine stereoisomers. Given the multiple pharmacological activities of Cinchona alkaloid stereoisomers, this article calls for subjecting them to comprehensive drug repurposing/chiral switch searches for new medical uses.

The role of peptides is nowadays relevant in fields such as drug discovery and biotechnology. Computational analyses are required to study their properties and gain insights into rational design strategies. Both natural and modified peptides containing non-natural amino acids require customized tools to run sequence and structure-based studies. PepFun 2.0 is a new version of the python package for the study of natural and modified peptides using a set of modules to analyze the sequence and structure of the molecules. PepFun 2.0 comprises five main modules for different tasks such as sequence alignments, prediction of properties, generation of conformers, modification of structures, detection of protein-peptide interactions, and extra functions to include peptides containing non-natural amino acids. The code and tutorial are available at: https://github.com/rochoa85/PepFun2

Metabolic reprogramming is a major hallmark of malignant transformation in cancer, and part of the so-called Warburg effect, in which the upregulation of glutamine catabolism plays a major role. The glutaminase enzymes convert glutamine to glutamate, which initiates this pathway. Inhibition of different forms of glutaminase (KGA, GAC, or LGA) demonstrated potential as an emerging anti-cancer therapeutic strategy. The regulation of these enzymes, and the molecular basis for their inhibition, have been the focus of much recent research. This review will explore the recent progress in understanding the molecular basis for activation and inhibition of different forms of glutaminase, as well as the recent focus on combination therapies of glutaminase inhibitors with other anti-cancer drugs.

Aim: This study aimed to develop a chemoinformatic tool for extracting natural product information from academic literature. Materials & methods: Machine learning graph embeddings were used to extract knowledge from a knowledge graph, connecting properties, molecular data and BERTopic topics. Results: Metapath2Vec performed best in extracting compound names and showed improvement over evaluation stages. Embedding Propagation on Heterogeneous Networks achieved the best performance in extracting bioactivity information. Metapath2Vec excelled in extracting species information, while DeepWalk and Node2Vec performed well in one stage for species location extraction. Embedding Propagation on Heterogeneous Networks consistently improved performance and achieved the best overall scores. Unsupervised embeddings effectively extracted knowledge, with different methods excelling in different scenarios. Conclusion: This research establishes a foundation for frameworks in knowledge extraction, benefiting sustainable resource use.

Aim: This work aims to contribute toward development of preventive measures for the control of monkeypox (mpox) virus disease through computational design of a multiepitope vaccine. Methods: To accomplish this, we employed a robust immunoinformatics approach to design a putative chimeric vaccine candidate from 18 viral transmembrane proteins. Results: The resulting chimeric vaccine candidate is a 76.4 kDa protein containing 687 amino acids with an estimated isoelectric point of 9.39. In addition, it was predicted to adopt a stable 3D conformation that harbors discontinuous B-cell epitopes and strongly interacts with key immune receptors. Conclusion: The designed hypothetical antigen is a valuable addition to the collection of prospective vaccine candidates for future development and trials against the re-emerging mpox disease.

Aim: A bacterial genetics-guided approach was utilized for the discovery of new compounds affecting bacterial genome stability. Materials & methods: Fungal extracts and fractions were tested for genome instability-mediated antibacterial activity. Interaction assays and RT-qPCR were used to identify compounds that boost the activity of sub-minimum inhibitory concentration streptomycin and obtain insights on the molecular mechanisms of the primary hit compound, respectively. Results: Several extracts and fractions caused bacterial genome instability. Codeine, in synergy with streptomycin, regulates double-strand break (DSB) repair and causes bacterial ribosome dysfunction in the absence of DSBs, and dysregulation of ribosome biogenesis in a DSB-dependent manner. Conclusion: This study demonstrates a potential viable strategy that we are exploring for the discovery of new chemical entities with activities against Escherichia coli and other bacterial pathogens.

The immune checkpoint inhibitors (ICIs) have revolutionized the treatment of advanced melanoma by significantly increasing survival rates, with the promise of durable disease remission in some patients. Herein we review the role of immune checkpoints in melanoma; the history of melanoma immunotherapy; pivotal clinical trial data for ipilimumab, pembrolizumab, nivolumab and relatlimab; and the current clinical role of each ICI. We discuss the challenges that accompany these triumphs in the treatment of melanoma, including: how to distinguish between responders and nonresponders; how to optimize ICI dosing and combinatorial approaches; and the best practices for monitoring response and managing immune-related toxicities. We offer our perspective on the financial toxicity of ICIs and new developments that could deliver answers to current challenges.

Future Drug DiscoveryAhead of Print CommentaryOpen AccessComputational approaches to targeting protein–protein interactions in cancer: a pathway to drug discoveryMelody Okereke, Kenneth Bitrus David & Oluwakorede Joshua AdedejiMelody Okereke *Author for correspondence: Tel.: +2348039209527; E-mail Address: melokereke30@gmail.comhttps://orcid.org/0000-0003-2533-6785Faculty of Pharmaceutical Sciences, University of Ilorin, Ilorin, Kwara State, Nigeria, Kenneth Bitrus David https://orcid.org/0000-0002-4688-5591Faculty of Pharmaceutical Sciences, Kaduna State University, Kaduna, Nigeria & Oluwakorede Joshua Adedeji https://orcid.org/0000-0002-7859-1889Faculty of Pharmaceutical Sciences, University of Ilorin, Ilorin, Kwara State, NigeriaPublished Online:5 May 2023https://doi.org/10.4155/fdd-2023-0003AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: artificial intelligencecancerdrug discoveryinteractionsPPIproteintargetingProtein–protein interactions (PPIs) are physical links that exist between proteins and their partners [1]. They are essential for many cellular processes, including protein folding, gene expression, signal transduction and cell differentiation. PPIs have been demonstrated to be dysregulated in cancer [1]. Therefore, understanding the structure and dynamics of PPIs is crucial for developing new therapeutic strategies for these diseases.PPIs are essential in regulating various signaling pathways, including the Hedgehog pathway, the MAPK pathway, the Notch pathway, the Wnt signaling pathway and the TGF-beta pathway [2]. In the MAPK pathway, PPIs mediate the sequential phosphorylation of several kinases, leading to apoptosis, differentiation and proliferation of cells [2]. In the Wnt pathway, PPIs mediate the binding of Wnt proteins to Frizzled receptors, activating intracellular signaling cascades [2]. Dysregulation of these pathways due to aberrant PPIs has been implicated in cancer and other diseases [1].The large and often flat interfaces involved in interactions make it challenging to target PPIs [2]. However, advances in computational methods and structural biology have enabled the rational design of small-molecule inhibitors that can disrupt PPIs in disease states [3]. These inhibitors can target specific protein–protein interfaces or allosteric sites, disrupting interactions and restoring normal cellular function.Role of PPIs in cancer development & progressionPPIs are involved in the development of cancer in a number of ways, one of which is the abnormal activation of oncogenic signaling pathways [4]. An instance worth highlighting is colorectal cancer. Here, mutations in the APC gene are a common cause of Wnt signaling pathway activation, resulting in the build-up of β-catenin and aberrant activation of downstream target genes [5]. PPIs between β-catenin and its binding partners, such as TCF/LEF transcription factors, are critical for the activation of the pathway [5]. Similarly, in chronic myeloid leukemia, the constitutive activation of the BCR-ABL fusion protein results in the aberrant activation of the Ras/MAPK and PI3K/Akt signaling pathways, which contribute to cell survival and proliferation [6]. PPIs between Bcr-Abl and its downstream effectors, such as Grb2 and Gab2, are critical for the activation of these pathways [6].In addition to activating oncogenic pathways, dysregulated PPIs can also contribute to tumor suppression by disrupting the function of tumor suppressor proteins. In the case of the regulation of the cell cycle and the response to DNA damage, for instance, the tumor suppressor protein p53 plays an essential role [7]. PPIs with their binding partners, such as Mdm2, regulate the stability and activity of p53, which facilitates its degradation and ubiquitination [7]. Dysregulation of these PPIs can result in the loss of p53 function, leading to the accumulation of genomic instability and the development of cancer [8].Targeting PPIs in cancer is challenging due to the large and often flat interfaces involved in these interactions [2]. However, advances in computational methods and structural biology have enabled the rational design of small-molecule inhibitors that can disrupt PPIs in disease states [3]. For example, small-molecule inhibitors that target PPIs between Bcr-Abl and its downstream effectors have been developed and are currently used in the treatment of chronic myeloid leukemia [9]. Similarly, small-molecule inhibitors that target PPIs between Mdm2 and p53 are being developed as potential cancer therapies [7].Traditional approaches to targeting PPIs: traditional small-molecule inhibitorsSmall-molecule inhibitors have been widely used as therapeutics for a variety of diseases, including cancer. These inhibitors typically target enzymes or receptors, and work by binding to specific sites on these proteins and disrupting their activity [10]. The efficacy of traditional small-molecule inhibitors, however, may be constrained by a number of factors, which can also result in the development of drug resistance. Resistance can arise through various mechanisms, including mutations in the target protein that prevent inhibitor binding, upregulation of alternative signaling pathways that bypass the inhibited pathway and the selection of pre-existing drug-resistant cells within the tumor [11].One limitation of traditional small-molecule inhibitors is their lack of specificity. Many inhibitors target proteins that are involved in multiple signaling pathways, and they can therefore have off-target effects that lead to unintended consequences [12]. For example, the widely used tyrosine kinase inhibitor imatinib was initially developed to target the Bcr-Abl fusion protein in chronic myeloid leukemia, but it also inhibits other kinases such as c-Kit and PDGFR, which can lead to side effects such as gastrointestinal toxicity and fluid retention [12]. Another limitation of traditional small-molecule inhibitors is their inability to target PPIs notwithstanding their potential as a viable therapeutic approach [8]. However, PPIs typically involve large and flat interfaces that are difficult to target with small molecules [2].Alternative approaches to PPI targetingWhile traditional small-molecule inhibitors have limitations in targeting PPIs, there are alternative approaches that are being explored to target these interactions. One approach is the use of biologics, such as monoclonal antibodies, that can specifically bind to PPI interfaces and disrupt the interaction [12]. For example, rituximab (a monoclonal antibody) has been successfully used in the treatment of B-cell lymphomas due to its ability to target the CD20 protein on B-cells and induce their death [13].Another approach is the use of stapled peptides, which are synthetic peptides that are stabilized with a covalent bond between two amino acids to maintain a specific conformation for binding to PPI interfaces. Stapled peptides have shown promise in preclinical studies for targeting PPIs involved in cancer, such as the interaction between MDM2 and p53 [13].In addition to biologics and stapled peptides, other approaches to PPI targeting include the use of protein degradation strategies, such as the use of proteolysis-targeting chimeras (PROTACs), which can induce the degradation of specific target proteins by recruiting them to an E3 ubiquitin ligase for ubiquitination and subsequent degradation by the proteasome. PROTACs have shown promise in targeting PPIs involved in cancer, such as the interaction between the estrogen receptor and the coregulator protein SRC-3 [3].Computational approaches to PPI targeting in cancerComputational approaches involve the use of computer simulations, algorithms and data analysis techniques to study biological systems and their interactions [3]. Computational methods have several advantages over traditional experimental approaches for PPI targeting, including their ability to rapidly screen large numbers of compounds, predict binding affinities and selectivity and identify potential off-target effects [3]. Computational methods can also reduce the need for expensive and time-consuming experimental assays. Various computational methods developed for PPI targeting in cancer include molecular docking, molecular dynamics simulations and virtual screening [14]. The following are examples of successful computational approaches that have been used in PPI targeting in cancer:Inhibitors of MDM2-p53 interactionThe growth and survival of cells are regulated by the MDM2-p53 interaction, which is often dysregulated in cancer [14]. Small-molecule inhibitors of this interaction, such as the US FDA-approved drug nutlin-3, have been identified using computational approaches [15]. To determine the binding location and mechanism of action of Nutlin-3, as well as to design inhibitors that are more potent and selective, molecular docking and dynamics simulations, respectively, were used.Inhibitors of Bcl-2 family of proteinsThe proteins of the Bcl-2 family regulate apoptosis and are often dysregulated in cancer [16]. Small-molecule inhibitors of this family, such as the US FDA-approved drugs venetoclax and navitoclax, have been identified using computational approaches [15]. The identification of the binding site and mechanism of action of these inhibitors and the design of more potent and selective inhibitors were carried out using molecular dynamics simulations.Inhibitors of NF-κB signaling pathwayThe NF-κB signaling pathway regulates inflammatory and immunological responses, and its activity is often dysregulated in cancer [17]. Computational methods have been used to identify small-molecule inhibitors of this pathway, including the US FDA-approved drug bortezomib [15]. The identification of the binding site and mechanism of action of bortezomib and the design of more potent and selective inhibitors were carried out using virtual screening and molecular dynamics simulations.With the aforementioned examples, computational approaches have proven to be successful in identifying potential PPI inhibitors in cancer. Through the combination of molecular docking, molecular dynamics simulations and virtual screening, researchers can rapidly identify potential inhibitors, predict their binding affinity and selectivity and optimize their activity, making computational approaches a powerful tool in PPI drug discovery. However, despite significant progress in this field, several challenges and limitations remain.Challenges & limitations of computational approaches to PPI targeting in cancerComputational approaches offer several advantages over traditional approaches for targeting PPIs in cancer. However, accurately predicting the binding affinity between a small molecule or biologic and its target PPI remains a significant challenge [8]. Different scoring functions used by different docking programs can yield varying results, and accurately predicting binding affinity is particularly difficult for more complex PPI interfaces [8]. Furthermore, experimental validation is necessary to confirm the predicted binding and efficacy. The absence of experimental data risks false positives and wasted resources.The accuracy of computational predictions depends heavily on the availability and quality of structural data for the target PPI [3]. If high-quality structural data is not available, computational methods may not accurately predict the binding interface and affinity [3]. Additionally, computational approaches may not account for important factors such as protein flexibility, water-mediated interactions and post-translational modifications that can affect PPI formation and stability [3,8]. These factors are especially important for PPIs that undergo significant conformational changes upon binding or involve partially disordered regions.Furthermore, computational methods may miss potential inhibitors or biologics that are not included in the library. This limitation highlights the need for rational design approaches that leverage computational methods to guide the synthesis of novel compounds with optimal properties for PPI targeting. However, potential challenges to these approaches exist, such as the difficulty of accurately modeling the effects of protein flexibility, the need for large computational resources and the potential for overfitting models to specific datasets. These challenges must be considered and addressed to ensure that computational approaches continue to advance the field of PPI targeting in cancer.To overcome these challenges and limitations of computational approaches for PPI targeting in cancer, several strategies can be employed. It is important to integrate experimental data with computational predictions to confirm the predicted binding and efficacy. This can help reduce the risk of false positives and improve the accuracy of predictions. Additionally, developing more accurate scoring functions and improving the resolution and quality of structural data for target PPIs can improve the accuracy of computational predictions. Furthermore, incorporating protein flexibility, water-mediated interactions and post-translational modifications in computational models can improve their predictive power. Finally, the use of machine learning algorithms can help overcome the limitations of small libraries and improve the identification of potential inhibitors or biologics. Overall, a combination of experimental and computational methods can overcome the challenges and limitations of computational approaches for PPI targeting in cancer.Future directions in PPI targeting & cancer therapyOver the years, PPI targeting has been of major interest to cancer drug researchers leading to various advancements in the field. The future is thus limitless owing to recent advances and the potential of computational drug discovery in understanding and accelerating the development of treatments that influence PPIs. Since the US FDA approved the kinase inhibitor imatinib in 2001 [15], almost 50% of cancer therapeutic discovery efforts are focused on developing new medications that target kinases. While protein kinase inhibition constitutes only one of the mechanisms in PPI targeting, increasing interest has highlighted its benefits in numerous cancers. Since the capacity of many aberrantly produced proteins to interact with a protein-binding partner directly contributes to their ability to promote tumor growth in the malignant state, addressing PPIs important to cancer therapy discovery is essential. Of such importance is the development of novel inhibitors by Cheng et al. where the computer-aided designed Complex 1 showed potential as the first molecule capable of inhibiting the CDK9-cyclin T1 PPI and improving the outcomes commonly associated with triple-negative breast cancer [18]. Compared with conventional chemotherapy involving cytotoxic drugs, protein-targeted anticancer drugs like kinase inhibitors have been shown to cause fewer side effects, thus increasing their potential for more use in the future.Despite the tolerability and effectiveness of PPI-targeted therapy, drug resistance remains an issue that could be further explored in future research [11]. Primary resistance to direct inhibitors may theoretically come from the presence of particular comutations or mutational heterogeneity in a tumor. Knowledge in this area is quite limited and requires more research in terms of large-scale omics analyses to understand, identify and utilize key factors that occur during pretreatment and are responsible for such resistance. Acquired resistance, referring to the de novo development of early resistance on the administration of tyrosine kinase inhibitors, is another issue in PPI therapy requiring further research. As ERK activity is suppressed in RAS-targeted therapy, MYC target genes, such as those encoding RTKs and their ligands, are often derepressed, which leads to the rapid reactivation of the RAS-MAPK pathway to some degree and is referred to as adaptive resistance [19]. In addition, the development of adaptive resistance in anaplastic lymphoma kinase-tyrosine kinase inhibitors is partially understood and requires further study. Given its predominant role in treatment failure, the novel concept of drug-tolerant persister (DTP) cells has garnered many studies and still requires more. In this regard, it is claimed that DTP cells continue to function after being exposed to anticancer drugs, and their DNA repair processes are changed to promote adaptive mutation, which explains the establishment of drug-resistant mutations [20]. All of these resistance mechanisms require further research to enable progress in PPI targeting for cancer therapy.With less than 20% of protein kinases targeted in cancer treatment, this presents an opportunity for more research to explore useful alternative kinases for inhibition in cancer therapy. More research is also anticipated in the production of next-generation kinase inhibitors with enhanced selectivity and CNS penetration [15]. Poor selectivity of PPI therapies is associated with a wide range of side effects that reduce tolerability and the role of CNS penetration is essential in bypassing the blood–brain barrier (BBB) in the management of brain metastases. A number of PPI therapies are quite limited in their ability to cross the BBB, hence the need for more developments in the use of modified formulations and drug administrations to enhance BBB penetration. To find and create small-molecule inhibitors that can target kinases like the TAM kinases, significant research will also be focused on the involvement of the tumor stromal microenvironment and immune biology in the defense of cancer cells [9]. While PPI treatments are currently effective in early-stage cancers, understanding how they can be improved to be beneficial in late-stage treatments as well is crucial.ConclusionPPIs play important roles in cellular signaling, a number of which play significant roles in cancer pathogenesis and progression. While they were formerly regarded as "undruggable targets," current progress has debunked that and continuous advances have accelerated the development of novel therapies effective in various cancer types. Compared with the traditional drug development process, rational computational drug design, which uses molecular modeling methods like pharmacophore modeling, molecular dynamics, virtual screening and molecular docking to describe the activity of biomolecules and defines molecular determinants for interaction with the drug target, aids in the development of more effective drug candidates with less time and resources. The future of PPI targeting is predicated on the modern use of computational tools and approaches. Artificial intelligence and machine learning can undoubtedly provide immense benefits in the analysis of protein structures, PPIs and targeting in drug design and discovery for cancer therapy.Author contributionsM Okereke conceptualized the idea for this paper. M Okereke, KB David and OJ Adedeji wrote the first drafts of the paper. M Okereke wrote the final draft and reviewed the paper for intellectual content, accuracy and comprehension. All authors agreed and approved the final manuscriptFinancial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.Open accessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/References1. Cheng SS, Yang GJ, Wang W, Leung CH, Ma DL. Correction to: the design and development of covalent protein-protein interaction inhibitors for cancer treatment. J. Hematol. Oncol. 13(1), 102 (2020).Crossref, Google Scholar2. Lu H, Zhou Q, He J et al. Recent advances in the development of protein-protein interactions modulators: mechanisms and clinical trials. Signal Transduct. Target Ther. 5(1), 213 (2020).Crossref, Google Scholar3. Marchand A, Van Hall-Beauvais AK, Correia BE. Computational design of novel protein-protein interactions–an overview on methodological approaches and applications. Curr. Opin. Struct. Biol. 74, 102370 (2022).Crossref, CAS, Google Scholar4. Nusse R, Clevers H. Wnt/β-catenin signaling, disease, and emerging therapeutic modalities. Cell 169(6), 985–999 (2017).Crossref, CAS, Google Scholar5. Druker BJ. Translation of the Philadelphia chromosome into therapy for CML. Blood 112(13), 4808–4817 (2008).Crossref, CAS, Google Scholar6. Aubrey BJ, Strasser A, Kelly GL. 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EBioMedicine 89, 104483 (2023).Crossref, CAS, Google ScholarFiguresReferencesRelatedDetails Ahead of Print STAY CONNECTED Metrics History Received 19 March 2023 Accepted 29 March 2023 Published online 5 May 2023 Information© 2023 The AuthorsKeywordsartificial intelligencecancerdrug discoveryinteractionsPPIproteintargetingAuthor contributionsM Okereke conceptualized the idea for this paper. M Okereke, KB David and OJ Adedeji wrote the first drafts of the paper. M Okereke wrote the final draft and reviewed the paper for intellectual content, accuracy and comprehension. All authors agreed and approved the final manuscriptFinancial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.Open accessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/PDF download

Future Drug DiscoveryVol. 4, No. 3 CommentaryOpen AccessmiRNA-205: a future therapeutic molecule for liver diseasesMarco Cabrera, Meghana Kolli, Meena Jaggi, Subhash C Chauhan & Murali M YallapuMarco CabreraDepartment of Immunology & Microbiology, School of Medicine, The University of Texas Rio Grande Valley, McAllen, TX 78504, USASouth Texas Center of Excellence in Cancer Research, School of Medicine, The University of Texas Rio Grande Valley, McAllen, TX 78504, USA, Meghana KolliDepartment of Immunology & Microbiology, School of Medicine, The University of Texas Rio Grande Valley, McAllen, TX 78504, USASouth Texas Center of Excellence in Cancer Research, School of Medicine, The University of Texas Rio Grande Valley, McAllen, TX 78504, USA, Meena JaggiDepartment of Immunology & Microbiology, School of Medicine, The University of Texas Rio Grande Valley, McAllen, TX 78504, USASouth Texas Center of Excellence in Cancer Research, School of Medicine, The University of Texas Rio Grande Valley, McAllen, TX 78504, USA, Subhash C ChauhanDepartment of Immunology & Microbiology, School of Medicine, The University of Texas Rio Grande Valley, McAllen, TX 78504, USASouth Texas Center of Excellence in Cancer Research, School of Medicine, The University of Texas Rio Grande Valley, McAllen, TX 78504, USA & Murali M Yallapu*Author for correspondence: Tel.: +1 956 296 1734; E-mail Address: murali.yallapu@utrgv.eduDepartment of Immunology & Microbiology, School of Medicine, The University of Texas Rio Grande Valley, McAllen, TX 78504, USASouth Texas Center of Excellence in Cancer Research, School of Medicine, The University of Texas Rio Grande Valley, McAllen, TX 78504, USAPublished Online:27 Jan 2023https://doi.org/10.4155/fdd-2022-0012AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Keywords: chemotherapyliver cancerliver diseasesliver fibrosismiRNAnonalcohol fatty livermiR-205 is consistently downregulated in liver fibrosis, liver cirrhosis, and liver cancer, which indicates it may be a potential therapeutic restoration molecule to tackle liver diseases.The spectrum of liver diseases presents a major public health concern that is widespread across the globe, accounting for 2 million deaths per year [1]. Among all liver diseases, the most common, nonalcoholic fatty liver disease (NAFLD), has been globally estimated to account for a quarter of all cases, ranging regionally from 14 to 32% [2]. In addition, liver cancer is the 16th most common cause of mortality worldwide [1]. Hence the imperativeness of novel therapeutic developments toward liver diseases is currently a clinically unmet need.miRNAs have gained significant attention after their discovery in Caenorhabditis elegans. miRNAs have offered a new area of investigation ranging from the understanding of post-transcription regulatory genes to their role in various disease processes, including tumorigenesis. miRNAs are small noncoding RNA molecules that regulate post-transcriptional gene expression. Approximately 52.5% of human genes that code for miRNA are linked to cancer, hence the interest for those searching for potential therapeutic targets. There are about 38,589 miRNAs that have been identified specifically in 271 organisms. Such quantification has been gathered from miRbase, an online data base for miRNA sequences.Biogenesis of miRNAs varies from organism to organism, depending on factors, such as the site where miRNAs are processed and the presence or absence of Drosha and DGCR8. Such processes follow a canonical pathway, albeit a noncanonical pathway has been suggested. It embarks its process at the nucleus and adjourns in the cytoplasm via Drosha and Dicer, respectively. As these processes continue, expression of argonaute (Ago) plays an imperative role in increasing miRNA stability post-transcriptionally. This suggests that Ago acts as a homeostatic component for miRNA maturity concentrations.miRNAs with their respective classification will be named as miR-X, X being the classification. miRNAs found within the human body have been associated with a myriad of biochemical functions. For instance, the up and down regulation of miR-29a and miR-29b are associated with high glucose and insulin resistance [3]. In addition, miRNA-320 has also been found to have an effect on insulin resistance [4]. In terms of their role in lipids within the liver and hepatocytes, miRNA-10b has been proven to be a pioneer in abnormal retention of lipids. However, when it comes to healing after liver-damaging agents, miR-21 aids in liver regeneration and tissue repair when exposed to alcohol [5]. Furthermore, miR-122 and miR-155 are both associated with liver homeostasis [6] and autoimmune hepatitis [7] while miR-186 and miR-205 show their action in proliferation of hepatocellular carcinoma (HCC) [8]. In hepatitis B viral infections and liver diseases, various miRNAs, such as miR-15b, miR-16-1, miR-17, miR-18a, miR-19a, miR-19b, miR-20a, miR-21, miR-22, miR-23a, miR-23b, miR-25, miR-26a, miR-29b, miR-30, miR-31, miR-34a, miR-92a, miR-125b, miR-130a, miR-132, miR-133a, miR-141, miR-145, miR-146, miR-148a, miR-150, miR-152, miR-155, miR-181b, miR-192, miR-196a, miR-199b, miR-200, miR-200b, miR-200c, miR-205, miR-214, miR-219, miR-221, miR-223, miR-323, miR-372, miR-373, miR-375, miR-455, miR-501, miR-548, miR-602 and miR-604 have been implemented as biomarkers, therapeutics, direct/indirect inhibitors, and immunomodulators [9]. Altogether, various disease pathological processes including liver diseases are significantly influenced by miRNA dysregulation and miR-205 is consistently downregulated in almost all liver diseases. Hepatitis B is a viral infection (HBV X, HBx protein play crucial role) that attacks the liver and can cause both acute and chronic disease. A significantly lower expression of miR-205 was observed in HBV infected patients [10]. In addition, supplementation of miR-205 was able to remarkably inhibit the HBx-enhanced proliferation of hepatoma cells [11]. Considering these links, the prognostic and therapeutic role of miR-205 in liver diseases is the focus of this commentary article.Role of miR-205 in liver diseasesmiRNAs are small noncoding RNAs that act as a regulator of target messenger RNAs expression which is performed post-transcriptionally. miR-205 is localized in two locations in the human genome, Chr1 and Chr12 [12]; and its function is regulated with expressions of other genes through collaboration of various intertwined mechanisms [13]. Such miRNAs are relevant as its mode of mechanism either as oncogenes or tumor suppressors. Dysregulation of miR-205 in breast, prostate, skin, liver, gliomas, pancreatic, colorectal and renal cancer have been reported [14]. Furthermore, miR-205 sensitizes gemcitabine resistant pancreatic cancer cells and reduces tumor weight and growth [15], while in cervical cancer it induces lymph node metastasis when upregulated [16]. Therefore, delineating the specific role of miR-205 in liver diseases is highly warranted.Liver fibrosis & cirrhosisThe development of liver fibrosis (LF) is aided by increased amounts of fibrillar extracellular matrix due to exposure a variety of liver tissue damaging agents. If untreated, LF progresses to liver cirrhosis (LCi). When the liver damaging agents emerge and stay prevalent, the healing process, hepatic fibrogenesis, subsequently acts opposite of its function, healing. When such liver damaging substances are chronically present long-term, so is the chronic-healing process, activating hepatic stellate cells to hyperactively secrete extracellular matrix. As a result, a chronic-cycle translating from LF to LCi and portal hypertension (a serious complication of LCi).A highly sensitive serum miRNA panel evaluation confirmed there is no significant variation in miRNA expression in patients with chronic hepatitis C with LF (early or advanced stage) compared with no fibrosis patients [17]. miRNAs, such as miR-124, miR-200a, miR-200c, miR-205, miR-103a, and miR-15, were downregulated while miR-141, miR-155, miR-208a, miR-499, and miR-574 were upregulated in early-stage fibrosis (F0-F2). Similarly, miR-124, miR-155, miR-200c, miR-205, miR-103a, and miR-15 were downregulated whereas miR-141, miR-200a, miR-208a, miR-499, and miR-574 were upregulated in advanced stage fibrosis (F3-F4). In another study, profiles of differential expression of circulating miRNAs in LCi samples, miR-205 was downregulated while miR-195, miR-25, and miR-16 were upregulated. Interestingly, miR-205 is consistently downregulated in chronic hepatitis, LCi and HBV-positive HCC patient samples. A study on the progression of steatosis in liver of mice up on dietary responses of tissue-specific miR-205 confirmed that it regulates the signaling and metabolism in fibrosis and progresses to steatosis [18]. In one word, it can be stated that miR-205 is absent in LF and LCi conditions.Nonalcoholic fatty liver diseaseNAFLD is one of the most common liver-associated diseases and metabolic syndrome. NAFLD is an excessive accumulation and infiltration of adipocytes in the liver with ramifications similar to that of alcohol related fatty liver disease despite the absence of alcohol or lack thereof. There are various factors that influence the disease progression of NAFLD, some being known and others unknown. One such factor is that of hepatic fat accumulation and its lipotoxicity. The initial pathologic observation of NAFLD is the gradual increase of steatosis within hepatocytes. Factors that contribute to steatosis such as excessive triglyceride accumulation and an increased calorie diet, are some of the components that affect such hepatic fat accumulation and dramatically predisposes the individual to insulin resistance. As insulin resistance builds up an additional complication, lipolysis, arises, which leads to excessive free fatty acids circulating via blood vessels, steatosis and hyperinsulinemia.Hu et al. [19], delineated the role and underlying mechanism of miR-205 in NAFLD. This study confirmed that over expression of miR-205 leads to the downregulation of liver triglycerides and enhancement of glycerol concentration, and thus suppressed lipid accumulation in high-fat diet-fed mice. Additionally, the miR-205 supplementation decreased body weight and liver mass. Further investigation also demonstrates that the overexpression of miR-205 alleviated lipid accumulation in OA-induced HepG2 and PH cells by targeting NEU1. Together, this study results suggest that miR-205/NEU1 can be a viable therapeutic target for treatment of NAFLD. In the series of experiments conducted on mouse models, it was found that miR-205 has an effect on transcription factor ZEBI, which has a role in liver metabolism and insulin resistance [20]. On the other hand, when NZ10 mice were predisposed with obesity, Type 2 diabetes and hepatic steatosis; fed with high protein fish oil diet and examined for the biochemical and physical functions, results showed that the diet prevented steatosis and reduced serum cholesterol and triglycerides, and the diet repressed the hepatic expression of fatty acid metabolic regulators like peroxisome proliferator activated receptor gamma co-activator-1, fatty acid synthase, fatty acid binding protein-4 and apolipoprotein A4 genes in these mice [18]. To prove miR-205 has the potential to act as a biomarker for other diseases such as Hidradenitis suppurativa, it was found that this, along with other RNAs, could be important for regulating skin and wound repair and age-related alterations [21].Liver cancerLiver cancer (LC), malignant components are linked to viral infections (hepatitis B and hepatitis C virus), obesity, and inflammation. However, viral infections do not lead directly to liver cancer. More frequently, such infections develop into LF and LCi which, when left untreated can progress to LC.HCC is the most common type of primary liver cancer and one of the most aggressive malignances, indicating the imperative course of action in terms of prognosis and an improved treatment strategy. miRNA markers in HCC diagnosis evaluation suggests that five miRNAs (miR-124, miR-141, miR-205, miR-208a, and miR-499a) were significantly upregulated in the serum of HCC patients compared with chronic hepatitis with advanced LF [17]. Receiver operating characteristic curves were prepared for these miRNAs to identify the best miRNA for the diagnosis of HCC. This data disclosed that miR-205 exhibited highest under receiver operating characteristic: 0.9 (0.81–0.99) along with good sensitivity (85%) and specificity (85.19). Sun et al. [22], reported that miR-205 expression is low while CDKL3 is high in liver cancer tissues compared with normal healthy people. The in vitro and in vivo tumorigenic inhibitory effects of miR-205 is achieved via bone marrow mesenchymal stem cells-secreted exosomal delivery mechanism. It was shown that miR-205 exhibits an inhibitory effect in the progression of LC through the regulation of CDKL3 [22]. Heat shock protein family of Hsp40, its member being A1 (DNAJA1), have shown higher cell proliferation, invasion and angiogenesis in liver cancer cells. The mRNA levels of miR-205 and DNAJA1 are negatively corelated in liver cancer [23]. DNAJA1 along with EF1A1, has been suggested to promote proliferation of liver cancer cells, while miR-205 regulates DNAJA1 induced proliferation and metastasis of liver cancer cells. Together, this study advice miR-205-5p/EF1A1 axis may be a potential biomarker to predict the prognosis for liver cancer patients.Interestingly enough, when miR-205 is downregulated, it promotes stem cell inhibition of HCC [24]. Similar effects have been observed in ubiquitin specific peptidase 7 (UPS7), an essential component for LC progression and regulation of p53, in that miR-205 acts as an inhibitor of UPS7 and helps channeling p53 into check of cell's proliferation levels [25]. Furthermore, when the expression of miR-205 host gene role was investigated, it showed that miR-205-5p activates the pathway signaling of PI3K/AKT and helps in the suppression, migration, proliferation and invasion of hepatoblastoma cells [26].HCC is highly correlated with the HBV infection. HBV X (HBx) protein plays crucial role in HCC development. In LC development, it has been suggested that HBx inhibits miR-205, enhancing tumor cell proliferation via hypermethylation of miR-205 [27]. HBx raises the levels of cellular cholesterol (metabolite of ACSL4), which can be blocked by miR-205 [28]. Low levels of miR-205 were negatively corelated with ACSL1 levels in clinical HCC patient tissue samples. miR-205 restoration is liver cancer cells involved in deregulation of lipid metabolism through targeting acyl-CoA synthetase long-chain family member 1 (ACSL1) [29]. All these events elucidate that miR-205 may play a role as a tumor suppressing miRNA whose down regulation may induce hepatocarcinogenesis [30].ConclusionThe continuum of liver disease has been a critical component of global public health interest as its mortality and morbidity reaches millions of individuals each year. The function and activity miR-205 varies in various liver diseases; however, if applied optimally, it can act as a biomarker and therapeutic agent. Sufficient clinic trial investigations are required to confirm the biomarker/therapeutic potential of miR-205 in due course.Author contributionsM Cabrera, M Kolli and MM Yallapu conceived the idea and wrote the major portion of the manuscript. The manuscript was also written and edited by M Cabrera, M Kolli, M Jaggi, SC Chauhan and MM Yallapu. The final manuscript has been read and approved by all authors.Financial & competing interests disclosureThis work was partially supported by UTRGV startup, CPRIT (RP210180), and NIH (GM139727, CA210192, CA206069, and CA204552) to MM Yallapu, M Jaggi and SC Chauhan. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. This work was supported by National Institutes of Health (GM139727, CA210192, CA206069, and CA204552). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.References1. Asrani SK, Devarbhavi H, Eaton J, Kamath PS. Burden of liver diseases in the world. J. Hepatol. 70(1), 151–171 (2019).Crossref, Google Scholar2. Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M. Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology 64(1), 73–84 (2016).Crossref, Google Scholar3. He A, Zhu L, Gupta N, Chang Y, Fang F. Overexpression of micro ribonucleic acid 29, highly up-regulated in diabetic rats, leads to insulin resistance in 3T3-L1 adipocytes. 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Commun. 444(2), 270–275 (2014).Crossref, CAS, Google Scholar30. Lai X, Gupta SK, Schmitz U et al. MiR-205-5p and miR-342-3p cooperate in the repression of the E2F1 transcription factor in the context of anticancer chemotherapy resistance. Theranostics 8(4), 1106–1120 (2018).Crossref, CAS, Google ScholarFiguresReferencesRelatedDetails Vol. 4, No. 3 Follow us on social media for the latest updates Metrics History Received 1 December 2022 Accepted 6 December 2022 Published online 27 January 2023 Published in print September 2022 Information© 2023 Future Science LtdKeywordschemotherapyliver cancerliver diseasesliver fibrosismiRNAnonalcohol fatty liverAuthor contributionsM Cabrera, M Kolli and MM Yallapu conceived the idea and wrote the major portion of the manuscript. The manuscript was also written and edited by M Cabrera, M Kolli, M Jaggi, SC Chauhan and MM Yallapu. The final manuscript has been read and approved by all authors.Financial & competing interests disclosureThis work was partially supported by UTRGV startup, CPRIT (RP210180), and NIH (GM139727, CA210192, CA206069, and CA204552) to MM Yallapu, M Jaggi and SC Chauhan. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. This work was supported by National Institutes of Health (GM139727, CA210192, CA206069, and CA204552). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download

The standard drug discovery paradigm of single molecule – single biological target – single biological effect is perhaps particularly unsuitable for anti-infective drug discovery. This is due to the rapid evolution of resistance likely to be observed with single target drugs. Multitargeted anti-infective drugs are likely to be superior due to their lower susceptibility to target-related resistance mechanisms. Strathclyde minor groove binders are a class of compounds which have been developed by adopting the multitargeted anti-infective drugs paradigm, and their effectiveness against a wide range of pathogenic organisms is discussed. The renaming of this class to Strathclyde nucleic acid binders is also presented due to their likely targets including both DNA and RNA.

Future Drug DiscoveryVol. 4, No. 2 EditorialOpen AccessShedding light on the dark genome: drugging long non-coding RNAStefan Schiesser, Werngard Czechtizky & Rhona J CoxStefan Schiesser *Author for correspondence: Tel.: +46 0 73 093 7442; E-mail Address: stefan.schiesser@astrazeneca.comhttps://orcid.org/0000-0002-8668-2844Department of Medicinal Chemistry, Research & Early Development, Respiratory & Immunology (R&I), BioPharmaceuticals R&D, AstraZeneca, Pepparedsleden 1, Mölndal, 43183, Sweden, Werngard CzechtizkyDepartment of Medicinal Chemistry, Research & Early Development, Respiratory & Immunology (R&I), BioPharmaceuticals R&D, AstraZeneca, Pepparedsleden 1, Mölndal, 43183, Sweden & Rhona J CoxDepartment of Medicinal Chemistry, Research & Early Development, Cardiovascular, Renal & Metabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca, Pepparedsleden 1, Mölndal, 43183, SwedenPublished Online:15 Jul 2022https://doi.org/10.4155/fdd-2022-0009AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: long non-coding RNAlncRNAantisense RNAdark genomeRNA structurerisdiplamsmall moleculedrug discoveryThe 2020 US FDA approval of risdiplam – a small molecule altering the splicing pattern of the pre-mRNA of SMN2 – proved that modulating disease-associated proteins at the coding RNA level with small molecules can be a successful clinical strategy to treat disease, until then the arena of oligonucleotide therapeutics. However, only around 1.5% of the genome encodes for proteins, with the remainder long viewed as 'junk RNA'. Recent advancements in biology now indicate that this so-called 'dark genome' does in fact contain non-coding RNA which is biologically relevant and can be associated with disease. Targeting non-coding RNA might therefore open up innovative mechanisms of action to modulate disease.Non-coding RNAs are arbitrarily divided in small non-coding RNA containing less than 200 nucleotides and long non-coding RNA (lncRNA) with more than 200 nucleotides (with some lncRNAs having tens of thousands). Recent drug discovery efforts have mainly been focused on modulating the biology of small non-coding RNA [1], but substantial progress has also been made in elucidating the structure and structure-function relationships of lncRNAs, paving the way for the discovery of functional small molecule binders. In this editorial we discuss the state-of-the-art of small molecule binders to lncRNAs and challenges to be solved.Humans are estimated to have more than 15,000 lncRNAs [2] although recent studies show that some lncRNAs might have been misannotated and possess small open reading frames encoding for functional peptides [3]. lncRNAs can be classified according to their localization in relation to genes as intergenic lncRNA, intronic lncRNA, sense lncRNA, antisense lncRNA (including natural antisense transcripts), bidirectional lncRNA, enhancer RNA and promotor upstream transcripts [4]. Like mRNA, lncRNAs are mainly transcribed by RNA polymerase II. They can undergo 5′ m7G-capping, splicing and 3′-polyadenylation [5], and are often associated with epigenetic modifications similar to protein coding genes.lncRNAs are involved in the development of various cancers, neurodegenerative disorders, inflammatory diseases, neuromuscular diseases and the innate immune response to viral infections [5]. They exert their function by interacting with DNA, RNA or proteins. They act as signals, decoys, guides and/or scaffolds to modulate transcription, translation, splicing patterns, protein/RNA stability and chromatin remodeling [5].Having at least one structurally defined region with a suitable binding pocket – either already present in the apo form or induced upon binding of the small molecule – can be beneficial for finding suitable small molecule modulators of lncRNA function. At this point, one can speculate that targeting of tertiary structure will give higher propensity for selective binders than targeting of secondary structure alone. Encouragingly, several reports prove that lncRNAs do have defined secondary and tertiary structures consisting of independent structural domains/modules with multiple helices, loops and junctions, and a considerable percentage of nucleotides engaged in base pairing [6]. Additionally, a deeper understanding of the structure–function relationship has been obtained for several lncRNAs [7] such as the impact of their structure on cellular localization [8] and the domains involved in protein binding. Consequently several modes of action for small molecules altering the pharmacological effect of lncRNAs can be envisioned. For example, a small molecule could (de)stabilize certain conformations of the lncRNA or compete with effector molecules (proteins, DNA or RNA) for binding to the lncRNA to drive the desired pharmacological effect.One of the best characterized lncRNA in the literature is HOTAIR, which is deregulated in various types of cancer. It facilitates chromatin remodeling through interaction of the 5′-end with EZH2, the catalytic subunit of the protein PRC2. Modeling of the lncRNA suggested that the binding region contains several hairpin loops, and a virtual screen was used to propose compounds [9] that could block the interaction with EZH2. Screening using a luciferase assay for silencing of NLK, a transcriptional target of HOTAIR, confirmed the activity of a handful of small molecules including ADQ (Figure 1); further selectivity analysis then confirmed that ADQ indeed binds to the lncRNA. A classical small molecule medicinal chemist may not regard ADQ as an ideal starting point for a drug discovery program, and we may question whether the downstream effects seen are all due to specific HOTAIR binding, but ADQ is a rare and early example of a small molecule with confirmed binding to a lncRNA.Figure 1. Selected structures of small molecules binding to long non-coding RNAs.MALAT1 is another well-characterized lncRNA. Overexpression is associated with breast cancer, and gene knock-out or the use of an inhibitory antisense oligonucleotide have been shown to have beneficial effects in preclinical oncology models. The 3′-end appears to be associated with function, and an X-ray [10,11] of this region shows a stem loop wrapped around the tail to form a triple helix. DPFp8 [12] was identified as a stabilizer of this triple helix with selectivity over four other RNA secondary structures, while no cellular assay data were reported. Other selective MALAT1 triple helix binders such as 'compound 5' and 'compound 16' [13] have also been identified and fall into more typical drug-like space. They have been evaluated in an oncology cell model, and appear to have different mechanisms, although the precise mechanism of action has not been elucidated.GAS5 is a lncRNA which is decreased in some Type 2 diabetes patients. It contains a premature stop codon, making it susceptible to nonsense-mediated decay. In order to stabilize GAS5 levels, compounds were identified which block the interaction between GAS5 and the protein UPF1, which under normal conditions binds to the stop codon region and tags it for decay. Using a relevant oligonucleotide fragment rather than the full lncRNA and a combinatorial library of cyclic peptidomimetics, screening was carried out in the presence of excess tRNA as a competitor to eliminate nonspecific binding. NP-C86 was identified and shown to bind to GAS5 with high affinity and to increase GAS5 concentration and increase glucose uptake in relevant patient-derived cells [14].In an alternative approach to identify small molecule binders to lncRNA, a group at Merck [15] screened 42 RNA targets, including 11 lncRNAs, against an array of chemically diverse small molecules, looking for those which bind to a single RNA target but no protein target. In comparison with previous methods, this approach requires no prior knowledge of RNA folding. They identified selective binders (structures not published), and concluded that molecules binding lncRNAs cover a similar space as those binding other RNA targets. Very recently, a detailed study on the identification of small molecule binders to the lncRNA Xist was published by Merck [16]. The lead compound X1 was shown to bind specifically to a single nucleotide domain of Xist. Investigations using small angle X-ray scattering followed by ab initio modeling suggested that binding of X1 reduces Xist's conformational flexibility and thus prevents binding of interacting proteins. Specific binding was demonstrated both in vitro and in vivo, and downstream effects of binding were observed in cellular systems.So what are the challenges to make lncRNAs attractive drug discovery targets, and where are the future opportunities? On the upside, lncRNAs are often easily detectable, function at their expression levels without amplification, could serve as biomarkers and can be specifically expressed in certain disease states and tissues. However, there are challenges due to their often low abundance, presence of isoforms – dependent on disease state – and poor conservation across species. Many human lncRNAs are, for example, not identified in mice which makes use of animal models more complicated [17]. In recent decades, RNAs as drug targets have been addressed predominantly through antisense oligonucleotides and small interfering RNAs; and more than ten oligo therapeutics have gained FDA approval. The design of therapeutic oligonucleotides is RNA sequence-based and highly effective, but there remain challenges regarding formulation, delivery to specific tissues, off-target liabilities, cost of goods and sometimes immunogenicity. Some of these challenges could be addressed with small molecules. As outlined above, for small molecules to impact RNA function, it is helpful if the RNA function is mediated by a secondary or tertiary RNA structure [15]. Prediction of secondary structure is increasingly sophisticated, but elucidating the tertiary structure is still a challenge with the complexity increasing with the length of the RNA. However, recent progress has been made in using RNA structure determination assays, such as selective 2′-hydroxyl acylation and primer extension (SHAPE) and psoralen analysis of RNA interactions and structures (PARIS) to map the secondary and tertiary structure of lncRNAs even in living cells [18,19]. Methods used to probe small molecule interactions with RNA such as fluorescent indicator displacement [20] and SHAPE [19] are now complemented by direct binding measurements using, for example, affinity-based mass spectrometry [15]. The knowledge of secondary and tertiary structural motifs combined with upcoming expertise with tools, such as Alphafoid, as well as the identification of an increasing number of small molecule binders to these motifs, will further facilitate the use of machine learning algorithms to predict binders and compose dedicated screening collections.At this point, the functions and roles of many lncRNAs in disease are still a matter of investigation. There are promising first results across several lncRNA targets, but it will be critical to understand the structure and biological function of broader sets of lncRNAs in much more depth to fully exploit their diagnostic, prognostic and therapeutic potential.Author contributionsThis manuscript was written through contributions of all authors. All authors have approved the final version of the manuscript.Financial & competing interests disclosureThe authors declare the following conflict of interest: S Schiesser, W Czechtizky and RJ Cox are employees of AstraZeneca, may own stock or stock options and be a co-inventor of AstraZeneca patents and/or patent applications. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.Open accessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/References1. Meyer S, Williams C, Akahori Y et al. Small molecule recognition of disease-relevant RNA structures. Chem. Rev. 49, 7167–7199 (2020).CAS, Google Scholar2. Derrien T, Johnson R, Bussotti G et al. The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome Res. 22(9), 1775–1789 (2012).Crossref, CAS, Google Scholar3. Hartford C, Lal A. When long noncoding becomes protein coding. Mol. Cell. 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RNA duplex map in living cells reveals higher-order transcriptome structure. Cell 165(5), 1267–1276 (2016).Crossref, CAS, Google Scholar19. Mustoe AM, Busan S, Rice GM et al. Pervasive regulatory functions of mRNA structure revealed by high-resolution SHAPE probing. Cell 173(1), 181–195 (2018).Crossref, CAS, Google Scholar20. Wicks SL, Hargrove AE. Fluorescent indicator displacement assays to identify and characterize small molecule interactions with RNA. Methods 167, 3–14 (2019).Crossref, CAS, Google ScholarFiguresReferencesRelatedDetails Vol. 4, No. 2 Follow us on social media for the latest updates Metrics History Received 9 May 2022 Accepted 16 June 2022 Published online 15 July 2022 Published in print June 2022 Information© 2022 Schiesser, StefanKeywordslong non-coding RNAlncRNAantisense RNAdark genomeRNA structurerisdiplamsmall moleculedrug discoveryAuthor contributionsThis manuscript was written through contributions of all authors. All authors have approved the final version of the manuscript.Financial & competing interests disclosureThe authors declare the following conflict of interest: S Schiesser, W Czechtizky and RJ Cox are employees of AstraZeneca, may own stock or stock options and be a co-inventor of AstraZeneca patents and/or patent applications. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.Open accessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/PDF download

The use of psychedelics as medicines and for overall better brain health is potentially one of the most transformative developments given their immediate and long-lasting therapeutic effects across a plethora of neuropsychiatric disorders and, more recently, some neurodegenerative diseases. The US psychedelic drugs market is forecasted to grow by 16.3% by 2027 due to the increasing prevalence of treatment-resistant depression and mental health disorders. Decades-long restrictions, which date back to when psychedelics were declared controlled substances in 1970, have been lifted to allow researchers to publish on the therapeutic benefits of psychedelics. This review will feature the incredible depth of research underway revealing how psychedelics impact brain structure and function to treat mental health and other neurological disorders.

Future Drug DiscoveryVol. 4, No. 2 EditorialOpen AccessThe possible drug for cancer and metastasis preventionVladimir N PakVladimir N Pak *Author for correspondence: E-mail Address: oncoshut@gmail.comhttps://orcid.org/0000-0002-2009-0416Freelance Researcher, Toronto, ON, CanadaPublished Online:18 May 2022https://doi.org/10.4155/fdd-2022-0008AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: alpha-fetoproteincancerdendritic cellimmunotherapymyeloid-derived suppressor cellnatural killer cellneonatal Fc receptorOnly those who attempt the absurd can achieve the impossible.– Albert EinsteinOne in two people will be diagnosed with cancer in their lifetime in developed countries. Prophylactics and early diagnostics are recommended to everyone. On the other hand, even after the treatments, 90% of cancer patients die of metastasis. Early cancer and metastasis prevention drugs are in demand.Natural killer (NK) cells are key effector cells in cancer immune surveillance. They eliminate tumors, particularly at the initial stages, and play an essential role in tumors and metastases prevention. These cells rapidly kill any foreign cells without prior immunization or MHC I restrictions. NK cells are the primary effectors of innate immunity, as they can destroy low-differentiated cancer stem cells and metastasis.During pregnancy and cancer, myeloid-derived suppressor cells (MDSCs) suppress both innate (including NK cells) and adaptive immunity [1]. MDSCs are a small heterogeneous cell population of immature myeloid progenitors of granulocytes, macrophages and dendritic cells (DCs) at different stages of differentiation generated from a common hematopoietic stem cell in the bone marrow. Targeting MDSCs through multiple approaches increases the antitumor activity of NK cells and increases the efficacy of other therapies.Surprisingly the 'magic target' for cancer treatment/prevention is not the cancer cell itself. Depleting MDSCs unleashes the natural processes of NK and cytotoxic T-cells activation to erase cancer cells and metastases [2]. MDSCs were specifically depleted by the α-fetoprotein (AFP)-daunorubicin conjugate [3]. Next, AFP-maytansine conjugate lead to a statistically significant reduction in tumor volume in mice compared with control groups. There was 100% survival in the AFP-maytansine group of ten animals at day 60, compared with 0% survival in the control group by day 38 [4]. It comes out that AFP-toxin is powerful cancer immunotherapy and, to a less extent, a cancer cells-targeted magic bullet [5].AFP binds compounds naturallyToxins can be chemically conjugated to AFP or its fragments [6]. On the other hand, the serum carrier/transport function of mammalian AFPs was found to be more than 50 different biochemical known compounds [7]. During pregnancy, AFP transfers polyunsaturated fatty acid (PUFA) from the mother to the embryo. The transportation through the placenta goes without AFP-PUFA complex dissociation. Unlike in the placenta, in embryo cells, PUFA is released while AFP goes back for the next PUFA shuttle delivery.Small amounts of AFP naturally exist in the blood because an AFP/AFP receptor (AFPR) autocrine system operates in normal and malignant blood mononuclear cells [8]. Like during pregnancy, AFP can potentiate the activity of compounds with a known high binding affinity, such as diethylstilbestrol, warfarin, quercetin, etc. [7]. The known antitumor activity of moderate toxins: ajoene, capsaicin, sinigrin, gossypol, astaxanthin, etc. can be explained by AFP shuttle delivery to AFP-binding immune and cancer cells. For example, AFP can potentiate paclitaxel antitumor action. In low doses, paclitaxel is not able to directly suppress tumor cell proliferation, induce apoptosis or alter the bone marrow hematopoiesis, but it significantly decreased the accumulation and immunosuppressive activities of tumor-infiltrating MDSCs. It has also reversed immunosuppression and chronic inflammation. Low non cytotoxic doses of paclitaxel modulate the functions of MDSCs in primary skin tumors and lymphoid organs, affect the production of mediators of chronic inflammation and T-cell activities, prolong mice survival and reduce the melanoma burden. They have also been used for enhancing the efficacy of accompanying anticancer therapies [9].Exogenous AFP complexes with dioxin, acetoxychavicol, genistein, curcumin, paclitaxel and other selected toxins inhibited tumor growth in mice and some of them can be applied to cancer patients [10].AFP can shuttle AFP-binding drugs present in the patient's blood. AFP and amphotericin B have shown response in six out of eight cancer patients. The cytokine storm-like reaction with fever and shivering observed immediately during the drugs infusions in patients indicates the immunotherapy action of the complex that depleted MDSCs in the blood ahead of cancer cells. The unleashed immune system eliminates the cancer cells [10].Nevertheless, injection is not an option for cancer and metastasis prevention in everyone during a lifetime. When you ask what patients want – obviously, they want appropriate therapy that works. But if you ask if they want an infusion or an oral drug, most would want an oral drug.However, oral administration is in general not feasible for protein drugs. Unenhanced bioavailability for oral administration for protein/peptide pharmaceuticals accounts for no more than 0–1% of the primary dose [11]. It seems absurd to take AFP-toxin orally.AFP–toxin oral administrationSome stomach cancers, as well as colon and gastric carcinomas, produce AFP. A high level of AFPR has been detected in gastric cancers. Therefore, AFP–toxin oral formulations can be used at least to treat AFPR-positive GI tract cancers [10].A small amount of AFP is produced by almost all cells of the human GI tract. There should be cells that reabsorb the AFP or AFP-nutrient complex back. These can be AFPR-positive stem or regenerating cells of the GI tract. On the other hand, very little albumin is lost in the GI tract. It is possible that any proximal transport of albumin into the intestinal lumen might be compensated for by the neonatal Fc receptor (FcRn)-reuptake or alternately by reabsorption. Such mechanisms might explain the progressive increase in FcRn expression levels from the duodenum to the proximal colon [12]. An adult human gut enterocytes transport the compounds absorbed from the intestine through FcRn-mediated endocytosis. The FcRn controls the fate of three very distinct delivery proteins: IgG, albumin and AFP, through a highly similar mode of binding [13]. FcRn-dependent transcytosis in the gut has been established for IgG and albumin [14].AFP competes with IgG for FcRn binding and has an apparent effect on decreasing IgG levels in preclinical studies. AFP is a high-value FcRn antagonist and can be applied in more than forty autoimmune diseases driven by autoantibodies. It has a similar clinical value to other ligands and monoclonal antibodies in a development (argenx, UCB, affibody) that blocks FcRn activity. The two common side effects reported for FcRn modulators – a headache or a decrease in albumin levels – were not detected during AFP treatments. Recombinant AFP (ACT-101) is currently undergoing the process of drug registration for inflammatory bowel disease, multiple sclerosis, myasthenia gravis, Hashimoto disease and others.IgG loaded with antigens is absorbed from the intestine and reaches FcRn-positive DCs in the lymph nodes. AFP that demonstrates a higher than albumin binding affinity to FcRn [13] can be assumed to transfer its load through enterocytes without dissociation, similar to the IgG antigen complex [15].Cancer treatment with oral porcine AFP & AFP-binding toxinsPorcine AFP (pAFP) is close to but not identical to AFP, with a high homology of the amino acid structure and similar immunologic properties. PAFP-rotenone complex has shown tumor-inhibiting properties in mice models [10]. Nevertheless, an investigation conducted addressing the possible transport properties of pAFP or pAFP-rotenone has shown their absence in both the free form and in the complex with the toxin in the blood after mice gavage. So, unlike injectable, oral pAFP-toxin complex could not reach the blood and deplete MDSCs or distant cancer cells through the AFPR.MDSCs are abundant in the peripheral blood of cancer patients and suppress NK cell antitumor activity, but they are barely detectable in the lymph nodes of healthy subjects. In peripheral lymphoid organs, MDSCs differentiates into DCs and macrophages [16]. In the lamina propria, human monocyte-derived DCs have FcRn, which transports the IgG-antigen to degrade compartments involved in the antigen presentation. DCs processes intestinal antigens, inducing the production of proper suppressor cells that, in turn, induce tolerance to food antigens. The intestinal DCs secrete proteins that suppress the activation of helper cells; thereby, preventing intestinal inflammations.By reducing the antigen-presenting capacity of monocytes/macrophages, AFP functions as an essential factor in the downregulation of the entire immune system in cancer [17]. AFP action on DCs is known to impair the activation of NK cells, similar to MDSCs [18]. On the opposite, like MDSC in the blood, the depletion of FcRn-positive DCs/macrophages [19] in the lamina propria and the gut lymph nodes leads to immune system activation and eventually to distant metastasis elimination. This mechanism of the pAFP-toxin complex action needs research. In any case, oral pAFP complexes with atractyloside, thapsigargin, rotenone, betulinic acid, ajoene and to the least extent with isotretinoin, tocotrienol and vitamin D3 work as cancer immunotherapy in mice [10].The suboptimal doses of the oral pAFP-atractyloside complex have shown a response in six out of 12 metastatic colorectal cancer patients [20]. Unlike injections, prolonged absorption of the complex in the gut does not induce cytokine storm-like reactions. A complete response was observed with small metastases. A better outcome can be expected from an early intake of the complex. In the additional trial, the doses of the pAFP-atractyloside complex were elevated, and the patient with stage IV ovarian cancer survived over 10 years [10].The pAFP-betulinic acid and pAFP-ajoene complexes demonstrated better tumor-inhibiting activity among those that can be permitted as supplements. Taken once or twice a year course, they can serve as a check-up of the immune system and prevent cancer and metastasis at the early stages.Future perspectiveAFP will pass the drug registration and be available for autoimmune, cancer and other diseases treatments. AFP chemical conjugate or AFP non-covalent complex with potent toxin will be used as injectable or oral drug for cancer and metastasis treatment. Porcine AFP with moderate toxins from traditional medicine can be manufactured as supplements for cancer prophylactics.Financial & competing interests disclosureThe author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.Open accessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/References1. Ostrand-Rosenberg S, Fenselau C. Myeloid-derived suppressor cells immune-suppressive cells that impair antitumor immunity and are sculpted by their environment. J. Immunol. 200, 422–431 (2018).Crossref, CAS, Google Scholar2. Pak VN. Selective targeting of myeloid-derived suppressor cells in cancer patients through AFP-binding receptors. Future Sci. OA 5, FSO321 (2018).Link, Google Scholar3. Belyaev NN, Abdolla N, Perfilueva Yu et al. Daunorubicin conjugated with alpha-fetoprotein selectively eliminates myeloid-derived suppressor cells MDSCs and inhibits experimental tumor growth. Cancer Immunol. Immunother. 67, 101–111 (2017).Crossref, Google Scholar4. Sherman I, Boohaker R, Stinson K, Griffin P, Hill W. AFP-maytansine conjugate - a novel targeted cancer immunotherapy. European Society of Medical Oncology (ESMO) Annual Meeting being Held Virtually September 16–21., Abstract 2838 (2021) (Poster 523P).Google Scholar5. Pak VN. The perfect combination of the most powerful cancer immunotherapy with the best targeted chemotherapy. Canc. Ther. Oncol. Int J. 20(5), 556050 (2022).Google Scholar6. Lin B, Dong X, Wang Q, Li W, Zhu M, Li M. AFP-Inhibiting fragments for drug delivery: the promise and challenges of targeting therapeutics to cancers. Front. Cell Dev. Biol. 9, 635476 (2021).Crossref, Google Scholar7. Mizejewski GJ. A compendium of ligands reported to bind alpha-fetoprotein: a comprehensive review and metaanalysis. Canc. Ther. Oncol. Int. J. 20(5), 556047 (2022).Google Scholar8. Esteban C, Trojan J, Macho A, Mishal Z, Lafarge-Frayssinet C, Uriel J. Activation of an alpha-fetoprotein/receptor pathway in human normal and malignant peripheral blood mononuclear cells. Leukemia 7(11), 1807–1816 (1993).CAS, Google Scholar9. Sevko A, Michels T, Vrohlings M et al. Antitumor effect of paclitaxel is mediated by inhibition of myeloid-derived suppressor cells and chronic inflammation in the spontaneous melanoma model. J. Immunol. 190(5), 2464–2471 (2013).Crossref, CAS, Google Scholar10. Pak VN. In: Alpha-fetoprotein and Its Receptor in Fixing the Cancer Brakes. Cambridge Scholars Publishing, Tyne, England, 209 (2021).Google Scholar11. McCrudden MTC, Singh TRR, Migalska K, Donnelly RF. Strategies for enhanced peptide and protein delivery. Ther. Deliv. 4(5), 593–614 (2013).Link, CAS, Google Scholar12. Hornby PJ, Cooper PR, Kliwinski C et al. Human and non-human primate intestinal FcRn expression and immunoglobulin G transcytosis. Pharm. Res. 31, 908–922 (2014).Crossref, CAS, Google Scholar13. Blumberg R, Baker SK, Pyzik M, Gandhi A. Methods to manipulate alpha-fetoprotein (AFP). US0200031928 (2020).Google Scholar14. Pyzik M, Sand KMK, Hubbard JJ, Andersen JT, Sandlie I, Blumberg RS. The neonatal Fc receptor (FcRn): a misnomer? Front. Immunol. 10, 1540 (2019).Crossref, CAS, Google Scholar15. Pyzik M, Rath T, Lencer WI, Baker K, Blumberg RS. FcRn: the architect behind the immune and nonimmune functions of IgG and albumin. J. Immunol. 194(10), 4595–4603 (2015).Crossref, CAS, Google Scholar16. Kwak T, Wang F, Deng H, Condamin T, Kumar V, Perego M et al. Distinct populations of immune-suppressive macrophages differentiate from monocytic myeloid-derived suppressor Cells in Cancer. Cell Rep. 33, 108571 (2020).Crossref, CAS, Google Scholar17. Laan-Pütsep K, Wigzell H, Cotran P, Gidlund M. Human α-fetoprotein (AFP) causes a selective down regulation of monocyte MHC class II molecules without altering other induced or noninduced monocyte markers or functions in monocytoid cell lines. Cell. Immunol. 133(2), 506–1810 (1991).Crossref, CAS, Google Scholar18. Yamamoto M, Tatsumi T, Miyagi T et al. α-Fetoprotein impairs activation of natural killer cells by inhibiting the function of dendritic cells. Clini. Experim. Immunol. 165(2), 211–219 (2011).Crossref, CAS, Google Scholar19. Latvala S, Jacobsen B, Otteneder MB, Herrmann A, Kronenberg S. Distribution of FcRn across species and tissues. J. Histochem. Cytochem. 65(6), 321–333 (2017).Crossref, CAS, Google Scholar20. Pak V, Molchanov O, Vincent M. Treatment of metastatic colorectal cancer with aimpila a glycoside/alphafetoprotein complex. J. Clin. Oncol. 25, 3589–3589 (2007).Crossref, Google ScholarFiguresReferencesRelatedDetails Vol. 4, No. 2 Follow us on social media for the latest updates Metrics Downloaded 463 times History Received 11 April 2022 Accepted 25 April 2022 Published online 18 May 2022 Published in print June 2022 Information© 2022 Pak, VladimirKeywordsalpha-fetoproteincancerdendritic cellimmunotherapymyeloid-derived suppressor cellnatural killer cellneonatal Fc receptorFinancial & competing interests disclosureThe author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.Open accessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/PDF download

Future Drug DiscoveryVol. 4, No. 1 CommentaryOpen AccessBreakthrough medicines during the COVID-19 pandemic eraEswara Naga Hanuma Kumar Ghali, Vijian Dhevan, Shravan K Narmala, Meena Jaggi, Subhash C Chauhan & Murali M YallapuEswara Naga Hanuma Kumar GhaliDepartment of Immunology & Microbiology, School of Medicine, The University of Texas Rio Grande Valley, McAllen, TX 78504, USA South Texas Center of Excellence in Cancer Research, School of Medicine, University of Texas Rio Grande Valley, McAllen, TX 78504, USA, Vijian DhevanDepartment of Surgery, University of Texas Rio Grande Valley, Harlingen, TX 78550, USA, Shravan K NarmalaDHR Health Hematology Oncology Institute, DHR Health, Edinburg, TX 78539, USA, Meena JaggiDepartment of Immunology & Microbiology, School of Medicine, The University of Texas Rio Grande Valley, McAllen, TX 78504, USA South Texas Center of Excellence in Cancer Research, School of Medicine, University of Texas Rio Grande Valley, McAllen, TX 78504, USA, Subhash C Chauhan https://orcid.org/0000-0002-3347-5120Department of Immunology & Microbiology, School of Medicine, The University of Texas Rio Grande Valley, McAllen, TX 78504, USA South Texas Center of Excellence in Cancer Research, School of Medicine, University of Texas Rio Grande Valley, McAllen, TX 78504, USA & Murali M Yallapu *Author for correspondence: Tel.: +956 296 1734; E-mail Address: murali.yallapu@utrgv.eduhttps://orcid.org/0000-0002-0073-8828Department of Immunology & Microbiology, School of Medicine, The University of Texas Rio Grande Valley, McAllen, TX 78504, USA South Texas Center of Excellence in Cancer Research, School of Medicine, University of Texas Rio Grande Valley, McAllen, TX 78504, USAPublished Online:8 Feb 2022https://doi.org/10.4155/fdd-2022-0002AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Keywords: antiviralsbiopharmaceuticschemotherapyfood and drug administrationmedicinenatural productsoncologyoral drugsSARS-CoV-2vaccinesThe COVID-19 pandemic has presented an unprecedented challenge by imposing a dramatic loss to human life worldwide. It has had devastating effects on social and economic status [1]. COVID-19 was caused by a zoonotic virus, namely novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) [2]. Globally, as of 23 December 2021, more than 5.3 million deaths were reported to the WHO. The COVID-19 pandemic has not yet ended, and because of constant viral mutations it poses a challenge to the medical community. Recently there have been major breakthroughs in developing mRNA vaccines, monoclonal antibodies, and various other therapeutic agents for COVID-19 [3].Besides the pandemic, it was also focused towards medical and research innovation efforts in the context of development of vaccines, medications, diagnostic/prognostic predictors and biomarkers, screening, transplantation, testing and point-of-care kits, wearable technologies, artificial intelligence, etc. During the last 2 years, notable and unprecedented research resulting in major breakthroughs includes artificial intelligence models for improving cancer and autism detection, discovering cellular mechanisms on genetic mutations in amyotrophic lateral sclerosis, genomic-based targeted and genetic-inhibited cancer treatment, cancer cell hijack mechanisms, immunotherapies, CAR-T therapies, and so on. The pandemic has not slowed the discoveries that led to unprecedented research resulting in major breakthroughs in various human diseases. In this commentary article, we present the most impactful breakthrough medicine innovations for tackling COVID-19, Ebola virus disease (EVD), heart diseases, cancer, Alzheimer's disease, diabetes, and malaria during the pandemic period that have a profound meaning and positive impact on human lives.Breakthrough medicinesMany therapeutic agents or drug molecules are available in the market to protect mankind from dreadful diseases. However, a diverse array of adverse effects are brought into account with drug usage at therapeutic or non therapeutic doses [4]. There is a need to consider the benefit-risk balance. Drug safety has earned a lot of attention as it plays a pivotal role in the development of various medical therapies and novel treatments. Drugs with a high-risk profile should be avoided unless there is an emergency use. Pregnant and lactating women, children and old people are considered as vulnerable groups and appropriate precautions need to be taken before administering medications for these groups [5]. In children, it is very important to use the recommended dose to avoid side effects. Protection of public health by ensuring the efficacy, safety, and security of human drugs is inevitable. The US FDA is taking this responsibility as a key component [6]. In the midst of the pandemic, there were about 5587 (2020) and 4810 (2021) approvals granted by FDA for number of indications. These approvals included as approved supplements, tentative approvals, orphan drugs, emergency use authorization (EUA), and full approvals. In this article, we have mentioned a brief outline of the most important and novel breakthrough medicines during the COVID-19 pandemic. This article also explains the composition and mechanism of action of these FDA approved breakthrough medicines against intended diseases.Coronavirus diseaseCOVID-19, an infectious disease caused by the SARS-CoV-2 virus, is a serious problem, responsible for the current pandemic with extensive morbidity and mortality throughout the world. SARS-CoV-2 uses the same receptor as SARS-CoV, ACE2 [7]. The COVID-19 vaccine drive has made marked progress in minimizing hospitalizations and deaths. RNA and DNA vaccines used genetically engineered RNA or DNA that encode a protein, which provides prompt immune response [8]. According to the 'Our World in Data' report, as of 23 December 2021, about 61% of the US population is fully vaccinated in addition to about 11% of the US population that is partially vaccinated against COVID-19 disease [9]. The FDA approved COVID-19 vaccines (Pfizer-BioNTech, Moderna, and Johnson & Johnson) protect patients against severe illness and hospitalizations due to COVID-19 disease. Pfizer-BioNTech COVID-19 vaccine has an active ingredient, nucleoside-modified mRNA encoding the viral spike (S) glycoprotein of SARS-CoV-2. On 23 August 2021, the FDA approved the Pfizer-BioNTech COVID-19 vaccine after initial EUA in December 2020. The Moderna COVID-19 vaccine is composed of nucleoside-modified mRNA encoding the viral spike (S) glycoprotein of SARS-CoV-2 as an active ingredient [10]. Both the Pfizer and Moderna vaccines provide a blueprint for the cells by using mRNA, and buildup the body's defense against the virus by allowing the generation of an antibody response which can attack the virus if the vaccinated individual is exposed. Johnson & Johnson's COVID-19 vaccine contains recombinant replication-incompetent adenovirus type 26 vector, that encodes a stabilized variant of the SARS-CoV-2 spike (S) protein as an active ingredient. It triggers an immune response and mimics natural infection; thereby, protecting people against future infections [11]. The FDA approved the first antiviral oral drugs, Paxlovid™ (ritonavir, Pfizer) and Lagevrio Lagevrio (molnupiravir, Merck & Co) for the treatment of mild-to-moderate COVID-19 adults and pediatric patients under an EUA. These drug molecules act as polymerase inhibitors and work against replication of the virus genetic material. In addition, Veklury® (remdesivir, Gilead), an intravenous infusion medication of SARS-CoV-2 nucleotide analog RNA polymerase inhibitor chemically named as 2-ethylbutyl N-{(S)-[2-C-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2,5-anhydro-d-altrononitril-6-Oyl]phenoxyphosphoryl}-L-alaninate, has been authorized under EUA for COVID-19 treatment. Besides, some drugs and non vaccine biological products, like Evusheld™ (tixagevimab copackaged with cilgavimab, AstraZeneca), Actemra® (tocilizumab, an IL-6 receptor antagonist, Genentech), Xevudy™ (sotrovimab, GlaxoSmithKline plc and Vir Biotechnology, Inc.), bamlanivimab and etesevimab together (for post exposure prophylaxis in subjects who meet high-risk criteria, Lilly), REGEN-COV (casirivimab and imdevimab, Regeneron Pharmaceuticals), and baricitinib (Olumiant, Lilly) were also permitted to use under EUAs.EVDEVD is a viral hemorrhagic fever disease that can be transmitted through direct contact with a deceased person or a sick person infected with the Ebola virus, or with an infected animal. EVD is a potentially fatal disease in humans. Inmazeb™ (Regeneron Pharmaceuticals Inc.), a mixture of three monoclonal antibodies, namely maftivimab, atoltivimab, and odesivimab-ebgn, is the first FDA approved drug to treat Zaire ebolavirus (EBOV) infection in pediatric and adult patients [12]. This triple antibody combination efficiently neutralizes the virus by blocking the virus entry into host cells. Similarly, Ebanga (Ansuvimab-zykl), is a single-dose intravenous recombinant human IgG1k monoclonal antibody used to treat EBOV infection in children and adults. It prevents the virus from entering into the cells by binding the virus to the glycoprotein 1 subunit of EBOV, preventing the binding of EBOV to host cells.Heart diseasesCardiovascular disease produces immense health and economic burdens. Ischemic heart disease, the leading cause of death in the world, is responsible for 16% of the world's total deaths, whereas stroke is the second leading cause of death that is responsible for approximately 11% of total deaths. Hypertension, cardiovascular disease, diabetes mellitus, and severe obesity are the main risk factors for heart disease with varying impacts depending on the age of the individual and the country of origin [13]. In 2021, FDA approved the usage of Kerendia® (finerenone, non steroid mineralocorticoid receptor agonist, Bayer) for the reduction of heart attacks, hospitalization for heart failure and cardiovascular deaths for adults with chronic kidney disease associated with Type 2 diabetes [14]. This drug is demonstrated to be helpful in people with Type 2 diabetes at various stages of kidney disease to improve their heart health. On 20 January 2021, Merck announced that Verquvo® (vericiguat), a soluble guanylate cyclase (sGC) stimulator, is approved to mitigate the risk of cardiovascular death and hospitalization by chronic heart failure. Nexletol® (bempedoic acid), is a newly approved drug that efficiently reduces cholesterol synthesis via inhibition of adenosine triphosphate citrate lyase (an enzyme upstream from 3-hydroxy-3-methylglutaryl-coenzyme A). This prescription is being used along with a proper diet and other medications to help lower 'bad' cholesterol in the blood (reducing the amount of cholesterol made by the liver), ultimately minimizing the risk of heart disease and lowering the incidence of strokes and heart attacks.CancerCancer is a major public health problem worldwide and is the second leading cause of death in the US. According to the American Cancer Society, there are about 1,898,160 new cancer cases diagnosed and approximately 608,570 people died from cancer in the US in 2021 [15]. A large number of agents, antibodies and drug-conjugates are being developed for the treatment of various cancers. For example, Cytalux™ (Target Laboratories, Inc.), a fluorescent drug that targets folate receptors, is a new FDA approved drug that is composed of pafolacianine and is used to identify ovarian cancer lesions during surgery in adult patients. On 29 October 2021, Scemblix® (asciminib, Novartis), a kinase inhibitor, was approved to treat Philadelphia chromosome-positive chronic myeloid leukemia. Asciminib has a distinct mechanism of action that binds to the BCR-ABL1 Myristoyl Pocket. Thus, it helps to address resistance in patients with chronic myeloid leukemia (CML) previously treated with two or more tyrosine kinase inhibitors (TKIs) and overcome mutations at the defective BCR-ABL1 gene. Besides, other kinase inhibitors such as Retevmo® (selpercatinib, Eli Lilly and Company) and Tabrecta® (capmatinib, Novartis) are approved to treat lung, thyroid, and non-small cell lung cancer (NSCLC), respectively.Tivdak™ (tisotumab vedotin-tftv, Seagen), is a microtubule inhibitor conjugate and a tissue factor-directed antibody, indicated to treat recurrent or metastatic cervical cancer with disease progression on or after chemotherapy. Likewise, Exkivity™ (mobocertinib, Takeda), a kinase inhibitor, was approved on 15 September 2021, for the treatment of metastatic or locally advanced NSCLC. This drug is effective in patients with EGFR exon 20 insertion mutations and whose disease has progressed on or after platinum-based chemotherapy. Rylaze™ (asparaginase erwinia chrysanthemi (recombinant)-rywn, Jazz Pharmaceuticals Ireland Limited), an asparagine-specific enzyme, used to treat lymphoblastic lymphoma and acute lymphoblastic leukemia in E. coli derived asparaginase products-allergic patients. Lumakras™ (sotorasib), an inhibitor of the RAS GTPase family (KRAS G12C inhibitor), is indicated for the treatment of various types of NSCLC (KRAS G12C positive) are locally advanced or metastatic and who have received at least one prior treatment. Pylarify® (piflufolastat F 18 or 8F-DCFPyL or PyL, Progenics Pharmaceuticals, Inc.), a radioactive diagnostic agent, approved to recognize prostate-specific membrane antigen (PSMA) positive lesions in prostate cancer. Rybrevant® (amivantamab-vmjw, Janssen Biotech, Inc.), a bispecific EGFR antibody treatment, is recommended to treat a subset of NSCLC (EGFR exon 20 insertion mutations). Under accelerated approval, Zynlonta (loncastuximab tesirine-lpyl, ADC Therapeutics SA), an alkylating agent conjugate and CD19-directed antibody, meant to treat various types of refractory or relapsed large B-cell lymphomas. In addition, Jemperli (dostarlimab-gxly, GlaxoSmithKline), a programmed cell death receptor-1 (PD-1) blocking antibody, approved for the treatment of adult patients with mismatch repair-deficient (dMMR) recurrent or advanced solid tumors. While, Fotivda® (tivozanib, an oral VEGF receptor tyrosine kinase inhibitor, AVEO Pharmaceuticals, Inc.) and Tepmetko® (tepotinib, Merck KGaA) are the kinase inhibitors that were approved to treat endometrial cancer, refractory or relapsed advance renal cell carcinoma and NSCLC, respectively. Other kinase inhibitors like Cosela™ (trilacicilib, G1 Therapeutics, Inc.) and Ukoniq® (umbralisib, TG Therapeutics Inc.) are used to reduce the chemotherapy-induced myelosuppression in small cell lung cancer, and treat follicular lymphoma and marginal zone lymphoma respectively. Similarly, Tukysa® (tucatinib, Seagen Inc.) and Ayvakit™ (avapritinib, Blueprint Medicines Corporation), also a kinase inhibitor, have been clinically used for the treatment of advanced unresectable or metastatic HER2-positive breast cancer in combination with trastuzumab and capecitabine, and metastatic gastrointestinal stromal tumors.In 2020, the FDA approved Orgovyx™ (relugolix, androgen deprivation therapy, Myovant Sciences GmbH and Pfizer Inc.), a gonadotropin-releasing hormone (GnRH) receptor antagonist, which is indicated for the treatment of advanced prostate cancer. In addition, Margenza® (margetuximab [anti-HER2 mAb], MacroGenics, Inc.), a HER2/neu receptor antagonist, in combination with chemotherapy, is used to treat HER2+ breast cancer. Similarly, Gallium 68 PSMA-11 (Gallium 68 PSMA-11), a radioactive diagnostic agent, is indicated to identify the positive lesions of PSMA in men using positron emission tomography (PET). Gavreto® (pralsetinib, Blueprint Medicines Corporation and Genentech, Inc.), a kinase inhibitor, is used to treat NSCLC. Monjuvi® (tafasitamab-cxix, MorphoSys AG), a CD19-directed cytolytic antibody, in combination with lenalidomide, is used for the treatment of relapsed or refractory diffuse large B-cell lymphoma. Zepzelca™ (lurbinectedin, Jazz Pharmaceuticals), an alkylating drug, is indicated to treat metastatic small cell lung cancer. In May 2020, Cerianna™ (fluoroestradiol F18, Zionexa USA), a radioactive diagnostic agent, is used for the detection of estrogen receptor (ER)-positive lesions with PET in patients with metastatic breast cancer. Trodelvy™ (sacituzumab govitecan-hziy, Gilead Sciences, Inc.), a Trop-2-directed antibody and topoisomerase inhibitor conjugate, is used to treat adult patients with metastatic triple-negative breast cancer who received at least two prior therapies for metastatic disease.Alzheimer's diseaseAn estimated 6.2 million Americans (≥65 years) are surviving with Alzheimer's dementia in 2021 [16]. Women are inordinately affected and, in 2019, it was ranked as the 7th leading cause of death. FDA approved the drugs such as Aduhelm® (aducanumab-avwa, Biogen)- an amyloid beta-directed antibody (approved in 2021) and Tauvid™ (flortaucipir F18, Eli Lilly and Company) a radioactive diagnostic agent (approved in 2020) for the treatment and diagnostic purposes.DiabetesDiabetes, a chronic disease characterized by elevated levels of blood glucose, has entered the top ten leading causes of death. It is also responsible for the largest rise in deaths in males among the top ten, with an 80% increase, since 2000. In 2021, Kerendia (Bayer), a non steroidal mineralocorticoid receptor antagonist, is a drug approved for the usage by FDA to reduce the risk of heart and kidney complications associated with Type 2 diabetes. It is composed of finerenone [17].MalariaHuman malaria is one of the most rampant human infectious diseases worldwide [18]. As per the Centers for Disease Control and Prevention, approximately 2000 cases of malaria were being diagnosed in the US each year. Patients often experience fever, flu-like illness, chills and chronic conditions that may cause severe complications like seizures, kidney failure, mental confusion and death. Artesunate (Amivas US, LLC) is the first-line treatment for children or adults with severe malaria, in combination with other antiviral agents.In addition to the above notable breakthrough medicines, NTLA-2001 (Intellia Therapeutics), an Investigational CRISPR therapy to treat transthyretin amyloidosis, acts by editing targeted human TTR gene [19]. Zokinvy (lonafarnib, Eiger BioPharmaceuticals) is an orally active farnesyltransferase inhibitor used to prevent premature aging and to treat progeroid and progeria laminopathies [20]. A triterpenoid antifungal, Brexafemme® (ibrexafungerp, Scynexis, Inc.), is used for the treatment of post menarchal pediatric females and adults with vulvovaginal candidiasis [21].ConclusionThe past 2 years, marked as the COVID-19 pandemic era, posed an unprecedented challenge to all human beings across the globe. The innovations and hard work of pharmaceutical and medical sciences in 2020 and 2021 resulted in many therapeutic lead molecules making to the list of FDA approvals. This includes several small molecules, monoclonal antibodies, imaging agents and antibody-drug conjugates for numerous unmet clinical needs. Together, the efforts of this commentary article are to provide some novel breakthrough medicines that are evident for successful clinical use for coronavirus disease, EVD, heart disease, cancer, Alzheimer's disease, diabetes and malaria. However, some emergency authorized use recommendations have to be analyzed further for their long-term clinical safety and continued usage.Author contributionsENHK Ghali and MM Yallapu conceived the idea and wrote the major portion of the manuscript and editing. The manuscript was also written and edited by V Dhevan, SK Narmala, M Jaggi, and SC Chauhan. The final manuscript has been read and approved by all authors.Financial & competing interests disclosureThis work was partially supported by UTRGV startup and CPRIT (RP210180) and NIH (SC1 GM139727, R01 CA210192, R01 CA206069 and R01 CA204552) to SC Chauhan, M Jaggi and MM Yallapu. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.Open accessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/References1. Nicola M, Alsafi Z, Sohrabi C et al. The socio-economic implications of the coronavirus pandemic (COVID-19): a review. Int. J. Surg. 78, 185–193 (2020).Crossref, Google Scholar2. Brix TH, Hegedüs L. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and thyroid disease. An update. Curr. Opin. Endocrinol. Diabetes Obes. 28(5), 525 (2021).Crossref, CAS, Google Scholar3. Excler JL, Saville M, Berkley S, Kim JH. Vaccine development for emerging infectious diseases. Nat. Med. 27(4), 591–600 (2021).Crossref, CAS, Google Scholar4. Keni R, Alexander A, Nayak PG, Mudgal J, Nandakumar K. COVID-19: emergence, spread, possible treatments, and global burden. Public Health Front. 8, 216 (2020).Crossref, Google Scholar5. Dathe K, Schaefer C. The use of medication in pregnancy. Dtsch. Arztebl. Int. 116(46), 783 (2019).Google Scholar6. Nielsen J, Eckstein L, Nicol D, Stewart C. Integrating public participation, transparency and accountability into governance of marketing authorisation for genome editing products. Front. Polit. Sci. 3, 1–13 (2021).Crossref, Google Scholar7. Hu B, Guo H, Zhou P, Zh LS. Characteristics of SARS-CoV-2 and COVID-19.. Nat. Rev. Microbiol. 19, 141–154 (2021).Crossref, CAS, Google Scholar8. Mascellino MT, Di Timoteo F, De Angelis M, Oliva A. Overview of the main anti-SARS-CoV-2 vaccines: mechanism of action, efficacy and safety. Infect. Drug Resist. 14, 3459 (2021).Crossref, Google Scholar9. Ritchie H, Mathieu E, Rodés-Guirao L et al. Coronavirus Pandemic (COVID-19) (2020). https://ourworldindata.org/coronavirusGoogle Scholar10. Moderna Information on COVID-19 Vaccine (2022). https://www.modernatx.com/covid19vaccine-eua/providers/about-vaccineGoogle Scholar11. Janssen COVID-19 Vaccine (Ad26.COV2.S) (2022). https://www.janssenmd.com/janssen-covid19-vaccine/pharmacology/mechanism-of-action/mechanism-of-action-of-the-janssen-covid19-vaccineGoogle Scholar12. Markham A. REGN-EB3: first approval. Drugs 81(1), 1–4 (2021).Crossref, Google Scholar13. Logette E, Lorin C, Favreau C et al. A machine-generated view of the role of blood glucose levels in the severity of COVID-19. Public Health Front. 9, 1–52 (2021).Crossref, Google Scholar14. Bakris GL, Agarwal R, Anker SD et al. Effect of finerenone on chronic kidney disease outcomes in Type 2 diabetes. N. Engl. J. Med. 383(23), 2219–2229 (2020).Crossref, CAS, Google Scholar15. Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2021. CA Cancer J. Clin. 71(1), 7–33 (2021).Crossref, Google Scholar16. Alzheimer's Association Report. 2021 Alzheimer's disease facts and figures. Alzheimer's & Demnetia 17(3), 327–406 (2021).Crossref, Google Scholar17. Filippatos G, Anker SD, Agarwal R et al. Finerenone and cardiovascular outcomes in patients with chronic kidney disease and Type 2 diabetes. Circulation 143(6), 540–552 (2021).Crossref, CAS, Google Scholar18. Dye-Braumuller KC, Kanyangarara M. Malaria in the USA:how vulnerable are we to future outbreaks? Curr. Trop. Med. Rep. 14, 1–9 (2021).Google Scholar19. Griffin JM, Rosenthal JL, Grodin JL, Maurer MS, Grogan M, Cheng RK. ATTR amyloidosis: current and emerging management strategies: JACC: cardio oncology state-of-the-art review. Cardio. Oncol. 3(4), 488–505 (2021).Google Scholar20. Dhillon S. Lonafarnib: first approval. Drugs 81(2), 283–289 (2021).Crossref, CAS, Google Scholar21. Lee A. Ibrexafungerp: first approval. Drugs 81(12), 1445–1450 (2021).Crossref, CAS, Google ScholarFiguresReferencesRelatedDetails Vol. 4, No. 1 Follow us on social media for the latest updates Metrics History Received 5 January 2022 Accepted 27 January 2022 Published online 8 February 2022 Published in print March 2022 Information© 2022 Murali M. YallapuKeywordsantiviralsbiopharmaceuticschemotherapyfood and drug administrationmedicinenatural productsoncologyoral drugsSARS-CoV-2vaccinesAuthor contributionsENHK Ghali and MM Yallapu conceived the idea and wrote the major portion of the manuscript and editing. The manuscript was also written and edited by V Dhevan, SK Narmala, M Jaggi, and SC Chauhan. The final manuscript has been read and approved by all authors.Financial & competing interests disclosureThis work was partially supported by UTRGV startup and CPRIT (RP210180) and NIH (SC1 GM139727, R01 CA210192, R01 CA206069 and R01 CA204552) to SC Chauhan, M Jaggi and MM Yallapu. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.Open accessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/PDF download

Future Drug DiscoveryVol. 4, No. 1 ForewordOpen AccessWelcome to Volume 4 of Future Drug DiscoveryHarriet WallHarriet Wall *Author for correspondence: E-mail Address: h.wall@future-science.comhttps://orcid.org/0000-0002-7785-1925Future Science Group, Unitec House, 2 Albert Place, London, N3 1QB, UKPublished Online:13 May 2022https://doi.org/10.4155/fdd-2022-0006AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: 2021artificial intelligenceCOVID-19drug discoveryforewordWelcome to Volume 14 of Future Drug Discovery! We would like to take this opportunity to discuss the highlights seen last year as well as what we can expect throughout 2022. We are grateful to our editorial board members, authors, peer reviewers and readers and we look forward to continuing to work with all of our contributors again in 2022.Content highlightsSARS-CoV-2 (COVID-19) remained a major issue throughout 2021, and the magnitude of its impact on drug discovery is reflected by our continued publication of articles relating to this topic. The journal's most-read article published in 2021 was 'High-throughput approaches of diagnosis and therapies for COVID-19: antibody panels, proteomics and metabolomics' by Rwik Sen (Active Motif, Inc., CA, USA) [1]. This review discussed how studies based on antibodies, proteomics and metabolomics contribute to the development of diagnostics and therapeutics against COVID-19.Additionally, Benedetta Spadaro (University of Cambridge, Cambridge, UK) discussed global strategies for COVID-19 vaccine development in their editorial titled 'COVID-19 vaccines: challenges and promises of trials, manufacturing and allocation of doses' [2]. This popular piece highlighted the challenges and breakthroughs in the scientific community's quest to develop, manufacture and distribute life saving vaccines.Adding to the discussion surrounding COVID-19, Clare L Box (Gifford Bioscience Limited, Birmingham, UK) examined possible avenues of drug development, including drug repurposing, antibody-based therapies, anti-inflammatory drugs and vaccine approaches in their editorial titled 'Evaluation of potential anti-COVID-19 therapies' [3].Following on from this, Mario Cazzola (University of Rome, Italy) discussed the need for new drugs to treat chronic obstructive pulmonary disorder in their editorial, 'Is it time to look beyond bronchodilators and corticosteroids in treating chronic obstructive pulmonary disorder?' [4].Artificial intelligence is becoming a key element in the drug discovery toolbox. In 2021, several articles covering this immense landscape were published in the journal.Our second most read review last year was 'Artificial intelligence in drug design: algorithms, applications, challenges and ethics' [5]. In this article, Alya A Arabi (University College London, London, UK) discussed artificial intelligence and machine learning in drug discovery, as well as touching on the ethical issues that this area presents.Additionally, Jürgen Bajorath (Rheinische Friedrich-Wilhelms-Universität, Bonn, Germany) explained how we can exploit machine learning to differentiate the properties of multitarget and single-target drugs in his editorial titled 'Structural characteristics of compounds with multitarget activity' [6].The use of 3D culture systems such as tumor organoids and tumor-on-a-chip devices has increased tremendously in the past decade. Aleksander Skardal (Ohio State University, OH, USA) evaluated this topic in his review titled 'Biofabrication of advanced in vitro and ex vivo cancer models for disease modeling and drug screening' [7]. This article focuses organoid and organ-on-a-chip metastasis models – noting that developments in these areas are imperative to the future of personalized medicine.Complementing our core content of reviews, editorials and commentaries, in 2021 the journal also published articles on topical areas of debate relevant to academia and industry in the form of interviews with experts in the field. One popular piece was 'An interview with Dr Mike Robertson on the development of novel drugs for HIV treatment in diverse populations' [8], where Mike Robertson (MSD Research Laboratories, NJ, USA) gave his insight on the global burden of HIV in different patient populations, and the development of islatravir – MSD's investigational NRTTI, which is currently in Phase III for HIV treatment and prevention.Further, we spoke to Melanie Leveridge (GlaxoSmithKline, London, UK) in the article 'Melanie Leveridge on ELRIG and a career in drug discovery' [9], where Melanie Leveridge gave advice to early career researchers, and discussed her new position as ELRIG chair.Now more than ever, it is important to invest in drug discovery and bridge the gap across generations within the field. If you are in the first 6 years of your career in drug discovery and are interested in joining Future Drug Discovery's Early Career Panel, please do not hesitate to get in touch [10].Demographics'Future Drug Discovery' has seen a continued increase in readership over the past year, with the majority of readers based in North America, followed by Asia and Europe (Figure 1).Figure 1. Percentage readership by location.Future Drug Discovery continues to receive submissions from around the globe. In 2021, the majority of our authors submitted from Europe, North America and Asia (Figure 2).Figure 2. Percentage authorship by location.Article outreach & online presenceFuture Drug Discovery continues to be active across social media and we are keen for our readers to engage. Follow us on Twitter at @fsdrugdiscovery to stay updated regarding our latest content, including our journal highlights and the latest drug discovery news [11].ConclusionWe appreciate the support and engagement that has been provided over the past year and look forward to what is to come in 2022. As always, we are happy to receive unsolicited articles to the journal, whether they are original research, reviews or opinion pieces. If you have a submission that you would like us to consider, you can find details on article preparation and submission online [12]. Finally, we would like to thank everyone for their continued feedback and support. We look forward to working with all of you over the coming year.Financial & competing interests disclosureH Wall is an employee of Future Science Ltd. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.Open accessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/References1. Sen R. High-throughput approaches of diagnosis and therapies for COVID-19: antibody panels, proteomics and metabolomics. Future Drug Discov. 3(1), 2631–3316 (2021).Link, Google Scholar2. Spadaro B. COVID-19 vaccines: challenges and promises of trials, manufacturing and allocation of doses. Future Drug Discov. 3(1), 2631–3316 (2021).Link, Google Scholar3. Box CL, Thompson KSJ. Evaluation of potential anti-COVID-19 therapies. Future Drug Discov. 3(1), 2631–3316 (2021).Link, Google Scholar4. Cazzola M, Matera MG. Is it time to look beyond bronchodilators and corticosteroids in treating COPD? Future Drug Discov. 3(2), 2631–3316 (2021).Link, Google Scholar5. Arabi AA. Artificial intelligence in drug design: algorithms, applications, challenges and ethics. Future Drug Discov. 3(2), 2631–3316 (2021).Link, Google Scholar6. Bajorath J. Structural characteristics of compounds with multitarget activity. Future Drug Discov. 3(2), 2631–3316 (2021).Link, Google Scholar7. Nairon KG, Skardal A. Biofabrication of advanced in vitro and ex vivo cancer models for disease modeling and drug screening. Future Drug Discov. 3(3), 2631–3316 (2021).Link, Google Scholar8. Robertson M. An interview with Dr. Mike Robertson on the development of novel drugs for HIV treatment in diverse populations. Future Drug Discov. 3(3), 2631–3316 (2021).Link, Google Scholar9. Leveridge M. Melanie Leveridge on ELRIG and a career in drug discovery. Future Drug Discov. 3(1), 2631–3316 (2021).Link, Google Scholar10. Future Drug Discovery. Future Drug Discovery, Early Career Panel. https://www.future-science.com/journal/fdd/earlycareerresearch/panelmembers Google Scholar11. @fsdrugdiscovery on twitter. Future Drug Discovery, Twitter page. http://www.twitter.com/fsdrugdiscovery Google Scholar12. Future Drug Discovery. Future Drug Discovery. https://www.future-science.com/journal/fdd Google ScholarFiguresReferencesRelatedDetails Vol. 4, No. 1 Follow us on social media for the latest updates Metrics History Received 8 April 2022 Accepted 8 April 2022 Published online 13 May 2022 Published in print March 2022 Information© 2022 Newlands Press LtdKeywords2021artificial intelligenceCOVID-19drug discoveryforewordFinancial & competing interests disclosureH Wall is an employee of Future Science Ltd. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.Open accessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/PDF download

Future Drug DiscoveryVol. 4, No. 1 InterviewOpen AccessInterview with Hunterian CEO Dr Vinod Jaskula-RangaVinod Jaskula-RangaVinod Jaskula-Ranga *Author for correspondence: E-mail Address: vinny@hunterian.comhttps://orcid.org/0000-0003-1202-634X100 Pacific Street, Cambridge, MA 02139, USAPublished Online:17 Jan 2022https://doi.org/10.4155/fdd-2021-0011AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Keywords: CRISPRcystic fibrosisgene editinginterviewPlease tell us about your career to date & what inspired you to take this path?I am the Founder and CEO of Hunterian Medicine, a gene editing and gene therapy start-up with locations in Cambridge, Massachusetts and Baltimore, Maryland. Before founding Hunterian, I was at Johns Hopkins for many years. I trained in the laboratory of Dr Carol Greider at the Johns Hopkins School of Medicine, receiving my doctorate in molecular biology and genetics. It was especially exciting to be part of her lab when she won the Nobel Prize. I then completed a fellowship in genetic engineering and molecular ophthalmology at the Wilmer Eye Institute of Johns Hopkins Medicine, where I initially used TALENs to edit the genes of human embryonic stem cells. I began to work on CRISPR technologies in 2012/2013 after reading a series of seminal papers by Jennifer Doudna, Emmanuelle Charpentier, Feng Zhang, George Church and others and translating the technology into therapeutics has been the entire focus of my work ever since.Please give an overview of the technology platform Hunterian Medicine has developed?Hunterian's platform technology solves the adeno-associated virus (AAV) in vivo delivery problem for CRISPR gene editing, enabling delivery using a single AAV, as well as select gene therapy applications. AAV is the gold standard for gene delivery, with both US FDA and EMA approvals, but its small size means large genes cannot fit inside using existing technologies. For CRISPR, the commonly used SpCas9 nuclease and the guide RNA are thought to be too large to fit inside a single AAV.Hunterian's platform technology is based on the discovery of novel genetic elements – compact promoters – that significantly reduce the length of the sequence required for in vivo expression. This enables in vivo delivery of CRISPR-SpCas9, as well as high-fidelity variants with undetectable off-targets, PAM variants and other CRISPR systems such as Cas12a, all through a single AAV. The result is that literally billions more sites in the genome can be targeted using CRISPR and therefore many more diseases.The in vivo delivery problem is best illustrated by the rapid development of ex vivo therapeutic applications with CRISPR, such as sickle cell disease. Here, a patient's bone marrow can be extracted, with gene editing taking place in a GMP facility and then reintroduced back to the patient without the challenge of in vivo delivery. However, most genetic diseases will require in vivo rather than ex vivo delivery of CRISPR – a patient's muscles, brain, lungs, etc. cannot be removed and worked on in a lab – so the vast potential of CRISPR to cure human diseases remains unrealized. We believe Hunterian's in vivo delivery technology has the potential to cure many genetic diseases affecting millions of people.Please tell us more about the work you are doing with your technology to develop a gene therapy for cystic fibrosisCystic fibrosis (CF) is a debilitating and ultimately fatal disease caused by mutations in the CFTR-encoded chloride channel. Despite remarkable therapeutic advances that have increased life expectancy, many patients do not respond to current medications, in particular for those carrying nonsense or splicing mutations. Hunterian was recently awarded the Therapeutics Development Award by the Cystic Fibrosis Foundation to develop a gene therapy for CF using our compact promoters. Unlike current treatments, a gene therapy for CF could benefit every CF patient irrespective of the patient's particular type of CFTR mutation.The same AAV capacity problem that I mentioned for CRISPR pertains to large genes such as CFTR. Hunterian's technology uses compact promoters to enable packaging of the full-length CFTR gene within a single AAV vector. Early gene therapy trials omitted a promoter to enable packaging of the full-length CFTR gene; however, these trials failed to detect expression, and as a result, a focus over the past couple of decades has been around the use of truncated genes, and this is not an ideal approach for a number of reasons.Beyond CFTR, it is widely assumed that all relevant genes that can be delivered by AAV are being developed into therapeutics; however, we have identified several genes of interest that were previously believed to be too large to fit into AAV.How are you hoping to collaborate with other research institutions to develop these therapies & what is the advantage of such collaborations?Our strengths center around our platform technology, which is applicable to a wide range of diseases, so we consider it essential to engage with disease-specific and clinical experts. These types of collaborations allow us to focus on developing our platform technology, while drawing on the deep expertise of scientists and clinicians who have dedicated their careers to understanding and treating particular diseases. A wonderful benefit from my training and many years at Johns Hopkins is that I can easily reach out to former colleagues, many who are world-renowned experts. For example, as part of our work on CF, Hunterian is collaborating with CF experts from Johns Hopkins Medicine and the Johns Hopkins Cystic Fibrosis Center. While our current collaborations are with Johns Hopkins scientists and clinicians, we are excited to work with experts from anywhere.I strongly believe in patient-driven drug development, so working with patients and disease foundations is vitally important to us. This is another form of collaboration that we engage in. It is something that I am particularly fond of – nothing is more motivating that speaking with patients.How are you hoping the use of CRISPR technology will change the medical field over the next few years?We are on the cusp of an unprecedented biomedical revolution that has been decades in the making. It is likely to touch nearly every aspect of our lives, but its most significant impact will be on human health. We can already see the enormous impact of CRISPR on therapeutic development, which will be fewer failures and shorter development timelines. New gene editing and cell therapies are already demonstrating cures for chronic genetic conditions for the first time, and more individualized treatments and therapeutics tailored to a person's specific genome are on the horizon. The intersection of technologies like CRISPR gene editing, artificial intelligence and synthetic biology will have broad-ranging impacts, transforming everything from disease models and early diagnosis to novel genetic interventions and cures for diseases.Development of in vivo therapeutics will be the next area of therapeutic disruption, and it has the potential to treat vast numbers of genetic diseases affecting millions of people. I cannot think of a more exciting technology to be working on now.DisclaimerThe opinions expressed in this interview are those of the interviewee and do not necessarily reflect the views of Future Medicine Ltd.Financial & competing interests disclosureThe interviewee is an employee of Hunterian Medicine. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.Open accessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/FiguresReferencesRelatedDetails Vol. 4, No. 1 Follow us on social media for the latest updates Metrics History Received 6 December 2021 Accepted 9 December 2021 Published online 17 January 2022 Published in print March 2022 Information© 2022 Vinod Jaskula-RangaKeywordsCRISPRcystic fibrosisgene editinginterviewDisclaimerThe opinions expressed in this interview are those of the interviewee and do not necessarily reflect the views of Future Medicine Ltd.Financial & competing interests disclosureThe interviewee is an employee of Hunterian Medicine. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.Open accessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/PDF download

Peptides have traditionally been perceived as poor drug candidates due to unfavorable characteristics mainly regarding their pharmacokinetic behavior, including plasma stability, membrane permeability and circulation half-life. Nonetheless, in recent years, general strategies to tackle those shortcomings have been established, and peptides are subsequently gaining increasing interest as drugs due to their unique ability to combine the advantages of antibodies and small molecules. Macrocyclic peptides are a special focus of drug development efforts due to their ability to address so called ‘undruggable’ targets characterized by large and flat protein surfaces lacking binding pockets. Here, the main strategies developed to date for adapting peptides for clinical use are summarized, which may soon help usher in an age highly shaped by peptide-based therapeutics. Nonetheless, limited membrane permeability is still to overcome before peptide therapeutics will be broadly accepted.

CXCR4 has been a target of interest in drug discovery for numerous years. However, so far, most if not all studies focused on finding antagonists of CXCR4 function. Recent studies demonstrate that targeting a minor allosteric pocket of CXCR4 induces an immunomodulating effect in immune cells expressing CXCR4, connected to the TLR pathway. Compounds binding in this minor pocket seem to be functionally selective with inverse agonistic properties in selected GPCR signaling pathways (Gi activation), but additional signaling pathways are likely to be involved in the immunomodulating effects. In depth research into these CXCR4-targeted immunomodulators could lead to novel treatment options for (auto)-immune diseases.

The recent emergence of COVID-19 influenced the layman's knowledge of drugs. Although several drugs have been discovered serendipitously, research has moved to the next-generation era of drug discovery. The use of drugs is inevitable and they have become lifesavers in the present era. Although research from different scientific backgrounds has supported the translational research of drug discovery, the prime role of pharmacy has to be remembered. Here we have summarized the role of some important subjects in pharmacy education, which have paved different ways in drug discovery and development.

Future Drug DiscoveryVol. 3, No. 4 EditorialOpen AccessKinetic intracellular assay measures compound binding kinetics at intracellular targets within living cellsCharles S Lay, Daniel A Thomas & Peter D CraggsCharles S Lay *Author for correspondence: E-mail Address: charles.lay@nottingham.ac.ukhttps://orcid.org/0000-0002-3060-6762University of Nottingham, Queens Medical Centre, Derby Road, NG7 2UH, UKMedicine Design, Medicinal Science & Technology, GlaxoSmithKline, Stevenage, SG1 2NY, UK, Daniel A ThomasArctoris, Oxford, OX14 4SA, UK & Peter D CraggsMedicine Design, Medicinal Science & Technology, GlaxoSmithKline, Stevenage, SG1 2NY, UKGSK-Francis Crick Institute Linklabs, Medicinal Science & Technology, GlaxoSmithKline, Stevenage, SG1 2NY, UKPublished Online:21 Dec 2021https://doi.org/10.4155/fdd-2021-0010AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: BET proteinintracellularKICAkineticsMotulsky–MahanNanoBRETnanoluciferasepharmacologyresidence timescreeningThe importance of measuring kineticsThe significance of measuring association and dissociation rates (kon and koff) in addition the dissociation constant (KD) when designing and developing new chemical entities is now widely recognized [1,2]. Dynamic changes in local drug concentration or target availability in vivo, mean that kinetic parameters may be better predictors of target occupancy. To date however, most drug discovery efforts have been driven by in vitro affinity measurements, conducted in closed systems where the total drug and target concentration remains constant. Effective drug pharmacodynamics depend on the sustained occupancy of the target, given that drugs can only elicit their effects when bound to a target. While affinity is in many cases a good predictor of the lifetime of the drug–target complex, increased target rebinding or reduced rates of drug dissociation could be equally or more important depending on the target's molecular context. Despite this, high throughput determination of kinetic binding values for test compounds has not been widely pursued in the early stages of drug discovery and development. The level of late-stage compound attrition due to lack of efficacy remains high [3] and the development of compounds with diverse kinetic profiles could be a useful way to improve therapeutic profiles and reduce off target effects.Current kinetic techniquesTechniques for the measurement of the binding kinetics of compounds to membrane proteins using cellular assays are now well established, including radioligand binding and nanoluciferase resonance energy transfer (NanoBRET) [4]. These assays enable the kinetics of drug interactions to be measured in more biologically and contextually relevant environments than those of solubilized receptors or membrane preparations.Conversely, kinetic assays for intracellular targets are commonly carried out on purified proteins using techniques such as time-resolved fluorescence energy transfer (TR-FRET) or surface plasmon resonance [5].As kinetic assays monitor the approach to an equilibrium between compound and target, these measurements are slowed by the presence of a membrane when targets are expressed within the cell. While the removal of the target from the intracellular environment simplifies the system to enable measurements to be made, this simplification may lead to the loss of important information on how the molecule would act in its native cellular context.The capability to measure live cell-binding kinetics at membrane receptors has allowed researchers to identify several cell-specific effects on compound binding. These include membrane mediated binding mechanisms, cell-specific conformations and effects such as rebinding; whereby, the accumulation of compound in the target vicinity increases the perceived association rate of compounds [4,6]. The inability to measure live cell-binding rates for intracellular targets may well mean that similar important effects are yet to be discovered. The lack of a cell-based quantitative assay for intracellular binding kinetics therefore leaves a gap in understanding between pharmacokinetic/pharmacodynamic models and purified protein-based methodologies [7,8].NanoBRET is a technique that utilizes the bioluminescent enzyme nanoluciferase (NL) as a reporter to enable bioluminescent resonance energy transfer (BRET) to fluorescent molecules [9]. The methodology can be used in recombinant cellular systems to measure interactions between NL-fused proteins and cell-permeable, fluorophore labeled probes. The utility of this technique for kinetic characterization studies was first explored using a methodology which added a fluorescent probe to washed cells that had previously been incubated with unlabeled compound. Comparative target residence times of unlabeled compounds could then be determined by the relative slowing of the association of the fluorescent tracer [9]. While this technique was a crucial advance, enabling the comparison of intracellular residence times within live cells, the methodology is low throughput and nonquantitative and does not measure association rates. Despite these limitations the technique has since been utilized for diverse drug-target families including, CDKs, JAKs, PARP, PYK2, BET and FAK proteins [10–14].The KICA methodologyIn a recent publication [15], a NanoBRET-based competition assay was used to measure intracellular kinetic binding rates of unlabeled compounds to bromo and extra-terminal domain containing (BET) protein family members. In this new approach, termed kinetic intracellular assay (KICA), the composite rate constants of effective forward (kforward) and reverse (kreverse) were measured. These rates incorporated cell penetrance, diffusion to the target and drug: target complex formation.The assay follows a simple protocol whereby cells are transiently transfected with an NL fusion expression construct, incubated overnight and then resuspended and combined with the NL substrate furimazine. These cells are subsequently added to microplates containing a fixed concentration of a pre characterized fluorescent-probe for the target of interest together with titrations of unlabeled test compounds. A luminescence microplate reader is then used to measure the BRET signal over time as the tracer and compound compete for the target.Using KICA at the BET protein BRD4, it was possible to reproducibly measure the differentiated binding kinetics of a range of test compounds. Composite kforward and kreverse rates are slower than equivalent association and dissociation rates measured using purified-protein methodologies. However, for cell-permeable compounds, the measured KICA rates correlate well, with association and dissociation rates determined using equivalent competition methodologies utilizing both protein and cells lysates.Matched compounds containing single atomic differences were screened with the BRD4 KICA methodology to simulate traditional compound development. In one example, a single atomic change led to a 3.4-fold decrease in reverse rate, demonstrating how KICA can be utilized in structural–kinetic relationship studies to guide the development of kinetically differentiated molecules.Compounds with low cellular permeability will have slowed KICA rates and as such it is suggested that parallel permeability or lysate kinetic assays should be conducted to identify slow association rate false positives. The KICA approach is ideally suited to the identification of, and subsequent optimization of kinetically selective compounds. In such a study, parallel KICA methods can be rapidly developed for homologous proteins; whereby, the only variance between protocols is the NL-target expressed in the cell line of interest. As compound and assay set-up remain constant, any differences in the KICA kforward and kreverse rates can be attributed solely to divergences in the formation of the specific drug: target complex. This approach was demonstrated in the study by screening a 31-compound test set through KICA assays configured for the BET proteins BRDT, 2, 3 and 4.A successful KICA must utilize a cell-permeable tracer that is a competitive binder for the binding site of interest. In addition, as in other kinetic competition assays, limitations are placed on the compound kinetics that can be monitored, defined by the relative kinetics of the tracer selected to develop the methodology [16].Future perspectiveAn improved fitting model could be developed in which, lysate and KICA data can be used to generate permeability values. If successful, this approach would generate a holistic model which incorporates permeability and binding in a single intact cell system.There is also the potential to carry out the KICA technique in disease relevant cell lines using CRISPR-Cas9 genetic engineering to introduce NL at the chromosomal location of the target gene. The CRISPR mediated addition of a high affinity fragment of a split NL to BRD4, in HEK293T cells expressing the corresponding NL fragment, has already been demonstrated [17,18] and preliminary KICA data using this cell line corresponds well to the values measured in the transient transfection-based system (unpublished observations).There is still much to understand about the chemical and structural drivers that modulate kon and koff. Improvements in assay technology and molecular dynamics simulations will result in a better understanding of how to optimize compounds to have differentiated kinetic properties through physical and simulated structural–kinetic relationship studies. KICA panels utilizing binding pocket mutations could help the understanding of which interactions are important for driving a specific kinetic profile.The value of a compound's kinetic binding profile is likely to be highly target specific, as exemplified by D2 antagonists, whereby rapid kon was found to be the most desirable kinetic binding parameter [19]. Developing compounds that exhibit differentiated, cellularly validated, binding kinetics should therefore increase the likelihood of success when compounds are tested within in vivo models.The KICA methodology is a high-throughput, recombinant cell-based approach that can be used to bridge the gap between purified protein-based techniques and PK-PD models. Using kinetic assays to develop kinetically differentiated compounds, rather than characterizing the binding profile of candidate molecules after selection for PK-PD models may reduce late-stage compound failure. Measuring the kinetics of compounds binding in live cells, makes the kinetic rates measured more representative of those in vivo.AcknowledgmentsWe would like to thank GSK for funding the original KICA research study.Open AccessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/Financial & competing interests disclosurePD Craggs is an employee and shareholder of GSK. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.References1. Copeland RA, Pompliano DL, Meek TD. Drug-target residence time and its implications for lead optimization. Nat. Rev. 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Dixon AS, Schwinn MK, Hall MP et al. NanoLuc complementation reporter optimized for accurate measurement of protein interactions in cells. ACS Chem. Biol. 11(2), 400–408 (2016).Crossref, CAS, Google Scholar19. Sykes DA, Moore H, Stott L et al. Extrapyramidal side effects of antipsychotics are linked to their association kinetics at dopamine D2 receptors. Nat. Commun. 8(1), 1–11 (2017).Crossref, Google ScholarFiguresReferencesRelatedDetails Vol. 3, No. 4 Follow us on social media for the latest updates Metrics Downloaded 466 times History Received 30 November 2021 Accepted 6 December 2021 Published online 21 December 2021 Published in print December 2021 Information© 2021 Charles LayKeywordsBET proteinintracellularKICAkineticsMotulsky–MahanNanoBRETnanoluciferasepharmacologyresidence timescreeningAcknowledgmentsWe would like to thank GSK for funding the original KICA research study.Open AccessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/Financial & competing interests disclosurePD Craggs is an employee and shareholder of GSK. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download