Seeking to identify a new chemotype for the development of NLRP3 inhibitors, inspired by the structure of the tool compound MCC950 (9), we designed and synthesized new derivatives by choosing the indole heterocycle as spacer between the furanyl moiety and the chemically handy sulfonamide chain core. Four compounds (19c, 19e, 19g, and 19j) selectively reduced NLRP3-dependent IL-1β levels with micromolar inhibitory activity in THP-1 cells, and were safe at the same concentrations. Moreover, they inhibited lactate dehydrogenase release, caspase-1 enzymatic activity, and ASC speck formation with a dose-response effect. Indoles 19c and 19g confirmed their biological activity in primary human macrophages, with IC50 of 19 and 15 µM, respectively. Furthermore, no off-target effects were observed as the compounds did not inhibit LPS-induced TNF release. In silico studies helped us rationalize the binding mode and showed that these derivatives can accommodate the NACHT domain and make several interactions with crucial key residues of the protein. Remarkably, the target engagement assay displayed that 19c and 19g can displace MCC950, thus confirming their direct binding to the NACHT domain. These preliminary results suggest the potential for future development of this new class of indoles as NLRP3 inhibitors.

Hepatitis C viral (HCV) infection is the leading cause of liver failure and still represents a global health burden. Over the past decade, great advancements made HCV curable, and sustained viral remission significantly improved to more than 98%. Historical treatment with pegylated interferon alpha and ribavirin has been displaced by combinations of direct-acting antivirals. These regimens include drugs targeting different stages of the HCV life cycle. However, the emergence of viral resistance remains a big concern. The design of peptidomimetic inhibitors (PIs) able to fit and fill the conserved substrate envelope region within the active site helped avoid contact with the vulnerable sites of the most common resistance-associated substitutions Arg155, Ala156, and Asp168. Herein, we give an overview of HCV NS3 PIs discovered during the past decade, and we deeply discuss the rationale behind the structural optimization efforts essential to achieve pangenotypic activity.

EDITORIAL article Front. Chem., 26 April 2023Sec. Medicinal and Pharmaceutical Chemistry Volume 11 - 2023 | https://doi.org/10.3389/fchem.2023.1202192

Pyrrolomycins (PMs) are a family of naturally occurring antibiotic agents, isolated from the fermentation broth of Actinosporangium and Streptomyces species. Pursuing our studies on pyrrolomycins, we performed the total synthesis of the F-series pyrrolomycins (1-4) by microwave-assisted synthesis (MAOS), thus obtaining the title compounds in excellent yields (63-69%). Considering that there is no evidence so far of the anticancer effect of this class of compounds, we investigated PMs for their antiproliferative activity against HCT116 and MCF-7 cancer cell lines. PMs showed anticancer activity at submicromolar level with a minimal effect on normal epithelial cell line (hTERT RPE-1), and they were able to induce several morphological changes including elongated cells, cytoplasm vacuolization, long and thin filopodia as well as the appearance of tunneling nanotubes (TNTs). These data suggest that PMs could act by impairing the cell membranes and the cytoskeleton organization, with subsequent increase of ROS generation and the activation of different forms of non-apoptotic cell death.

The use of peptides as therapeutics has often been associated with several drawbacks such as poor absorption, low stability to proteolytic digestion, and fast clearance. Peptidomimetics are developed by modifications of native peptides with the aim of obtaining molecules that are more suitable for clinical development and, for this reason, are widely used as tools in medicinal chemistry programs. The effort to disclose innovative peptidomimetic therapies is recurrent and constantly evolving as demonstrated by the new lead compounds in clinical trials. Synthetic strategies for the development of peptidomimetics have also been implemented with time. This perspective highlights some of the most recent efforts for the design and synthesis of peptidomimetic agents together with their biological evaluation toward a panel of targets.

The five-membered pyrrolidine ring is one of the nitrogen heterocycles used widely by medicinal chemists to obtain compounds for the treatment of human diseases. The great interest in this saturated scaffold is enhanced by (1) the possibility to efficiently explore the pharmacophore space due to sp3-hybridization, (2) the contribution to the stereochemistry of the molecule, (3) and the increased three-dimensional (3D) coverage due to the non-planarity of the ring-a phenomenon called "pseudorotation". In this review, we report bioactive molecules with target selectivity characterized by the pyrrolidine ring and its derivatives, including pyrrolizines, pyrrolidine-2-one, pyrrolidine-2,5-diones and prolinol described in the literature from 2015 to date. After a comparison of the physicochemical parameters of pyrrolidine with the parent aromatic pyrrole and cyclopentane, we investigate the influence of steric factors on biological activity, also describing the structure-activity relationship (SAR) of the studied compounds. To aid the reader's approach to reading the manuscript, we have planned the review on the basis of the synthetic strategies used: (1) ring construction from different cyclic or acyclic precursors, reporting the synthesis and the reaction conditions, or (2) functionalization of preformed pyrrolidine rings, e.g., proline derivatives. Since one of the most significant features of the pyrrolidine ring is the stereogenicity of carbons, we highlight how the different stereoisomers and the spatial orientation of substituents can lead to a different biological profile of drug candidates, due to the different binding mode to enantioselective proteins. We believe that this work can guide medicinal chemists to the best approach in the design of new pyrrolidine compounds with different biological profiles.

The discovery of novel synthetic compounds with drug-like properties is an ongoing challenge in medicinal chemistry. Natural products have inspired the synthesis of compounds for pharmaceutical application, most of which are based on N-heterocyclic motifs. Among these, the pyrrole ring is one of the most explored heterocycles in drug discovery programs for several therapeutic areas, confirmed by the high number of pyrrole-based drugs reaching the market. In the present review, we focused on pyrrole and its hetero-fused derivatives with anticancer, antimicrobial, and antiviral activities, reported in the literature between 2015 and 2019, for which a specific target was identified, being responsible for their biological activity. It emerges that the powerful pharmaceutical and pharmacological features provided by the pyrrole nucleus as pharmacophore unit of many drugs are still recognized by medicinal chemists.

: Pyrrolomycins (PMs) are polyhalogenated antibiotics known as powerful biologically active compounds, yet featuring high cytotoxicity. The present study reports the antibacterial and antitumoral properties of new chemically synthesized PMs, where the three positions of the pyrrolic nucleus were replaced by nitro groups, aiming to reduce their cytotoxicity while maintaining or even enhancing the biological activity. Indeed, the presence of the nitro substituent in diverse positions of the pyrrole determined an improvement of the minimal bactericidal concentration (MBC) against Gram-positive (i.e., Staphylococcus aureus) or -negative (i.e., Pseudomonas aeruginosa) pathogen strains as compared to the natural PM-C. Moreover, some new nitro-PMs were as active as or more than PM-C in inhibiting the proliferation of colon (HCT116) and breast (MCF 7) cancer cell lines and were less toxic towards normal epithelial (hTERT RPE-1) cells. Altogether, our findings contribute to increase the knowledge of the mode of action of these promising molecules and provide a basis for their rationale chemical or biological manipulation.

A new series of eighteen imidazo [2,1-b] [1,3,4]thiadiazole derivatives was efficiently synthesized and screened for antiproliferative activity against the National Cancer Institute (NCI-60) cell lines panel. Two out of eighteen derivatives, compounds 12a and 12h, showed remarkably cytotoxic activity with the half maximal inhibitory concentration values (IC50) ranging from 0.23 to 11.4 mu M, and 0.29-12.2 mu M, respectively. However, two additional compounds, 12b and 13g, displayed remarkable in vitro antiproliferative activity against pancreatic ductal adenocarcinoma (PDAC) cell lines, including immortalized (SUIT-2, Capan-1, Panc-1), primary (PDAC-3) and gemcitabine-resistant (Panc-1R), eliciting IC50 values ranging from micromolar to sub-micromolar level, associated with significant reduction of cell-migration and spheroid shrinkage. These remarkable results might be explained by modulation of key regulators of epithelial-to-mesenchymal transition (EMT), including E-cadherin and vimentin, and inhibition of metalloproteinase-2/-9. High-throughput arrays revealed a significant inhibition of the phosphorylation of 45 tyrosine kinases substrates, whose visualization on Cytoscape highlighted PTK2/FAK as an important hub. Inhibition of phosphorylation of PTK2/FAK was validated as one of the possible mechanisms of action, using a specific ELISA. In conclusion, novel imidazothiadiazoles show potent antiproliferative activity, mediated by modulation of EMT and PTK2/FAK. (C) 2020 Elsevier Masson SAS. All rights reserved.

Background Expression of proton-coupled folate transporter (PCFT) is associated with survival of mesothelioma patients treated with pemetrexed, and is reduced by hypoxia, prompting studies to elucidate their correlation. Methods Modulation of glycolytic gene expression was evaluated by PCR arrays in tumour cells and primary cultures growing under hypoxia, in spheroids and after PCFT silencing. Inhibitors of lactate dehydrogenase (LDH-A) were tested in vitro and in vivo. LDH-A expression was determined in tissue microarrays of radically resected malignant pleural mesothelioma (MPM, N = 33) and diffuse peritoneal mesothelioma (DMPM, N = 56) patients. Results Overexpression of hypoxia marker CAIX was associated with low PCFT expression and decreased MPM cell growth inhibition by pemetrexed. Through integration of PCR arrays in hypoxic cells and spheroids and following PCFT silencing, we identified the upregulation of LDH-A, which correlated with shorter survival of MPM and DMPM patients. Novel LDH-A inhibitors enhanced spheroid disintegration and displayed synergistic effects with pemetrexed in MPM and gemcitabine in DMPM cells. Studies with bioluminescent hypoxic orthotopic and subcutaneous DMPM athymic-mice models revealed the marked antitumour activity of the LDH-A inhibitor NHI-Glc-2, alone or combined with gemcitabine. Conclusions This study provides novel insights into hypoxia/PCFT-dependent chemoresistance, unravelling the potential prognostic value of LDH-A, and demonstrating the preclinical activity of LDH-A inhibitors.

Background/Aim: A new class of imidazo[2,1-b][1,3,4]thiadiazole compounds have recently been evaluated as inhibitors of phosphorylation of focal adhesion kinase (FAK) in pancreatic cancer. FAK is overexpressed in mesothelioma and has recently emerged as an interesting target for the treatment of this disease. Materials and Methods: Ten imidazo[2,1-b][1,3,4]thiadiazole compounds characterized by indole bicycle and a thiophene ring, were evaluated for their cytotoxic activity in two primary cell cultures of peritoneal mesothelioma, Mesoll and STO cells. Results: Compounds 1a and 1b showed promising antitumor activity with IC50 values in the range of 059 to 2.81 mu M in both cell lines growing as monolayers or as spheroids. Their antiproliferative and antimigratory activity was associated with inhibition of phospho-FAK, as detected by a specific ELISA assay in STO cells. Interestingly, these compounds potentiated the antiproliferative activity of gemcitabine, and these results might be explained by the increase in the mRNA expression of the key gemcitabine transporter human equilibrative nucleoside transporter-1 (hENT-1). Conclusion: These promising results support further studies on new imidazo[2,1-b][1,3,4]thiadiazole compounds as well as on the role of both FAK and hENT-1 modulation in order to develop new drug combinations for peritoneal mesothelioma.

A new series of imidazo[2,1-b][1,3,4]thiadiazole derivatives was efficiently synthesized and screened for their in vitro antiproliferative activity on a panel of pancreatic ductal adenocarcinoma (PDAC) cells, including SUIT-2, Capan-1 and Panc-1. Compounds 9c and 9l, showed relevant in vitro antiproliferative activity on all three pre-clinical models with half maximal inhibitory concentration (IC50) ranging from 5.11 to 10.8 µM, while the compounds 9e and 9n were active in at least one cell line. In addition, compound 9c significantly inhibited the migration rate of SUIT-2 and Capan-1 cells in the scratch wound-healing assay. In conclusion, our results will support further studies to increase the library of imidazo [2,1-b][1,3,4] thiadiazole derivatives for deeper understanding of the relationship between biological activity of the compounds and their structures in the development of new antitumor compounds against pancreatic diseases.

Heterocyclic rings are recognized as key components of many natural, semi-synthetic and synthetic molecules with a broad spectrum of biological activities. Among these molecules, the indole and imidazo[2,1-b][1,3,4]thiadiazole systems have recently been described as useful scaffolds for the design of anticancer agents. Herein the antitumor activity of a series of 3-(6-phenylimidazo[2,1-b][1,3,4]thiadiazol-2-yl)-1H-indoles, designed as hybrid structures, was assessed. Seven out of 10 compounds (1a-g) were submitted to National Cancer Institute (NCI). Remarkably, compound 1g showed antiproliferative activity against the full panel of sixty human cancer lines, with half-maximal inhibitory concentration of between 1.67 and 10.3 μM. Further studies showed antiproliferative activity of 1a-g and of three additional compounds 1h, 1i and 1l, with different substituents on the indole nucleus and phenyl ring, against three pancreatic cancer cell lines. In particular, derivatives 1g and 1h inhibited both proliferation and migration of SUIT-2 cells at concentrations lower than 10 μM. In conclusion, new indole derivatives are characterized by in vitro antitumor activity, supporting future mechanistic studies.

Introduction: Despite clinical efforts, pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. The scarcity of effective therapies can be reflected by the lack of reliable biomarkers to adapt anticancer drugs prescription to tumors’ and patients’ features.Areas covered: Pharmacogenetics should provide the way to select patients who may benefit from a specific therapy that best matches individual and tumor genetic profile, but it has not yet led to gains in outcome. This review describes PDAC pharmacogenetics findings, critically reappraising studies on polymorphisms and -omics profiles correlated to response to gemcitabine, FOLFIRINOX, and nab-paclitaxel combinations, as well as limitations of targeted therapies. Further, we question whether personalized approaches will benefit patients to any significant degree, supporting the need of new strategies within well-designed trials and validated genomic tests for treatment decision-making.Expert opinion: A major challenge in PDAC is the identification of subgroups of patients who will benefit from treatments. Minimally-invasive tests to analyze biomarkers of drug sensitivity/toxicity should be developed alongside anticancer treatments. However, progress might fall below expectations because of tumor heterogeneity and clonal evolution. Whole-genome sequencing and liquid biopsies, as well as prospective validation in selected cohorts, should overcome the limitations of traditional pharmacogenetic approaches.

A class of 36 new 2-(6-phenylimidazo[2,-1-b][1,3,4]thiadiazol-2-yl)-1H-indoles was efficiently synthesized and evaluated for their anti-biofilm properties against the Gram-positive bacterial reference strains Staphylococcus aureus ATCC 25923, S. aureus ATCC 6538 and Staphylococcus epidermidis ATCC 12228, and the Gram-negative strains Pseudomonas aeruginosa ATCC 15442 and Escherichia coli ATCC 25922. Many of these new compounds, were able to inhibit biofilm formation of the tested staphylococcal strains showing BIC50 lower than 10 μg/ml. In particular, derivatives 9c and 9h showed remarkable anti-biofilm activity against S. aureus ATCC 25923 with BIC50 values of 0.5 and 0.8 μg/ml, respectively, whereas compound 9aa was the most potent against S. aureus ATCC 6538, with a BIC50 of 0.3 μg/ml. Remarkably, these compounds showed effects in the early stages of the biofilm formation without affecting the mature biofilm of the same strains and the viability of the planktonic form. Their ability in counteracting a virulence factor (biofilm formation) without interfering with the bacterial growth in the free life form make them novel valuable anti-virulence agents.

Combination therapies are used in the clinic to achieve cure, better efficacy and to circumvent resistant disease in patients. Initial assessment of the effect of such combinations, usually of two agents, is frequently performed using in vitro assays. In this review, we give a short summary of the types of analyses that were presented during the Preclinical and Early-phase Clinical Pharmacology Course of the Pharmacology and Molecular Mechanisms Group, European Organization for Research and Treatment on Cancer, that can be used to determine the efficacy of drug combinations. The effect of a combination treatment can be calculated using mathematical equations based on either the Loewe additivity or Bliss independence model, or a combination of both, such as Chou and Talalay's median-drug effect model. Interactions can be additive, synergistic (more than additive), or antagonistic (less than additive). Software packages CalcuSyn (also available as CompuSyn) and Combenefit are designed to calculate the extent of the combined effects. Interestingly, the application of machine-learning methods in the prediction of combination treatments, which can include pharmacogenomic, genetic, metabolomic and proteomic profiles, might contribute to further refinement of combination regimens. However, more research is needed to apply appropriate rules of machine learning methods to ensure correct predictive models.

One aim of cell-based in vitro assays is to identify the best drug candidate to develop using the best tumor cell model. This is challenging in every anticancer drug discovery process. Briefly, we summarize the parameters to be taken into account when performing in vitro cell assays, in order to obtain reliable and reproducible results, which was fundamentally discussed by lecturers at the educational course on preclinical and early-phase clinical pharmacology studies, at the 40th Winter Meeting of the Pharmacology and Molecular Mechanisms Group of the European Organization for Research and Treatment of Cancer. Moreover, specific cellular sensitivity tests are described. In addition to monolayer in vitro cell models for the screening of new potential candidate drugs, three-dimensional tumor/cell tissue models are emerging as new pre-clinical tools that more closely reflect the in vivo microenvironment. Therefore, the use of different in vitro models for drug screening can enhance the predictability and reliability of preclinical drug-discovery phases and target validation.

PharmacogenomicsVol. 19, No. 10 EditorialFree AccessProton-coupled folate transporter as a biomarker of outcome to treatment for pleural mesotheliomaGiovanna Li Petri, Stella Cascioferro, Barbara Parrino, Godefridus J Peters, Patrizia Diana & Elisa GiovannettiGiovanna Li Petri Department of Medical Oncology, VU University Medical Center, Cancer Center Amsterdam, De Boelelaan 1117, 1081HV Amsterdam, The Netherlands Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, Sezione di Chimica e Tecnologie Farmaceutiche, Università degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy, Stella Cascioferro Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, Sezione di Chimica e Tecnologie Farmaceutiche, Università degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy, Barbara Parrino Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, Sezione di Chimica e Tecnologie Farmaceutiche, Università degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy, Godefridus J Peters Department of Medical Oncology, VU University Medical Center, Cancer Center Amsterdam, De Boelelaan 1117, 1081HV Amsterdam, The Netherlands, Patrizia Diana Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, Sezione di Chimica e Tecnologie Farmaceutiche, Università degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy & Elisa Giovannetti*Author for correspondence: Tel.: +31 20 444 2267; Fax: +31 20 444 3844; E-mail Address: elisa.giovannetti@gmail.com Department of Medical Oncology, VU University Medical Center, Cancer Center Amsterdam, De Boelelaan 1117, 1081HV Amsterdam, The Netherlands Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, Sezione di Chimica e Tecnologie Farmaceutiche, Università degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy Cancer Pharmacology Lab, AIRC Start-Up Unit, University of Pisa, via Paradisa, 56100, Pisa, ItalyPublished Online:19 Jun 2018https://doi.org/10.2217/pgs-2018-0071AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit Keywords: chemoresistancemesotheliomaPCFTpemetrexedThe main objective of pharmacogenetics is the identification of genetic features involved in clinically meaningful variations in drug responsiveness. Therefore, pharmacogenetics may reduce the variation in individual response to drugs and for tailoring therapies according to genetic profile, fulfilling the promise of precision medicine [1].The problem of interindividual variability in drug response is particularly important in anticancer regimens, which are characterized by a narrow therapeutic window. Small alterations in the transport or metabolism of anticancer agents may indeed cause large changes in their pharmacological effects, both in terms of toxicity and efficacy.As reported by the Pharmacology and Molecular Mechanisms group of the European Organization for the Research and Treatment of Cancer, the terms 'pharmacogenetics' and 'pharmacogenomics' are often used interchangeably and pharmacogenetics is considered as concerning the individual patient's characteristics and pharmacogenomics those of the tumor [2]. However, according to this position paper as well as to the definition by the European agency for the evaluation of medicinal products 'pharmacogenetics' focuses on the association of one gene or several genes with drug activity, while 'pharmacogenomics' considers the whole genome, through the broader application of new genomic technologies [3]. Thus, in oncology, a pharmacogenetic approach aims to customize the chemotherapy treatment according to individual/somatic as well as tumor genetic characteristics. This represents a modern and intriguing challenge and pharmacogenetic tools are warranted to maximize the therapeutic efficacy and minimize useless treatments, especially in patients affected by solid tumors with limited therapeutic approaches, such as malignant pleural mesothelioma (MPM).MPM is an aggressive tumor arising from the pleura, with grim prognosis. Its incidence is increasing throughout most of the world and it is predicted that it will rise in the next 10 years [4]. Most patients with MPM are not amenable to radical surgery and systemic therapy is the only potential treatment option. A number of prognostic factors have been described and are part of two prognostic scoring systems. In the European Organization for the Research and Treatment of Cancer score, poor prognosis is associated with a poor performance status, a high white blood cell count, male gender and having sarcomatoid histologic subtype, while In the Cancer and Leukemia Group B scoring system pleural involvement, LDH >500 IU/l, poor PS, chest pain, platelets >400,000/μl, nonepithelial histology and age older than 75 years, jointly predict poor overall survival (OS).Based on extensive genomic profiling, four MPM molecular subtypes were associated with OS [5]. However, up to now, there are no data about specific genetic biomarkers of response to chemotherapy in MPM patients. The identification of such molecular predictors is urgently needed in order to select patients for optimal treatment strategies and to improve clinical outcome.More than 14 years ago, US FDA approved the first ever (and the last so far) treatment drug to be used in the first-line treatment of MPM in combination with cisplatin, pemetrexed. In fact, this combination significantly improved the response rate (41.3 vs 16.7%; p < 0.0001), time to progression (5.7 vs 3.9 months; p = 0.001), OS (12.1 vs 9.3 months; p = 0.020) and quality of life compared with cisplatin alone. The combination with carboplatin gave similar results [6].Pemetrexed is a multitargeted antifolate agent that enters the cancer cells through different transporters and is then converted to a series of analogous polyglutamate derivatives by the enzyme folylpolyglutamate synthetase. This polyglutamylation leads to extended intracellular retention, resulting in more prolonged efficacy. Active metabolites of pemetrexed inhibit several folate-dependent enzymes such as thymidylate synthase, dihydrofolate reductase, glycinamide ribonucleotide formyl transferase and to a lesser extent, aminoimidazole carboxamide ribonucleotide tranformylase and CI-tetrahydrofolate synthase, thereby inhibiting both pyrimidine and purine biosynthesis [7].In vitro studies demonstrated that several factors increase the antitumor activity of pemetrexed, including low expression levels of the main target TS, high activity of folylpolyglutamate synthetase and the rapid transport across the plasma membrane mediated by influx transporters for which pemetrexed has different affinities [8]. In agreement with previous data in nonsquamous and squamous specimens from lung cancer patients treated with pemetrexed-based chemotherapy, as reviewed by Santarpia and collaborators [9], high TS expression has also been associated with poor response, as well as significantly shorter median progression-free survival and OS in the two largest multicenter studies on pemetrexed-based regimens in MPM [10,11].In a more recent translational study, the efficacy of pemetrexed was associated with both low TS and high proton-coupled folate transporter (PCFT/SLC46A1) expression levels [12]. In fact, a high mRNA expression of PCFT and a low-mRNA expression of TS were associated with lower risk of developing a progressive disease compared with disease-control as well as with significantly longer progression-free survival and OS. Low-PCFT protein levels were also associated with shorter OS and multivariate analysis confirmed PCFT-independent prognostic role.In mammalian cells, three distinct processes mediate membrane transport of folates and antifolates, including pemetrexed [13]. The reduced folate carrier (RFC) is a carrier-mediated anion exchanger, facilitating the translocation of a folate substrate across the cell membrane by the co-transport of another anion in the opposite direction. With a Ki for methotrexate influx ranging from 2 to 5 μmol/l in murine and human tumor cell lines, RFC has a higher affinity for methotrexate than for folic acid. Of note, the affinity for pemetrexed is twofold higher [14]. The folate receptors FR-α and FR-β are binding proteins anchored to the cell membranes by a glycosyl phosphatidylinositol tail that mediates transport by an endocytotic process. The maximum rate of transport into cells mediated by this mechanism is 100, the rate mediated by RFC. Hence, FR-mediated transport contributes little to the uptake of pemetrexed. The third folate transport system, characterized by a unique and distinct low-pH optimum that is present in the majority of human solid tumors, is mediated by PCFT. This transporter was initially characterized in mice, and only recently emerged as the main transporter mediating pemetrexed influx, with remarkable transport km values of 0.2–0.8 μm [15].The pivotal role of PCFT in the transport of folates and antifolates has been demonstrated in various models, showing that PCFT transfection in HeLa cells increased pemetrexed cytotoxicity [16], while PCFT silencing increased IC50 values by fourfold and threefold in the MSTO-211H and H2452 MPM cells, respectively [12]. Of note, PCFT promoter can be silenced via DNA methylation [17], resulting in reduced transcriptional activity. Indeed, H28 MPM cells, which displayed methylation of 85% of the CpG-islands of PCFT promoter had low- expression levels of PCFT and were relatively more resistant to pemetrexed [12]. Additional experiments were then performed to evaluate PCFT expression and cell proliferation after DNA demethylation. These experiments were performed with 5-Aza-2′-deoxycytidine, which significantly reduced the methylation of the PCFT promoter, down to -70% in the H28 cells and subsequently increased the mRNA expression of PCFT as well as the growth inhibitory activity of pemetrexed. This means that PCFT was not only identified as a new biomarker that can help predict pemetrexed's effectiveness or chemoresistance, but also a potential useful target to overcome such chemoresistance.Where do we go from here? We suggest the following rational preclinical and clinical development strategies. First of all, additional preclinical studies in appropriate in vivo models of MPM should evaluate the key role of PCFT in the antitumor activity of pemetrexed and other potential molecular mechanisms underlying the differential PCFT expression, such as, for instance, the hypoxic/metabolic status of these tumors, which has also been correlated to pemetrexed resistance [18] and might suggest novel antiglycolitic therapeutic strategies [19,20]. Second, translational studies with prospectively collected samples, are essential for the validation of the previous retrospective data as well as for the identification of the best cut-off expression level for a validated pharmacogenetic test. Finally, a Phase I basket trial should test 5-Aza-2′-deoxycytidine, which is a well-tolerated compound currently used to treat myelodysplastic syndrome, in patients with different tumor types, such as mesothelioma and lung cancer, to be treated with pemetrexed and cisplatin and stratified according to PCFT expression. Subsequently, Phase II and Phase III trials could then select the best tumor type and establish the role of this new potential pharmacogenetic-guided treatment versus the standard of care backbone chemotherapy, according to previously standardized and validated thresholds.Hopefully, the results of these studies should enable oncologists to stratify patients based on PCFT before anticancer treatment, allowing the optimization of clinical outcomes through effective personalization of treatment.Financial & competing interests disclosureThis work was partially supported by 'the Law Offices of Peter G Angelos Grant' from the Mesothelioma Applied ResearchFoundation (MARF), USA. 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 Sánchez NS, Mills GB, Mills Shaw KR. Precision oncology: neither a silver bullet nor a dream. Pharmacogenomics 18(16), 1525–1539 (2017).Link, CAS, Google Scholar2 Robert J, Le Morvan V, Giovannetti E, Peters GJ. On the use of pharmacogenetics in cancer treatment and clinical trials. Eur. J. Cancer. 50(15), 2532–2543 (2014).Crossref, Medline, CAS, Google Scholar3 European Medicines Agency for the Evaluation of Medicinal Products (EMEA). Position paper on terminology in pharmacogenetics (EMEA/CPMP/3070/01), London, UK (2002). www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003889.pdf.Google Scholar4 Carbone M, Kanodia S, Chao A et al. Consensus report of the 2015 Weinman international conference on mesothelioma. J. Thorac. Oncol. 11(8), 1246–1262 (2016).Crossref, Medline, Google Scholar5 Bueno R, Stawiski EW, Goldstein LD et al. Comprehensive genomic analysis of malignant pleural mesothelioma identifies recurrent mutations, gene fusions and splicing alterations. Nat. Genet. 48(4), 407–416 (2016).Crossref, Medline, CAS, Google Scholar6 Ceresoli GL, Zucali PA, Favaretto AG et al. Phase II study of pemetrexed plus carboplatin in malignant pleural mesothelioma. J. Clin. Oncol. 24(9), 1443–1448 (2006).Crossref, Medline, CAS, Google Scholar7 Chattopadhyay S, Moran RG, Goldman ID. Pemetrexed: biochemical and cellular pharmacology, mechanisms and clinical applications. Mol. Cancer Ther. 6(2), 404–417 (2007).Crossref, Medline, CAS, Google Scholar8 Giovannetti E, Toffalorio F, De Pas T, Peters GJ. Pharmacogenetics of conventional chemotherapy in non-small-cell lung cancer: a changing landscape. Pharmacogenomics 13(9), 1073–1086 (2012).Link, CAS, Google Scholar9 Santarpia M, Rolfo C, Peters GJ, Leon LG, Giovannetti E. On the pharmacogenetics of non-small-cell lung cancer treatment. Expert Opin. Drug Metab. Toxicol. 12(3), 307–317 (2016).Crossref, Medline, CAS, Google Scholar10 Righi L, Papotti MG, Ceppi P et al. Thymidylate synthase but not excision repair cross-complementation group 1 tumor expression predicts outcome in patients with malignant pleural mesothelioma treated with pemetrexed-based chemotherapy. J. Clin. Oncol. 28(9), 1534–1539 (2010).Crossref, Medline, CAS, Google Scholar11 Zucali PA, Giovannetti E, Destro A et al. Thymidylate synthase and excision repair cross-complementing group-1 as predictors of responsiveness in mesothelioma patients treated with pemetrexed/carboplatin. Clin. Cancer Res. 17(8), 2581–2590 (2011).Crossref, Medline, CAS, Google Scholar12 Giovannetti E, Zucali PA, Assaraf YG et al. Role of proton-coupled folate transporter in pemetrexed resistance of mesothelioma: clinical evidence and new pharmacological tools. Ann. Oncol. 28(11), 2725–2732 (2017).Crossref, Medline, CAS, Google Scholar13 Gonen N, Assaraf YG. Antifolates in cancer therapy: structure, activity and mechanisms of drug resistance. Drug Resist. Updat. 15(4), 183–210 (2012).Crossref, Medline, CAS, Google Scholar14 Mauritz R, Peters GJ, Priest DG et al. Multiple mechanisms of resistance to methotrexate and novel antifolates in human CCRF-CEM leukemia cells and their implications for folate homeostasis. Biochem. Pharmacol. 63(2), 105–115 (2002).Crossref, Medline, CAS, Google Scholar15 Zhao R, Goldman ID. The proton-coupled folate transporter: physiological and pharmacological roles. Curr. Opin. Pharmacol. 13(6), 875–880 (2013).Crossref, Medline, CAS, Google Scholar16 Diop-Bove NK, Wu J, Zhao R, Locker J, Goldman ID. Hypermethylation of the human proton-coupled folate transporter (SLC46A1) minimal transcriptional regulatory region in an antifolate-resistant HeLa cell line. Mol. Cancer Ther. 8(8), 2424–2431 (2009).Crossref, Medline, CAS, Google Scholar17 Gonen N, Bram EE, Assaraf YG. PCFT/SLC46A1 promoter methylation and restoration of gene expression in human leukemia cells. Biochem. Biophys. Res. Commun. 376(4), 787–792 (2008).Crossref, Medline, CAS, Google Scholar18 Raz S, Sheban D, Gonen N, Stark M, Berman B, Assaraf YG. Severe hypoxia induces complete antifolate resistance in carcinoma cells due to cell cycle arrest. Cell Death Dis. 5, e1067 (2014).Crossref, Medline, CAS, Google Scholar19 Nabavi N, Bennewith KL, Churg A, Wang Y, Collins CC, Mutti L. Switching off malignant mesothelioma: exploiting the hypoxic microenvironment. Genes Cancer 7(11–12), 340–354 (2016).Crossref, Medline, CAS, Google Scholar20 Giovannetti E, Leon LG, Gómez VE, Zucali PA, Minutolo F, Peters GJ. A specific inhibitor of lactate dehydrogenase overcame the resistance toward gemcitabine in hypoxic mesothelioma cells, and modulated the expression of the human equilibrative transporter-1. Nucleosides Nucleotides Nucleic Acids 35(10–12), 643–651 (2016).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByThe structure and function prediction of protein involved in toxin metabolism in Drosophila melanogasterLactate/GPR81 signaling and proton motive force in cancer: Role in angiogenesis, immune escape, nutrition, and Warburg phenomenonPharmacology & Therapeutics, Vol. 206 Vol. 19, No. 10 Follow us on social media for the latest updates Metrics History Received 7 May 2018 Accepted 8 May 2018 Published online 19 June 2018 Published in print July 2018 Information© 2018 Future Medicine LtdKeywordschemoresistancemesotheliomaPCFTpemetrexedFinancial & competing interests disclosureThis work was partially supported by 'the Law Offices of Peter G Angelos Grant' from the Mesothelioma Applied ResearchFoundation (MARF), USA. 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