DNA mimic proteins are relatively obscure control factors that resemble DNA by mimicking its negatively charged distribution. They achieve this using negatively charged amino acids like aspartic acid (ASP/D) and glutamic acid (GLU/E). Known DNA mimic proteins control various cellular mechanisms, such as transcription, DNA repair, and gene regulation, by intervening in the binding of DNA to effector proteins. In addition to their biological functions, DNA mimic proteins may also be applicable in biotechnology, for example, by regulating CRISPR-Cas9 activity to enhance gene editing precision. Therefore, DNA mimic proteins warrant further research. However, most DNA mimic proteins cannot be identified using traditional bioinformatics methods owing to their unique amino acid sequences and structural features. We developed a new protein fingerprint, called relative distance protein fingerprint (RD-PFP), that can be used to analyze the distribution of amino acids on a protein surface. We optimized our RD-PFP by using machine learning and the characteristic feature of DNA mimic proteins (namely, their DNA-like negatively charged distribution) to more accurately predict DNA mimicry from protein structures. Our pioneering study contributes to the development of machine learning-based bioinformatics methods for screening DNA mimic proteins.

Drug resistance in cancer therapy is the major reason for poor prognosis. Addressing this clinically unmet issue is important and urgent. In this study, we found that targeting USP24 by the specific USP24 inhibitors, USP24-i and its analogues, dramatically activated autophagy in the interphase and mitotic periods of lung cancer cells by inhibiting E2F4 and TRAF6, respectively. USP24 functional knockout, USP24C1695A, or targeting USP24 by USP24-i-101 inhibited drug resistance and activated autophagy in gefitinib-induced drug-resistant mice with doxycycline-induced EGFRL858R lung cancer, but this effect was abolished after inhibition of autophagy, indicating that targeting USP24-mediated induction of autophagy is required for inhibition of drug resistance. Genomic instability and PD-L1 levels were increased in drug resistant lung cancer cells and were inhibited by USP24-i-101 treatment or knockdown of USP24. In addition, inhibition of autophagy by bafilomycin-A1 significantly abolished the effect of USP24-i-101 on maintaining genomic integrity, decreasing PD-L1 and inhibiting drug resistance acquired in chemotherapy or targeted therapy. In summary, an increase in the expression of USP24 in cancer cells is beneficial for the induction of drug resistance and targeting USP24 by USP24-i-101 optimized from USP24-i inhibits drug resistance acquired during cancer therapy by increasing PD-L1 protein degradation and genomic stability in an autophagy induction-dependent manner.

Shrimp acute hepatopancreatic necrosis disease (AHPND) is one of the most devastating diseases to impact the global shrimp farming industry, with a mortality rate of 70 %–100 %. The key virulence factors are a pair of Photorhabdus insect-related (Pir)-like toxins, PirAvp and PirBvp. In this study, by using an in vitro transcription and translation assay, we first confirmed that the quorum sensing transcriptional regulator AphBvp could trigger the expression of its downstream genes after binding to the AphBvp binding sequence in the promoter region of the pirAvp/pirBvp operon. Next, we showed that AphBvp was essential for the expression of these toxins by using an aphBvp-deletion mutant (ΔaphBvp) derived from the AHPND-causing Vibrio parahaemolyticus. Lastly, we discovered that the expression levels of PirAvp and PirBvp were up-regulated under acidic conditions (pH 4.5), and further showed that an acidic environment promoted the binding of AphBvp to the pirABvp promoter. We speculate that this was because the acidic environment favored the formation of AphBvp tetramers, which is important for binding to DNA. Taken together, these findings improve our understanding of the gene regulatory mechanisms of pirAvp and pirBvp, and suggest that the pH value of the environment might affect the virulence of AHPND-causing V. parahaemolyticus.

Class IIa histone deacetylases (HDACs) have been linked to tumorigenesis in various cancers. Previously, we designed phenylhydroxamic acid LH4f as a potent class IIa HDAC inhibitor. However, it also unselectively inhibited class I and class IIb HDACs. To enhance the compound’s selectivity towards class IIa HDACs, the ortho-phenyl group from the selective HDAC7 inhibitor 1 is incorporated into ortho position of the phenylhydroxamic acid in LH4f. Compared to LH4f, most resulting compounds displayed substantially improved selectivity towards the class IIa HDACs. Notably, compound 7 g exhibited the strongest HDAC9 inhibition with an IC50 value of 40 nM. Molecular modelling further identified the key interactions of compound 7 g bound to HDAC9. Compound 7 g significantly inhibited several human cancer cells, induced apoptosis, modulated caspase-related proteins as well as p38, and caused DNA damage. These findings suggest the potential of class IIa HDAC inhibitors as lead compounds for the development of cancer therapeutics.

Pulmonary fibrosis is excessive scarring of the lung tissues. Transforming growth factor-beta (TGF-β) has been implicated in pulmonary fibrosis due to its ability to induce the epithelial-to-mesenchymal transition (EMT) and promote epithelial cell migration. Cyclin-dependent kinase 8 (CDK8) can mediate the TGF-β signaling pathways and could function as an alternative therapeutic target for treating pulmonary fibrosis. Here, we performed a structure-based virtual screening campaign to identify CDK8 inhibitors from a library of 1.6 million compounds. The screening process ended with the identification of a novel CDK8 inhibitor, P162-0948 (IC50: 50.4 nM). An interaction analysis highlighted important CDK8-ligand interactions that support its binding and inhibitory activity. Testing against a panel of 60 different kinases demonstrated P162-0948 selectivity toward CDK8. Crucially, the inhibitor was found to be structurally novel when compared to known CDK8 inhibitors. Testing in A549 human alveolar epithelial cell lines showed that the P162-0948 can reduce cell migration and protein expression of EMT-related proteins. When P162-0948 was treated in cells at 5 μM, phosphorylation of Smad in the nucleus was reduced, which suggests disruption of the TGF-β/Smad signaling pathway. The identification of P162-0948 shows that it is not only potent, but its structural novelty can inform future design studies for potential therapeutics targeting pulmonary fibrosis.

Dual-specificity tyrosine-regulated kinase 1 A (DYRK1A) is crucial in neurogenesis, synaptogenesis, and neuronal functions. Its dysregulation is linked to neurodegenerative disorders like Down syndrome and Alzheimer's disease. Although the development of DYRK1A inhibitors has significantly advanced in recent years, the selectivity of these drugs remains a critical challenge, potentially impeding further progress. In this study, we utilised structure-based virtual screening (SBVS) from NCI library to discover novel DYRK1A inhibitors. The top-ranked compounds were then validated through enzymatic assays to assess their efficacy towards DYRK1A. Among them, NSC361563 emerged as a potent and selective DYRK1A inhibitor. It was shown to decrease tau phosphorylation at multiple sites, thereby enhancing tubulin stability. Moreover, NSC361563 diminished the formation of amyloid beta and offered neuroprotective benefits against amyloid beta-induced toxicity. Our research highlights the critical role of selective DYRK1A inhibitors in treating neurodegenerative diseases and presents a promising starting point for the development of targeted therapies.

The rational installation of pharmacophores targeting HSP90 and LSD1 axes has achieved significant anti-cancer capacity in prostate and colorectal cancer. Among the series of hybrids, inhibitor 6 exhibited remarkable antiproliferative activity against prostate cancer cell lines PC-3 and DU145, with GI50 values of 0.24 and 0.30 mu M, respectively. It demonstrated notable efficacy in combinatorial attack and cell death initiation towards apoptosis. The cell death process was mediated by PARP induction and gamma H2AX signaling, and was also characterized as caspase-dependent and Bcl-xL/Bax-independent. Notably, no difference in eye size or morphology was observed in the zebrafish treated with compound 6 compared to the reference group (AUY922). The profound treatment response in docetaxel-resistant PC-3 cells highlighted the dual inhibitory ability in improving docetaxel sensitivity. Additionally, at a minimum concentration of 1.25 mu M, compound 6 effectively inhibited the growth of patient-derived colorectal cancer (CRC) organoids for up to 10 days in vitro. Together, the designed HSP90/LSD1 inhibitors present a novel route and significant clinical value for anti-cancer drug therapy.

Alzheimer's disease (AD) is an irreversible neurodegenerative disease, with tau pathology caused by abnormally activated dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) being one of the culprits. Coumestrol, a phytoestrogen and natural antioxidant found in various plants, has been reported to alleviate AD, but the underlying mechanism remains unclear. We confirmed coumestrol as a novel DYRK1A inhibitor through enzyme-based assays, X-ray crystallography, and cell line experiments. Coumestrol exhibited minimal cytotoxicity at concentrations up to 100 μM in cell types such as N2A and SH-SY5Y and reduced DYRK1A-induced phosphorylated tau protein levels by >50 % at 60 μM. In the tau protein phosphorylation and microtubule assembly assay, coumestrol at 30 μM reduced phosphorylated tau by >50 % and restored the microtubule assembly process. Coumestrol also significantly reduced amyloid-β (Aβ)-induced oxidative stress in microglia at 1 μM. In zebrafish larvae co-overexpressing DYRK1A and tau, coumestrol mitigated neuronal damage and protected motor function at 48 h-postfertilization. Our results suggest that coumestrol has potential therapeutic effects in AD by inhibiting DYRK1A, lowering p-Tau levels, restoring microtubule assembly, and protecting microglia cells from Aβ-induced cell death, providing new insights into the development of coumestrol as a potential AD treatment.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the current coronavirus disease pandemic. With the rapid evolution of variant strains, finding effective spike protein inhibitors is a logical and critical priority. Angiotensin-converting enzyme 2 (ACE2) has been identified as the functional receptor for SARS-CoV-2 viral entry, and thus related therapeutic approaches associated with the spike protein–ACE2 interaction show a high degree of feasibility for inhibiting viral infection. Our computer-aided drug design (CADD) method meticulously analyzed more than 260,000 compound records from the United States National Cancer Institute (NCI) database, to identify potential spike inhibitors. The spike protein receptor-binding domain (RBD) was chosen as the target protein for our virtual screening process. In cell-based validation, SARS-CoV-2 pseudovirus carrying a reporter gene was utilized to screen for effective compounds. Ultimately, compounds C2, C8, and C10 demonstrated significant antiviral activity against SARS-CoV-2, with estimated EC50 values of 8.8 μM, 6.7 μM, and 7.6 μM, respectively. Using the above compounds as templates, ten derivatives were generated and robust bioassay results revealed that C8.2 (EC50 = 5.9 μM) exhibited the strongest antiviral efficacy. Compounds C8.2 also displayed inhibitory activity against the Omicron variant, with an EC50 of 9.3 μM. Thus, the CADD method successfully discovered lead compounds binding to the spike protein RBD that are capable of inhibiting viral infection.

Shrimp acute hepatopancreatic necrosis disease (AHPND) is one of the most devastating diseases to impact the global shrimp farming industry, with a mortality rate of 70 %-100 %. The key virulence factors are a pair of Photorhabdus insect-related (Pir)-like toxins, PirAvp and PirBvp. In this study, by using an in vitro transcription and translation assay, we first confirmed that the quorum sensing transcriptional regulator AphBvp could trigger the expression of its downstream genes after binding to the AphBvp binding sequence in the promoter region of the pirAvp/pirBvp operon. Next, we showed that AphBvp was essential for the expression of these toxins by using an aphB vp-deletion mutant ( Delta aphB vp ) derived from the AHPND-causing Vibrio parahaemolyticus. Lastly, we discovered that the expression levels of PirAvp and PirBvp were up-regulated under acidic conditions (pH 4.5), and further showed that an acidic environment promoted the binding of AphBvp to the pirABvp promoter. We speculate that this was because the acidic environment favored the formation of AphBvp tetramers, which is important for binding to DNA. Taken together, these findings improve our understanding of the gene regulatory mechanisms of pirAvp and pirBvp, and suggest that the pH value of the environment might affect the virulence of AHPNDcausing V. parahaemolyticus.

The dual-specificity tyrosine-phosphorylation-regulated kinase 1 A (DYRK1A) presents a promising therapeutic target for neurological diseases. However, current inhibitors lack selectivity, which can lead to unexpected side effects and increase the difficulty of studying DYRK1A. Therefore, identifying selective inhibitors targeting DYRK1A is essential for reducing side effects and facilitating neurological disease research. This study aimed to discover DYRK1A inhibitors through a screening pipeline incorporating a deep neural network (DNN) model. Herein, we report an optimized model with an accuracy of 0.93 on a testing set. The pipeline was then performed to identify potential DYRK1A inhibitors from the National Cancer Institute (NCI) library. Four novel DYRK1A inhibitors were identified, and compounds NSC657702 and NSC31059 were noteworthy for their potent inhibition, with IC50 values of 50.9 and 39.5 nM, respectively. NSC31059 exhibited exceptional selectivity across 70 kinases. The compounds also significantly reduced DYRK1A-induced tau phosphorylation at key sites associated with the pathology of neurodegenerative diseases. Moreover, they promoted tubulin polymerization, suggesting a role in microtubule stabilization. Cytotoxicity assessments further confirmed the neuronal safety of the compounds. Together, the results demonstrated a promising screening pipeline and novel DYRK1A inhibitors as candidates for further optimization and development.

Marine natural products offer immense potential for drug development, but the limited supply of marine organisms poses a significant challenge. Establishing aquaculture presents a sustainable solution for this challenge by facilitating the mass production of active ingredients while reducing our reliance on wild populations and harm to local environments. To fully utilize aquaculture as a source of biologically active products, a cell-free system was established to target molecular components with protein-modulating activity, including topoisomerase II, HDAC, and tubulin polymerization, using extracts from aquaculture corals. Subsequent in vitro studies were performed, including MTT assays, flow cytometry, confocal microscopy, and Western blotting, along with in vivo xenograft models, to verify the efficacy of the active extracts and further elucidate their cytotoxic mechanisms. Regulatory proteins were clarified using NGS and gene modification techniques. Molecular docking and SwissADME assays were performed to evaluate the drug-likeness and pharmacokinetic and medicinal chemistry-related properties of the small molecules. The extract from Lobophytum crassum (LCE) demonstrated potent broad-spectrum activity, exhibiting significant inhibition of tubulin polymerization, and showed low IC50 values against prostate cancer cells. Flow cytometry and Western blotting assays revealed that LCE induced apoptosis, as evidenced by the increased expression of apoptotic protein-cleaved caspase-3 and the populations of early and late apoptotic cells. In the xenograft tumor experiments, LCE significantly suppressed tumor growth and reduced the tumor volume (PC3: 43.9%; Du145: 49.2%) and weight (PC3: 48.8%; Du145: 7.8%). Additionally, LCE inhibited prostate cancer cell migration, and invasion upregulated the epithelial marker E-cadherin and suppressed EMT-related proteins. Furthermore, LCE effectively attenuated TGF-β-induced EMT in PC3 and Du145 cells. Bioactivity-guided fractionation and SwissADME validation confirmed that LCE’s main component, 13-acetoxysarcocrassolide (13-AC), holds greater potential for the development of anticancer drugs.

Glaucoma is considered a neurodegenerative disease characterized by progressive visual field defects that may lead to blindness. Although controlling intraocular pressure (IOP) is the mainstay of glaucoma treatment, some glaucoma patients have unmet needs due to unclear pathogenic mechanisms. Recently, there has been growing evidence that neuroinflammation is a potential target for the development of novel antiglaucoma agents. In this study, we investigated the protective effects and cellular mechanisms of H7E, a novel small molecule inhibits HDAC8, using in vitro and in vivo glaucoma-like models. Importantly, H7E mitigated extracellular MMP-9 activity and MCP-1 levels in glutamate- or S100B-stimulated reactive Müller glia. In addition, H7E inhibited the upregulation of inflammation- and proliferation-related signaling pathways, particularly the ERK and JNK MAPK pathways. Under conditions of oxidative damage, H7E prevents retinal cell death and reduces extracellular glutamate released from stressed Müller glia. In a mouse model of NMDA-induced retinal degeneration, H7E alleviated functional and structural defects within the inner retina as assessed by electroretinography and optical coherence tomography. Our results demonstrated that the newly identified compound H7E protects against glaucoma damage by specifically targeting HDAC8 activity in the retina. This protective effect is attributed to the inhibition of Müller glial activation and the prevention of retinal cell death caused by oxidative stress.

Pancreatic ductal adenocarcinoma (PDAC) has limited treatment options, underscoring the urgent need for developing new therapies. The upregulation of TBK1 activity plays a crucial role in multiple pancreatic cancer-related signaling pathways, suggesting that inhibiting the kinase activity of TBK1 could be a promising strategy. Herein, we discovered a novel TBK1 inhibitor, LIB3S0280, using a structure-based virtual screening (SBVS) strategy. In the anti-proliferative and viability assays, LIB3S0280 showed significant inhibition against pancreatic cancer cell lines that highly express TBK1 with the GI50 values of 2.24 and 4.71 μM and IC50 values of 6.64 and 10.98 μM at 96 h. For the downstream targets, LIB3S0280 can inhibit TBK1 downstream signaling by decreasing the phosphorylation of IκBα and AKT better than a known TBK1 inhibitor, BX-795. Furthermore, PDAC cells were arrested in G2/M and underwent apoptosis or senescence with the treatment of LIB3S0280. These findings suggest that TBK1 inhibitor LIB3S0280 has great potential as a lead compound in the further development of a novel treatment for PDAC.

Coronaviruses (CoV) are highly pathogenic single-strand RNA viruses. CoV infections cause fatal respiratory symptoms and lung injuries in humans and significant economic losses in livestock. Since the SARS-2 outbreak in 2019, the highly conserved main protease (Mpro), also termed 3-chymotrypsin-like protease (3CLpro), has been considered an attractive drug target for treating CoV infections. Mpro mediates the proteolytic cleavage of eleven sites in viral polypeptides necessary for virus replication. Here, we report that disulfiram, an FDA-approved drug for alcoholic treatment, exhibits a broad-spectrum inhibitory effect on CoV Mpros. Analytical ultracentrifugation and circular dichroism analyses indicated that disulfiram treatment blocks the dimeric formation of SARS and PEDV Mpros and decreases the thermostability of SARS, SARS-2, and PEDV Mpros, whereas it facilitates the dimerization and stability of MERS Mpro. Furthermore, mass spectrometry and structural alignment revealed that disulfiram targets the Cys44 residue of Mpros, which is located at the substrate entrance and close to the catalytic His41. In addition, molecular docking analysis suggests that disulfiram conjugation interferes with substrate entry to the catalytic center. In agreement, mutation of Cys44 modulates the disulfiram sensitivity of CoV Mpros. Our study suggests a broad-spectrum inhibitory function of disulfiram against CoV Mpros.

Dysregulation of RNA splicing processes is intricately linked to tumorigenesis in various cancers, especially breast cancer. Cdc2-like kinase 2 (CLK2), an oncogenic RNA-splicing kinase pivotal in breast cancer, plays a significant role, particularly in the context of triple-negative breast cancer (TNBC), a subtype marked by substantial medical challenges due to its low survival rates. In this study, we employed a structure-based virtual screening (SBVS) method to identify potential CLK2 inhibitors with novel chemical structures for treating TNBC. Compound 670551 emerged as a novel CLK2 inhibitor with a 50% inhibitory concentration (IC 50 ) value of 619.7 nM. Importantly, Compound 670551 exhibited high selectivity for CLK2 over other protein kinases. Functionally, this compound significantly reduced the survival and proliferation of TNBC cells. Results from a cell-based assay demonstrated that this inhibitor led to a decrease in RNA splicing proteins, such as SRSF4 and SRSF6, resulting in cell apoptosis. In summary, we identified a novel CLK2 inhibitor as a promising potential treatment for TNBC therapy.

The identification of an effective inhibitor is an important starting step in drug development. Unfortunately, many issues such as the characterization of protein binding sites, the screening library, materials for assays, etc., make drug screening a difficult proposition. As the size of screening libraries increases, more resources will be inefficiently consumed. Thus, new strategies are needed to preprocess and focus a screening library towards a targeted protein. Herein, we report an ensemble machine learning (ML) model to generate a CDK8-focused screening library. The ensemble model consists of six different algorithms optimized for CDK8 inhibitor classification. The models were trained using a CDK8-specific fragment library along with molecules containing CDK8 activity. The optimized ensemble model processed a commercial library containing 1.6 million molecules. This resulted in a CDK8-focused screening library containing 1,672 molecules, a reduction of more than 99.90%. The CDK8-focused library was then subjected to molecular docking, and 25 candidate compounds were selected. Enzymatic assays confirmed six CDK8 inhibitors, with one compound producing an IC 50 value of ≤100 nM. Analysis of the ensemble ML model reveals the role of the CDK8 fragment library during training. Structural analysis of molecules reveals the hit compounds to be structurally novel CDK8 inhibitors. Together, the results highlight a pipeline for curating a focused library for a specific protein target, such as CDK8.

The overexpression of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), commonly observed in neurodegenerative diseases like Alzheimer's disease (AD) and Down syndrome (DS), can induce the formation of neurofibrillary tangles (NFTs) and amyloid plaques. Hence, designing a selective DYRK1A inhibitor would result in a promising small molecule for treating neurodegenerative diseases. Developing selective inhibitors for DYRK1A has been a difficult challenge due to the highly preserved ATP-binding site of protein kinases. In this study, we employed a structure-based virtual screening (SBVS) campaign targeting DYRK1A from a database containing 1.6 million compounds. Enzymatic assays were utilized to verify inhibitory properties, confirming that Y020-3945 and Y020-3957 showed inhibitory activity towards DYRK1A. In particular, the compounds exhibited high selectivity for DYRK1A over a panel of 120 kinases, reduced the phosphorylation of tau, and reversed the tubulin polymerization for microtubule stability. Additionally, treatment with the compounds significantly reduced the secretion of inflammatory cytokines IL-6 and TNF-α activated by DYRK1A-assisted NFTs and Aβ oligomers. These identified inhibitors possess promising therapeutic potential for conditions associated with DYRK1A in neurodegenerative diseases. The results showed that Y020-3945 and Y020-3957 demonstrated structural novelty compared to known DYRK1A inhibitors, making them a valuable addition to developing potential treatments for neurodegenerative diseases.