Structure-based Virtual Screening Strategy Research Articles

Discovery of a novel SHP2 allosteric inhibitor using virtual screening, FMO calculation, and molecular dynamic simulation.

SHP2 is a non-receptor protein tyrosine phosphatase to remove tyrosine phosphorylation. Functionally, SHP2 is an essential bridge to connect numerous oncogenic cell-signaling cascades including RAS-ERK, PI3K-AKT, JAK-STAT, and PD-1/PD-L1 pathways. This study aims to discover novel and potent SHP2 inhibitors using a hierarchical structure-based virtual screening strategy that combines molecular docking and the fragment molecular orbital method (FMO) for calculating binding affinity (referred to as the Dock-FMO protocol). For the SHP2 target, the FMO method prediction has a high correlation between the binding affinity of the protein-ligand interaction and experimental values (R2 = 0.55), demonstrating a significant advantage over the MM/PBSA (R2 = 0.02) and MM/GBSA (R2 = 0.15) methods. Therefore, we employed Dock-FMO virtual screening of ChemDiv database of ∼2,990,000 compounds to identify a novel SHP2 allosteric inhibitor bearing hydroxyimino acetamide scaffold. Experimental validation demonstrated that the new compound (E)-2-(hydroxyimino)-2-phenyl-N-(piperidin-4-ylmethyl)acetamide (7188-0011) effectively inhibited SHP2 in a dose-dependent manner. Molecular dynamics (MD) simulation analysis revealed the binding stability of compound 7188-0011 and the SHP2 protein, along with the key interacting residues in the allosteric binding site. Overall, our work has identified a novel and promising allosteric inhibitor that targets SHP2, providing a new starting point for further optimization to develop more potent inhibitors. All the molecular docking studies were employed to identify potential leads with Maestro v10.1. The protein-ligand binding affinities of potential leads were further predicted by FMO calculations at MP2/6-31G* level using GAMESS v2020 system. MD simulations were carried out with AmberTools18 by applying the FF14SB force field. MD trajectories were analyzed using VMD v1.9.3. MM/GB(PB)SA binding free energy analysis was carried out with the mmpbsa.py tool of AmberTools18. The docking and MD simulation results were visualized through PyMOL v2.5.0.

Unveiling Novel ERCC1-XPF Complex Inhibitors: Bridging the Gap from In Silico Exploration to Experimental Design.

Modifications in DNA repair pathways are recognized as prognostic markers and potential therapeutic targets in various cancers, including non-small cell lung cancer (NSCLC). Overexpression of ERCC1 correlates with poorer prognosis and response to platinum-based chemotherapy. As a result, there is a pressing need to discover new inhibitors of the ERCC1-XPF complex that can potentiate the efficacy of cisplatin in NSCLC. In this study, we developed a structure-based virtual screening strategy targeting the inhibition of ERCC1 and XPF interaction. Analysis of crystal structures and a library of small molecules known to act against the complex highlighted the pivotal role of Phe293 (ERCC1) in maintaining complex stability. This residue was chosen as the primary binding site for virtual screening. Using an optimized docking protocol, we screened compounds from various databases, ultimately identifying more than one hundred potential inhibitors. Their capability to amplify cisplatin-induced cytotoxicity was assessed in NSCLC H1299 cells, which exhibited the highest ERCC1 expression of all the cell lines tested. Of these, 22 compounds emerged as promising enhancers of cisplatin efficacy. Our results underscore the value of pinpointing crucial molecular characteristics in the pursuit of novel modulators of the ERCC1-XPF interaction, which could be combined with cisplatin to treat NSCLC more effectively.

Targeting Cathepsin L in Cancer Management: Leveraging Machine Learning, Structure-Based Virtual Screening, and Molecular Dynamics Studies.

Cathepsin L (CTSL) expression is dysregulated in a variety of cancers. Extensive empirical evidence indicates their direct participation in cancer growth, angiogenic processes, metastatic dissemination, and the development of treatment resistance. Currently, no natural CTSL inhibitors are approved for clinical use. Consequently, the development of novel CTSL inhibition strategies is an urgent necessity. In this study, a combined machine learning (ML) and structure-based virtual screening strategy was employed to identify potential natural CTSL inhibitors. The random forest ML model was trained on IC50 values. The accuracy of the trained model was over 90%. Furthermore, we used this ML model to screen the Biopurify and Targetmol natural compound libraries, yielding 149 hits with prediction scores >0.6. These hits were subsequently selected for virtual screening using a structure-based approach, yielding 13 hits with higher binding affinity compared to the positive control (AZ12878478). Two of these hits, ZINC4097985 and ZINC4098355, have been shown to strongly bind CTSL proteins. In addition to drug-like properties, both compounds demonstrated high affinity, ligand efficiency, and specificity for the CTSL binding pocket. Furthermore, in molecular dynamics simulations spanning 200 ns, these compounds formed stable protein-ligand complexes. ZINC4097985 and ZINC4098355 can be considered promising candidates for CTSL inhibition after experimental validation, with the potential to provide therapeutic benefits in cancer management.

Discovery of novel TLR4/MD-2 inhibitors: Receptor structure-based virtual screening studies and anti-inflammatory evaluation

In this study, a receptor structure-based virtual screening strategy was constructed using a computer-aided drug design. First, the compounds were filtered based on the Lipinski pentad and adsorption, distribution, metabolism, excretion, and toxicity profiles. Then, receptor structure-based pharmacophore models were constructed and screened. Finally, the in vitro toxicity and anti-inflammatory activities of hit compounds were initially evaluated to investigate their in vitro anti-inflammatory effects and mechanisms of action. The results revealed that hit 94 had the best anti-inflammatory activity and low toxicity while inhibiting the activation of Toll-like receptor (TLR) 4/myeloid differentiation factor 2 (MD2)-associated signaling pathways of nuclear factor-κB and mitogen-activated protein kinase. In vivo adjuvant arthritis results also revealed that hit 94 ameliorated foot swelling to a greater extent in rats compared with the positive control drug indomethacin. These results suggest that hit 94 can be used as a potential TLR/MD2 inhibitor for inflammatory diseases.

Identification of CBPA as a New Inhibitor of PD-1/PD-L1 Interaction.

Targeting of the PD-1/PD-L1 immunologic checkpoint is believed to have provided a real breakthrough in the field of cancer therapy in recent years. Due to the intrinsic limitations of antibodies, the discovery of small-molecule inhibitors blocking PD-1/PD-L1 interaction has gradually opened valuable new avenues in the past decades. In an effort to discover new PD-L1 small molecular inhibitors, we carried out a structure-based virtual screening strategy to rapidly identify the candidate compounds. Ultimately, CBPA was identified as a PD-L1 inhibitor with a KD value at the micromolar level. It exhibited effective PD-1/PD-L1 blocking activity and T-cell-reinvigoration potency in cell-based assays. CBPA could dose-dependently elevate secretion levels of IFN-γ and TNF-α in primary CD4+ T cells in vitro. Notably, CBPA exhibited significant in vivo antitumor efficacy in two different mouse tumor models (a MC38 colon adenocarcinoma model and a melanoma B16F10 tumor model) without the induction of observable liver or renal toxicity. Moreover, analyses of the CBPA-treated mice further showed remarkably increased levels of tumor-infiltrating CD4+ and CD8+ T cells and cytokine secretion in the tumor microenvironment. A molecular docking study suggested that CBPA embedded relatively well into the hydrophobic cleft formed by dimeric PD-L1, occluding the PD-1 interaction surface of PD-L1. This study suggests that CBPA could work as a hit compound for the further design of potent inhibitors targeting the PD-1/PD-L1 pathway in cancer immunotherapy.

Development of Novel Ecto-Nucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1) Inhibitors for Tumor Immunotherapy.

The cyclic guanosine monophosphate–adenosine monophosphate synthase–stimulator of interferon genes–TANK-binding kinase 1–interferon regulating factor 3 (cGAS-STING-TBK1-IRF3) axis is now acknowledged as the major signaling pathway in innate immune responses. However, 2′,3′-cGAMP as a STING stimulator is easily recognized and degraded by ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which reduces the effect of tumor immunotherapy and promotes metastatic progression. In this investigation, the structure-based virtual screening strategy was adopted to discover eight candidate compounds containing zinc-binding quinazolin-4(3H)-one scaffold as ENPP1 inhibitors. Subsequently, these novel inhibitors targeting ENPP1 were synthesized and characterized by NMR and high-resolution mass spectra (HRMS). In bioassays, 7-fluoro-2-(((5-methoxy-1H-imidazo[4,5-b]pyridin-2-yl)thio)methyl)quina-zolin-4(3H)-one(compound 4e) showed excellent activity against the ENPP1 at the molecular and cellular levels, with IC50 values of 0.188 μM and 0.732 μM, respectively. Additionally, compound 4e had superior selectivity towards metastatic breast cancer cells (4T1) than towards normal cells (LO2 and 293T) in comparison with cisplatin, indicating that compound 4e can potentially be used in metastatic breast cancer therapy. On the other hand, compound 4e upgraded the expression levels of IFN-β in vivo by preventing the ENPP1 from hydrolyzing the cGAMP to stimulate a more potent innate immune response. Therefore, this compound might be applied to boost antitumor immunity for cancer immunotherapy. Overall, our work provides a strategy for the development of a promising drug candidate targeting ENPP1 for tumor immunotherapy.

In Silico Drug Repositioning to Target the SARS-CoV-2 Main Protease as Covalent Inhibitors Employing a Combined Structure-Based Virtual Screening Strategy of Pharmacophore Models and Covalent Docking.

The epidemic caused by the SARS-CoV-2 coronavirus, which has spread rapidly throughout the world, requires urgent and effective treatments considering that the appearance of viral variants limits the efficacy of vaccines. The main protease of SARS-CoV-2 (Mpro) is a highly conserved cysteine proteinase, fundamental for the replication of the coronavirus and with a specific cleavage mechanism that positions it as an attractive therapeutic target for the proposal of irreversible inhibitors. A structure-based strategy combining 3D pharmacophoric modeling, virtual screening, and covalent docking was employed to identify the interactions required for molecular recognition, as well as the spatial orientation of the electrophilic warhead, of various drugs, to achieve a covalent interaction with Cys145 of Mpro. The virtual screening on the structure-based pharmacophoric map of the SARS-CoV-2 Mpro in complex with an inhibitor N3 (reference compound) provided high efficiency by identifying 53 drugs (FDA and DrugBank databases) with probabilities of covalent binding, including N3 (Michael acceptor) and others with a variety of electrophilic warheads. Adding the energy contributions of affinity for non-covalent and covalent docking, 16 promising drugs were obtained. Our findings suggest that the FDA-approved drugs Vaborbactam, Cimetidine, Ixazomib, Scopolamine, and Bicalutamide, as well as the other investigational peptide-like drugs (DB04234, DB03456, DB07224, DB7252, and CMX-2043) are potential covalent inhibitors of SARS-CoV-2 Mpro.

Virtual screening for novel inhibitors of human Histone Deacetylase 6: Promising new leads for Oral Squamous Cell Carcinoma

Over 90% of Oral Cancers are Oral Squamous Cell Carcinomas (OSCCs). Despite having advanced treatment modalities, there is no significant improvement in the survival rate of Oral Cancer patients over the years. Thus, there arises a need for the identification of new drug targets besides development of new and effective drugs for this disease. Since Histone Deacetylase 6 (HDAC6), a class IIB member of HDAC family, is known to be upregulated in this disease in addition to being associated with tumor growth, it can be considered as a promising drug target for this disease. In this study, a structure-based virtual screening strategy was used to screen a library of 1,539 natural compounds from Naturally Occurring Plant-based Anti-cancerous Compound– Activity–Target (NPACT) database. Upon filtering and docking, top 30 hits were identified and two of them, namely Camptothecin and Diosgenin, were tested experimentally on OSCCs cell lines for anti-proliferative effects. Both of these compounds exhibited inhibitory activity against Oral Cancer cells, therefore suggesting that they can be potential HDAC6 inhibitors, which can further serve as promising leads for OSCCs.

Combined treatment of mitoxantrone sensitizes breast cancer cells to rapalogs through blocking eEF-2K-mediated activation of Akt and autophagy

Oncogenic activation of the mTOR signaling pathway occurs frequently in tumor cells and contributes to the devastating features of cancer, including breast cancer. mTOR inhibitors rapalogs are promising anticancer agents in clinical trials; however, rapalogs resistance remains an unresolved clinical challenge. Therefore, understanding the mechanisms by which cells become resistant to rapalogs may guide the development of successful mTOR-targeted cancer therapy. In this study, we found that eEF-2K, which is overexpressed in cancer cells and is required for survival of stressed cells, was involved in the negative-feedback activation of Akt and cytoprotective autophagy induction in breast cancer cells in response to mTOR inhibitors. Therefore, disruption of eEF-2K simultaneously abrogates the two critical resistance signaling pathways, sensitizing breast cancer cells to rapalogs. Importantly, we identified mitoxantrone, an admitted anticancer drug for a wide range of tumors, as a potential inhibitor of eEF-2K via a structure-based virtual screening strategy. We further demonstrated that mitoxantrone binds to eEF-2K and inhibits its activity, and the combination treatment of mitoxantrone and mTOR inhibitor resulted in significant synergistic cytotoxicity in breast cancer. In conclusion, we report that eEF-2K contributes to the activation of resistance signaling pathways of mTOR inhibitor, suggesting a novel strategy to enhance mTOR-targeted cancer therapy through combining mitoxantrone, an eEF-2K inhibitor.

Structure-based virtual screening and biological evaluation of novel non-bisphosphonate farnesyl pyrophosphate synthase inhibitors

Farnesyl pyrophosphate synthase (FPPS) is known to participate in a variety of disease-related cell signaling pathway and bisphosphonates (BPs) are served as FPPS inhibitors. However, the high polarity of BPs often induces a series of side effects, limiting their applications. In the present study, novel non-BP FPPS inhibitors were discovered by in silico screening and experimental validation. From the structure-based virtual screening (SBVS) strategy combining molecular docking, pharmacophore and binding affinity prediction, 10 hits with novel scaffolds were filtered. The inhibition activity of hits against FPPS was identified and 7 hits showed comparable or higher inhibition activity than Zoledronate. The hit VS-4 with higher lipophilicity (XlogP = 1.81) and binding affinity (KD = 14.3 ± 2.63 μM) to FPPS was selected for further study on cancer cells with different FPPS expression level. Experimental results revealed that VS-4 could better target the FPPS high-expressing colon LoVo and HCT116 cancer cell lines with IC50 of 51.772 ± 0.473 and 43.553 ± 1.027 μM, respectively, whereas the IC50 value against FPPS low expressing MDA-MB-231 cells was >100 μM. The mechanism of VS-4 against colon cancer cells was investigated by flow cytometry and the results indicated that VS-4 induced cell apoptosis by increasing the intracellular reactive oxygen species (ROS) level. Taken together, the SBVS strategy could be used to discover promising non-BP FPPS inhibitors and the lead compound VS-4 might shed a light on designing more potent inhibitors as novel anticancer drugs.

Discovery of human TyrRS inhibitors by structure-based virtual screening, structural optimization, and bioassays.

The human tyrosyl transfer-RNA (tRNA) synthetase (TyrRS), which is well known for its essential aminoacylation function in protein synthesis, has been shown to translocate to the nucleus and protect against DNA damage caused by external stimuli. Small molecules that can fit into the active site pocket of TyrRS are thought to affect the nuclear role. The exploitation of TyrRS inhibitors has attracted attention recently. In this investigation, we adopted a structure-based virtual screening strategy and subsequent structure–activity relationship analysis to discover new TyrRS inhibitors, and identified a potent compound 5,7-dihydroxy-6,8-bis((3-hydroxyphenyl)thio)-2-phenyl-4H-chromen-4-one (compound 11, Ki = 8.8 μM). In intact HeLa cells, this compound showed a protective effect against DNA damage. Compound 11 is a good lead compound for the further development of drugs against disorders caused by DNA damage.

Docking- and pharmacophore-based virtual screening for the identification of novel Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB) inhibitor with a thiobarbiturate scaffold

Mycobacterium tuberculosis (Mtb) protein tyrosine phosphatase B (MptpB) is an important virulence factor for Mtb that contributes to survival of the bacteria in macrophages. The absence of a human ortholog makes MptpB an attractive target for new therapeutics to treat tuberculosis. MptpB inhibitors could be an effective treatment to overcome emerging TB drug resistance. Adopting a structure-based virtual screening strategy, we successfully identified thiobarbiturate-based drug-like MptpB inhibitor 15 with an IC50 of 22.4 μM, and as a non-competitive inhibitor with a Ki of 24.7 μM. Importantly, not only did it exhibit moderate cell membrane permeability, compound 15 also displayed potent inhibition of intracellular TB growth in the macrophage, making it an excellent lead compound for anti-TB drug discovery. To the best of our knowledge, this novel thiobarbiturate is the first class of MptpB inhibitor reported so far that leveraged docking- and pharmacophore-based virtual screening approaches. The results of preliminary structure-activity relationship demonstrated that compound 15 identified herein was not a singleton and may inspire the design of novel selective and drug-like MptpB inhibitors.

Discovery of Natural Products as Novel and Potent FXR Antagonists by Virtual Screening.

Farnesoid X receptor (FXR) is a member of nuclear receptor family involved in multiple physiological processes through regulating specific target genes. The critical role of FXR as a transcriptional regulator makes it a promising target for diverse diseases, especially those related to metabolic disorders such as diabetes and cholestasis. However, the underlying activation mechanism of FXR is still a blur owing to the absence of proper FXR modulators. To identify potential FXR modulators, an in-house natural product database (NPD) containing over 4,000 compounds was screened by structure-based virtual screening strategy and subsequent hit-based similarity searching method. After the yeast two-hybrid (Y2H) assay, six natural products were identified as FXR antagonists which blocked the CDCA-induced SRC-1 association. The IC50 values of compounds 2a, a diterpene bearing polycyclic skeleton, and 3a, named daphneone with chain scaffold, are as low as 1.29 and 1.79 μM, respectively. Compared to the control compound guggulsterone (IC50 = 6.47 μM), compounds 2a and 3a displayed 5- and 3-fold higher antagonistic activities against FXR, respectively. Remarkably, the two representative compounds shared low topological similarities with other reported FXR antagonists. According to the putative binding poses, the molecular basis of these antagonists against FXR was also elucidated in this report.

Identification of Substituted Naphthotriazolediones as Novel Tryptophan 2,3-Dioxygenase (TDO) Inhibitors through Structure-Based Virtual Screening.

A structure-based virtual screening strategy, comprising homology modeling, ligand-support binding site optimization, virtual screening, and structure clustering analysis, was developed and used to identify novel tryptophan 2,3-dioxygenase (TDO) inhibitors. Compound 1 (IC50 = 711 nM), selected by virtual screening, showed inhibitory activity toward TDO and was subjected to structural modifications and molecular docking studies. This resulted in the identification of a potent TDO selective inhibitor (11e, IC50 = 30 nM), making it a potential compound for further investigation as a cancer therapeutic and other TDO-related targeted therapy.

Modeling, molecular dynamics, and docking assessment of transcription factor rho: a potential drug target in Brucella melitensis 16M.

The zoonotic disease brucellosis, a chronic condition in humans affecting renal and cardiac systems and causing osteoarthritis, is caused by Brucella, a genus of Gram-negative, facultative, intracellular pathogens. The mode of transmission and the virulence of the pathogens are still enigmatic. Transcription regulatory elements, such as rho proteins, play an important role in the termination of transcription and/or the selection of genes in Brucella. Adverse effects of the transcription inhibitors play a key role in the non-successive transcription challenges faced by the pathogens. In the investigation presented here, we computationally predicted the transcription termination factor rho (TtFRho) inhibitors against Brucella melitensis 16M via a structure-based method. In view the unknown nature of its crystal structure, we constructed a robust three-dimensional homology model of TtFRho’s structure by comparative modeling with the crystal structure of the Escherichia coli TtFRho (Protein Data Bank ID: 1PVO) as a template in MODELLER (v 9.10). The modeled structure was optimized by applying a molecular dynamics simulation for 2 ns with the CHARMM (Chemistry at HARvard Macromolecular Mechanics) 27 force field in NAMD (NAnoscale Molecular Dynamics program; v 2.9) and then evaluated by calculating the stereochemical quality of the protein. The flexible docking for the interaction phenomenon of the template consists of ligand-related inhibitor molecules from the ZINC (ZINC Is Not Commercial) database using a structure-based virtual screening strategy against minimized TtFRho. Docking simulations revealed two inhibitors compounds – ZINC24934545 and ZINC72319544 – that showed high binding affinity among 2,829 drug analogs that bind with key active-site residues; these residues are considered for protein-ligand binding and unbinding pathways via steered molecular dynamics simulations. Arg215 in the model plays an important role in the stability of the protein-ligand complex via a hydrogen bonding interaction by aromatic-π contacts, and the ADMET (absorption, distribution, metabolism, and excretion) analysis of best leads indicate nontoxic in nature with good potential for drug development.

New scaffolds of natural origin as Integrase–LEDGF/p75 interaction inhibitors: Virtual screening and activity assays

The disruption of crucial interactions between HIV-1 Integrase and cellular cofactor LEDGF/p75 represents an emerging approach for the design and development of new antiretroviral agents. In this study we report the successful application of a structure-based virtual screening strategy for the discovery of natural hit structures able to inhibit Integrase–LEDGF/p75 interaction. The application of sequential filters (drug-likeness, 3D-pharmacophore mapping, docking, molecular dynamics simulations) yielded a hit list of compounds, out of which 9 were tested in the in vitro AlphaScreen assays and 8 exhibited a detectable inhibition of the interaction between the two proteins. The best inhibitors belong to different chemical classes and could be represent a good starting point for further optimization and structure–activity relationship studies.

Identification of a low–molecular weight TrkB antagonist with anxiolytic and antidepressant activity in mice

The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase B (TrkB) have emerged as key mediators in the pathophysiology of several mood disorders, including anxiety and depression. However, therapeutic compounds that interact with TrkB receptors have been difficult to develop. Using a combination of structure-based in silico screening and high-capacity functional assays in recombinant and neuronal cells, we identified a low-molecular weight TrkB ligand (ANA-12) that prevented activation of the receptor by BDNF with a high potency. ANA-12 showed direct and selective binding to TrkB and inhibited processes downstream of TrkB without altering TrkA and TrkC functions. KIRA-ELISA analysis demonstrated that systemic administration of ANA-12 to adult mice decreased TrkB activity in the brain without affecting neuronal survival. Mice administered ANA-12 demonstrated reduced anxiety- and depression-related behaviors on a variety of tests predictive of anxiolytic and antidepressant properties in humans. This study demonstrates that structure-based virtual screening strategy can be an efficient method for discovering potent TrkB-selective ligands that are active in vivo. We further propose that ANA-12 may be a valuable tool for studying BDNF/TrkB signaling and may constitute a lead compound for developing the next generation of therapeutic agents for the treatment of mood disorders.

Targeting the BH3 Domain Mediated Protein−Protein Interaction of Bcl-xL through Virtual Screening

Apoptosis, or programmed cell death, forms an important part of the cellular regulation machinery. The Bcl-2 protein family, comprising of proapoptotic and antiapoptotic members, forms an important part of the cells internal apoptotic pathway. Overexpression of the antiapoptotic members of the family in a number of cancer cell lines renders them immune to apoptosis and the ability to survive under conditions of cellular stress. Inhibition of the antiapoptotic members of the Bcl-2 family are, therefore, an interesting target for the development of anticancer therapy. An innovative structure-based virtual screening strategy was developed to identify inhibitors of Bcl-xL, an antiapoptotic member of the Bcl-2 family. Various innovative filters, such as receptor-based pharmacophore, cascade docking approach, cross-docking, and composite scoring with docking pose based descriptors were designed through exhaustive validation studies and implemented in the screening funnel. The 1.8 million 'big-n-greasy' subset from ZINC was screened using the protocol, and 45 compounds were finally selected for biological evaluation against Bcl-xL. The evaluation led to the identification of one low-micromolar and two weaker inhibitors belonging to novel scaffolds. Further evaluation of structure-activity relationships around these scaffolds could help in the development of anticancer leads against Bcl-xL.