Consensus Pharmacophore Strategy For Identifying Novel SARS-Cov-2 Mpro Inhibitors from Large Chemical Libraries.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main Protease (Mpro) is an enzyme that cleaves viral polyproteins translated from the viral genome and is critical for viral replication. Mpro is a target for anti-SARS-CoV-2 drug development, and multiple Mpro crystals complexed with competitive inhibitors have been reported. In this study, we aimed to develop an Mpro consensus pharmacophore as a tool to expand the search for inhibitors. We generated a consensus model by aligning and summarizing pharmacophoric points from 152 bioactive conformers of SARS-CoV-2 Mpro inhibitors. Validation against a library of conformers from a subset of ligands showed that our model retrieved poses that reproduced the crystal-binding mode in 77% of the cases. Using models derived from a consensus pharmacophore, we screened >340 million compounds. Pharmacophore-matching and chemoinformatics analyses identified new potential Mpro inhibitors. The candidate compounds were chemically dissimilar to the reference set, and among them, demonstrating the relevance of our model. We evaluated the effect of 16 candidates on Mpro enzymatic activity finding that seven have inhibitory activity. Three compounds (1, 4, and 5) had IC50 values in the midmicromolar range. The Mpro consensus pharmacophore reported herein can be used to identify compounds with improved activity and novel chemical scaffolds against Mpro. The method developed for its generation is provided as an open-access code (https://github.com/AngelRuizMoreno/ConcensusPharmacophore) and can be applied to other pharmacological targets.

Host genetic regulation of human gut microbial structural variation.

Although the impact of host genetics on gut microbial diversity and the abundance of specific taxa is well established1-6, little is known about how host genetics regulates the genetic diversity of gut microorganisms. Here we conducted a meta-analysis of associations between human genetic variation and gut microbial structural variation in 9,015 individuals from four Dutch cohorts. Strikingly, the presence rate of a structural variation segment in Faecalibacterium prausnitzii that harbours an N-acetylgalactosamine (GalNAc) utilization gene cluster is higher in individuals who secrete the type A oligosaccharide antigen terminating in GalNAc, a feature that is jointly determined by human ABO and FUT2 genotypes, and we could replicate this association in a Tanzanian cohort. In vitro experiments demonstrated that GalNAc can be used as the sole carbohydrate source for F. prausnitzii strains that carry the GalNAc-metabolizing pathway. Further in silico and in vitro studies demonstrated that other ABO-associated species can also utilize GalNAc, particularly Collinsella aerofaciens. The GalNAc utilization genes are also associated with the host's cardiometabolic health, particularly in individuals with mucosal A-antigen. Together, the findings of our study demonstrate that genetic associations across the human genome and bacterial metagenome can provide functional insights into the reciprocal host-microbiome relationship.

Identification and characterization of a small molecule that activates thiosulfate sulfurtransferase and stimulates mitochondrial respiration.

The enzyme Thiosulfate sulfurtransferase (TST, EC 2.8.1.1), is a positive genetic predictor of diabetes type 2 and obesity. As increased TST activity protects against the development of diabetic symptoms in mice, an activating compound for TST may provide therapeutic benefits in diabetes and obesity. We identified a small molecule activator of human TST through screening of an inhouse small molecule library. Kinetic studies in vitro suggest that two distinct isomers of the compound are required for full activation as well as an allosteric mode of activation. Additionally, we studied the effect of TST protein and the activator on TST activity through mitochondrial respiration. Molecular docking and molecular dynamics (MD) approaches supports an allosteric site for the binding of the activator, which is supported by the lack of activation in the Escherichia coli. mercaptopyruvate sulfurtransferase. Finally, we show that increasing TST activity in isolated mitochondria increases mitochondrial oxygen consumption.

Phage display sequencing reveals that genetic, environmental, and intrinsic factors influence variation of human antibody epitope repertoire.

Phage-displayed immunoprecipitation sequencing (PhIP-seq) has enabled high-throughput profiling of human antibody repertoires. However, a comprehensive overview of environmental and genetic determinants shaping human adaptive immunity is lacking. In this study, we investigated the effects of genetic, environmental, and intrinsic factors on the variation in human antibody repertoires. We characterized serological antibody repertoires against 344,000 peptides using PhIP-seq libraries from a wide range of microbial and environmental antigens in 1,443 participants from a population cohort. We detected individual-specificity, temporal consistency, and co-housing similarities in antibody repertoires. Genetic analyses showed the involvement of the HLA, IGHV, and FUT2 gene regions in antibody-bound peptide reactivity. Furthermore, we uncovered associations between phenotypic factors (including age, cell counts, sex, smoking behavior, and allergies, among others) and particular antibody-bound peptides. Our results indicate that human antibody epitope repertoires are shaped by both genetics and environmental exposures and highlight specific signatures of distinct phenotypes and genotypes.

Synthetic Peptides That Antagonize the Angiotensin-Converting Enzyme-2 (ACE-2) Interaction with SARS-CoV-2 Receptor Binding Spike Protein.

The SARS-CoV-2 viral spike protein S receptor-binding domain (S-RBD) binds ACE2 on host cells to initiate molecular events, resulting in intracellular release of the viral genome. Therefore, antagonists of this interaction could allow a modality for therapeutic intervention. Peptides can inhibit the S-RBD:ACE2 interaction by interacting with the protein-protein interface. In this study, protein contact atlas data and molecular dynamics simulations were used to locate interaction hotspots on the secondary structure elements α1, α2, α3, ß3, and ß4 of ACE2. We designed a library of discontinuous peptides based upon a combination of the hotspot interactions, which were synthesized and screened in a bioluminescence-based assay. The peptides demonstrated high efficacy in antagonizing the SARS-CoV-2 S-RBD:ACE2 interaction and were validated by microscale thermophoresis which demonstrated strong binding affinity (∼10 nM) of these peptides to S-RBD. We anticipate that such discontinuous peptides may hold the potential for an efficient therapeutic treatment for COVID-19.

Characterization of gut microbial structural variations as determinants of human bile acid metabolism.

Bile acids (BAs) facilitate intestinal fat absorption and act as important signaling molecules in host-gut microbiota crosstalk. BA-metabolizing pathways in the microbial community have been identified, but it remains largely unknown how the highly variable genomes of gut bacteria interact with host BA metabolism. We characterized 8,282 structural variants (SVs) of 55 bacterial species in the gut microbiomes of 1,437 individuals from two cohorts and performed a systematic association study with 39 plasma BA parameters. Both variations in SV-based continuous genetic makeup and discrete clusters showed correlations with BA metabolism. Metagenome-wide association analysis identified 809 replicable associations between bacterial SVs and BAs and SV regulators that mediate the effects of lifestyle factors on BA metabolism. This is the largest microbial genetic association analysis to demonstrate the impact of bacterial SVs on human BA composition, and it highlights the potential of targeting gut microbiota to regulate BA metabolism through lifestyle intervention.

In Silico Design and Selection of New Tetrahydroisoquinoline-Based CD44 Antagonist Candidates.

CD44 promotes metastasis, chemoresistance, and stemness in different types of cancer and is a target for the development of new anti-cancer therapies. All CD44 isoforms share a common N-terminal domain that binds to hyaluronic acid (HA). Herein, we used a computational approach to design new potential CD44 antagonists and evaluate their target-binding ability. By analyzing 30 crystal structures of the HA-binding domain (CD44HAbd), we characterized a subdomain that binds to 1,2,3,4-tetrahydroisoquinoline (THQ)-containing compounds and is adjacent to residues essential for HA interaction. By computational combinatorial chemistry (CCC), we designed 168,190 molecules and compared their conformers to a pharmacophore containing the key features of the crystallographic THQ binding mode. Approximately 0.01% of the compounds matched the pharmacophore and were analyzed by computational docking and molecular dynamics (MD). We identified two compounds, Can125 and Can159, that bound to human CD44HAbd (hCD44HAbd) in explicit-solvent MD simulations and therefore may elicit CD44 blockage. These compounds can be easily synthesized by multicomponent reactions for activity testing and their binding mode, reported here, could be helpful in the design of more potent CD44 antagonists.

Repositioning of Etravirine as a Potential CK1ε Inhibitor by Virtual Screening.

CK1ε is a key regulator of WNT/ß-catenin and other pathways that are linked to tumor progression; thus, CK1ε is considered a target for the development of antineoplastic therapies. In this study, we performed a virtual screening to search for potential CK1ε inhibitors. First, we characterized the dynamic noncovalent interactions profiles for a set of reported CK1ε inhibitors to generate a pharmacophore model, which was used to identify new potential inhibitors among FDA-approved drugs. We found that etravirine and abacavir, two drugs that are approved for HIV infections, can be repurposed as CK1ε inhibitors. The interaction of these drugs with CK1ε was further examined by molecular docking and molecular dynamics. Etravirine and abacavir formed stable complexes with the target, emulating the binding behavior of known inhibitors. However, only etravirine showed high theoretical binding affinity to CK1ε. Our findings provide a new pharmacophore for targeting CK1ε and implicate etravirine as a CK1ε inhibitor and antineoplastic agent.

Pranlukast Antagonizes CD49f and Reduces Stemness in Triple-Negative Breast Cancer Cells.

INTRODUCTION: Cancer stem cells (CSCs) drive the initiation, maintenance, and therapy response of breast tumors. CD49f is expressed in breast CSCs and functions in the maintenance of stemness. Thus, blockade of CD49f is a potential therapeutic approach for targeting breast CSCs. In the present study, we aimed to repurpose drugs as CD49f antagonists. MATERIALS AND METHODS: We performed consensus molecular docking using a subdomain of CD49f that is critical for heterodimerization and a collection of pharmochemicals clinically tested. Molecular dynamics simulations were employed to further characterize drug-target binding. Using MDA-MB-231 cells, we evaluated the effects of potential CD49f antagonists on 1) cell adhesion to laminin; 2) mammosphere formation; and 3) cell viability. We analyzed the effects of the drug with better CSC-selectivity on the activation of CD49f-downstream signaling by Western blot (WB) and co-immunoprecipitation. Expressions of the stem cell markers CD44 and SOX2 were analyzed by flow cytometry and WB, respectively. Transactivation of SOX2 promoter was evaluated by luciferase reporter assays. Changes in the number of CSCs were assessed by limiting-dilution xenotransplantation. RESULTS: Pranlukast, a drug used to treat asthma, bound to CD49f in silico and inhibited the adhesion of CD49f+ MDA-MB-231 cells to laminin, indicating that it antagonizes CD49f-containing integrins. Molecular dynamics analysis showed that pranlukast binding induces conformational changes in CD49f that affect its interaction with ß1-integrin subunit and constrained the conformational dynamics of the heterodimer. Pranlukast decreased the clonogenicity of breast cancer cells on mammosphere formation assay but had no impact on the viability of bulk tumor cells. Brief exposure of MDA-MB-231 cells to pranlukast altered CD49f-dependent signaling, reducing focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K) activation. Further, pranlukast-treated cells showed decreased CD44 and SOX2 expression, SOX2 promoter transactivation, and in vivo tumorigenicity, supporting that this drug reduces the frequency of CSC. CONCLUSION: Our results support the function of pranlukast as a CD49f antagonist that reduces the CSC population in triple-negative breast cancer cells. The pharmacokinetics and toxicology of this drug have already been established, rendering a potential adjuvant therapy for breast cancer patients.

Scaffolding-Induced Property Modulation of Chemical Space.

Physicochemical property switching of chemical space is of great importance for optimization of compounds, for example, for biological activity. Cyclization is a key method to control 3D and other properties. A two-step approach, which involves a multicomponent reaction followed by cyclization, is reported to achieve the transition from basic moieties to charge neutral cyclic derivatives. A series of multisubstituted oxazolidinones, oxazinanones, and oxazepanones as well as their thio and sulfur derivatives are synthesized from readily available building blocks with mild conditions and high yields. Like a few other methods, MCR and cyclization allow for the collective transformation of a large chemical space into a related one with different properties.

Guide for Selection of Relevant Cell Lines During the Evaluation of new Anti-Cancer Compounds.

BACKGROUND: Human cancer cell lines are valuable models for anti-cancer drug development. Although all cancer cells share common biological features, each cancer cell line has unique genotypic/ phenotypic characteristics that affect drug response. Thus, the information obtained with a specific cancer cell line cannot be easily extrapolated to other cancer cells. Consequently, cell line selection during experimental design is critical for providing proper and clinically relevant structure-activity analysis. METHODS: Herein, we critically review the use of cancer cell lines as tools for activity analysis by comparing two different scenarios: i) the use of multiple cancer cell lines, with the NCI-60 Program as the most representative example; and, ii) the selection of a single cell line with specific biological characteristics that match the rationale of compound design. RESULTS: Considering that most laboratories evaluate the activity of new compounds using few cell lines, we provide a systematic strategy for selection based on the expression levels and genetic status of the target and the effectiveness of target inhibition or silencing. We exemplify the use of public databases for data retrieval and analysis as well as the critical comparison of such information with published results. CONCLUSION: This approach refines cell line selection, avoiding the perpetuation of published poor selection and enhancing the relevance of the results.

Virtual screening-driven repositioning of etoposide as CD44 antagonist in breast cancer cells.

CD44 is a receptor for hyaluronan (HA) that promotes epithelial-to-mesenchymal transition (EMT), induces cancer stem cell (CSC) expansion, and favors metastasis. Thus, CD44 is a target for the development of antineoplastic agents. In order to repurpose drugs as CD44 antagonists, we performed consensus-docking studies using the HA-binding domain of CD44 and 11,421 molecules. Drugs that performed best in docking were examined in molecular dynamics simulations, identifying etoposide as a potential CD44 antagonist. Ligand competition and cell adhesion assays in MDA-MB-231 cells demonstrated that etoposide decreased cell binding to HA as effectively as a blocking antibody. Etoposide-treated MDA-MB-231 cells developed an epithelial morphology; increased their expression of E-cadherin; and reduced their levels of EMT-associated genes and cell migration. By gene expression analysis, etoposide reverted an EMT signature similarly to CD44 knockdown, whereas other topoisomerase II (TOP2) inhibitors did not. Moreover, etoposide decreased the proportion of CD44+/CD24- cells, lowered chemoresistance, and blocked mammosphere formation. Our data indicate that etoposide blocks CD44 activation, impairing key cellular functions that drive malignancy, thus rendering it a candidate for further translational studies and a potential lead compound in the development of new CD44 antagonists.