Target identification for repurposed drugs active against SARS-CoV-2 via high-throughput inverse docking.

Screening already approved drugs for activity against a novel pathogen can be an important part of global rapid-response strategies in pandemics. Such high-throughput repurposing screens have already identified several existing drugs with potential to combat SARS-CoV-2. However, moving these hits forward for possible development into drugs specifically against this pathogen requires unambiguous identification of their corresponding targets, something the high-throughput screens are not typically designed to reveal. We present here a new computational inverse-docking protocol that uses all-atom protein structures and a combination of docking methods to rank-order targets for each of several existing drugs for which a plurality of recent high-throughput screens detected anti-SARS-CoV-2 activity. We demonstrate validation of this method with known drug-target pairs, including both non-antiviral and antiviral compounds. We subjected 152 distinct drugs potentially suitable for repurposing to the inverse docking procedure. The most common preferential targets were the human enzymes TMPRSS2 and PIKfyve, followed by the viral enzymes Helicase and PLpro. All compounds that selected TMPRSS2 are known serine protease inhibitors, and those that selected PIKfyve are known tyrosine kinase inhibitors. Detailed structural analysis of the docking poses revealed important insights into why these selections arose, and could potentially lead to more rational design of new drugs against these targets.

Synthesis of homochiral sulfanyl- and sulfoxide-substituted naphthyltriazoles and study of the conformational stability.

The preparation of a series of novel homochiral atropisomeric sulfanyl- and sulfoxide-substituted naphthyltriazoles is described. The triazolization methodology used presents a new way towards novel and highly stable 1,2,3-triazole-based atropisomers, and introduces a new and complementary synthetic pathway towards 4-sulfanyl substituted 1,2,3-triazoles. Starting from sulfanyl-substituted naphthyl ketones, enantiopure amines, and 4-nitrophenyl azide, a collection of 16 sulfanyl-substituted naphthyltriazoles were obtained via the triazolization reaction in which the homochiral diastereomers are readily isolated. Subsequent monooxidation results in the preparation of several sulfoxide-substituted naphthyltriazoles. The absolute configuration of a set of diastereomeric sulfanyl- and sulfoxide-appended naphthyltriazoles was deduced via X-ray crystallography. Furthermore, the conformational stability of the atropisomers was determined experimentally, and further confirmed and analyzed with the aid of computational DFT calculations.

Vitamin D receptor exhibits different pharmacodynamic features in tumoral and normal microenvironments: A molecular modeling study.

The vitamin D receptor (VDR) constitutes a promising therapeutic target for the treatment of cancer. Unfortunately, its natural agonist calcitriol does not have clinical utility due to its potential to induce hypercalcemic effects at the concentrations required to display antitumoral activity. For this reason, the search for new calcitriol analogues with adequate therapeutic profiles has been actively pursued by the scientific community. We have previously reported the obtaining and the biological activity evaluation of new calcitriol analogues by modification of its sidechain, which exhibited relevant antiproliferative and selectivity profiles against tumoral and normal cells. In this work we conducted molecular modeling studies (i.e. molecular docking, molecular dynamics, constant pH molecular dynamics (CpHMD) and free energy of binding analysis) to elucidate at an atomistic level the molecular basis related to the potential of the new calcitriol analogues to achieve selectivity between tumoral and normal cells. Two histidine residues (His305 and His397) were found to exhibit a particular tautomeric configuration that produces the observed bioactivity. Also, different acid-based properties were observed for His305 and His307 with His305 showing an increased acidity (pKa 5.2) compared to His397 (pKa 6.8) and to the typical histidine residue. This behavior favored the pharmacodynamic interaction of the calcitriol analogues exhibiting selectivity for tumoral cells when VDR was modeled at the more acidic tumoral environment (pH ≅ 6) compared to the case when VDR was modeled at pH 7.4 (normal cell environment). On the other hand, non-selective compounds, including calcitriol, exhibited a similar interaction pattern with VDR when the receptor was modeled at both pH conditions. The results presented constitute the first evidence on the properties of the VDR receptor in different physicochemical environments and thus represent a significant contribution to the in silico screening and design of new calcitriol analogues.

Identification of the potential biological target of N-benzenesulfonyl-1,2,3,4-tetrahydroquinoline compounds active against gram-positive and gram-negative bacteria.

The development of new antibiotics with activity towards a broad spectrum of bacteria, including multiresistant strains, is a very important topic for global public health. As part of previous works, N-benzenesulfonyl-1,2,3,4-tetrahydroquinoline (BSTHQ) derivatives were described as antimicrobial agents active against gram-positive and gram-negative pathogens. In this work, experimental and molecular modelling studies were performed in order to identify their potential biological target in the light of structure-based design efforts towards further BSTHQ derivatives. First, a carboxyfluorescein leakage assay was performed using liposomes to mimic bacterial membranes, which found no significative membrane disruption effects with respect to control samples. These results support a non-surfactant antimicrobial activity of the tested compounds. In a second stage, the inhibition of potential antimicrobial targets was screened using molecular modelling methods, taking into account previously reported druggable targets deposited in the ChEMBL database for Escherichia coli and Staphylococcus aureus. Two enzymes, namely D-glutamic acid-adding enzyme (MurD) and N-acetylglucosamine-1-phophate-uridyltransferase (GlmU), both involved in bacterial membrane synthesis, were identified as potential targets. Pharmacodynamic interaction models were developed using known MurD and GlmU inhibitors by applying state-of-the-art chemoinformatic methods (molecular docking, molecular dynamics and free energy of interaction analyses). These models were further extended to the analysis of the studied BSTHQ derivatives. Overall, our results demonstrated that the studied BSTHQ derivatives elicit their antibacterial activity by interacting with a specific molecular target, GlmU being the highly feasible one. Based on the presented results, further structure-aided design efforts towards the obtaining of novel BSTHQ derivatives are envisioned.Communicated by Ramaswamy H. Sarma.

Design, synthesis and molecular docking studies of novel N-arylsulfonyl-benzimidazoles with anti Trypanosoma cruzi activity.

Currently, only two drugs (i.e. benznidazole (BZN) and nifurtimox (NFX)) have been approved for the treatment of Trypanosoma cruzi (Tc) infection, the etiological agent causing Chagas disease. Since both drugs exhibit severe side effects, patients frequently abandon therapy, resulting in an inefficient pharmacotherapeutic treatment. In this context, there is an urgent need to develop new, safer and optimised anti-Tc agents. In this report, we present the synthesis and biological activity of 11 novel and 3 already reported N-arylsulfonyl-benzimidazole derivatives (NBSBZD,1-14) currently in development as potential anti-Tc compounds. These compounds were designed as part of a library of synthetic arylsulfonyl heterocycle derivatives constructed from privileged structures exhibiting drug-like properties. Based on bioactivity assays against Tc, (in both the extracellular and intracellular forms), we observed that 10 compounds exhibited bioactivity against the epimastigote form, while six of them exhibited activity against the amastigote counterpart. Also, the compounds showed less cytotoxicity compared to the reference drug BZN as measured in Vero cell culture. In order to elucidate the potential mechanism of action, metabolite excretion profiles studies were performed, and complemented with molecular modeling studies performed over known Tc druggable targets. Consistency was observed between experimental and theoretical findings, with metabolic profiles showing that compounds 1, 2, 9, 12 and 14 interfered with the normal glycolysis cycle of Tc, while molecular modeling studies were able to establish a solid structure-activity relationship towards the inhibition of 6-phospho-1-fructokinase, a key enzyme involved in the parasite glycolytic cascade. Overall, the present study constitutes a multidisciplinary contribution to the development of new anti-Chagas compounds.

Synthesis, biological evaluation and molecular modeling of a novel series of fused 1,2,3-triazoles as potential anti-coronavirus agents.

Synthesis and biological evaluation of a novel library of fused 1,2,3-triazole derivatives are described. The in-house developed multicomponent reaction based on commercially available starting materials was applied and broad biological screening against various viruses was performed, showing promising antiviral properties for compounds 14d, 14n, 14q, 18f and 18i against human coronavirus 229E. Further in silico studies identified the key molecular interactions between those compounds and the 3-chymotrypsin-like protease, which is essential to the intracellular replication of the virus, supporting the hypothesis that the protease is the target molecule of the potential antiviral derivatives.

Stability and plasmatic protein binding of novel zidovudine prodrugs: Targeting site ii of human serum albumin.

Despite its vastly demonstrated clinical efficacy, zidovudine (AZT) exhibits several suboptimal pharmacokinetic properties. In particular, its short plasmatic half-life (t1/2 ~ 1 h) is related to its low bound fraction to whole plasmatic proteins and in particular to human serum albumin (HSA). The design of prodrugs constitutes a promising strategy to enhance AZT pharmacokinetic properties, including its affinity for HSA. Recently, we reported the synthesis and chemical stability evaluation of three novel prodrugs of AZT obtained by derivatization with dicarboxylic acids (1-3). In this work, we present the design, synthesis and evaluation of chemical and enzymatic stabilities of a novel series of double prodrugs of AZT obtained by derivatization of 1-3 with a methylated l-phenylalanine moiety (4-6). In addition, the plasmatic protein binding properties were studied both by experimental and theoretical techniques. Prodrugs 4-6 were found to be relatively stable at pH 7.4 (t1/2 between 4.1 and 57.8 h), while also demonstrated adequate stabilities in human plasma at 37 °C (t1/2 between 1.0 and 2.1 h). Also, prodrugs 4-6 were able to regenerate AZT at a rate that depended on the length of the alkyl chain in 1-3. Additionally, 4-6 exhibited a significantly increased binding to plasmatic proteins (between 52.1 and 72.5%) with respect to AZT (12%) and 1-3 (between 26 and 34%). It is noteworthy that the displacement experiments with HSA site I and II markers, demonstrated that 4-6 bound to a different site than that of AZT and 1-3. Molecular modeling studies (i.e. molecular docking and free energy of binding analysis) were applied to shed light at an atomistic level on the pharmacodynamic properties driving the interaction of 4-6 with HSA. Overall, the present work provides a state of the art contribution to the design and development of novel prodrugs of AZT with optimized pharmacokinetic properties.

Structural basis for the potent inhibition of the HIV integrase-LEDGF/p75 protein-protein interaction.

Integrase (IN) constitutes one of the key enzymes involved in the lifecycle of the Human Immunodeficiency Virus (HIV), the etiological agent of AIDS. The biological role of IN strongly depends on the recognition and binding of cellular cofactors belonging to the infected host cell. Thus, the inhibition of the protein-protein interaction (PPI) between IN and cellular cofactors has been envisioned as a promising therapeutic target. In the present work we explore a structure-activity relationship for a set of 14 compounds reported as inhibitors of the PPI between IN and the lens epithelium-derived growth factor (LEDGF/p75). Our results demonstrate that the possibility to adopt the bioactive conformation capable of interacting with the hotspots IN-LEDGF/p75 hotspots residues constitutes a critical feature to obtain a potent inhibition. A ligand efficiency (|Lig-Eff|) quantitative descriptor combining both interaction energetics and conformational requirements was developed and correlated with the reported biological activity. Our results contribute to the rational development of IN-LEDGF/p75 interaction inhibitors providing a solid quantitative structure-activity relationship aimed for the screening of new IN-LEDGF/p75 interaction inhibitors.

Development of a receptor model for efficient in silico screening of HIV-1 integrase inhibitors.

Integrase (IN) is a key viral enzyme for the replication of the type-1 human immunodeficiency virus (HIV-1), and as such constitutes a relevant therapeutic target for the development of anti-HIV agents. However, the lack of crystallographic data of HIV IN complexed with the corresponding viral DNA has historically hindered the application of modern structure-based drug design techniques to the discovery of new potent IN inhibitors (INIs). Consequently, the development and validation of reliable HIV IN structural models that may be useful for the screening of large databases of chemical compounds is of particular interest. In this study, four HIV-1 IN homology models were evaluated respect to their capability to predict the inhibition potency of a training set comprising 36 previously reported INIs with IC50 values in the low nanomolar to the high micromolar range. Also, 9 inactive structurally related compounds were included in this training set. In addition, a crystallographic structure of the IN-DNA complex corresponding to the prototype foamy virus (PFV) was also evaluated as structural model for the screening of inhibitors. The applicability of high throughput screening techniques, such as blind and ligand-guided exhaustive rigid docking was assessed. The receptor models were also refined by molecular dynamics and clustering techniques to assess protein sidechain flexibility and solvent effect on inhibitor binding. Among the studied models, we conclude that the one derived from the X-ray structure of the PFV integrase exhibited the best performance to rank the potencies of the compounds in the training set, with the predictive power being further improved by explicitly modeling five water molecules within the catalytic side of IN. Also, accounting for protein sidechain flexibility enhanced the prediction of inhibition potencies among the studied compounds. Finally, an interaction fingerprint pattern was established for the fast identification of potent IN inhibitors. In conclusion, we report an exhaustively validated receptor model if IN that is useful for the efficient screening of large chemical compounds databases in the search of potent HIV-1 IN inhibitors.

Synthesis, biological evaluation and molecular modeling of 4,6-diarylpyrimidines and diarylbenzenes as novel non-nucleosides HIV-1 reverse transcriptase inhibitors.

A series of novel 4,6-diarylpyrimidines (4,6-DAPY) and diarylbenzenes (DABE) compounds were synthesized and evaluated as inhibitors of human immunodeficiency virus type-1 (HIV-1). Among them, the most potent HIV-1 inhibitors were 8b, 8d, 14b and 18 (EC(50) = 0.049, 0.381, 0.599 and 0.398 µM, respectively), with HIV-1 inhibitory activity improved or similar to nevirapine (NVP, EC(50) = 0.097 µM) and delavirdine (DEV, EC(50) = 0.55 µM). The other compounds displayed moderate activity (8c, EC(50) = 5.25 µM) or were inactive (8a and 14a) against HIV-1 replication. Molecular modeling studies were performed with the synthesized compounds in complex with the wild-type reverse transcriptase (RT). A correlation was found between the anti-HIV activity and the electrostatic energy of interaction with Lys101 residue. These findings enrich the SAR of these Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) families.

Rational approaches for the design of effective human immunodeficiency virus type 1 nonnucleoside reverse transcriptase inhibitors.

The binding of several classes of nonnucleoside reverse transcriptase inhibitors (NNRTIs) to wild-type (wtRT) and K103N mutant (mRT) human immunodeficiency virus type 1 (HIV-1) reverse transcriptase is studied by molecular dynamics and energy decomposition techniques. The imidoylthiourea (ITU), diaryltriazine (DATA), and diarylpyrimidine (DAPY) NNRTIs studied maintain the hydrogen bond with Lys101 during the 3 ns molecular dynamics trajectories. When bound to mRT, all the DAPYs studied establish hydrogen bonds with Glu138; among these, those of the potent inhibitors TMC120 and TMC125 are water-mediated. The molecular interactions of the NNRTIs in the binding pocket are correlated to the drugs' potency. Quantitative free energy analyses show a linear relationship between the van der Waals energetic component and the potency against wtRT. The molecular basis of the interaction between NNRTIs and RT presented here provide quantitative approaches for the design of novel effective anti-HIV drugs.

Binding to human serum albumin of zidovudine (AZT) and novel AZT derivatives. Experimental and theoretical analyses.

This work presents the binding of AZT and nine novel AZT derivatives to human serum albumin (HSA), both defatted (HSA(D)) and complexed with fatty acids (HSA(FA)). The bound fractions and binding site were determined by applying an ultrafiltration procedure, with an increased affinity for the majority of these derivatives to HSA(D) being found with respect to that of AZT, while only one derivative exhibited an increased affinity for HSA(FA). By means of computational methods, we observed that specific electrostatic interactions are responsible for the increased affinity for HSA(D), while the presence of fatty acids complexed to HSA caused an intense electrostatic repulsion with negatively charged ligands located in Sudlow site I, thus diminishing their bound fractions. A strong relationship between the calculated energetic components and the observed experimental affinity was identified.