'All That Glitters Is Not Gold': High-Resolution Crystal Structures of Ligand-Protein Complexes Need Not Always Represent Confident Binding Poses.

Our understanding of the structure-function relationships of biomolecules and thereby applying it to drug discovery programs are substantially dependent on the availability of the structural information of ligand-protein complexes. However, the correct interpretation of the electron density of a small molecule bound to a crystal structure of a macromolecule is not trivial. Our analysis involving quality assessment of ~0.28 million small molecule-protein binding site pairs derived from crystal structures corresponding to ~66,000 PDB entries indicates that the majority (65%) of the pairs might need little (54%) or no (11%) attention. Out of the remaining 35% of pairs that need attention, 11% of the pairs (including structures with high/moderate resolution) pose serious concerns. Unfortunately, most users of crystal structures lack the training to evaluate the quality of a crystal structure against its experimental data and, in general, rely on the resolution as a 'gold standard' quality metric. Our work aims to sensitize the non-crystallographers that resolution, which is a global quality metric, need not be an accurate indicator of local structural quality. In this article, we demonstrate the use of several freely available tools that quantify local structural quality and are easy to use from a non-crystallographer's perspective. We further propose a few solutions for consideration by the scientific community to promote quality research in structural biology and applied areas.

Desolvation of Peptide Bond by O to S Substitution Impacts Protein Stability.

Amino acid side chains are key to fine-tuning the microenvironment polarity in proteins composed of polar amide bonds. Here, we report that substituting an oxygen atom of the backbone amide bond with sulfur atom desolvates the thioamide bond, thereby increasing its lipophilicity. The impact of such local desolvation by O to S substitution in proteins was tested by synthesizing thioamidated variants of Pin1 WW domain. We observe that a thioamide acts in synergy with nonpolar amino acid side chains to reduce the microenvironment polarity and increase protein stability by more than 14 °C. Through favorable van der Waals and hydrogen bonding interactions, this single atom substitution significantly stabilizes proteins without altering the amino acid sequence and structure of the native protein.

NU-6027 Inhibits Growth of Mycobacterium tuberculosis by Targeting Protein Kinase D and Protein Kinase G.

Tuberculosis (TB) is a global health concern, and this situation has further worsened due to the emergence of drug-resistant strains and the failure of BCG vaccine to impart protection. There is an imperative need to develop highly sensitive, specific diagnostic tools, novel therapeutics, and vaccines for the eradication of TB. In the present study, a chemical screen of a pharmacologically active compound library was performed to identify antimycobacterial compounds. The phenotypic screen identified a few novel small-molecule inhibitors, including NU-6027, a known CDK-2 inhibitor. We demonstrate that NU-6027 inhibits Mycobacterium bovis BCG growth in vitro and also displayed cross-reactivity with Mycobacterium tuberculosis protein kinase D (PknD) and protein kinase G (PknG). Comparative structural and sequence analysis along with docking simulation suggest that the unique binding site stereochemistry of PknG and PknD accommodates NU-6027 more favorably than other M. tuberculosis Ser/Thr protein kinases. Further, we also show that NU-6027 treatment induces the expression of proapoptotic genes in macrophages. Finally, we demonstrate that NU-6027 inhibits M. tuberculosis growth in both macrophage and mouse tissues. Taken together, these results indicate that NU-6027 can be optimized further for the development of antimycobacterial agents.

An amide to thioamide substitution improves the permeability and bioavailability of macrocyclic peptides.

Solvent shielding of the amide hydrogen bond donor (NH groups) through chemical modification or conformational control has been successfully utilized to impart membrane permeability to macrocyclic peptides. We demonstrate that passive membrane permeability can also be conferred by masking the amide hydrogen bond acceptor (>C = O) through a thioamide substitution (>C = S). The membrane permeability is a consequence of the lower desolvation penalty of the macrocycle resulting from a concerted effect of conformational restriction, local desolvation of the thioamide bond, and solvent shielding of the amide NH groups. The enhanced permeability and metabolic stability on thioamidation improve the bioavailability of a macrocyclic peptide composed of hydrophobic amino acids when administered through the oral route in rats. Thioamidation of a bioactive macrocyclic peptide composed of polar amino acids results in analogs with longer duration of action in rats when delivered subcutaneously. These results highlight the potential of O to S substitution as a stable backbone modification in improving the pharmacological properties of peptide macrocycles.

NOD: a web server to predict New use of Old Drugs to facilitate drug repurposing.

Computational methods accelerate the drug repurposing pipelines that are a quicker and cost-effective alternative to discovering new molecules. However, there is a paucity of web servers to conduct fast, focussed, and customized investigations for identifying new uses of old drugs. We present the NOD web server, which has the mentioned characteristics. NOD uses a sensitive sequence-guided approach to identify close and distant homologs of a protein of interest. NOD then exploits this evolutionary information to suggest potential compounds from the DrugBank database that can be repurposed against the input protein. NOD also allows expansion of the chemical space of the potential candidates through similarity searches. We have validated the performance of NOD against available experimental and/or clinical reports. In 65.6% of the investigated cases in a control study, NOD is able to identify drugs more effectively than the searches made in DrugBank. NOD is freely-available at http://pauling.mbu.iisc.ac.in/NOD/NOD/ .

Identification of Potential Binders of Mtb Universal Stress Protein (Rv1636) Through an in silico Approach and Insights Into Compound Selection for Experimental Validation.

Millions of deaths caused by Mycobacterium tuberculosis (Mtb) are reported worldwide every year. Treatment of tuberculosis (TB) involves the use of multiple antibiotics over a prolonged period. However, the emergence of resistance leading to multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) is the most challenging aspect of TB treatment. Therefore, there is a constant need to search for novel therapeutic strategies that could tackle the growing problem of drug resistance. One such strategy could be perturbing the functions of novel targets in Mtb, such as universal stress protein (USP, Rv1636), which binds to cAMP with a higher affinity than ATP. Orthologs of these proteins are conserved in all mycobacteria and act as "sink" for cAMP, facilitating the availability of this second messenger for signaling when required. Here, we have used the cAMP-bound crystal structure of USP from Mycobacterium smegmatis, a closely related homolog of Mtb, to conduct a structure-guided hunt for potential binders of Rv1636, primarily employing molecular docking approach. A library of 1.9 million compounds was subjected to virtual screening to obtain an initial set of ~2,000 hits. An integrative strategy that uses the available experimental data and consensus indications from other computational analyses has been employed to prioritize 22 potential binders of Rv1636 for experimental validations. Binding affinities of a few compounds among the 22 prioritized compounds were tested through microscale thermophoresis assays, and two compounds of natural origin showed promising binding affinities with Rv1636. We believe that this study provides an important initial guidance to medicinal chemists and biochemists to synthesize and test an enriched set of compounds that have the potential to inhibit Mtb USP (Rv1636), thereby aiding the development of novel antitubercular lead candidates.

Repurposing drugs against the main protease of SARS-CoV-2: mechanism-based insights supported by available laboratory and clinical data.

The ongoing global pandemic of COVID-19 has brought life to almost a standstill with the implementation of lockdowns and social distancing as some of the preventive measures in the absence of any approved specific therapeutic interventions. To combat this crisis, research communities worldwide are falling back on the existing repertoire of approved/investigational drugs to probe into their anti-coronavirus properties. In this report, we describe our unique efforts in identifying potential drugs that could be repurposed against the main protease of SARS-CoV-2 (SARS-CoV-2 Mpro). To achieve this goal, we have primarily exploited the principles of 'neighbourhood behaviour' in the protein 3D (workflow-I) and chemical 2D structural space (workflow-II) coupled with docking simulations and insights into the possible modes of action of the selected candidates from the available literature. This integrative approach culminated in prioritizing 29 potential repurpose-able agents (20 approved drugs and 9 investigational molecules) against SARS-CoV-2 Mpro. Apart from the approved/investigational anti-viral drugs, other notable hits include anti-bacterial, anti-inflammatory, anti-cancer and anti-coagulant drugs. Our analysis suggests that some of these drugs have the potential to simultaneously modulate the functions of viral proteins and the host response system. Interestingly, many of these identified candidates (12 molecules from workflow-I and several molecules, belonging to the chemical classes of alkaloids, tetracyclines, peptidomimetics, from workflow-II) are suggested to possess anti-viral properties, which is supported by laboratory and clinical data. Furthermore, this work opens a new avenue of research to probe into the molecular mechanism of action of many drugs, which are known to demonstrate anti-viral activity but are so far not known to target viral proteases.

Mutations in SARS-CoV-2 viral RNA identified in Eastern India: Possible implications for the ongoing outbreak in India and impact on viral structure and host susceptibility.

Direct massively parallel sequencing of SARS-CoV-2 genome was undertaken from nasopharyngeal and oropharyngeal swab samples of infected individuals in Eastern India. Seven of the isolates belonged to the A2a clade, while one belonged to the B4 clade. Specific mutations, characteristic of the A2a clade, were also detected, which included the P323L in RNA-dependent RNA polymerase and D614G in the Spike glycoprotein. Further, our data revealed emergence of novel subclones harbouring nonsynonymous mutations, viz. G1124V in Spike (S) protein, R203K, and G204R in the nucleocapsid (N) protein. The N protein mutations reside in the SR-rich region involved in viral capsid formation and the S protein mutation is in the S2 domain, which is involved in triggering viral fusion with the host cell membrane. Interesting correlation was observed between these mutations and travel or contact history of COVID-19 positive cases. Consequent alterations of miRNA binding and structure were also predicted for these mutations. More importantly, the possible implications of mutation D614G (in SD domain) and G1124V (in S2 subunit) on the structural stability of S protein have also been discussed. Results report for the first time a bird's eye view on the accumulation of mutations in SARS-CoV-2 genome in Eastern India.

Repurposing Drugs Based on Evolutionary Relationships Between Targets of Approved Drugs and Proteins of Interest.

Drug repurposing has garnered much interest as an effective method for drug development among biopharmaceutical companies. The availability of information on complete sequences of genomes and their associated biological data, genotype-phenotype-disease relationships, and properties of small molecules offers opportunities to explore the repurpose-able potential of existing pharmacopoeia. This method gains further importance, especially, in the context of development of drugs against infectious diseases, some of which pose serious complications due to emergence of drug-resistant pathogens. In this article, we describe computational means to achieve potential repurpose-able drug candidates that may be used against infectious diseases by exploring evolutionary relationships between established targets of FDA-approved drugs and proteins of pathogen of interest.

Increasing the bioactive space of peptide macrocycles by thioamide substitution.

We show that substituting a single atom, O to S (amide to thioamide), in a peptide bond results in global restriction of the conformational flexibility in peptide macrocycles with minimal perturbation of the parent conformation. The van der Waals interactions between the C[double bond, length as m-dash]S group and the surrounding atoms are the major driving force in inducing the conformational restriction, resulting in well-defined structures of these cyclic peptides with static 3-D presentation of the pharmacophores. Utilizing this property of thioamides, we report the development of a superactive antagonist of pro-angiogenic αvß3, αvß5 and α5ß1 integrins, which are responsible for cancer cell proliferation and survival. Using simple thio-scanning and spatial screening of a non-efficacious and conformationally flexible cyclic peptide, we could achieve a more than 105 fold enhancement in its efficacy in cellulo via a single O to S substitution. The developed peptide shows better efficacy in inhibiting the pro-angiogenic integrins than the drug candidate cilengitide, with a significantly enhanced serum half-life of 36 h compared to that of cilengitide (12 h). The long shelf-life, absence of non-specific toxicity and resistance to degradation of the thioamidated macrocyclic peptides in human serum suggest the promise of thioamides in markedly improving the affinity, efficacy and pharmacology of peptide macrocycles.