Search

Abstract In this study, a set of dietary polyphenols was comprehensively studied for the selective identification of the potential inhibitors/modulators for galectin-1. Galectin-1 is a potent prognostic indicator of tumor progression and a highly regarded therapeutic target for various pathological conditions. This indicator is composed of a highly conserved carbohydrate recognition domain (CRD) that accounts for the binding affinity of β-galactosides. Although some small molecules have been identified as galectin-1 inhibitors/modulators, there are limited studies on the identification of novel compounds against this attractive therapeutic target. The extensive computational techniques include potential drug binding site recognition on galectin-1, binding affinity predictions of ~ 500 polyphenols, molecular docking, and dynamic simulations of galectin-1 with selective dietary polyphenol modulators, followed by the estimation of binding free energy for the identification of dietary polyphenol-based galectin-1 modulators. Initially, a deep neural network-based algorithm was utilized for the prediction of the druggable binding site and binding affinity. Thereafter, the intermolecular interactions of the polyphenol compounds with galectin-1 were critically explored through the extra-precision docking technique. Further, the stability of the interaction was evaluated through the conventional atomistic 100 ns dynamic simulation study. The docking analyses indicated the high interaction affinity of different amino acids at the CRD region of galectin-1 with the proposed five polyphenols. Strong and consistent interaction stability was suggested from the simulation trajectories of the selected dietary polyphenol under the dynamic conditions. Also, the conserved residue (His44, Asn46, Arg48, Val59, Asn61, Trp68, Glu71, and Arg73) associations suggest high affinity and selectivity of polyphenols toward galectin-1 protein. Graphic Abstract

© 2021 The Authors., [Background and Purpose]: Local anaesthetics block sodium and a variety of potassium channels. Although previous studies identified a residue in the pore signature sequence together with three residues in the S6 segment as a putative binding site, the precise molecular basis of inhibition of Kv channels by local anaesthetics remained unknown. Crystal structures of Kv channels predict that some of these residues point away from the central cavity and face into a drug binding site called side pockets. Thus, the question arises whether the binding site of local anaesthetics is exclusively located in the central cavity or also involves the side pockets. [Experimental Approach]: A systematic functional alanine mutagenesis approach, scanning 58 mutants, together with in silico docking experiments and molecular dynamics simulations was utilized to elucidate the binding site of bupivacaine and ropivacaine. [Key Results]: Inhibition of Kv1.5 channels by local anaesthetics requires binding to the central cavity and the side pockets, and the latter requires interactions with residues of the S5 and the back of the S6 segments. Mutations in the side pockets remove stereoselectivity of inhibition of Kv1.5 channels by bupivacaine. Although binding to the side pockets is conserved for different local anaesthetics, the binding mode in the central cavity and the side pockets shows considerable variations. [Conclusion and Implications]: Local anaesthetics bind to the central cavity and the side pockets, which provide a crucial key to the molecular understanding of their Kv channel affinity and stereoselectivity, as well as their spectrum of side effects., The study was supported by the Ministerio de Ciencia e Innovación (MICINN, Spain) Grants SAF2016-75021-R and PID2019-104366RB-C21 (to C.V. and T.G.); the European Regional Development Fund (Fondo Europeo de Desarrollo Regional [FEDER]) and the Instituto de Salud Carlos III CIBERCV programme CB/11/00222 (to C.V. and T.G.); the Consejo Superior de Investigaciones Científicas (CSIC) Grants PIE201820E104 and 2019AEP148 (to C.V.); the Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) 1191133 and the Fondo de Equipamiento Científico y Tecnológico (FONDEQUIP) 160063 grants from ANID (to W.G.); and the Deutsche Forschungsgemeinschaft (DFG) Grant DE1482-4/1 to N.D.

Multi-Drug Resistance (MDR) exerted by tumor cells is majorly due to the overexpression of ATP Binding cassette transporters such as ABCB1/P-glycoprotein (P-gp). Annonaceous acetogenins (AGEs) exert anticancer activity by strongly inhibiting NADH oxidase of cancer cells. The present in silico study aims at screening a potent MDR inhibitor among acetogenins from the plant Annona muricata. Twenty-four AGEs were selected and screened for their pharmacokinetic properties. An inward facing conformation of P-gp is required for understanding the interaction of AGEs at the drug binding region and hence the human P-gp protein was modeled. The selected compounds were then docked with the ATP binding site and the drug binding site of modeled human P-gp. Annonacin A.1, Annohexocin.1 and Annomuricin E.1 docked better with high MM/GBSA dG binding in the drug binding region as compared with the conventional drugs. These compounds had a better docking score as compared with control inhibitor drugs at the ATP binding region. The complexes were subjected to MD simulation and Annonacin A was stable throughout the simulation period. Therefore, Annonacin A might act as a competitive inhibitor for the chemo drugs for binding at the drug binding region of P-gp. Hence it is capable of decreasing the efflux of chemo drugs out of the cells by P-Glycoprotein/ABCB1/MDR1. With this computational study, it is concluded that this compound might potentially reverse MDR, and hence can be taken forward for validation studies. Communicated by Ramaswamy H. Sarma.

Background: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the oldest known and most dangerous diseases. Although the spread of TB was controlled in the early 20th century using antibiotics and vaccines, TB has again become a threat because of increased drug resistance. There is still a lack of effective treatment regimens for a person who is already infected with multidrug-resistant Mtb (MDR-Mtb) or extensively drug-resistant Mtb (XDRMtb). In the past decades, many research groups have explored the drug resistance profiles of Mtb based on sequence data by GWAS, which identified some mutations that were significantly linked with drug resistance, and attempted to explain the resistance mechanisms. However, they mainly focused on several significant mutations in drug targets (e.g. rpoB, katG). Some genes which are potentially associated with drug resistance may be overlooked by the GWAS analysis. Objective: In this article, our motivation is to detect potential drug resistance genes of Mtb using a heat diffusion model. Methods: All sequencing data, which contained 127 samples of Mtb, i.e. 34 ethambutol-, 65 isoniazid-, 53 rifampicin- and 45 streptomycin-resistant strains. The raw sequence data were preprocessed using Trimmomatic software and aligned to the Mtb H37Rv reference genome using Bowtie2. From the resulting alignments, SAMtools and VarScan were used to filter sequences and call SNPs. The GWAS was performed by the PLINK package to obtain the significant SNPs, which were mapped to genes. The P-values of genes calculated by GWAS were transferred into a heat vector. The heat vector and the Mtb protein-protein interactions (PPI) derived from the STRING database were inputted into the heat diffusion model to obtain significant subnetworks by HotNet2. Finally, the most significant (P < 0.05) subnetworks associated with different phenotypes were obtained. To verify the change of binding energy between the drug and target before and after mutation, the method of molecular dynamics simulation was performed using the AMBER software. Results: We identified significant subnetworks in rifampicin-resistant samples. Excitingly, we found rpoB and rpoC, which are drug targets of rifampicin. From the protein structure of rpoB, the mutation location was extremely close to the drug binding site, with a distance of only 3.97 Å. Molecular dynamics simulation revealed that the binding energy of rpoB and rifampicin decreased after D435V mutation. To a large extent, this mutation can influence the affinity of drug-target binding. In addition, topA and pyrG were reported to be linked with drug resistance, and might be new TB drug targets. Other genes that have not yet been reported are worth further study. Conclusion: Using a heat diffusion model in combination with GWAS results and protein-protein interactions, the significantly mutated subnetworks in rifampicin-resistant samples were found. The subnetwork not only contained the known targets of rifampicin (rpoB, rpoC), but also included topA and pyrG, which are potentially associated with drug resistance. Together, these results offer deeper insights into drug resistance of Mtb, and provides potential drug targets for finding new antituberculosis drugs.

The COVID-19 pandemic has been responsible for several deaths worldwide. The causative agent behind this disease is the Severe Acute Respiratory Syndrome – novel Coronavirus 2 (SARS-nCoV2). SARS-nCoV2 belongs to the category of RNA viruses. The main protease, responsible for the cleavage of the viral polyprotein is considered as one of the hot targets for treating COVID-19. Earlier reports suggest the use of HIV anti-viral drugs for targeting the main protease of SARS-CoV, which caused SARS in the year 2002-03. Hence, drug repurposing approach may prove to be useful in targeting the main protease of SARS-nCoV2. The high-resolution crystal structure of 3CLpro (main protease) of SARS-nCoV2 (PDB ID: 6LU7) was used as the target. The Food and Drug Administration (FDA) approved and SWEETLEAD database of drug molecules were screened. The apo form of the main protease was simulated for a cumulative of 150 ns and 10 μs open source simulation data was used, to obtain conformations for ensemble docking. The representative structures for docking were selected using RMSD-based clustering and Markov State Modeling analysis. This ensemble docking approach for main protease helped in exploring the conformational variation in the drug binding site of the main protease leading to efficient binding of more relevant drug molecules. The drugs obtained as best hits from the ensemble docking possessed anti-bacterial and anti-viral properties. Small molecules with these properties may prove to be useful to treat symptoms exhibited in COVID-19. This in-silico ensemble docking approach would support identification of potential candidates for repurposing against COVID-19.

Protein-drug interactions play important roles in many biological processes and therapeutics. Prediction of the active binding site of a protein helps discover and optimise these interactions leading to the design of better ligand molecules. The tertiary structure of a protein determines the binding sites available to the drug molecule. A quick and accurate prediction of the binding site from sequence alone without utilising the three-dimensional structure is challenging. Deep Learning has been used in a variety of biochemical tasks and has been hugely successful. In this paper, a Residual Neural Network (leveraging skip connections) is implemented to predict a protein's most active binding site. An Annotated Database of Druggable Binding Sites from the Protein DataBank, sc-PDB, is used for training the network. Features extracted from the Multiple Sequence Alignments (MSAs) of the protein generated using DeepMSA, such as Position-Specific Scoring Matrix (PSSM), Secondary Structure (SS3), and Relative Solvent Accessibility (RSA), are provided as input to the network. A weighted binary cross-entropy loss function is used to counter the substantial imbalance in the two classes of binding and non-binding residues. The network performs very well on single-chain proteins, providing a pocket that has good interactions with a ligand.

Current small molecule inhibitors of KRAS(G12C) bind irreversibly in the switch-II pocket, exploiting the strong nucleophilicity of the acquired cysteine as well as the preponderance of the GDP-bound form of this mutant. Nevertheless, many oncogenic KRAS mutants lack these two features, and it remains unknown whether targeting the switch-II pocket is a practical therapeutic approach for KRAS mutants beyond G12C. Here we use NMR spectroscopy and a novel cellular KRAS engagement assay to address this question by examining a collection of SII-P ligands from the literature and from our own laboratory. We show that the switch-II pockets of many GTP hydrolysis-deficient KRAS hotspot (G12, G13, Q61) mutants are accessible using non-covalent ligands, and that this accessibility is not necessarily coupled to the GDP state of KRAS. The results we describe here emphasize the switch-II pocket as a privileged drug binding site on KRAS and unveil new therapeutic opportunities in RAS-driven cancer.

Low voltage-activated calcium currents are mediated by T-type calcium channels CaV3.1, CaV3.2, and CaV3.3, which modulate a variety of physiological processes including sleep, cardiac pace-making, pain, and epilepsy. CaV3 isoforms’ biophysical properties, overlapping expression and lack of subtype-selective pharmacology hinder the determination of their specific physiological roles in health and disease. Notably, CaV3.3’s contribution to normal and pathophysiological function has remained largely unexplored. We have identified Pn3a as the first subtype-selective spider venom peptide inhibitor of CaV3.3, with >100-fold lower potency against the other T-type isoforms. Pn3a modifies CaV3.3 gating through a depolarizing shift in the voltage dependence of activation thus decreasing CaV3.3-mediated currents in the normal range of activation potentials. Paddle chimeras of KV1.7 channels bearing voltage sensor sequences from all four CaV3.3 domains revealed preferential binding of Pn3a to the S3-S4 region of domain II (CaV3.3DII). This novel T-type channel pharmacological site was explored through computational docking simulations of Pn3a into all T-type channel isoforms highlighting it as subtype-specific pharmacophore with therapeutic potential. This research expands our understanding of T-type calcium channel pharmacology and supports the suitability of Pn3a as a molecular tool in the study of the physiological roles of CaV3.3 channels.

MitoNEET (gene cisd1) is a mitochondrial outer membrane [2Fe-2S] protein and is a potential drug target in several metabolic diseases. Previous studies have demonstrated that mitoNEET functions as a redox-active and pH-sensing protein that regulates mitochondrial metabolism, although the structural basis of the potential drug binding site(s) remains elusive. Here we report the crystal structure of the soluble domain of human mitoNEET with a sulfonamide ligand, furosemide. Exploration of the high-resolution crystal structure is used to design mitoNEET binding molecules in a pilot study of molecular probes for use in future development of mitochondrial targeted therapies for a wide variety of metabolic diseases, including obesity, diabetes and neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. MitoNEET is an iron-sulfur protein found in the outer mitochondrial membrane and implicated in several metabolic disorders. Here the crystal structure of mitoNEET with a bound sulfonamide ligand is reported and used to design new mitoNEET ligands.

Background Amoxicillin-resistant H. pylori strains are increasing worldwide. To explore the potential resistance mechanisms involved, the 3D structure modeling and access tunnel prediction for penicillin-binding proteins (PBP1A) was performed, based on the Streptococcus pneumoniae, PBP 3D structure. Molecular covalent docking was used to determine the interactions between amoxicillin (AMX) and PBP1A. Results The AMX-Ser368 covalent complex interacts with the binding site residues (Gly367, Ala369, ILE370, Lys371, Tyr416, Ser433, Thr541, Thr556, Gly557, Thr558, and Asn560) of PBP1A, non-covalently. Six tunnel-like structures, accessing the PBP1A binding site, were characterized, using the CAVER algorithm. Tunnel-1 was the ultimate access route, leading to the drug catalytic binding residue (Ser368). This tunnel comprises of eighteen amino acid residues, 8 of which are shared with the drug binding site. Subsequently, to screen the presence of PBP1A mutations, in the binding site and tunnel residues, in our clinical strains, in vitro assays were performed. H. pylori strains, isolated under gastroscopy, underwent AMX susceptibility testing by E-test. Of the 100 clinical strains tested, 4 were AMX-resistant. The transpeptidase domain of the pbp1a gene of these resistant, plus 10 randomly selected AMX-susceptible strains, were amplified and sequenced. Of the amino acids lining the tunnel-1 and binding site residues, three (Ser414Arg, Val469Met and Thr556Ser) substitutions, were detected in 2 of the 4 resistant and none of the sequenced susceptible strains, respectively. Conclusions We hypothesize that mutations in amino acid residues lining the binding site and/or tunnel-1, resulting in conformational/spatial changes, may block drug binding to PBP1A and cause AMX resistance.

PoxtA and OptrA are ATP binding cassette (ABC) proteins of the F subtype (ABCF) that confer resistance to oxazolidinone, such as linezolid, and phenicol antibiotics, such as chloramphenicol. PoxtA/OptrA are often encoded on mobile genetic elements, facilitating their rapid spread amongst Gram-positive bacteria. These target protection proteins are thought to confer resistance by binding to the ribosome and dislodging the antibiotics from their binding sites. However, a structural basis for their mechanism of action has been lacking. Here we present cryo-electron microscopy structures of PoxtA in complex with the Enterococcus faecalis 70S ribosome at 2.9–3.1 Å, as well as the complete E. faecalis 70S ribosome at 2.2–2.5 Å. The structures reveal that PoxtA binds within the ribosomal E-site with its antibiotic resistance domain (ARD) extending towards the peptidyltransferase center (PTC) on the large ribosomal subunit. At its closest point, the ARD of PoxtA is still located >15 Å from the linezolid and chloramphenicol binding sites, suggesting that drug release is elicited indirectly. Instead, we observe that the ARD of PoxtA perturbs the CCA-end of the P-site tRNA causing it to shift by ∼4 Å out of the PTC, which correlates with a register shift of one amino acid for the attached nascent polypeptide chain. Given that linezolid and chloramphenicol are context-specific translation elongation inhibitors, we postulate that PoxtA/OptrA confer resistance to oxazolidinones and phenicols indirectly by perturbing the P-site tRNA and thereby altering the conformation of the attached nascent chain to disrupt the drug binding site.

SWI/SNF (BAF) chromatin remodelling complexes are key regulators of gene expression programs, and attractive drug targets for cancer therapies. Here we show that the N-terminus of the BAF155/SMARCC1 subunit contains a putative DNA-binding MarR-like domain, a chromodomain and a BRCT domain that are interconnected to each other to form a distinct module. In this structure the chromodomain makes interdomain interactions and has lost its canonical function to bind to methylated lysines. The structure provides new insights into the missense mutations that target this module in cancer. This study also reveals two adjacent, highly-conserved pockets in a cleft between the domains that form a potential binding site, which can be targeted with small molecules, offering a new strategy to target SWI/SNF complexes., Allen et al. determine crystal structure of the N-terminus of SWI/SNF chromatin remodelling factor subunit BAF155. They identify an interconnected structure of MarR-like, BRCT and chromodomains, visualise the potential effect of cancer-associated missense mutations and suggest a binding site and target for small molecule inhibitors.

Tuberculosis (TB) is the leading cause of death from a single infectious agent and in 2019 an estimated 10 million people worldwide contracted the disease. Although treatments for TB exist, continual emergence of drug-resistant variants necessitates urgent development of novel antituberculars. An important new target is the lipid transporter MmpL3, which is required for construction of the unique cell envelope that shields Mycobacterium tuberculosis (Mtb) from the immune system. However, a structural understanding of the mutations in Mtb MmpL3 that confer resistance to the many preclinical leads is lacking, hampering efforts to circumvent resistance mechanisms. Here, we present the cryoelectron microscopy structure of Mtb MmpL3 and use it to comprehensively analyze the mutational landscape of drug resistance. Our data provide a rational explanation for resistance variants local to the central drug binding site, and also highlight a potential alternative route to resistance operating within the periplasmic domain.

Lung cancer caused by mutations in the epidermal growth factor receptor (EGFR) is a major cause of cancer deaths worldwide. EGFR Tyrosine kinase inhibitors (TKIs) have been developed, and have shown increased survival rates and quality of life in clinical studies. However, drug resistance is a major issue, and treatment efficacy is lost after about an year. Therefore, predicting the response to targeted therapies for lung cancer patients is a significant research problem. In this work, we address this issue and propose a personalized model to predict the drug-response of lung cancer patients. This model uses clinical information, geometrical properties of the drug binding site, and the binding free energy of the drug-protein complex. The proposed model achieves state of the art performance with 97.5% accuracy, 100% recall, 95% precision, and 96.3% F1-score with a random forest classifier. This model can also be tested on other types of cancer and diseases, and we believe that it may help in taking optimal clinical decisions for treating patients with targeted therapies

Cyclin-dependent kinase 8 (CDK8) and its regulatory partner Cyclin C (CycC) play conserved roles in modulating RNA polymerase II (Pol II)-dependent gene expression. To understand the structure and function relations of CDK8, we analyzed the structures of human and Drosophila CDK8 proteins using molecular dynamics simulations, combined with functional analyses in Drosophila. Specifically, we evaluated the structural differences between hCDK8 and dCDK8 to predict the effects of the LXXLL motif mutation (AQKAA), the P154L mutations, and drug binding on local structures of the CDK8 proteins. First, we have observed that both the LXXLL motif and the kinase activity of CDK8 are required for the normal larval-to-pupal transition in Drosophila. Second, our molecular dynamic analyses have revealed that hCDK8 has higher hydrogen bond occupation of His149-Asp151 and Asp151-Asn156 than dCDK8. Third, the substructure of Asp282, Phe283, Arg285, Thr287 and Cys291 can distinguish human and Drosophila CDK8 structures. In addition, there are two hydrogen bonds in the LXXLL motif: a lower occupation between L312 and L315, and a relatively higher occupation between L312 and L316. Human CDK8 has higher hydrogen bond occupation between L312 and L316 than dCDK8. Moreover, L312, L315 and L316 in the LXXLL motif of CDK8 have the specific pattern of hydrogen bonds and geometries, which could be crucial for the binding to nuclear receptors. Furthermore, the P154L mutation dramatically decreases the hydrogen bond between L312 and L315 in hCDK8, but not in dCDK8. The mutations of P154L and AQKAA modestly alter the local structures around residues 154. Finally, we identified the inhibitor-induced conformational changes of hCDK8, and our results suggest a structural difference in the drug-binding site between hCDK8 and dCDK8. Taken together, these results provide the structural insights into the roles of the LXXLL motif and the kinase activity of CDK8 in vivo.

Transient receptor potential vanilloid 2 (TRPV2) plays a critical role in a variety of physiological and pathophysiological processes, putting TRPV2 on the list of important drug targets. Yet, specific TRPV2 agonists and antagonists are currently unavailable. Their development requires a precise knowledge of how the currently known non-specific small molecules interact with TRPV2 at the molecular level. Here we present TRPV2 structures in ligand-bound states resolved by cryo-electron microscopy in the presence of 2-aminoethoxydiphenyl borate (2-ABP), 2-APB with doxorubicin (DOXO), and ruthenium red (RR). We identified a novel 2-APB drug binding site between the S5 helix and S4-S5 linker on two adjacent TRPV2 monomers and determined the mechanism of TRPV2 pore block by RR. We also showed that a large organic molecule like DOXO can enter the TRPV2 pore in the presence of 2-APB. Additionally, we discovered a structural lipid bound in a unique position in the vanilloid pocket, which is absent in the 2-APB-bound state of the channel, allowing us to propose a model for TRPV2 channel gating. Together, this work provides a further understanding of TRPV2 function and a structural framework for the development of TRPV2-specific modulators.

Neisseria gonorrhoeae has become a highly antimicrobial-resistant Gram-negative pathogen. Multidrug efflux is a major mechanism that N. gonorrhoeae uses to counteract the action of multiple classes of antibiotics. It appears that gonococci bearing mosaic-like sequences within the gene mtrD, encoding the most predominant and clinically important transporter of any gonococcal multidrug efflux pump, significantly elevate drug resistance and enhance transport function. Here, we report cryo-electron microscopy (EM) structures of N. gonorrhoeae MtrD carrying a mosaic-like sequence that allow us to understand the mechanism of drug recognition. Our work will ultimately inform structure-guided drug design for inhibiting these critical multidrug efflux pumps., Neisseria gonorrhoeae is an obligate human pathogen and causative agent of the sexually transmitted infection (STI) gonorrhea. The most predominant and clinically important multidrug efflux system in N. gonorrhoeae is the multiple transferrable resistance (Mtr) pump, which mediates resistance to a number of different classes of structurally diverse antimicrobial agents, including clinically used antibiotics (e.g., β-lactams and macrolides), dyes, detergents and host-derived antimicrobials (e.g., cationic antimicrobial peptides and bile salts). Recently, it has been found that gonococci bearing mosaic-like sequences within the mtrD gene can result in amino acid changes that increase the MtrD multidrug efflux pump activity, probably by influencing antimicrobial recognition and/or extrusion to elevate the level of antibiotic resistance. Here, we report drug-bound solution structures of the MtrD multidrug efflux pump carrying a mosaic-like sequence using single-particle cryo-electron microscopy, with the antibiotics bound deeply inside the periplasmic domain of the pump. Through this structural approach coupled with genetic studies, we identify critical amino acids that are important for drug resistance and propose a mechanism for proton translocation.

SARS CoV 2 has spread worldwide and caused a major outbreak of coronavirus disease 2019 (COVID-19). To date, no licensed drug or a vaccine is available against COVID19.Starting from all of the resolved SARS CoV2 crystal structures, this study aims to find inhibitors for all of the SARS CoV2 proteins. To achieve this, I used PocketMatch to test the similarity of approved drugs binding sites against all of the binding sites found on SARS CoV 2 proteins. After that docking was used to confirm the results.I found drugs that inhibit the main protease, Nsp12 and Nsp3. The discovered drugs are either in clinical trials (Sildenafil, Lopinavir, Ritonavir) or have in vitro antiviral activity (Nelfinavir, Indinavir, Amprenavir, depiqulinum , Gemcitabine, Raltitrexed, Aprepitant, montelukast, Ouabain, Raloxifene) whether against SARS CoV 2 or other viruses. In addition to this, further analysis of pockets revealed a steroidal pocket that might open the door to hypotheses on why the mortality of men is higher than women.Many of the in silico repurposing studies test binding of the compound to the target using docking. The significance of this study adds to the similarity between the drug binding site and the target binding site. This takes into consideration the dynamic behaviour of the pocket after ligand binding.

Arabinosyltransferase B (EmbB) belongs to a family of membrane-bound glycosyltransferases that build the lipidated polysaccharides of the mycobacterial cell envelope, and are targets of anti-tuberculosis drug ethambutol. We present the 3.3 Å resolution single-particle cryo-electron microscopy structure of Mycobacterium smegmatis EmbB, providing insights on substrate binding and reaction mechanism. Mutations that confer ethambutol resistance map mostly around the putative active site, suggesting this to be the location of drug binding., The cryo-EM structure of Mycobacterium smegmatis arabinosyltransferase B EmbB involved in mycobacterial cell wall biosynthesis provides insights into the substrate binding and reaction mechanism. Mapping of the ethambutol resistance associated mutations onto the structure suggests the location of the drug binding site.

Introduction: Flavonoids are widely used as dietary supplements, and thus, play a significant role in the research field. In recent time, the interaction of flavonoid-metal complexes with serum albumin (SA) has widely been studied since the complexation poses a significant impact on biological activities. Additionally, the binding nature of flavonoids with SA gets modified in the presence of metal ions. Methods: In the present review, we studied the interaction of quercetin (Qu), a well-known flavonoid, and its Cu2+ complexes with SA to provide sufficient information about the beneficial role of metal-flavonoid complexes over free flavonoids. Results: Complexation with Cu(II) ion may alter the mode of binding of Qu with SAs. The strength of binding might be increased in the presence of Cu(II) as evidenced by the binding constant calculation. However, the drug binding site in bovine serum albumin (BSA) and human serum albumin (HSA) are not altered during the complexation process. Conclusion: To enhance the pharmaceutical outcomes of Qu molecules, one may use Qu-Cu(II) complex for the development and delivery of the small molecules.

DNA topoisomerases are key enzyme responsible for modulating the topological state of the DNA by breaking and rejoining of DNA strand. Characterization of a Gly717Asp mutation in the human topoisomerase was performed using several catalytic assays. The mutant enzyme was shown to have comparable cleavage and fast religation rate as compared to the wild-type protein. Addition of the anticancer drug camptothecin significantly reduced the religation step. The simulative approaches and analysis of the cleavage/religation equilibrium indicate that the mutation is able to modify the architecture of the drug binding site, increasing the persistence of the drug for the enzyme DNA covalent complex. Taken together these results indicate that the structure modification of the drug binding site is the key reason for the increasing CPT persistence and furthermore provide the possibility for new anti-cancer drug discovery.

On January 2020, a new coronavirus (officially named SARS-CoV-2) was associated with alarming outbreak of a pneumonia-like illness, which was later named by the WHO as COVID-19, originating from Wuhan City, China. Although many clinical studies involving antiviral and immunomodulatory drug treatments for SARS-CoV-2 all without reported results, no approved drugs have been found to effectively inhibit the virus so far. Full genome sequencing of the virus was done, and uploaded to be freely available for the world scientists to explore. A promising target for SARS-CoV-2 drug design is a chymotrypsin-like cysteine protease (3CLpro), a main protease responsible for the replication and maturation of functional proteins in the life cycle of the SARS coronavirus. Here we aim to explore SARS-CoV-2 3CLpro as possible drug targets based on ligand- protein interactions. In addition, ADME properties of the ligands were also analyzed to predict their drug likeliness. The results revealed Out of 9 ligands, 8 ligands (JFM, X77, RZG, HWH, T8A, 0EN, PEPTIDE and DMS) showed best ADME properties. These findings suggest that these ligands can be used as potential molecules for developing potent inhibitors against SARS-CoV-2 3CLpro, which could be helpful in inhibiting the propagation of the COVID-19. Furthermore, 10 potential amino acids residues were recognized as potential drug binding site (THR25, HIS41, GLY143, SER144, CYS145, MET165, GLU166, GLN189, ASP295 and ARG298). All those amino acid residues were subjected to missense SNP analysis were recognized to affect the structure and function of the protein. These characteristics provide them the promising to be target sites for the fresh generation inhibitors to work with and overcome drug resistance. These findings would be beneficial for the drug development for inhibiting SARS-CoV-2 3CLpro hence assisting the pharmacogenomics effort to manage the infection. of SARS-CoV-2.

The human proteome contains 826 G protein-coupled receptors (GPCR), which control a wide array of key physiological functions, making them important drug targets. GPCR functions are based on allosteric coupling from the extracellular orthosteric drug binding site across the cell membrane to intracellular binding sites for partners such as G proteins and arrestins. This signaling process is related to dynamic equilibria in conformational ensembles that can be observed by NMR in solution. A previous high-resolution NMR study of the A2A adenosine receptor (A2AAR) resulted in a qualitative characterization of a network of such local polymorphisms. Here, we used 19F-NMR experiments with probes at the A2AAR intracellular surface, which provides the high sensitivity needed for a refined description of different receptor activation states by ensembles of simultaneously populated conformers and the rates of exchange among them. We observed two agonist-stabilized substates that are not measurably populated in apo-A2AAR and one inactive substate that is not seen in complexes with agonists, suggesting that A2AAR activation includes both induced fit and conformational selection mechanisms. Comparison of A2AAR and a constitutively active mutant established relations between the 19F-NMR spectra and signaling activity, which enabled direct assessment of the difference in basal activity between the native protein and its variant.

Labdane diterpenes are important substances due to their remarkable biological activities such as, antibacterial, antiprotozoal, antifungal and cytostatic and cytotoxic effects against human cancer cells. We have isolated a labdane diterpene named “leoheterin” from the aerial parts of the Otostegia fruticosa Forssk (Briq) obtained from south west Arabian mountains of Saudi Arabia. The isolated compound was characterized by 1HNMR, 13CNMR, IR and UV–visible spectroscopies. Due to the pharmaceutical importance of this class of compounds we have studied the interaction of HSA with leoheterin by using several spectroscopic methods. The change in the UV spectrum of HSA in presence of leoheterin gives a primary idea about the interaction between them. Congruently, leoheterin quenches the fluorescence of HSA with a prominent blue shift of 5 nm, reminiscent of involvement of hydrophobic interactions. There was 1:1 binding between leoheterin and albumin which was taken place via static quenching mechanism. From CD it was revealed that leoheterin induces the secondary structure of HSA which is further supported by 3-d fluorescence measurements which shows a decrease in the size of the HSA-leoheterin complex as compared to the HSA alone. Molecular docking simulations presented that among the first three conformers, which have been arranged according to the least binding energies and are also in good corroboration with the free energies of binding obtained experimentally, the first two conformers shown the binding in hemin binding site of subdomain IB while in third conformer the binding site was near to the drug binding site 1 located in subdomain IIA. All conformers exhibited the involvement of hydrogen bonding as well as hydrophobic interactions.

Drug-induced Immune thrombocytopenia (DIT) is a common complication of several medications, including commonly used antibiotics. The most widely studied DIT is caused by quinine. In DIT, antibodies predominantly bind to the platelet membrane glycoprotein (GP) IX in a drug-dependent fashion resulting in increased platelet clearance. Binding of the sensitizing drug, such as quinine, to GPIX has been proposed but is yet to be established. This work demonstrates that quinine is retained specifically by human GPIX. Quinine binding was first analyzed in wild-type mouse platelets and in transgenic mouse platelet expressing human GPIX using high performance liquid chromatography. Binding of quinine to GPIX was then measured in Chinese hamster ovary (CHO) cells expressing a combination of wild type, human or mouse, three human/mouse chimeric constructs and six mutant GPIX proteins. Quinine was retained by human GPIX. No detectable absorption was observed with mouse GPIX or human GPIbα. The quinine binding site was mapped to residues 110-115 of human GPIX suggesting that quinine interacts with specific residues of the GP. These findings provide further insights into the molecular mechanisms of DIT.

Antibiotics can cause dormancy (bacteriostasis) or induce death (cidality) of the targeted bacteria. The bactericidal capacity is one of the most important properties of antibacterial agents. However, the understanding of the fundamental differences in the mode of action of bacteriostatic or bactericidal antibiotics, especially those belonging to the same chemical class, is very rudimentary. Here, by examining the activity and binding properties of chemically distinct macrolide inhibitors of translation, we have identified a key difference in their interaction with the ribosome, which correlates with their ability to cause cell death. While bacteriostatic and bactericidal macrolides bind in the nascent peptide exit tunnel of the large ribosomal subunit with comparable affinities, the bactericidal antibiotics dissociate from the ribosome with significantly slower rates. The sluggish dissociation of bactericidal macrolides correlates with the presence in their structure of an extended alkyl-aryl side chain, which establishes idiosyncratic interactions with the ribosomal RNA. Mutations or chemical alterations of the rRNA nucleotides in the drug binding site can protect cells from macrolide-induced killing, even with inhibitor concentrations that significantly exceed those required for cell growth arrest. We propose that the increased translation downtime due to slow dissociation of the antibiotic may damage cells beyond the point where growth can be reinitiated upon the removal of the drug due to depletion of critical components of the gene-expression pathway.