Structural insights into the potential binding sites of Cathepsin D using molecular modelling techniques.

Alzheimer's disease (AD) is the most prevalent type of dementia caused by the accumulation of amyloid beta (Aß) peptides. The extracellular deposition of Aß peptides in human AD brain causes neuronal death. Therefore, it has been found that Aß peptide degradation is a possible therapeutic target for AD. CathD has been known to breakdown amyloid beta peptides. However, the structural role of CathD is not yet clear. Hence, for the purpose of gaining a deeper comprehension of the structure of CathD, the present computational investigation was performed using virtual screening technique to predict CathD's active site residues and substrate binding mode. Ligand-based virtual screening was implemented on small molecules from ZINC database against crystal structure of CathD. Further, molecular docking was utilised to investigate the binding mechanism of CathD with substrates and virtually screened inhibitors. Localised compounds obtained through screening performed by PyRx and AutoDock 4.2 with CathD receptor and the compounds having highest binding affinities were picked as; ZINC00601317, ZINC04214975 and ZINCC12500925 as our top choices. The hydrophobic residues Viz. Gly35, Val31, Thr34, Gly128, Ile124 and Ala13 help stabilising the CathD-ligand complexes, which in turn emphasises substrate and inhibitor selectivity. Further, MM-GBSA approach has been used to calculate binding free energy between CathD and selected compounds. Therefore, it would be beneficial to understand the active site pocket of CathD with the assistance of these discoveries. Thus, the present study would be helpful to identify active site pocket of CathD, which could be beneficial to develop novel therapeutic strategies for the AD.

Molecular insights into the inhibition of early stages of Aß peptide aggregation and destabilization of Alzheimer's Aß protofibril by dipeptide D-Trp-Aib: A molecular modelling approach.

Amyloid beta (Aß) peptide aggregates rapidly into the soluble oligomers, protofibrils and fibrils to form senile plaques, a neurotoxic component and pathological hallmark of Alzheimer's disease (AD). Experimentally, it has been demonstrated the inhibition of an early stages of Aß aggregation by a dipeptide D-Trp-Aib inhibitor, but its molecular mechanism is still unclear. Hence, in the present study, we used molecular docking and molecular dynamics (MD) simulations to explore the molecular mechanism of inhibition of an early oligomerization and destabilization of preformed Aß protofibril by D-Trp-Aib. Molecular docking study showed that the D-Trp-Aib binds at the aromatic (Phe19, Phe20) region of Aß monomer, Aß fibril and hydrophobic core of Aß protofibril. MD simulations revealed the binding of D-Trp-Aib at the aggregation prone region (Lys16-Glu22) resulted in the stabilization of Aß monomer by π-π stacking interactions between Tyr10 and indol ring of D-Trp-Aib, which decreases the ß-sheet content and increases the α-helices. The interaction between Lys28 of Aß monomer to D-Trp-Aib could be responsible to block the initial nucleation and may impede the fibril growth and elongation. The loss of hydrophobic contacts between two ß-sheets of Aß protofibril upon binding of D-Trp-Aib at the hydrophobic cavity resulted in the partial opening of ß-sheets. This also disrupts a salt bridge (Asp23-Lys28) leading to the destabilization of Aß protofibril. Binding energy calculations revealed that van der Waals and electrostatic interactions maximally favours the binding of D-Trp-Aib to Aß monomer and Aß protofibril respectively. The residues Tyr10, Phe19, Phe20, Ala21, Glu22, Lys28 of Aß monomer, whereas Leu17, Val18, Phe19, Val40, Ala42 of protofibril contributing for the interactions with D-Trp-Aib. Thus, the present study provides structural insights into the inhibition of an early oligomerization of Aß peptides and destabilization of Aß protofibril, which could be useful to design novel inhibitors for the treatment of AD.

Structure based virtual screening, molecular dynamic simulation to identify the oxadiazole derivatives as inhibitors of Enterococcus D-Ala-D-Ser ligase for combating vancomycin resistance.

Vancomycin resistance in enterococci mainly arises due to alteration in terminal peptidoglycan dipeptide. A comprehensive structural analysis for substrate specificity of dipeptide modifying d-Alanine: d-Serine ligase (Ddls) is essential to screen its inhibitors for combating vancomycin resistance. In this study modeled 3D structure of EgDdls from E. gallinarum was used for structure based virtual screening (SBVS) of oxadiazole derivatives. Initially, fifteen oxadiazole derivatives were identified as inhibitors at the active site of EgDdls from PubChem database. Further, four EgDdls inhibitors were evaluated using pharmacokinetic profile and molecular docking. The results of molecular docking showed that oxadiazole inhibitors could bind preferentially at ATP binding pocket with the lowest binding energy. Further, molecular dynamics simulation results showed stable behavior of EgDdls in complex with screened inhibitors. The residues Phe172, Lys174, Glu217, Phe292, and Asn302 of EgDdls were mainly involved in interactions with screened inhibitors. Furthermore, MM-PBSA calculation showed electrostatic and van der Waals interactions mainly contribute to overall binding energy. The PCA analysis showed motion of central domain and omega loop of EgDdls. This is involved in the formation of native dipeptide and stabilized after binding of 2-(1-(Ethylsulfonyl) piperidin-4-yl)-5-(furan-2-yl)-1,3,4-oxadiazole, which could be reason for the inhibition of EgDdls. Hence, in this study we have screened inhibitors of EgDdls which could be useful to alleviate the vancomycin resistance problem in enterococci, involved in hospital-acquired infections, especially urinary tract infections (UTI).

In vivo developmental studies of Helicoverpa armigera and in silico molecular interactions with trypsin reveal the bio-insecticidal potential of trypsin inhibitor (SSTI) isolated from Solanum surattense.

We report the impact of gut protease inhibition on the development of Helicoverpa armigera by trypsin inhibitor and the use of molecular modeling to understand the mechanism of trypsin inhibition. Larvae of H. armigera fed on an artificial diet containing 150 and 300 µg/ml SSTI showed a negative impact on the insects' development in terms of mean larval weight, larval fatality, survival rate, and nutritional indices. Prominent physical abnormalities like curled wings, malformed appendages, and small body size were observed during the development. Gene expression studies revealed down regulation in trypsin (HaTry 1, 2, 3, 4, 6, 8) and chymotrypsin (HaChy 1, 2, 3, 4) genes of the larval gut upon treatment of SSTI. Homology modeling has been used to build the three-dimensional structure of SSTI, which showed ß-sheets having a stable canonical inhibitory loop (CIL) with conserved lysine residue. Molecular docking studies showed the strong binding of SSTI at the active site of trypsin. Molecular dynamic (MD) simulation revealed the stable interactions of the rigid CIL of SSTI at the active site of trypsin, leading to its destabilization. Conserved lysine63 of the P1 site in SSTI forms a strong hydrogen bonding network with residues Asp189 and Ser190 of trypsin.

Interrogating the substrate specificity landscape of UvrC reveals novel insights into its non-canonical function.

Although it is relatively unexplored, accumulating data highlight the importance of tripartite crosstalk between nucleotide excision repair (NER), DNA replication, and recombination in the maintenance of genome stability; however, elucidating the underlying mechanisms remains challenging. While Escherichia coli uvrA and uvrB can fully complement polAΔ cells in DNA replication, uvrC attenuates this alternative DNA replication pathway, but the exact mechanism by which uvrC suppresses DNA replication is unknown. Furthermore, the identity of bona fide canonical and non-canonical substrates for UvrCs are undefined. Here, we reveal that Mycobacterium tuberculosis UvrC (MtUvrC) strongly binds to, and robustly cleaves, key intermediates of DNA replication/recombination as compared with the model NER substrates. Notably, inactivation of MtUvrC ATPase activity significantly attenuated its endonuclease activity, thus suggesting a causal link between these two functions. We built an in silico model of the interaction of MtUvrC with the Holliday junction (HJ), using a combination of homology modeling, molecular docking, and molecular dynamic simulations. The model predicted residues that were potentially involved in HJ binding. Six of these residues were mutated either singly or in pairs, and the resulting MtUvrC variants were purified and characterized. Among them, residues Glu595 and Arg597 in the helix-hairpin-helix motif were found to be crucial for the interaction between MtUvrC and HJ; consequently, mutations in these residues, or inhibition of ATP hydrolysis, strongly abrogated its DNA-binding and endonuclease activities. Viewed together, these findings expand the substrate specificity landscape of UvrCs and provide crucial mechanistic insights into the interplay between NER and DNA replication/recombination.

Purification and characterization of antibacterial surfactin isoforms produced by Bacillus velezensis SK.

Bacillus velezensis SK having broad-spectrum antimicrobial activity has been isolated from soil. The efficient extraction of antimicrobial compounds produced in various mediums has been done using Diaion HP-20 resin. Further, characterization of an antimicrobial compound by TLC, FTIR, in-situ bioautography analysis revealed the presence of cyclic lipopeptides, which is then purified by the combination of silica gel, size exclusion, dual gradient, and RP-HPLC chromatography techniques. Growth kinetic studies showed that Bacillus velezensis SK produces a mixture of lipopeptides (1.33 gL-1). The lipopeptide exhibits good pH (2-10) and temperature stability up to 80 °C. LC-ESI-MS analysis of partially purified lipopeptide identified variant of surfactin, further analysis of purified chromatographic fractions revealed the occurrence of most abundant C15-surfactin homologues (m/z 1036.72 Da). The isolated surfactin exhibits good antimicrobial activity (1600 AU/ml) against drug-resistant food-born B. cereus and human pathogen Staphylococcus aureus. Hence, identified strain B. velezensis SK and its potent antibacterial surfactin lipopeptide could be used in various food and biomedical applications.

Structural insights and inhibition mechanism of TMPRSS2 by experimentally known inhibitors Camostat mesylate, Nafamostat and Bromhexine hydrochloride to control SARS-coronavirus-2: A molecular modeling approach.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has been responsible for the cause of global pandemic Covid-19 and to date, there is no effective treatment available. The spike 'S' protein of SARS-CoV-2 and ACE2 of the host cell are being targeted to design new drugs to control Covid-19. Similarly, a transmembrane serine protease, TMPRSS2 of the host cell plays a significant role in the proteolytic cleavage of viral 'S' protein helpful for the priming of ACE2 receptors and viral entry into human cells. However, three-dimensional structural information and the inhibition mechanism of TMPRSS2 is yet to be explored experimentally. Hence, we have used a molecular dynamics (MD) simulated homology model of TMPRSS2 to study the inhibition mechanism of experimentally known inhibitors Camostat mesylate, Nafamostat and Bromhexine hydrochloride (BHH) using molecular modeling techniques. Prior to docking, all three inhibitors were geometry optimized by semi-empirical quantum chemical RM1 method. Molecular docking analysis revealed that Camostat mesylate and its structural analogue Nafamostat interact strongly with residues His296 and Ser441 present in the catalytic triad of TMPRSS2, whereas BHH binds with Ala386 along with other residues. Comparative molecular dynamics simulations revealed the stable behavior of all the docked complexes. MM-PBSA calculations also revealed the stronger binding of Camostat mesylate to TMPRSS2 active site residues as compared to Nafamostat and BHH. Thus, this structural information could be useful to understand the mechanistic approach of TMPRSS2 inhibition, which may be helpful to design new lead compounds to prevent the entry of SARS-Coronavirus 2 in human cells.

Antibiotic resistance and inhibition mechanism of novel aminoglycoside phosphotransferase APH(5) from B. subtilis subsp. subtilis strain RK.

Bacterial resistance towards aminoglycoside antibiotics mainly occurs because of aminoglycoside phosphotransferases (APHs). It is thus necessary to provide a rationale for focusing inhibitor development against APHs. The nucleotide triphosphate (NTP) binding site of eukaryotic protein kinases (ePKs) is structurally conserved with APHs. However, ePK inhibitors cannot be used against APHs due to cross reactivity. Thus, understanding bacterial resistance at the atomic level could be useful to design new inhibitors against such resistant pathogens. Hence, we carried out in vitro studies of APH from newly deposited multidrug-resistant organism Bacillus subtilis subsp. subtilis strain RK. Enzymatic modification studies of different aminoglycoside antibiotics along with purification and characterization revealed a novel class of APH, i.e., APH(5), with molecular weight 27 kDa approximately. Biochemical analysis of virtually screened inhibitor ZINC71575479 by coupled spectrophotometric assay showed complete enzymatic inhibition of purified APH(5). In silico toxicity study comparison of ZINC71575479 with known inhibitor of APH, i.e., tyrphostin AG1478, predicted its acceptable values for 96 h fathead minnow LC50, 48 h Tetrahymena pyriformis IGC50, oral rat LD50, and developmental toxicity using different QSAR methodologies. Thus, the present study gives novel insight into the aminoglycoside resistance and inhibition mechanism of APH(5) by applying experimental and computational techniques synergistically.