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Abstract Tuberculosis is a highly contagious disease caused by Mycobacterium tuberculosis (Mtb), which is one of the prominent reasons for the death of millions worldwide. The bacterium has a substantially higher mortality rate than other bacterial diseases, and the rapid rise of drug-resistant strains only makes the situation more concerning. Currently, the only licensed vaccine BCG (Bacillus Calmette–Guérin) is ineffective in preventing adult pulmonary tuberculosis prophylaxis and latent tuberculosis re-activation. Therefore, there is a pressing need to find novel and safe vaccines that provide robust immune defense and have various applications. Vaccines that combine epitopes from multiple candidate proteins have been shown to boost immunity against Mtb infection. This study applies an immunoinformatic strategy to generate an adequate multi-epitope immunization against Mtb employing five antigenic proteins. Potential B-cell, cytotoxic T lymphocyte, and helper T lymphocyte epitopes were speculated from the intended proteins and coupled with 50 s ribosomal L7/L12 adjuvant, and the vaccine was constructed. The vaccine’s physicochemical profile demonstrates antigenic, soluble, and non-allergic. In the meantime, docking, molecular dynamics simulations, and essential dynamics analysis revealed that the multi-epitope vaccine structure interacted strongly with Toll-like receptors (TLR2 and TLR3). MM-PBSA analysis was performed to ascertain the system’s intermolecular binding free energies accurately. The immune simulation was applied to the vaccine to forecast its immunogenic profile. Finally, in silico cloning was used to validate the vaccine’s efficacy. The immunoinformatics analysis suggests the multi-epitope vaccine could induce specific immune responses, making it a potential candidate against Mtb. However, validation through the in-vivo study of the developed vaccine is essential to assess its efficacy and immunogenicity profile, which will assure active protection against Mtb.

Inhibitor of DNA binding protein 3 (ID3) has long been characterized as an oncogene that implicates its functional role through its Helix–Loop–Helix (HLH) domain upon protein–protein interaction. An insight into the dimerization brought by this domain helps in identifying the key residues that favor the mechanism behind it. Molecular dynamics (MD) simulations were performed for the HLH proteins ID3 and Transcription factor E2-alpha (E2A/E12) and their ensemble complex (ID3-E2A/E12) to gather information about the HLH domain region and its role in the interaction process. Further evaluation of the results by Principal Component Analysis (PCA) and Free Energy Landscape (FEL) helped in revealing residues of E2A/E12: Lys570, Ala595, Val598, and Ile599 and ID3: Glu53, Gln63, and Gln66 buried in their HLH motifs imparting key roles in dimerization process. Furthermore the T-pad analysis results helped in identifying the key fluctuations and conformational transitions using the intrinsic properties of the residues present in the domain region of the proteins thus specifying their crucial role towards molecular recognition. The study provides an insight into the interacting mechanism of the ID3-E2A/E12 complex and maps the structural transitions arising in the essential conformational space indicating the key structural changes within the helical regions of the motif. It thereby describes how the internal dynamics of the proteins might regulate their intrinsic structural features and its subsequent functionality.

The C(4) N‐heteroaryl 2‐amino‐3‐nitro‐4H‐chromenes are podophyllotoxin (POD) mimics as they possess the pharmacophore features of the anticancer natural product. We have achieved a facile synthesis of several such 4H‐chromenes by substitution of C(4) SMe in N‐alkyl 4‐(methylthio)‐3‐nitro‐4H‐chromen‐2‐amines with heteroaromatic amines like 2‐aminopyridines and 2‐aminopyrimidines. Resulting 4H‐chromenes were evaluated for anticancer activity against prostate and lung cancer cell lines. Two of the 4H‐chromenes showed significant anti‐cancer activity, even better than POD. Both of them were non‐toxic towards normal cell‐lines. In silico ADMET studies revealed drug‐likeliness of all the 4H‐chromenes. Molecular docking studies with αβ tubulin concurred with in vivo results and revealed that the 4H‐chromenes bind to the active site of POD. Molecular dynamics simulation studies on the complexes of αβ tubulin with the two best 4H‐chromenes revealed high stability. Overall, our studies revealed that the two 4H‐chromenes could act as lead compounds for further development of potent anticancer drugs. All the newly synthesized 4H‐chromenes were subjected to in vivo anti‐cancer activity against prostate and lung cancer cell lines. The studies revealed that two of them 18 q and 18 s exhibited IC50values better than POD. [ABSTRACT FROM AUTHOR]

The enzyme β-Ketoacyl ACP synthase I (KasA) is a potent drug target in mycolic acid pathway of Mycobacterium tuberculosis (Mtb). In the present study, we investigated the structural dynamics of wild-type (WT) and mutants KasA (D66N, G269S, G312S, and F413L) in both monomer and dimer form to provide insight into protein structural stability. To gain better understanding of structural flexibility of KasA, combined molecular dynamics and essential dynamics were employed to analyze the conformational changes induced by non-active site mutations. The results confirm that non-active site mutations lower the structural stability in dimer KasA as compared to WT. The protein network topology and close residue interactions of WT and mutant residues of KasA have been predicted through residue interaction network analysis (RIN). Non-active site mutations distort RIN architecture and subsequently affect the drug binding landscape. T-pad associated with mode vector analysis comprehensively pronounces the structural impact caused by non-active site mutations. It also identified the critical fluctuating residues present in the gate segment (GS) region (115–147). The non-active site mutations altered the structural stability of the mutant protein structures, and these mutations may be a cause for the resistance mechanism of KasA against anti-tuberculosis drugs. Further, it is observed that dimer mutant KasA proteins display much more structural flexibility than WT at the ligand binding site which is evident from the binding site analysis and hydrogen bond interaction patterns. This study provides a better understanding of the structural dynamic behaviour of KasA mutants, thereby facilitating the need to find a novel and potent inhibitor against Mtb. Unlabelled Image • KasA protein is one of the potential drug targets in the treatment of tuberculosis • Variations in conformational behaviour of WT and mutant systems were observed and investigated through PCA and FEL analysis • The significant conformational changes in gate segment (GS) region established its high vibrant nature in monomer KasA • Residue interaction network analysis depicted the impact of non-active site mutations in network architecture • Non-active site mutations caused the protein to exhibit more structural flexibility as compared to the WT protein [ABSTRACT FROM AUTHOR]

ARHI, a putative tumor suppressor protein with unique 32 amino acid extension in the N-terminal region, differs from oncogenes Ras and Rap, negatively regulates STAT3 signaling and inhibits the migration of ovarian cancer cells. ARHI associates directly with STAT3, also forms complex with importinβ, and prevents formation of RanGTPase–importinβ complex, which is essential for transporting STAT3 into the nucleus. Hence, the structural aspects pertaining to ARHI mediated inhibition of STAT3 translocation can provide hints on the regulation of STAT3 signaling mechanism. Accordingly, in the present study, the structure of ARHI was predicted and its transition from inactive to active state studied using MD simulations and free energy landscape analysis. The transition of ARHI is marked by the movement of switch I region towards γ-phosphate of GTP, in addition, the hydrophobic interaction between N-terminal helix and switch II region of ARHI accounts for its low intrinsic GTPase activity. Further, the protein–protein interaction studies reveal that the residues of N-terminal helix, effector domain, P-loop and G box motif of ARHI actively form polar and non-polar interaction with NTD of STAT3 and make them compact thereby rendering STAT3 inaccessible for Ran–importinβ mediated translocation. On the other hand, ARHI competes with RanGTPase and interacts with importinβ via basic–acidic patch interaction, which leads to inhibition of STAT3 translocation. The interacting residues involved for this structural mechanism would be instrumental in designing inhibitors for STAT3, which mimics ARHI thereby leading to the suppression of cancer cell growth. [ABSTRACT FROM AUTHOR]

Background: Bacterial lipases find so many industrial applications. Therefore, new source of lipase suitable for industrial conditions is always required. Lipase zymography methods use costly chromogenic substrates and indicator dyes and are few in numbers., Objective: The objectives of this work include lipase purification and its characterization from Acinetobacter radioresiens PR8 and development of new zymography method for lipase detection., Method: The lipase was purified using conventional method and cation exchange chromatography and it was characterized biochemically and analytically. Based on these characterization new in-gel lipase zymography method was developed., Results: In this present work, an alkalophilic lipase producing bacterium was isolated from soil; screened for extracellular lipase activity and identified to be Acinetobacter radioresistens PR8 (Genbank accession ID: MF073322). Enzyme production kinetics showed maximum production (4.16 U/ml at pH 9) of enzyme after 72 h. The lipase activity was found to be highest in olive oil (1% v/v; 8.1 U/ml). Low molecular weight (27 kDa) alkaline (pH 9) cold active (20 °C) lipase was purified from Acinetobacter radioresistens PR8. Lipase was characterized using PMF, FT-IR and its high conformational stability (Transition temperature: 122.3 °C) was attributed from its DSC spectrum. The importance of magnesium and sodium ions for enhancing lipase activity was obtained from flux balance analysis., Conclusion: Based on the lipase activating role of Mn2+ and Na+ ions, optimum temperature, pH with no chromogenic substrates and indicator dyes, a new in gel zymography method for lipase detection was developed., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Enzymes involved in the pyrimidine biosynthesis pathway have become an important target for the pharmacological intervention. One among those enzymes, Aspartate Trans Carbamoylase (ATCase), catalyses the condensation of aspartate and carbamoyl phosphate to form N-carbamoyl-l-aspartate and inorganic phosphate. The present study provides the molecular insights into the enzyme ATCase. The three-dimensional structure of ATCase from Thermus thermophilus HB8 was modeled based on the crystal structure of ATCase in Pyrococcus abyssi (PDB ID:1ML4). Molecular dynamics simulation was performed to identify the conformational stability of TtATCase with and without its ligand complexes. Based on the pharmacokinetic properties and the glide-docking scores of ligands from four databases (Maybridge, Binding, Asinex and Technology for Organic Synthesis (TOS laboratory) for the screening of ligands, we identified four potential ligand molecules for TtATCase. From the molecular docking results, we proposed that the residues Thr53, Arg104, and Gln219 are consistently involved in strong hydrogen-bonding interactions and play a vital role in the TtATCase activity. From the results of molecular dynamics simulation, the ligand molecules are found to bind appropriately to the target enzyme. However, the structure of TtATCase needs to be determined experimentally to confirm this.