Structure-guided computational insecticide discovery targeting ß-N-acetyl-D-hexosaminidase of Ostrinia furnacalis.

Ostrinia furnacalis is a species of moth in the Crambidae family that is harmful to maize and other corn crops in Southeast Asia and the Western Pacific regions. Ostrinia furnacalis causes devastating losses to economically important corn fields. The ß-N-acetyl-D-hexosaminidase is an essential enzyme in O. furnacalis and its substrate binding +1 active site is different from that of the plants and humans ß-N-acetyl-D-hexosaminidases. To develop environment-friendly insecticides against OfHex1, we conducted structure-guided computational insecticide discovery to identify potential inhibitors that can bind the active site and inhibit the substrate binding and activity of the enzyme. We adopted a three-pronged strategy to conduct virtual screening using Glide and virtual screening workflow (VSW) in Schrödinger Suite-2022-3, against crystal structures of OfHex1 (PDB Id:3NSN), its homologue in humans (PDB Id: 1NP0) and Alphafold model of ß-N-acetyl-D-hexosaminidase from Trichogramma pretiosum, an egg parasitoid that protects the crops from O. furnacalis. A library of 20,313 commercially available and "insecticide-like" compounds was extracted from published literature. LigPrep enabled 44,943 ready-to-dock conformers generation. Glide docking revealed 18 OfHex1-specific hits that were absent in human and T. pretiosum screens. Reference docking was conducted using inhibitors/natural ligands in the crystal structures and hits with better docking scores than the reference were selected for MD simulations using Desmond to understand the stability of hit-target interactions. We noted five compounds that bound to OfHex1 TMX active-site based on their docking scores, consistent binding as noted by MD simulations and their insecticide/pesticide likeliness as noted by the Comprehensive Pesticide Likeness Analysis.Communicated by Ramaswamy H. Sarma.

Identification of selective inhibitors for phosphodiesterase 5A using e-pharmacophore modelling and large-scale virtual screening-based structure guided drug discovery approaches.

The inhibition of Phosphodiesterase 5A (PDEA5) has the potential to modulate pulmonary arterial hypertension and cardiovascular diseases. Exploring the cross-reactivity of clinically available PDE5A therapeutics with PDE6A is intriguing in order to develop highly selective PDE5A compounds in cardiovascular arena. In the current study, we leveraged e-pharmacophore based screening and molecular dynamics (MD) simulation to discover more selective PDE5A inhibitors as compared to the PDE6A catalytic domain. e-Pharmacophore based mapping of the CoCoCo database (7 million compounds: ∼ 150,000,000 conformers), followed by Glide docking, MM-GBSA, and protein-inhibitor interaction analysis, revealed 1536427, 4832637 and 6788240 as stable, tight binders of PDE5A instead of PDE6A. These compounds adhere to Lipinski Rule of Five (RO5) and ADME/Tox criteria. MD simulations analysis showed that 1536427 stays stable and tightly binds to catalytic (Q-region) core of PDE5A catalytic domain as compared to sildenafil. Pronounced inward motions of the hydrophobic (H-region) and Lid region indicate the closure of PDE5A-1536427 complex, whereas this region in PDE6A-1536427 is more open. Significant differences in the interactions, stability, and dynamics of 1536427 were observed in the catalytic domain of PDE6A, demonstrating less specificity for PDE6A in comparison to PDE5A. After lead optimization and therapeutic interventions, this proposed lead may emerge as a promising PDE5A selective inhibitor.Communicated by Ramaswamy H. Sarma.

SARS-CoV-2 Omicron subvariant spike N405 unlikely to rapidly deamidate.

The RGD motif on the SARS-CoV-2 spike protein has been suggested to interact with RGD-binding integrins αVß3 and α5ß1 to enhance viral cell entry and alter downstream signaling cascades. The D405N mutation on the Omicron subvariant spike proteins, resulting in an RGN motif, has recently been shown to inhibit binding to integrin αVß3. Deamidation of asparagines in protein ligand RGN motifs has been demonstrated to generate RGD and RGisoD motifs that permit binding to RGD-binding integrins. Two asparagines, N481 and N501, on the Wild-type spike receptor-binding domain have been previously shown to have deamidation half-lives of 16.5 and 123 days, respectively, which may occur during the viral life cycle. Deamidation of Omicron subvariant N405 may recover the ability to interact with RGD-binding integrins. Thus, herein, all-atom molecular dynamics simulations of the Wild-type and Omicron subvariant spike protein receptor-binding domains were conducted to investigate the potential for asparagines, the Omicron subvariant N405 in particular, to assume the optimized geometry for deamidation to occur. In summary, the Omicron subvariant N405 was primarily found to be stabilized in a state unfavourable for deamidation after hydrogen bonding with downstream E406. Nevertheless, a small number of RGD or RGisoD motifs on the Omicron subvariant spike proteins may restore the ability to interact with RGD-binding integrins. The simulations also provided structural clarification regarding the deamidation rates of Wild-type N481 and N501 and highlighted the utility of tertiary structure dynamics information in predicting asparagine deamidation. Further work is needed to characterize the effects of deamidation on spike-integrin interactions.

Report of rpoB mutation in clinically suspected cases of drug resistant leprosy: a study from Eastern India.

BACKGROUND: The current strategy for leprosy control depends mainly on early case detection and providing the recommended multidrug therapy (MDT) dosage. Understanding the molecular mechanisms of drug resistance to each of these drugs is essential in providing effective treatment and preventing the spread of resistant strains in the community. The progress of molecular biology research provides a very efficient opportunity for the diagnosis of drug resistance by in vitro method. AIM: We aimed to investigate the point mutations within the rpoB gene region of the Mycobacterium leprae genome, which are responsible for resistance to rifampicin, in order to determine the emergence of drug resistance in leprosy in the Kolkata region of West Bengal. METHODS: A total of 50 patients with a relapse of leprosy were enrolled in the study. Skin smears were obtained for estimation of bacillary index and biopsies were obtained in 70% alcohol for extraction of DNA. The extracted DNA was amplified by M. leprae-polymerase chain reaction (PCR) targeting rpoB gene region. Every single nucleotide base in the sequence is aligned to reference sequence and identity gaps were determined by NCBI - BLAST. Later in-silico analysis was done to identify the changes in the translated protein sequences. RESULTS: A mutation at the base pair position 2275405 where G is replaced by C in the M. leprae genome, which corresponds to the coding region of rpoB gene (279 bp - 2275228 to2275506), was observed in two patients. This missense mutation in CAC codon brings about a glutamic acid to histidine change in the amino acid sequence of RNA polymerase beta subunit at the position 442 (Glu442His), a region specific for rifampicin interaction, which might be responsible for unresponsiveness to rifampicin by manifesting a stable bacteriological index in these 2 patients even after completion of 24 months of multibacillary multi-drug therapy (MB-MDT). LIMITATIONS: The major limitations of multiple-primer PCR amplification refractory mutation system (MARS) assay is that it capable of detecting mutation at codon 425 and cannot distinguish any silent amino acid changes. CONCLUSION: The study indicates the existence of rifampicin drug resistance in Eastern India.

Drug resistance patterns in Mycobacterium leprae isolates from relapsed leprosy patients attending The Leprosy Mission (TLM) Hospitals in India.

Implementation of multidrug therapy (MDT) in leprosy control programmes has significantly reduced the global prevalence of the disease in the last two decades. After many years of use of MDT, it is expected that drug resistance in Mycobacterium leprae may emerge. This is a major concern, especially during the stage of elimination. In the present study, slit-skin smears were collected from 140 leprosy relapse cases from different Leprosy Mission hospitals across India. DNA extracted from 111 (79%) of these samples was analysed for the genes associated with drug resistance in M. leprae. More than 90% of the patients relapsed as multibacillary (MB) cases. In our study, four (3.6%) of the DNA samples analysed showed mutations associated with rifampicin resistance. We also observed that mutations associated with resistance to dapsone and ofloxacin were observed in 9 (8.1%) of the DNA samples each; two samples had both dapsone and ofloxacin resistance. Further surveillance and appropriate interventions are needed to ensure the continued success of chemotherapy for leprosy.