Pandemic COVID-19 infections have spread throughout the world. There is no effective treatment against this disease. Viral RNA-dependent RNA polymerase (RdRp) catalyzes the replication of RNA from RNA and the main protease (M pro) has a role in the processing of polyproteins that are translated from the RNA of SARS-CoV-2, and thus these two enzymes are strong candidates for targeting by anti-viral drugs. Small molecules such as lopinavir and favipiravir significantly inhibit the activity of M pro and RdRp in vitro. Studies have shown that structurally modified lopinavir, favipiravir, and other similar compounds can inhibit COVID-19 main protease (M pro) and RNA-dependent RNA polymerase (RdRp). In this study, lopinavir and its structurally similar compounds were chosen to bind the main protease, and favipiravir was chosen to target RNA-dependent RNA polymerase. Molecular docking and the quantitative structure-activity relationships (QSAR) study revealed that the selected candidates have favorable binding affinity but less druggable properties. To improve the druggability, four structural analogues of lopinavir and one structural analogue of favipiravir was designed by structural modification. Molecular interaction analyses have displayed that lopinavir and favipiravir analogues interact with the active site residues of M pro and RdRp, respectively. Absorption, distribution, metabolism, excretion and toxicity (ADMET) properties, medicinal chemistry profile, and physicochemical features were shown that all structurally modified analogues are less toxic and contain high druggable properties than the selected candidates. Subsequently, 50 ns molecular dynamics simulation of the top four docked complexes demonstrated that CID44271905, a lopinavir analogue, forms the most stable complex with the M pro. Further MMPBSA analyses using the MD trajectories also confirmed the higher binding affinity of CID44271905 towards M pro. In summary, this study demonstrates a new way to identify leads for novel anti-viral drugs against COVID-19.

Seamless (i.e., without unwanted DNA sequences) mutant bacterial artificial chromosomes (BACs) generated via recombination-mediated genetic engineering (recombineering) are better suited to study gene function compared to complementary DNA (cDNA) because they contain only the specific mutation and provide all the regulatory sequences required for in vivo gene expression. However, precisely mutated BACs are typically rare (∼1:1,000 to 1:100,000), making their isolation quite challenging. Although these BACs have been classically isolated by linking the mutation to additional genes, i.e., selectable markers, this approach is prone to false positives and is labor-intensive because it requires the subsequent removal of the selectable marker. We created Founder Principle-driven Enrichment (FPE), a method based on the population genetics "founder principle," to directly isolate rare mutant BACs, without any selectable marker, from liquid cultures via the polymerase chain reaction (PCR). Here, we provide a detailed description of FPE, including protocols for BAC recombineering and PCR screening. © 2017 by John Wiley & Sons, Inc.

In this study, we have designed and synthesized pyrazoline analogues that partially mimic the structure of mycobactin, to address the requirement of novel therapeutics to tackle the emerging global challenge of antimicrobial resistance (AMR). Our investigation resulted in the identification of novel lead compounds 44 and 49 as potential mycobactin biosynthesis inhibitors against mycobacteria. Moreover, candidates efficiently eradicated intracellularly surviving mycobacteria. Thermofluorimetric analysis and molecular dynamics simulations suggested that compounds 44 and 49 bind to salicyl-AMP ligase (MbtA), a key enzyme in the mycobactin biosynthetic pathway. To the best of our knowledge, these are the first rationally designed mycobactin inhibitors to demonstrate an excellent in vivo pharmacokinetic profile. In addition, these compounds also exhibited more potent whole-cell efflux pump inhibition than known efflux pump inhibitors verapamil and chlorpromazine. Results from this study pave the way for the development of 3-(2-hydroxyphenyl)-5-(aryl)-pyrazolines as a new weapon against superbug-associated AMR challenges.

As newer variants of SARS-CoV-2 continue to pose major threats to global human health and economy, identifying novel druggable antiviral targets is the key towards sustenance. Here, we identify an evolutionary conserved “E-L-L” motif present within the HR2 domain of all human and non-human coronavirus spike (S) proteins that play a crucial role in stabilizing its post-fusion six-helix bundle (6-HB) structure and thus, fusion-mediated viral entry. Mutations within this motif reduces the fusogenicity of the S protein without affecting its stability or membrane localization. We found that posaconazole, an FDA-approved drug, binds to this “E-L-L” motif and impedes the formation of 6-HB, thus effectively inhibiting SARS-CoV-2 infection in cells. While posaconazole exhibits high efficacy in blocking S protein-mediated viral entry, mutations within the “E-L-L” motif rendered the protein completely resistant to the drug, establishing its specificity towards this motif. Our data demonstrate that posaconazole restricts early stages of infection through specific inhibition of membrane fusion and viral genome release into the host cell and is equally effective towards all major variants of concerns of SARS-CoV-2 including beta, kappa, delta, and omicron. Together, we show that this conserved essential “E-L-L” motif is an ideal target for the development of prophylactic and therapeutic interventions against SARS-CoV-2.

Glycolipids like phosphatidylinositol hexamannosides (PIM 6 ) and lipoglycans, such as lipomannan (LM) and lipoarabinomannan (LAM), play crucial roles in virulence, survival, and antibiotic resistance of various mycobacterial species. Phosphatidyl-myo-inositol mannosyltransferase A (PimA) catalyzes the transfer of the mannose moiety (M) from GDP-mannose (GDPM) to phosphatidyl-myo-inositol (PI) to synthesize GDP and phosphatidyl-myo-inositol monomannoside (PIM). This PIM is mannosylated, acylated, and further modified to give rise to the higher PIMs, LM, and LAM. It is yet to be known how PI, PIM, PI-GDPM, and PIM-GDP interact with PimA. Here, we report the docked structures of PI and PIM to understand how the substrates and the products interact with PimA. Using molecular dynamics (MD) simulations for 300 ns, we have investigated how various ligand-bound conformations change the dynamics of PimA. Our studies demonstrated the “open to closed” motions of PimA. We observed that PimA is least dynamic when bound to both GDPM and PI. MD simulations indicated that the loop residues 59–70 and the α-helical residues 73–86 of PimA play important roles while interacting with both PI and PIM. MD analyses also suggested that the residues Y9, P59, R68, L69, N97, R196, R201, K202, and R228 of PimA play significant roles in the mannose transfer reaction. Overall, docking studies and MD simulations provide crucial insights to design future therapeutic drugs against mycobacterial PimA.