Discovery of Nirmatrelvir Resistance Mutations in SARS-CoV-2 3CLpro: A Computational-Experimental Approach.

The COVID-19 pandemic has emphasized the urgency for effective antiviral therapies against SARS-CoV-2. Targeting the main protease (3CLpro) of the virus has emerged as a promising approach, and nirmatrelvir (PF-07321332), the active component of Pfizer's oral drug Paxlovid, has demonstrated remarkable clinical efficacy. However, the emergence of resistance mutations poses a challenge to its continued success. In this study, we employed alchemical free energy perturbation (FEP) alanine scanning to identify nirmatrelvir-resistance mutations within SARS-CoV-2 3CLpro. FEP identified several mutations, which were validated through in vitro IC50 experiments and found to result in 8- and 72-fold increases in nirmatrelvir IC50 values. Additionally, we constructed SARS-CoV-2 omicron replicons containing these mutations, and one of the mutants (S144A/E166A) displayed a 20-fold increase in EC50, confirming the role of FEP in identifying drug-resistance mutations. Our findings suggest that FEP can be a valuable tool in proactively monitoring the emergence of resistant strains and guiding the design of future inhibitors with reduced susceptibility to drug resistance. As nirmatrelvir is currently widely used for treating COVID-19, this research has important implications for surveillance efforts and antiviral development.

Synergistic Inhibition Guided Fragment-Linking Strategy and Quantitative Structure-Property Relationship Modeling To Design Inhalable Therapeutics for Asthma Targeting CSF1R.

The colony-stimulating factor-1 receptor (CSF1R) is a tyrosine-protein kinase that is a potential target for asthma therapeutics. We have applied a fragment-lead combination approach to identify small fragments that act synergistically with GW2580, a known inhibitor of CSF1R. Two fragment libraries were screened in combination with GW2580 by surface plasmon resonance (SPR). Binding affinity measurements confirmed that thirteen fragments bind specifically to the CSF1R, and a kinase activity assay further validated the inhibitory effect of these fragments. Several fragment compounds enhanced the inhibitory activity of the lead inhibitor. Computational solvent mapping, molecular docking, and modeling studies suggest that some of these fragments bind adjacent to the binding site of the lead inhibitor and further stabilize the inhibitor-bound state. Modeling results guided the computational fragment-linking approach to design potential next-generation compounds. The inhalability of these proposed compounds was predicted using quantitative structure-property relationships (QSPR) modeling based on an analysis of 71 drugs currently on the market. This work provides new insights into the development of inhalable small molecule therapeutics for asthma.

Identification of small molecule inhibitors against MMP-14 via High-Throughput screening.

Matrix metalloproteinases (MMPs) are involved in various cellular events in physiology and pathophysiology through endopeptidases activity. The expression levels and activities of most MMPs remain minimal in the normal conditions, whereas some MMPs are significantly activated in pathological conditions such as cancer and neovascularization. Hence, MMPs are considered as both diagnostic markers and potential targets for therapeutic agents. Twenty-three known human MMPs share a similar active site structure with a zinc-binding motif, resulting in lack of specificity. Therefore, the enhancement of target specificity is a primary goal for the development of specific MMP inhibitors. MMP-14 regulates VEGFA/VEGFR2-system through cleavage of the non-functional VEGFR1 in vascular angiogenesis. In this study, we developed a fluorescence-based enzymatic assay using a specific MMP-14 substrate generated from VEGFR1 cleavage site. This well optimized assay was used as a primary screen method to identify MMP-14 specific inhibitors from 1,200 Prestwick FDA-approved drug library. Of ten initial hits, two compounds showed IC50 values below 30 µM, which were further validated by direct binding analysis using surface plasmon resonance (SPR). Clioquinol and chloroxine, both of which contain a quinoline structure, were identified as MMP-14 inhibitors. Five analogs were tested, four of which were found to be completely devoid of inhibitory activity. Clioquinol exhibited selectivity towards MMP-14, as it showed no inhibitory activity towards four other MMPs.

Nonactive-Site Mutations in S. aureus FabI That Induce Triclosan Resistance.

The wide use of the antimicrobial agent/biocide, triclosan, promotes triclosan-resistant bacterial strains, including Staphylococcus aureus, as well as leads to accumulation in the aquatic and terrestrial environments. Knowledge of the molecular actions of triclosan on S. aureus is needed to understand the consequence of triclosan resistance and environmental accumulation of triclosan on S. aureus resistant strains, as well as to develop biphenyl ether analogs as antibiotic candidates. Triclosan inhibits an essential enzyme in the fatty acid biosynthetic pathway, the reduced nicotinamide adenine dinucleotide (NADH)/reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent enoyl-acyl carrier protein (enoyl-ACP) reductase, or FabI. In this study, we used error-prone polymerase chain reaction (epPCR) to generate mutations in the S. aureus FabI enzyme. Instead of using an elaborate FabI enzyme activity assay that involves ACP-linked substrates to determine whether triclosan inhibits the enzyme activities of individual FabI mutants, we used an efficient and economical assay that we developed, based on thermal shift principles, to screen for triclosan binding to FabI mutants in cells. We identified four active-site mutations. More interestingly, we also identified nine triclosan-resistant mutations distant from the active site (G113V, Y123H, S166N, N220I, G227C, A230T, V241I, F252I, and H253P) but located in disparate positions in the monomer-monomer and dimer-dimer interface regions in S. aureus FabI. We suggest that these sites may serve as potential allosteric sites for designing potential therapeutic inhibitors that offer advantages in selectivity since allosteric sites are less evolutionarily conserved.

Determination of absolute configuration and binding efficacy of benzimidazole-based FabI inhibitors through the support of electronic circular dichroism and MM-GBSA techniques.

We have previously reported benzimidazole-based compounds to be potent inhibitors of FabI for Francisella tularensis (FtFabI), making them promising antimicrobial hits. Optically active enantiomers exhibit markedly differing affinities toward FtFabI. The IC50 of benzimidazole (-)-1 is ∼100× lower than the (+)-enantiomer, with similar results for the 2 enantiomers. Determining the absolute configuration for these optical compounds and elucidating their binding modes is important for further design. Electronic circular dichroism (ECD) quantum calculations have become important in determining absolute configurations of optical compounds. We determined the absolute configuration of (-)/(+)-1 and (-)/(+)-2 by comparing experimental spectra and theoretical density functional theory (DFT) simulations of ECD spectra at the B3LYP/6-311+G(2d, p) level using Gaussian09. Comparison of experimental and calculated ECD spectra indicates that the S configuration corresponds to the (-)-rotation for both compounds 1 and 2, while the R configuration corresponds to the (+)-rotation. Further, molecular dynamics simulations and MM-GBSA binding energy calculations for these two pairs of enantiomers with FtFabI show much tighter binding MM-GBSA free energies for S-1 and S-2 than for their enantiomers, R-1 and R-2, consistent with the S configuration being the more active one, and with the ECD determination of the S configuration corresponding to (-) and the R configuration corresponding to (+). Thus, our computational studies allow us to assign (-) to (S)- and (+) to (R)- for compounds 1 and 2, and to further evaluate structural changes to improve efficacy.

An Efficient and Economical Assay to Screen for Triclosan Binding to FabI.

Triclosan is an effective inhibitor for enoyl acyl carrier protein reductase (ENR) in fatty acid biosynthesis. Triclosan-resistant mutants of ENR have emerged. Thus, it is important to detect these triclosan-resistant mutations in ENR. Generally, enzyme activity assays on the mutants are used to determine the effect of triclosan on ENR activity. Since the substrates are linked to acyl carrier protein (ACP), the assays are challenging due to the need to prepare the ACP and link it to the substrates. Non-ACP-linked (coenzyme A [CoA]-linked) substrates can be used in some ENR, but not in all. Consequently, screening for triclosan-resistant mutants is also challenging. We have developed a simple thermal shift assay, which does not use ACP-linked substrates, to determine the binding ability of triclosan to the ENR active site, and thus it can be used for screening for triclosan-resistant mutants. Staphylococcus aureus FabI enzyme and its mutants were used to demonstrate the binding ability of triclosan with NADP(+) to FabI. The direct correlation between the binding ability and enzyme activity was demonstrated with Francisella tularensis FabI. This method may also be applied to select effective triclosan analogues that inhibit ENR activity.