Computational Investigation of a Series of Small Molecules as Potential Compounds for Lysyl Hydroxylase-2 (LH2) Inhibition

The catalytic function of lysyl hydroxylase-2 (LH2), a member of the Fe(II)/αKG-dependent oxygenase superfamily, is to catalyze the hydroxylation of lysine to hydroxylysine in collagen, resulting in stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs). Reports show that high amounts of LH2 lead to the accumulation of HLCCs, causing fibrosis and specific types of cancer metastasis. Some members of the Fe(II)/αKG-dependent family have also been reported to have intramolecular

Computational Insights into the Inhibition Mechanism of Xanthine Oxidoreductase by Oxipurinol

Xanthine oxidoreductase (XOR) is a molybdopterin-containing enzyme found in many living organisms. Its function is to convert hypoxanthine to xanthine and subsequently to urate, which are the final steps in purine elimination in humans. Elevated uric acid levels in the human body can cause gout and hyperuricemia. Therefore, drug development efforts targeting this enzyme are a primary focus not only to treat these conditions but also for other diseases. Oxipurinol, an analog of xanthine, was the drug that emerged as a “gold standard” inhibitor of XO in the late sixties. Crystallographic studies have shown direct coordination of oxipurinol to the molybdenum cofactor (MoCo). The proposed inhibition mechanism based on available crystal structures posits that the oxipurinol’s nitrogen replaces a water-exchangeable OH ligand of the Mo atom. However, the detailed steps involved in the inhibition mechanism remain undefined, which would provide important insights for designing more efficacious drugs with similar inhibition functions. In this study, the inhibition mechanism of XOR by oxipurinol is investigated via molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations. The structural and dynamical effects of oxipurinol on the pre-catalytic structure of the metabolite-bound system are presented, as well as the modeled reaction mechanism catalyzed by the MoCo center in the active site. The kinetics and thermodynamics of the proposed reaction mechanism as well as the non-covalent interactions with the binding cavity align with experimental findings. Our results also suggest the suitability of the inhibition reaction via another tautomer of oxipurinol other than the experimentally predominant tautomer. This might suggest possible routes for designing new analogs of oxipurinol with a similar coordination mode to the latter tautomer, which could lead to more energetically favorable inhibitors

Computational Characterization of the Inhibition Mechanism of Xanthine Oxidoreductase by Topiroxostat

Xanthine oxidase (XO) is a member of the molybdopterin-containing enzyme family. It interconverts xanthine to uric acid as the last step of purine catabolism in the human body. The high uric acid concentration in the blood directly leads to human diseases like gout and hyperuricemia. Therefore, drugs that inhibit the biosynthesis of uric acid by human XO have been clinically used for many years to decrease the concentration of uric acid in the blood. In this study, the inhibition mechanism of XO and a new promising drug, Topiroxostat (code: FYX-051), is investigated by employing molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations. This drug has been reported to act as both a non-covalent and covalent inhibitor and undergoes a stepwise inhibition by all its hydroxylated metabolites, which include 2-hydroxy-FYX-051, dihydroxy-FYX-051, and trihydroxy-FYX-051. However, the detailed mechanism of inhibition of each metabolite remains elusive and can be useful for designing more effective drugs with similar inhibition functions. Hence, herein we present the computational investigation of the structural and dynamical effects of FYX-051 and the calculated reaction mechanism for all the oxidation steps catalyzed by the molybdopterin center in the active site. Calculated results for the proposed reaction mechanisms for each metabolite’s inhibition reaction in the enzyme’s active site, binding affinities, and the non-covalent interactions with surrounding amino acid residues are consistent with previously reported experimental findings. Analysis of the non-covalent interactions via EDA and NCI suggests residues L648, K771, E802, R839, L873, R880, R912, F914, F1009, L1014, and A1079 can be used as key interacting residues for further hybrid-type inhibitor development

Effects of the Y432S Cancer-Associated Variant on the Reaction Mechanism of Human DNA Polymerase κ

Human polymerases are vital for genetic information management. Their function involves catalyzing the synthesis of DNA strands with unparalleled accuracy, which ensures the fidelity and stability of the human genomic blueprint. Several disease-associated mutations and their functional impact on DNA polymerases have been reported. One particular polymerase, human DNA polymerase kappa (Pol κ), has been reported to be susceptible to several cancer-associated mutations. The Y432S mutation in Pol κ, which is associated with various cancers, is of interest due to its impact on polymerization activity and markedly reduced thermal stability. Here, we have used computational simulations to investigate the functional consequences of the Y432S by means of classical molecular dynamics (MD) and coupled quantum mechanics/molecular mechanics (QM/MM) methods. Our results suggest that Y432S results in structural effects on domains involved in nucleotide addition and ternary complex stabilization while maintaining catalytic competence. Calculation of the minimum energy path associated with the reaction mechanism of wild type (WT) and Y432S Pol κ indicate that while both enzymes are catalytically competent, the cancer mutation results in a slightly endoergic reaction and an increase in the catalytic barrier. Interactions with a third magnesium ion and environmental effects on non-bonded interactions, particularly involving key residues, contribute to the kinetic and thermodynamic distinctions between the WT and mutant during the catalytic reaction. The energetics and electronic findings suggest that active site residues favor the catalytic reaction with dCTP3– over dCTP4–

Computational Characterization of the Inhibition Mechanism of Xanthine Oxidoreductase by Topiroxostat

Xanthine oxidase (XO) is a member of the molybdopterin-containing enzyme family. It interconverts xanthine to uric acid as the last step of purine catabolism in the human body. The high uric acid concentration in the blood directly leads to human diseases like gout and hyperuricemia. Therefore, drugs that inhibit the biosynthesis of uric acid by human XO have been clinically used for many years to decrease the concentration of uric acid in the blood. In this study, the inhibition mechanism of XO and a new promising drug, topiroxostat (code: FYX-051), is investigated by employing molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations. This drug has been reported to act as both a noncovalent and covalent inhibitor and undergoes a stepwise inhibition by all its hydroxylated metabolites, which include 2-hydroxy-FYX-051, dihydroxy-FYX-051, and trihydroxy-FYX-051. However, the detailed mechanism of inhibition of each metabolite remains elusive and can be useful for designing more effective drugs with similar inhibition functions. Hence, herein we present the computational investigation of the structural and dynamical effects of FYX-051 and the calculated reaction mechanism for all of the oxidation steps catalyzed by the molybdopterin center in the active site. Calculated results for the proposed reaction mechanisms for each metabolite’s inhibition reaction in the enzyme’s active site, binding affinities, and the noncovalent interactions with the surrounding amino acid residues are consistent with previously reported experimental findings. Analysis of the noncovalent interactions via energy decomposition analysis (EDA) and noncovalent interaction (NCI) techniques suggests that residues L648, K771, E802, R839, L873, R880, R912, F914, F1009, L1014, and A1079 can be used as key interacting residues for further hybrid-type inhibitor development

Computational Characterization of the Inhibition Mechanism of Xanthine Oxidoreductase by Topiroxostat

Xanthine oxidase (XO) is a member of the molybdopterin-containing enzyme family. It interconverts xanthine to uric acid as the last step of purine catabolism in the human body. The high uric acid concentration in the blood directly leads to human diseases like gout and hyperuricemia. Therefore, drugs that inhibit the biosynthesis of uric acid by human XO have been clinically used for many years to decrease the concentration of uric acid in the blood. In this study, the inhibition mechanism of XO and a new promising drug, topiroxostat (code: FYX-051), is investigated by employing molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations. This drug has been reported to act as both a noncovalent and covalent inhibitor and undergoes a stepwise inhibition by all its hydroxylated metabolites, which include 2-hydroxy-FYX-051, dihydroxy-FYX-051, and trihydroxy-FYX-051. However, the detailed mechanism of inhibition of each metabolite remains elusive and can be useful for designing more effective drugs with similar inhibition functions. Hence, herein we present the computational investigation of the structural and dynamical effects of FYX-051 and the calculated reaction mechanism for all of the oxidation steps catalyzed by the molybdopterin center in the active site. Calculated results for the proposed reaction mechanisms for each metabolite’s inhibition reaction in the enzyme’s active site, binding affinities, and the noncovalent interactions with the surrounding amino acid residues are consistent with previously reported experimental findings. Analysis of the noncovalent interactions via energy decomposition analysis (EDA) and noncovalent interaction (NCI) techniques suggests that residues L648, K771, E802, R839, L873, R880, R912, F914, F1009, L1014, and A1079 can be used as key interacting residues for further hybrid-type inhibitor development

Computational Characterization of the Inhibition Mechanism of Xanthine Oxidoreductase by Topiroxostat

Xanthine oxidase (XO) is a member of the molybdopterin-containing enzyme family. It interconverts xanthine to uric acid as the last step of purine catabolism in the human body. The high uric acid concentration in the blood directly leads to human diseases like gout and hyperuricemia. Therefore, drugs that inhibit the biosynthesis of uric acid by human XO have been clinically used for many years to decrease the concentration of uric acid in the blood. In this study, the inhibition mechanism of XO and a new promising drug, topiroxostat (code: FYX-051), is investigated by employing molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations. This drug has been reported to act as both a noncovalent and covalent inhibitor and undergoes a stepwise inhibition by all its hydroxylated metabolites, which include 2-hydroxy-FYX-051, dihydroxy-FYX-051, and trihydroxy-FYX-051. However, the detailed mechanism of inhibition of each metabolite remains elusive and can be useful for designing more effective drugs with similar inhibition functions. Hence, herein we present the computational investigation of the structural and dynamical effects of FYX-051 and the calculated reaction mechanism for all of the oxidation steps catalyzed by the molybdopterin center in the active site. Calculated results for the proposed reaction mechanisms for each metabolite’s inhibition reaction in the enzyme’s active site, binding affinities, and the noncovalent interactions with the surrounding amino acid residues are consistent with previously reported experimental findings. Analysis of the noncovalent interactions via energy decomposition analysis (EDA) and noncovalent interaction (NCI) techniques suggests that residues L648, K771, E802, R839, L873, R880, R912, F914, F1009, L1014, and A1079 can be used as key interacting residues for further hybrid-type inhibitor development

Leveraging QM/MM and Molecular Dynamics Simulations to Decipher the Reaction Mechanism of the Cas9 HNH Domain to Investigate off-Target Effects

The clustered regularly interspaced short palindromic repeats (CRISPR) technology is an RNA-guided targeted genome-editing tool using Cas family proteins. Two magnesium-dependent nuclease domains of this enzyme, termed HNH and RuvC, are responsible for cleaving the target DNA (t-DNA) and non-target DNA (nt-DNA) strands, respectively. The HNH domain is believed to determine the DNA cleavage activity of both endonuclease domains and is sensitive to complementary RNA-DNA base pairing. However, the underlying molecular mechanisms of CRISPR-Cas9, by which it rebukes or accepts mismatches, are poorly understood. Thus, investigation of the structure and dynamics of the catalytic state of Cas9 with either matched or mismatched t-DNA can provide insights for improving its specificity by reducing off-target cleavages. Here, we focus on a recently discovered catalytic-active form of the Streptococcus pyogenes Cas9 (SpCas9) and employ classical molecular dynamics (MD) and coupled quantum mechanics/molecular mechanics (QM/MM) simulations to study two possible mechanisms of t-DNA cleavage reaction catalyzed by the HNH domain. Moreover, by designing a mismatched t-DNA structure called MM5 (C to G in the fifth position from the PAM region), the impact of single-guide RNA (sgRNA) and t-DNA complementarity on the catalysis process was investigated. Based on these simulations, our calculated binding affinities, minimum energy paths, and analysis of catalytically important residues provide atomic-level details of the differences between matched and mismatched cleavage reactions. In addition, several residues exhibit significant differences in their catalytic role for the two studied systems, including K253, K263, R820, K896, and K913

Impact of Remdesivir Incorporation Along the Primer Strand on SARS-CoV-2 RNA-dependent RNA polymerase

Remdesivir was the first antiviral drug that received emergency use authorization from the United States Food and Drug Administration and is now formally approved to treat COVID-19. Remdesivir is a nucleotide analogue that targets the RNA-dependent RNA polymerase (RdRp) of coronaviruses, including SARS-CoV-2. The solution of multiple RdRp structures has been one of the main axes of research in the race against the SARS-CoV-2 virus. Several hypotheses of the mechanism of inhibition of RdRp by remdesivir have been proposed, although open questions remain. This work uses molecular dynamics (MD) simulations to explore the impact of remdesivir and two analogues as incoming nucleotides, and of up to four incorporations of remdesivir along the primer strand on RdRp. The simulation results suggest that the overall structure and dynamical behavior of RdRp is destabilized by remdesivir and the two analogues in the incoming position. The incorporation of remdesivir along the primer strand im- pacts specific non-bonded interactions between the nascent RNA and the polymerase subunit, as well as overall dynamical networks on RdRp. The strongest impact on the structure and dynamics are observed after three incorporations, when remdesivir is located at position -A3, in agreement with previously reported experimental and computational results. Our results provide atomic-level detail on the role played by remdesivir on the disruption of RNA synthesis by RdRp, and the main drivers of these disruptions