Synthesis and fungicidal activity of novel 2-(2-alkylthio-6-phenylpyrimidin-4-yl)-1H-benzimidazoles.

With the aim of exploring new benzimidazole derivative with high fungicidal activity, a series of novel 2-(2-(alkylthio)-6-phenylpyrimidin-4-yl)-1H-benzimidazoles were designed and synthesized, and their in vitro fungicidal activities were evaluated. Compounds 5a, 5f, 5g, 5h, 5i and 5l exhibited excellent fungicidal activities against Botrytis cinerea, and 5c, 5f, 5h, 5i and 5l displayed notable fungicidal activities against Sclerotinia sclerotiorum. Among them, compound 5i (R1 = fluorine, R2 = benzyl) displayed the best activity towards the two tested fungi. Docking study of 5i with ß-tubulin protein revealed that the NH moiety of benzimidazole ring generated a hydrogen bond with Gln-11 residue, and the fluorine atom of benzene ring formed a hydrogen bond with Tyr-208 residue, respectively; the benzene ring of Tyr-222 and the pyrimidine ring of 5i yielded a π-π interaction. The molecular electrostatic potential analysis elucidated the nitrogen atom of benzimidazole ring, fluorine atom of benzene ring and sulfur atom of thioether moiety were located in the negative potential regions, whereas some hydrogen atoms of benzene, benzimidazole and pyrimidine rings were located in the positive potential regions. This analysis demonstrated the reason why 5i can form hydrogen bonds with amino acid residues of target protein.

Importance of the main chain of lysine for histone lysine methyltransferase catalysis.

Histone lysine methyltransferases (KMTs) are biomedicinally important class of epigenetic enzymes that catalyse methylation of lysine residues in histones and other proteins. Enzymatic and computational studies on the simplest lysine analogues that possess a modified main chain demonstrate that the lysine's backbone contributes significantly to functional KMT binding and catalysis.

Unraveling the Origins of Changing Product Specificity Properties of Arginine Methyltransferase PRMT7 by the E181D and E181D/Q329A Mutations through QM/MM MD and Free-Energy Simulations.

Arginine methylations can regulate important biological processes and affect many cellular activities, and the enzymes that catalyze the methylations are protein arginine methyltransferases (PRMTs). The biological consequences of arginine methylations depend on the methylation states of arginine that are determined by the PRMT's product specificity. Nevertheless, it is still unclear how different PRMTs may generate different methylation states for the target proteins. PRMT7 is the only known member of type III PRMT that produces monomethyl arginine (MMA) product. Interestingly, its E181D and E181D/Q329A mutants can catalyze, respectively, the formation of asymmetrically dimethylated arginine (ADMA) and symmetrically dimethylated arginine (SDMA). The reasons as to why the mutants have the abilities to add the second methyl group and E181D (E181D/Q329A) has the unique product specificity in generating ADMA (SDMA) have not been understood. Here, quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) and potential of mean force (PMF) free-energy simulations are performed for the E181D and E181D/Q329A mutants to understand the origin for their ability to generate, respectively, ADMA and SDMA. The simulations show that the free-energy barrier for adding the second methyl group to MMA in E181D (E181D/Q329A) to produce ADMA (SDMA) is considerably lower than the corresponding barriers in wild type and E181D/Q329A (wild type and E181D), consistent with experimental observations. Some important factors that contribute to the change of the activity and product specificity due to the E181D and E181D/Q329A mutations are identified based on the data from the simulations and analysis. It is shown that the transferable methyl group (from SAM) and Nη2 (the nitrogen atom that is methylated in the substrate MMA) can only form good near-attack conformations in the E181D reaction state for the methyl transfer (not in wild type and E181D/Q329A), while the transferable methyl group and Nη1 (the nitrogen atom that is not methylated in the substrate MMA) can only form good near-attack conformations in E181D/Q329A (not in wild type and E181D). The results suggest that the steric repulsions in the reaction state between the methyl group on MMA and active-site residues (e.g., Q329) and the release of such repulsions (e.g., from the Q329A mutation) may play an important role in generating specific near-attack conformations for the methyl transfer and controlling the product specificity for the mutants. The general principle identified in this work for PRMT7 is expected to be useful for understanding the activity and product specificity of other PRMTs as well.

Synthesis and fungicidal activity of novel benzimidazole derivatives bearing pyrimidine-thioether moiety against Botrytis cinerea.

BACKGROUND: Botrytis cinerea is a serious plant fungus and strongly affects the yield and quality of crops. The main control strategy is the employment of fungicides. To research for efficient fungicide with novel structure, a series of novel benzimidazole derivatives bearing pyrimidine and thioether moieties were designed and synthesized. RESULTS: Some target compounds such as 4h, 4i, 4k, 4l, 4m, 4s, 4t and 4u exhibited notable fungicidal activities, with half maximal effective concentration (EC50 ) values in the range 0.13-0.24 µg mL-1 , which means that their activities were comparable or higher than that of carbendazim (EC50 = 0.21 µg mL-1 ). Among them, N-(4-fluorophenyl)-2-((4-(1H-benzimidazol-2-yl)-6-(4-methoxyphenyl) pyrimidin-2-yl)thio)acetamide (4m) displayed the best activity (EC50 = 0.13 µg mL-1 ). Molecular electrostatic potential analysis of 4m elucidated that the NH moiety of benzimidazole ring was located in the positive potential region and may generate hydrogen bond with target amino acid residue. Molecular docking analysis revealed that there was one hydrogen bond and one 𝜋-𝜋 interaction between 4m and target protein. CONCLUSIONS: This study demonstrated that the benzimidazole derivatives bearing pyrimidine and thioether moieties can be further optimized as a lead compound for the control of B. cinerea. The combination of molecular electrostatic potential and molecular docking analyses may provide a valuable reference for studying the interaction between the ligand and target protein. © 2021 Society of Chemical Industry.

Catalytic Mechanism and Product Specificity of Protein Arginine Methyltransferase PRMT7: A Study from QM/MM Molecular Dynamics and Free Energy Simulations.

QM/MM molecular dynamics and potential of mean force (PMF) free-energy simulations are performed for wild-type PRMT7 and E172Q, E181Q, and Q329A mutants in this work, and the catalytic mechanism, product specificity, and the role of key residues for the PRMT7 activity are investigated. The main strategies of PRMT7 in reducing the activation barrier for methyl transfer that are found in this study include (1) formation of reactive (near attack) conformations for the substrate Arg, (2) strengthening the active-site interactions at the transition state, and (3) generation of more effective nucleophiles by changing charge distributions on the target Arg through active-site interactions. More importantly, it is shown that it is a combination of these different factors that determines the PRMT7 methylation activity and substrate/product specificity. By taking these factors into consideration, it is possible to provide explanations for the observed effects of some mutations. For E172Q, E181Q, and Q329A, the simulation results suggest that E172Q has the least activity among the three mutants. The free energy barrier increases by 7 and 3 kcal/mol, respectively, as a result of the E181 → Q and Q329 → A mutations. The results showed that PRMT7 has a preference of adding a methyl group to the ω-guanidino nitrogen Nη2 atom of the substrate Arg and that the second methylation reactions cannot occur, which are consistent with previous investigations.

Examining sterically demanding lysine analogs for histone lysine methyltransferase catalysis.

Methylation of lysine residues in histone proteins is catalyzed by S-adenosylmethionine (SAM)-dependent histone lysine methyltransferases (KMTs), a genuinely important class of epigenetic enzymes of biomedical interest. Here we report synthetic, mass spectrometric, NMR spectroscopic and quantum mechanical/molecular mechanical (QM/MM) molecular dynamics studies on KMT-catalyzed methylation of histone peptides that contain lysine and its sterically demanding analogs. Our synergistic experimental and computational work demonstrates that human KMTs have a capacity to catalyze methylation of slightly bulkier lysine analogs, but lack the activity for analogs that possess larger aromatic side chains. Overall, this study provides an important chemical insight into molecular requirements that contribute to efficient KMT catalysis and expands the substrate scope of KMT-catalyzed methylation reactions.