Discovery of efficient inhibitors against pyruvate dehydrogenase complex component E1 with bactericidal activity using computer aided design

https://doi.org/10.1016/j.pestbp.2021.104894Get rights and content

Highlights

  • Seventeen novel 2,6-dimethyl-4-aminopyrimidine derivatives containing acylhydrazone moiety were designed using computer-aided drug design.

  • Compound 6l had a strong binding capacity for E. coli PDHc-E1.

  • Compounds in series 6 displayed highly selective between E. coli PDHc-E1 and mammal PDHc-E1.

  • The binding models for 6l and E. coli PDHc-E1 and porcine PDHc-E1 were defined.

  • Compounds 6a, 6d, 6e, 6g, 6h, 6i, 6m, and 6n can effectively against Ralstonia solanacearum at 100 μg/mL.

Abstract

Computer aided optimization of lead compounds is of great significance to the design and discovery of new agrochemicals. A series of 2,6-dimethyl-4-aminopyrimidine acylhydrazones 6 was rationally designed as pyruvate dehydrogenase complex component E1 (PDHc-E1) inhibitors using computer aided drug design. Compounds in series 6 showed excellent inhibitory activity against Escherichia coli PDHc-E1, which was considerably higher than that of the lead compound A2. Compound 6l showed the best inhibitory activity (IC50 = 95 nM). Molecular docking, site-directed mutagenesis, and enzymatic assays revealed that the compounds bound in a “straight” conformation in the active site of E. coli PDHc-E1. Compounds 6b, 6e, and 6l showed negligible inhibition against porcine PDHc-E1. The in vitro antibacterial activity indicated that 6a, 6d, 6e, 6g, 6h, 6i, 6m, and 6n exhibited 61%–94% inhibition against Ralstonia solanacearum at 100 μg/mL, which was better than commercial thiodiazole‑copper (29%) and bismerthiazol (55%). These results demonstrated that a lead structure for a highly selective PDHc-E1 inhibitor as a bactericide could be obtained using computer aided drug design.

Introduction

The pyruvate dehydrogenase complex (PDHc) catalyzes the conversion of pyruvate into acetyl-coenzyme A and is a key component of the metabolic energy pathway of organisms (Patel and Korotchkina, 2003). The PDHc component E1 (E1p, EC 1.2.4.1, PDHc-E1) is the initial member of the PDHc and catalyzes the only irreversible reaction of the complex, in conjunction with the cofactors thiamine diphosphate (ThDP) and Mg2+ (Nemeria et al., 2004). Therefore, PDHc-E1 in microorganisms is an important target in the design of microbicides. Because the catalytic process of PDHc-E1 requires the cofactor, ThDP (Balakrishnan et al., 2012), the design and synthesis of ThDP analogs may be a good strategy for finding novel microbicides targeting PDHc-E1.

Currently, most of the reported microbial PDHc-E1 inhibitors are ThDP analogs (Fig. 1) (Schellenberger, 1990; Wittorf and Gubler, 1971; Shreve et al., 1983; Kluger et al., 1984; Tripatara et al., 1999; Hawksley et al., 2001; Leeper et al., 2005; Mann et al., 2004; He et al., 2012; He et al., 2015; Zhou et al., 2017; Zhou et al., 2019; He et al., 2016; Feng et al., 2019a; Feng et al., 2019b; He et al., 2014; Feng et al., 2014a, Feng et al., 2014b). Crystal structure and molecular docking analysis have revealed that these inhibitors have a single binding mode in the active site of PDHc-E1, and are bound in a “V” conformation with multiple flexible bonds that easily rotate in the cavity, similar to the binding of ThDP (Zhou et al., 2017; Zhou et al., 2019; He et al., 2016; Feng et al., 2019a; Feng et al., 2019b; He et al., 2014; Feng et al., 2014a; Arjunan et al., 2002; Pei et al., 2008; Arjunan et al., 2004; Pei et al., 2010). However, no ThDP analogs as PDHc-E1 inhibitors have been marketed as commercial microbicides. Therefore, it is an interesting challenge to find effective PDHc-E1 inhibitors as new microbicides. Compound A2 is a PDHc-E1 inhibitor that has shown certain fungicidal activity and is a useful lead compound for further study (He et al., 2015).

By analyzing the shape of the active cavity of Escherichia coli (E. coli) PDHc-E1, we found that the active pocket of E. coli PDHc-E1 (PDB ID: 1L8A) is located at the interface of the two chains of the dimer (chains A and B) (Fig. 2) (Arjunan et al., 2002). All the reported PDHc-E1 inhibitors, have a pyrimidine ring in the left part and a structural unit in the right part of the molecule that are bound in two sub-pockets, similar to the binding of ThDP (Fig. 2). The two parts are connected by a rotatable bond. However, even when these inhibitors are bound, there is still a lot of space remaining in the active cavity. Considering the aminopyrimidine ring and the benzene ring in the structure of the lead compound A2 are conjugated aromatic rings. To develop efficient PDHc-E1 inhibitors, the rotatable bond that connects the aminopyrimidine and benzene rings was replaced by a conjugated structural unit. Acylhydrazone derivatives with a conjugated structure (-C=N-NH-C=O-) usually show excellent microbicidal activity (Kodisundaram et al., 2013; Chen et al., 2016; Xie et al., 2020; Liu et al., 2019). Therefore, we attempted to replace the bridged bond, which connects the aminopyrimidine and benzene rings in A2, by an acylhydrazone moiety. It has been reported that the π-π interactions between the two aromatic rings can be further enhanced by the introduction of a methyl group to these aromatic rings (Watt et al., 2011). The aminopyrimidine ring of A2 can form π-π interactions with the amino acid residue F602 in the active site of E. coli PDHc-E1 as was demonstrated in our previous study (He et al., 2020). Based on the above considerations, a methyl group was introduced at the 6-position of the aminopyrimidine ring, resulting in the 2,6-dimethyl-4-aminopyrimidine acylhydrazone 6a (R = H) (Fig. 2). Compound 6a could bind in the active site of E. coli PDHc-E1 as confirmed by molecular dynamics (MD) simulation, which indicated that 6a might be a potential PDHc-E1 inhibitor. Then, a series of 2,6-dimethyl-4-aminopyrimidine acylhydrazones 6 was further designed using fragment-based drug design (FBDD) strategies. To our surprise, the molecular simulation results showed that every compound in series 6 was bound in the active cavity in a straight manner, with the right part of the molecule extended directly toward the outside of the cavity. This straight manner of binding was also indicated by the single-crystal X-ray diffraction data.

On the basis of the molecular design, we conducted a systematic study of series 6, including the synthesis, inhibitory effects against E. coli PDHc-E1, selectivity for the target enzyme, and bactericidal activity. Computer aided drug design (CADD) strategies were used throughout the study to assist in the design of compounds and verify our experimental results.

Section snippets

Molecular docking

The chemical structures of 6a and 6l were prepared using Sybyl 7.0 followed by the 2000 steepest descent minimizations and 2000 conjugate gradient minimizations, respectively. The PDHc-E1 three-dimensional (3D) crystal structure (PDB ID: 1L8A) derived from E. coli was obtained from the Research Collaboratory for Structural Bioinformatics (RCSB) protein data bank (PDB, https://www.rcsb.org/) (Rose et al., 2016.). Homology modeling was used to construct the 3D structure of porcine PDHc-E1. Human

Docking study of 6a with E. coli PDHc-E1

The binding mode of 6a with E. coli PDHc-E1 is shown in Fig. 3A, which was obtained by performing a 2500 ps molecular dynamics (MD) simulation after the molecular docking. The MD simulation was performed in AILDE server and the root-mean-square deviations (RMSD) for the protein backbone and ligand during the MD simulation are plotted in Fig. 3B. The RMSD of the backbone atoms (black solid line in Fig. 3B) fluctuated around 2 Å and the RMSD of 6a (red solid line in Fig. 3B) fluctuated around

Funding

The work was supported in part by the National Natural Science Foundation of China (21907035, 21877047, 31701820, and 21472062), the 111 Project B17019, the Postdoctoral Science Foundation of China (2019M662684).

Declaration of Competing Interest

The authors declare no competing financial interests.

Acknowledgments

The authors acknowledge Prof. Guangfu Yang, Central China Normal University, for providing computing resources.

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    Both authors contributed equally to this work.

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