Atomic Resolution Analyses of Isocoumarin Derivatives for Inhibition of Lysyl-tRNA Synthetase

Abstract

Aminoacyl-tRNA synthetases, the essential enzyme family for protein translation, are attractive targets for developing antibacterial, antifungal, and antiparasitic agents and for treating other human diseases. The antimalarial natural product cladosporin was discovered recently as a novel lysyl-tRNA synthetase (LysRS) specific inhibitor. Here, we report a thorough analysis of cladosporin derivatives using chemical synthesis, biophysical, and biochemical experiments. A series of isocoumarin derivatives with only one nonhydrogen atom/bond change per compound was synthesized. These changes include replacements of methyltetrahydropyran moiety by methylcyclohexane or cyclohexane, lactone by lactam, hydroxyl groups by methoxyl groups, and dismission of the chiral center at C3 with a Δ 3,4 double bond. We evaluated these compounds by thermal shift assays and enzymatic experiments and further studied their molecular recognition by the Plasmodium falciparum LysRS through total five high-resolution crystal structures. Our results showed that the methyltetrahydropyran moiety of cladosporin could be replaced by a more stable methylcyclohexane without reducing binding ability. Removing the methyl group from the methylcyclohexane moiety slightly decreased the interaction with LysRS. Besides, the replacement with a lactam group or a conjugated Δ 3,4 double bond within the scaffold could be two more options to optimize the compound. Lastly, the two phenolic hydroxyl groups were critical for the compounds to bind LysRS. The detailed analyses at atomic resolution in this study provide a foundation for the further development of new antibiotics from cladosporin derivatives.

Inhibition ofPlasmodium falciparumLysyl-tRNA synthetase via an anaplastic lymphoma kinase inhibitor

“…The complex structure of Pf LysRS-ASP3026 was largely consistent with the structure of Pf LysRS complexing with cladosporin and cladosporin analogs ( 22 , 36 , 37 , 41 ). The room-mean-square deviation (RMSD) between Pf LysRs-ASP3026 and Pf LysRs-cladosporin was 0.733 Å for 449 superimposed Cα atoms ( 36 , 41 ) (Figure 5A ).…”

“…Nonetheless, apparent decreases in the resonance units (RU) for all concentrations of ASP3026 are observed, which might be attributed to the competition between ATP and ASP3026. In addition, adding the cladosporin analog 3-(cyclohexylmethyl)-6,8-dihydroxy-1H-isochromen-1-one, which exhibits higher activity against Pf LysRS ( 25 , 37 ), completely suppressed the resonance ( Supplementary Figure S7C ), confirming that ASP3026 binds to the ATP pocket of Pf LysRS.…”

“…Because the synergistic binding of inhibitors with substrate amino acids and adenosine was previously found in Pf LysRS and ProRS ( 10 , 22 ), we performed the screening in the presence of 100 μM l -lysine, and 100 μM adenosine. Dimethyl sulfoxide (DMSO) and cladosporin analog 3-(cyclohexylmethyl)-6,8-dihydroxy-1H-isochromen-1-one were used as negative and positive controls, respectively ( Supplementary Figure S3B ) ( 37 ).…”

Recent Development of Aminoacyl tRNA Synthetase Inhibitors for Human Diseases: A Future Perspective.

“…Upon screening these libraries, the unsaturation of the isocoumarin core and transformation of the metabolically unstable tetrahydropyran ring to the cyclohexyl ring yielded a selective LysRS inhibitor with improved metabolic stability at the level of hepatic extraction. In line with a previous report by Rusch et al focusing on metabolic weak points of cladosporin, Zhou et al confirmed that the methyl tetrahydropyran moiety of cladosporin could be replaced by a more stable methylcyclohexane while maintaining potency in the ATP hydrolysis assay (compounds 15, 16) [28]. The methyl group in the cyclohexane ring was important for the hydrophobic interaction with Ser344, resulting in increased potency 4-fold compared to that of compound 15.…”

Recent Development of Aminoacyl-tRNA Synthetase Inhibitors for Human Diseases: A Future Perspective

“…Upon screening these libraries using the Transcreener AMP assay system to assess aminoacylation by LysRS [23], the unsaturation of the isocoumarin core and transformation of the metabolically unstable tetrahydropyran ring to the cyclohexyl ring yielded a selective LysRS inhibitor with improved metabolic stability at the level of hepatic extraction. In line with a previous report by Rusch et al [26] focusing on metabolic weak points of cladosporin, Zhou et al confirmed that the methyl tetrahydropyran moiety of cladosporin could be replaced by a more stable methylcyclohexane, while maintaining potency in the ATP hydrolysis assay (compounds 15 and 16) [28]. The methyl group in the cyclohexane ring was important for the hydrophobic interaction with Ser344, resulting in increased potency of 4-fold compared to that of compound 15.…”