Towards Novel 3-Aminopyrazinamide-Based Prolyl-tRNA Synthetase Inhibitors: In Silico Modelling, Thermal Shift Assay and Structural Studies

Abstract

Human cytosolic prolyl-tRNA synthetase (HcProRS) catalyses the formation of the prolyl-tRNAPro, playing an important role in protein synthesis. Inhibition of HcProRS activity has been shown to have potential benefits in the treatment of fibrosis, autoimmune diseases and cancer. Recently, potent pyrazinamide-based inhibitors were identified by a high-throughput screening (HTS) method, but no further elaboration was reported. The pyrazinamide core is a bioactive fragment found in numerous clinically validated drugs and has been subjected to various modifications. Therefore, we applied a virtual screening protocol to our in-house library of pyrazinamide-containing small molecules, searching for potential novel HcProRS inhibitors. We identified a series of 3-benzylaminopyrazine-2-carboxamide derivatives as positive hits. Five of them were confirmed by a thermal shift assay (TSA) with the best compounds 3b and 3c showing EC50 values of 3.77 and 7.34 µM, respectively, in the presence of 1 mM of proline (Pro) and 3.45 µM enzyme concentration. Co-crystal structures of HcProRS in complex with these compounds and Pro confirmed the initial docking studies and show how the Pro facilitates binding of the ligands that compete with ATP substrate. Modelling 3b into other human class II aminoacyl-tRNA synthetases (aaRSs) indicated that the subtle differences in the ATP binding site of these enzymes likely contribute to its potential selective binding of HcProRS. Taken together, this study successfully identified novel HcProRS binders from our anti-tuberculosis in-house compound library, displaying opportunities for repurposing old drug candidates for new applications such as therapeutics in HcProRS-related diseases.

Double drugging of prolyl-tRNA synthetase provides a new paradigm for anti-infective drug development

“…Differential loop movements induced by ligand binding (528–538, particularly Phe534-Ala535-Gly536 where in Phe534 adopts unique rotameric conformations) were observed within HFG-ANP bound, HFG-L95 bound and apo Tg PRS enzyme when compared to the L-pro-L95 bound structures ( S7 Fig ). Similar loop movements and side-chain conformation flips were also found between Hs PRS-HFG-ANP (PDB ID: 4HVC) and Hs PRS-ATP pocket ligand (PDB Id: 7OT3)[ 21 , 36 ]. The HFG binding loop (residues 405–420) is found to be disordered in almost all HFG bound structures ( Fig 3B ).…”

“…Hewitt et al in 2017 showed that glyburide and the ligand TCMDC124506 can target the PRS enzyme allosterically [ 27 ]. Recent advancements towards ATP mimetics targeting the ATP pocket of PRSs have opened a new window towards drug development [ 33 – 36 ]. A partner group recently published a series of inhibitors that targets the ATP pockets of both malaria and host PRSs [ 37 ].…”

Discovery of Novel 3-Hydroxyquinazoline-2,4(1H,3H)-Dione Derivatives: A Series of Metal Ion Chelators with Potent Anti-HCV Activities

“…The stability of a protein system is enhanced after binding to its specific ligand, leading to an increase in the melting temperature ( T m ). Taking advantage of this feature, the thermal shift assay was extensively used to assess the binding of small molecules to the protein targets [ 51 , 52 , 53 ]. The metal ion chelators were thought to exert an anti-HCV effect by chelating with Mg 2+ in the catalytic center of NS5B [ 34 , 36 , 38 ].…”

Adenosine-Mimicking Derivatives of 3-Aminopyrazine-2-Carboxamide: Towards Inhibitors of Prolyl-tRNA Synthetase with Antimycobacterial Activity

“…In our previous study, we demonstrated that similar 3-aminopyrazinamide derivatives with simplified substituents ( Figure 2 , in comparison to the Adachi ligand) could also bind to hsProRS (as demonstrated by thermal shift assay and crystallographic structures) [ 10 ], although with weaker affinity. The weaker affinity could have been due to the missing carbonyl oxygen.…”

“…The weaker affinity could have been due to the missing carbonyl oxygen. In our previous series, compounds with 2’-substitution on the benzene ring had affinity to hsProRS [ 10 ], but were devoid of significant antimycobacterial activity [ 11 ]. On the contrary, compounds with 4’-substituent did not bind to hsProRS [ 10 ] but possessed moderate in vitro growth inhibition activity against mycobacteria [ 11 ].…”