Multidrug-resistant tuberculosis (MDR-TB) poses a significant threat to mankind and as such earned its place on the WHO list of priority pathogens. New antimycobacterials with a mechanism of action different to currently used agents are highly required. This study presents the design, synthesis, and biological evaluation of 3-acylaminopyrazine-2-carboxamides derived from a previously reported inhibitor of human prolyl-tRNA synthetase. Compounds were evaluated in vitro against various strains of mycobacteria, pathogenic bacteria, and fungi of clinical significance. In general, high activity against mycobacteria was noted, while the antibacterial and antifungal activity was minimal. The most active compounds were 4'-substituted 3-(benzamido)pyrazine-2-carboxamides, exerting MIC (Minimum Inhibitory Concentration) from 1.95 to 31.25 µg/mL. Detailed structure-activity relationships were established and rationalized in silico with regard to mycobacterial ProRS as a probable target. The active compounds preserved their activity even against multidrug-resistant strains of Mycobacterium tuberculosis. At the same time, they were non-cytotoxic against HepG2 human hepatocellular carcinoma cells. This project is the first step in the successful repurposing of inhibitors of human ProRS to inhibitors of mycobacterial ProRS with antimycobacterial activity.

• Klíčová slova
• 3-aminopyrazinamide, antibacterial, antimycobacterial, homology model, molecular dynamics, prolyl-tRNA synthetase, repurposing, tuberculosis,
• MeSH
• adenosin farmakologie   MeSH
• aminoacyl-tRNA-synthetasy * MeSH
• antituberkulotika farmakologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• Mycobacterium tuberculosis * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosin MeSH
• aminoacyl-tRNA-synthetasy * MeSH
• antituberkulotika MeSH

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.

• Klíčová slova
• X-ray crystallographic studies, in silico modelling, inhibitor, prolyl-tRNA synthetase, thermal shift assay,
• MeSH
• adenosintrifosfát metabolismus   MeSH
• aminoacyl-tRNA-synthetasy antagonisté a inhibitory   MeSH
• biotest metody   MeSH
• inhibitory enzymů chemie   izolace a purifikace   farmakologie   MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• ligandy MeSH
• molekulární modely MeSH
• počítačová simulace * MeSH
• pyrazinamid chemie   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosintrifosfát MeSH
• aminoacyl-tRNA-synthetasy MeSH
• inhibitory enzymů MeSH
• ligandy MeSH
• prolyl T RNA synthetase MeSH Prohlížeč
• pyrazinamid MeSH

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) has become a frequently deadly infection due to increasing antimicrobial resistance. This serious issue has driven efforts worldwide to discover new drugs effective against Mtb. One research area is the synthesis and evaluation of pyrazinamide derivatives as potential anti-TB drugs. In this paper we report the synthesis and biological evaluations of a series of ureidopyrazines. Compounds were synthesized by reacting alkyl/aryl isocyanates with aminopyrazine or with propyl 5-aminopyrazine-2-carboxylate. Reactions were performed in pressurized vials using a CEM Discover microwave reactor with a focused field. Purity and chemical structures of products were assessed, and the final compounds were tested in vitro for their antimycobacterial, antibacterial, and antifungal activities. Propyl 5-(3-phenylureido)pyrazine-2-carboxylate (compound 4, MICMtb = 1.56 μg/mL, 5.19 μM) and propyl 5-(3-(4-methoxyphenyl)ureido)pyrazine-2-carboxylate (compound 6, MICMtb = 6.25 μg/mL, 18.91 μM) had high antimycobacterial activity against Mtb H37Rv with no in vitro cytotoxicity on HepG2 cell line. Therefore 4 and 6 are suitable for further structural modifications that might improve their biological activity and physicochemical properties. Based on the structural similarity to 1-(2-chloropyridin-4-yl)-3-phenylurea, a known plant growth regulator, two selected compounds were evaluated for similar activity as abiotic elicitors.

• Klíčová slova
• Mycobacterium tuberculosis, abiotic elicitors, anti-infectives, callus culture, ester, pyrazinoic acid, ureidopyrazine,
• MeSH
• antituberkulotika chemická syntéza   chemie   farmakologie   MeSH
• buňky Hep G2 MeSH
• Fagopyrum chemie   MeSH
• fyziologický stres účinky léků   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• molekulární struktura MeSH
• Mycobacterium tuberculosis účinky léků   MeSH
• proliferace buněk účinky léků   MeSH
• pyrazinamid chemie   farmakologie   MeSH
• pyraziny chemická syntéza   chemie   farmakologie   MeSH
• regulátory růstu rostlin chemická syntéza   chemie   farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antituberkulotika MeSH
• propyl 5-(3-(4-methoxyphenyl)ureido)pyrazine-2-carboxylate MeSH Prohlížeč
• propyl 5-(3-phenylureido)pyrazine-2-carboxylate MeSH Prohlížeč
• pyrazinamid MeSH
• pyraziny MeSH
• regulátory růstu rostlin MeSH