Lysyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization and Identification of Inhibitory Compounds

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial infections and has highly developed systems for acquiring resistance against numerous antibiotics. The gene ( lysS) encoding P. aeruginosa lysyl-tRNA synthetase (LysRS) was cloned and overexpressed, and the resulting protein was purified to 98% homogeneity. LysRS was kinetically evaluated, and the Km values for the interaction with lysine, adenosine triphosphate (ATP), and tRNALys were determined to be 45.5, 627, and 3.3 µM, respectively. The kcatobs values were calculated to be 13, 22.8, and 0.35 s−1, resulting in kcatobs/ KM values of 0.29, 0.036, and 0.11 s−1µM−1, respectively. Using scintillation proximity assay technology, natural product and synthetic compound libraries were screened to identify inhibitors of function of the enzyme. Three compounds (BM01D09, BT06F11, and BT08F04) were identified with inhibitory activity against LysRS. The IC50 values were 17, 30, and 27 µM for each compound, respectively. The minimum inhibitory concentrations were determined against a panel of clinically important pathogens. All three compounds were observed to inhibit the growth of gram-positive organisms with a bacteriostatic mode of action. However, two compounds (BT06F11 and BT08F04) were bactericidal against cultures of gram-negative bacteria. When tested against human cell cultures, BT06F11 was not toxic at any concentration tested, and BM01D09 was toxic only at elevated levels. However, BT08F04 displayed a CC50 of 61 µg/mL. In studies of the mechanism of inhibition, BM01D09 inhibited LysRS activity by competing with ATP for binding, and BT08F04 was competitive with ATP and uncompetitive with the amino acid. BT06F11 inhibited LysRS activity by a mechanism other than substrate competition.

Elucidating effects of single and multiple resistance mechanisms on bacterial response to meropenem by quantitative and systems pharmacology modeling and population genomics

“…Furthermore, there was overlap between the mutations seen in the colonies from the control arm and the colonies from the treated arms for most strains, consistent with the assumption of amplification of pre-existing resistant mutants. The aspS mutation in the PAOD1ΔmexR control colony can be considered equivalent to the lysS mutations in the treatment arms (50, 51). In PAΔmexR a commonly occurring large genomic deletion in galU caused resistance in a control mutant but was not selected by treatment because of its higher fitness cost compared to the oprD mutation which was selected by treatment (52).…”

Two Forms of Tyrosyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization and Discovery of Inhibitory Compounds

“…23 We also recently showed that bilirubin was a specific inhibitor of ATP binding LysRS. 24 In support of ATP competition specificity by these compounds in TyrRS, this same chemical compound library has been screened in our laboratory against several other aaRS enzymes (PheRS, GluRS, MetRS, GlnRS, LeuRS, LysRS, ProRS, ArgRS, AspRS, and HisRS) from P. aeruginosa , all of which have ATP as a substrate. In no instance in these screens were the TyrRS hit compounds observed to inhibit the activity of these additional aaRS enzymes.…”

“… Authors’ Note The authors disclose that portions of the Materials and Methods section were previously described in the following articles and are described here for clarity and descriptive purposes: Balboa et al, 24 Hu et al, 11 Pena et al, 12 Escamilla et al (Glutaminyl-tRNA Synthetase from Pseudomonas aeruginosa : Characterization, Structure, and Development as a Screening Platform. Protein Sci.…”

Structure-guided conversion from an anaplastic lymphoma kinase inhibitor into Plasmodium lysyl-tRNA synthetase selective inhibitors

“…Lysyl-tRNA synthetase (LysRS) has been validated as a promising target for antimalarial, anti-Cryptosporidium, anti-Pseudomonas aeruginosa, and anti-Mycobacterium tuberculosis therapies [23][24][25] . The ATP-binding pocket is the most promising area for the development of LysRS inhibitors.…”