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

Pseudomonas aeruginosa is a multidrug-resistant (MDR) pathogen and a causative agent of both nosocomial and community-acquired infections. The genes (tyrS and tyrZ) encoding both forms of P. aeruginosa tyrosyl-tRNA synthetase (TyrRS-S and TyrRS-Z) were cloned and the resulting proteins purified. TyrRS-S and TyrRS-Z were kinetically evaluated and the Km values for interaction with Tyr, ATP, and tRNATyr were 172, 204, and 1.5 μM and 29, 496, and 1.9 μM, respectively. The kcatobs values for interaction with Tyr, ATP, and tRNATyr were calculated to be 3.8, 1.0, and 0.2 s–1 and 3.1, 3.8, and 1.9 s–1, respectively. Using scintillation proximity assay (SPA) technology, a druglike 2000-compound library was screened to identify inhibitors of the enzymes. Four compounds (BCD37H06, BCD38C11, BCD49D09, and BCD54B04) were identified with inhibitory activity against TyrRS-S. BCD38C11 also inhibited TyrRS-Z. The IC50 values for BCD37H06, BCD38C11, BCD49D09, and BCD54B04 against TyrRS-S were 24, 71, 65, and 50 μM, respectively, while the IC50 value for BCD38C11 against TyrRS-Z was 241 μM. Minimum inhibitory concentrations (MICs) were determined against a panel of clinically important pathogens. All four compounds were observed to inhibit the growth of cultures of both Gram-positive and Gram-negative bacteria organisms with a bacteriostatic mode of action. When tested against human cell cultures, none of the compounds were toxic at concentrations up to 400 μg/mL. In mechanism of inhibition studies, BCD38C11 and BCD49D09 selectively inhibited TyrRS activity by competing with ATP for binding. BCD37H06 and BCD54B04 inhibited TyrRS activity by a mechanism other than substrate competition

Short treatment with antalarmin alters adrenal gland receptors in the rat model of endometriosis

Endometriosis is a chronic inflammatory disorder in which endometrial tissue is found outside the uterine cavity. Previous reports suggest that there is a dysregulation of the hypothalamic pituitary adrenal axis during the progression of endometriosis. Our previous report showed that a short-term treatment with antalarmin, a corticotrophin releasing hormone receptor type 1 (CRHR1) antagonist decreases the number and size of endometriotic vesicles in the auto-transplantation rat model of endometriosis. Our current goal was to examine the mRNA expression of intra-adrenal receptors to better understand the mechanisms of the hypothalamic pituitary adrenal (HPA) axis involvement in endometriosis. We used two groups of female rats. The first received sham surgery or endometriosis surgery before collecting the adrenals after 7 days of the disease progression. The second group of animals received endometriosis surgery and a treatment of either vehicle or antalarmin (20 mg/kg, i. p.) during the first 7 days after endometriosis induction and then the disease was allowed to progress until day 60. Rats with sham surgery served as controls. Results showed that the mRNA expression of the mineralocorticoid (MRC2) receptor was lower in the rats after 7 days of endometriosis surgery and in rats with endometriosis that received antalarmin. In addition, the CRHR1 was significantly elevated in animals that received antalarmin and this was counteracted by a non-significant elevation in CRHR2 mRNA. The glucocorticoid receptor mRNA within the adrenals was not affected by endometriosis or antalarmin treatment. This report is one of the first to explore intra-adrenal mRNA for receptors involved in the HPA axis signaling as well as in the sympatho-adrenal signaling, calling for additional research towards understanding the role of the adrenal glands in chronic inflammatory diseases such as endometriosis