Inhibition of a truncated form of human mitochondrial kidney-type glutaminase (hKGA124-551) by bis-2-(5-phenylactamido-1,2,4-thialdiazol-2-yl)ethyl sulfide (BPTES).

摘要

Mitochondrial glutaminase (GA) catalyzes the hydrolysis of glutamine producing glutamate and an ammonium ion. There are three isoforms of mammalian GA that are essential to hepatic ureagenesis, renal ammoniagenesis, synthesis of the neurotransmitter glutamate, and the catabolism of glutamine. Here we focus on the human KGA isoform that is predominantly expressed in kidney, brain, intestine, and tissues of the immune system. Recent publications suggest that GA is a novel target for developing new cancer therapeutics. These studies have indicated that inhibition of GA by small molecule inhibitors significantly reduces the size of tumors in rats and inhibits growth of transformed cells in culture. A truncated form of human KGA hKGA124-551 that contains amino acids 124-551, was produced to delete the C-terminal sequences that are unique to the KGA and GAC isoforms. This construct was assayed in the presence of (bis-2-(5-phenylactamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES). BPTES is a potent small molecule inhibitor of mammalian GA that was previously shown to inhibit rat KGA in microM concentrations. In the current study, we adapted the standard GA assay to a microtiter plate format and used it to characterize the inhibition of hKGA124-551 using microM amounts of BPTES.;Our data indicate that BPTES is a mixed non-competitive inhibitor at low concentrations of phosphate, but at higher phosphate concentrations the inhibition is predominantly uncompetitive. Lastly, gel filtration and dynamic light scattering experiments were performed to determine if hKGA 124-551 oligomers are formed in the presence of BPTES and to characterize the effect of increasing concentrations of phosphate. The data suggest that in low phosphate and in the absence of BPTES, the hKGA124-551 exists as a dimer, but in the presence of BPTES and higher phosphate concentrations the molecular weight shifts to a tetramer or higher oligomer. The combined data indicate that BPTES is a potent lead compound for the development of a therapeutic inhibitor of human GA that may be a potential cancer therapeutic.