A novel inhibitor of Mycobacterium tuberculosis pantothenate synthetase.

Pantothenate synthetase (PS; EC 6.3.2.1), encoded by the panC gene, catalyzes the essential adenosine triphosphate (ATP)-dependent condensation of D-pantoate and beta-alanine to form pantothenate in bacteria, yeast, and plants; pantothenate is a key precursor for the biosynthesis of coenzyme A (CoA) and acyl carrier protein (ACP). Because the enzyme is absent in mammals and both CoA and ACP are essential cofactors for bacterial growth, PS is an attractive chemotherapeutic target. An automated high-throughput screen was developed to identify drugs that inhibit Mycobacterium tuberculosis PS. The activity of PS was measured spectrophotometrically through an enzymatic cascade involving myokinase, pyruvate kinase, and lactate dehydrogenase. The rate of PS ATP utilization was quantitated by the reduction of absorbance due to the oxidation of NADH to NAD+ by lactate dehydrogenase, which allowed for an internal control to detect interference from compounds that absorb at 340 nm. This coupled enzymatic reaction was used to screen 4080 compounds in a 96-well format. This discussion describes a novel inhibitor of PS that exhibits potential as an antimicrobial agent.

Primary and secondary drug screening assays for Friedreich ataxia.

Friedreich ataxia (FRDA) is an autosomal recessive neuro- and cardiodegenerative disorder for which there are no proven effective treatments. FRDA is caused by decreased expression and/or function of the protein frataxin. Frataxin chaperones iron in the mitochondrial matrix for the assembly of iron-sulfur clusters (ISCs), which are prosthetic groups critical for the function of the Krebs cycle and the mitochondrial electron transport chain (ETC). Decreased expression of frataxin or the yeast frataxin orthologue, Yfh1p, is associated with decreased ISC assembly, mitochondrial iron accumulation, and increased oxidative stress, all of which contribute to mitochondrial dysfunction. Using yeast depleted of Yfh1p, a high-throughput screening (HTS) assay was developed in which mitochondrial function was monitored by reduction of the tetrazolium dye WST-1 in a growth medium with a respiration-only carbon source. Of 101 200 compounds screened, 302 were identified that effectively rescue mitochondrial function. To confirm activities in mammalian cells and begin understanding mechanisms of action, secondary screening assays were developed using murine C2C12 cells and yeast mutants lacking specific complexes of the ETC, respectively. The compounds identified in this study have potential relevance for other neurodegenerative disorders associated with mitochondrial dysfunction, such as Parkinson disease.