The development of new inhibitors and probes for the assay of pantothenate synthetase

Pantothenate (Vitamin B5) is a key precursor for coenzyme A (CoA) and acyl carrier proteins (ACP), both of which are vital for essential metabolic pathways in organisms. As pantothenate is biosynthesized in micro-organisms, plants, and fungi, but not in animals, recent drug discovery validated the enzymes of pantothenate pathway as a potential drug target to combat virulent pathogens, for example Mycobacterium tuberculosis (M. tuberculosis). The pathway is best understood in E. coli and involves four bioconversions using four enzymes. We are interested in developing inhibitors for the pantothenate synthetase (PS), the last enzyme in the pantothenate pathway, that catalyses the Mg2+-ATP dependent condensation of D-pantoate and β-alanine to give pantothenate. It is reported that sulfamoyl adenosine mimics of the intermediate formed during the enzymatic reaction are inhibitors of both E. coli and M. tuberculosis PS which are homologous. Variable potencies for inhibitors were reported based on the structures substituted at the pantoate moiety of the pantoyl adenylate intermediate. Consequently, the aim of this project was set to explore the accommodation potential of pantoate binding pocket for sulphonamide inhibitors that differs on the pantoate moiety. A series of pantoyl adenylate intermediate mimics 50-60 were proposed. The computational evaluation of these structures for the M. tuberculosis PS active site demonstrated seven of the compounds with high Glidescores (-10.0 to -11.0 kcalmol-1) with the structure 60 ranked at the top of the series (-13.9 kcalmol-1). Glidescores of all the sulfamates scored less than the natural pantoyl adenylate intermediate 22. The sulfamates were prepared according to the literature procedures. Higher yields of the sodium or tetra-butyl ammonium salt of the final products 50-58 (30–60%) were obtained using the silyl protected sulfamoyl adenosine 90. The synthesis of the isoxazole ester analogue 59 and the pantoyl sulfamate 60 were achieved in yields of 50% and 30% respectively. The inhibition kinetics of the prepared compounds was determined using the established coupled enzyme assay. The inhibitor assay results showed that aromatic replacement of the pantoate moiety is well tolerated by the enzyme. The aliphatic sulfamates were mostly poor inhibitors of E. coli PS and the pantoyl sulfmate 60 was ineffective as an inhibitor. The inhibitory results, in general, had a good correlation with the Glidescores. The second scope of the project investigated the development of alternate techniques to monitor enzymatic reactions that use ATP. Two approaches that can be monitored by fluorescence were explored. The properties of fluorescently labelled ATP substrate analogues 100-103 were studied with snake venom phosphodiesterase and hexokinase. The ATP surrogates responded well for the phosphodiesterase but not for hexokinase. It was observed that high concentrations of the chemosensor were required with PS. The higher binding affinities of these analogues to phosphodiesterase, resulted in investigation of their binding using ITC. However, reliable binding parameters could not be derived due to prominent kinetic conversion. The second method employed fluorescence-based pyrophosphate sensors for selective detection of pyrophosphate produced during the ATP hydrolysis. The results revealed that naphthalene-based sensor 112.2Zn selectively recognized pyrophosphate anion over other nucleotides and common ions Cl-, H2PO4-, HCO3-, and AcO-. The naphthalene-based sensor 112.2Zn showed the anticipated fluorescence change for pyrophosphate hydrolysis catalysed by pyrophosphatase. The anthracene-based sensor complexes 113.2Zn, 113.2Cd showed less selectivity toward pyrophosphate over mono nucleotide triphosphates, but could differentiate nucleotide polyphosphates from other ions.