PROBING BINDING REQUIREMENTS OF NAD KINASE WITH MODIFIED NAD ANALOGUES

NAD kinase (NADK) catalyzes a magnesium-dependent phosphorylation of the 2′-hydroxyl group of the adenosine ribose moiety of nicotinamide adenine dinucleotide (NAD) using ATP or inorganic polyphosphates as phosphoryl donors to give NADP. There are two classes of the enzyme, one is specific for NAD+ and the other also phosphorylates NADH. With the recent discovery of NADK from Mycobacterium tuberculosis and with the emergence of multidrug resistant and extensively drug resistant tuberculosis (TB) worldwide, NADK has became deeply important as attractive target for antibiotic discovery. Recently Boshoff et al. (1) reported that inhibition of enzymes involved in the early stages of NAD biosynthesis for the treatment of TB is often reversed by the unwanted involvement of alternative rescue pathways, whereas the enzymes comprising the last steps, such as NAD synthetase (NADS) and NADK, should be preferred. Furthermore, NADK cannot be complemented by rescue pathways, and being the only enzyme which supplies vital NADP in prokaryotic or eukaryotic cells, could be an even better target than NADS. As a proof of concept, following our recent discovery of NAD analogues, such as DTA 1 and 8-BrDTA 2 (2), that afforded potent but not selective inhibitors, we decided to design a new series of NAD analogues where the nicotinamide riboside moiety has been replaced by 2-thiopyridyl, benzyl and meta-nitro benzyl groups (compounds 3-5), maintaining the short di-sulfur linker instead of the pyrophosphate linkage of NAD. The synthesis and the biological evaluation against human and bacterial NADKs of these heterodisulfides will be presented in detail.