Compact NAD mimics as selective inhibitors of NAD kinases

We have examined sequence diversity of 147 co-crystal structures of proteins utilizing NAD as a cofactor or as a substrate. Most of these proteins bind NAD in an extended conformation (e.g. the distance between the adenine 6-amino group of NAD and the nicotinamide amide carbon is 16 Å or more). However, there are clear exceptions to this general trend. Several bacterial reductases bind NAD in an extremely folded conformation (the distance of 6 Å) and NAD kinases bind NAD in bent (compact) conformations with the distance of 11-12 Å. Although NAD can be accommodated in all of the above conformations it is likely that compact NAD mimics (with the distance of 11-12 Å) should not fit to the binding domains of majority of NAD-utilizing enzymes that require an extended conformation of NAD. It may be expected, therefore, that such compounds could be selective against NAD kinases. Tiazofurin adenine dinucleotide (TAD) is a potent inhibitor (Ki = 100 nM) of NAD-dependent IMP-dehydrogenase. This NAD mimic binds at the cofactor binding domain in the extended conformation. As a proof of concept, we prepared tiazofurin-5’-yl adenosine-5’-yl disulfide (1) by replacing the pyrophosphate (-O-P-O-P-O-) linkage by a short (-S-S-) disulfide bridge. We found that this compact disulfide analogue of TAD has lost its activity against IMPDH and became a moderate inhibitor of M. tuberculosis (IC50 = 80 μM) NAD kinase. We also found that di(adenosine-5’yl) disulfide (2), a by-product in the synthesis of 1, showed slightly better activity against mycobacterium (IC50 = 45 μM) as well as human (IC50 = 87 μM) NAD kinase. Introduction of bromine at the C-8 of adenine ring results in restriction of conformation of 8-bromo-adenosine to the syn conformation. We prepared the corresponding 8-bromo disulfide (3) and found further improvement in the inhibitory activity against human (IC50 = 6 μM) and mycobacterium (IC50 = 14) NAD kinase. Compound 3 is the most potent inhibitor of NAD kinases reported so far. Using a simple method for preparation of disulfide 2 we synthesized a number of nucleobase and sugar modified analogues that have been evaluated against these enzymes, and we will discuss our results.