Synthesis, Biological, and Computational Evaluations of Conformationally Restricted NAD-Mimics as Discriminant Inhibitors of Human NMN-Adenylyltransferase Isozymes

Abstract

Nicotinamide adenine dinucleotide (NAD) cofactor metabolism plays a significant role in cancer development. Tumor cells have an increased demand for NAD and ATP to support rapid growth and proliferation. Limiting the amount of available NAD by targeting critical NAD biosynthesis enzymes has emerged as a promising anticancer therapeutic approach. In mammals, the enzyme nicotinamide/nicotinic acid adenylyltransferase (NMNAT) catalyzes a crucial downstream reaction for all known NAD synthesis routes. Novel nicotinamide/nicotinic acid adenine dinucleotide (NAD/NaAD) analogues 1–4, containing a methyl group at the ribose 2′-C and 3′-C-position of the adenosine moiety, were synthesized as inhibitors of the three isoforms of human NMN-adenylyltransferase, named hNMNAT-1, hNMNAT-2, and hNMNAT-3. An NMR-based conformational analysis suggests that individual NAD-analogues (1–4) have distinct conformational preferences. Biological evaluation of dinucleotides 1–4 as inhibitors of hNMNAT isoforms revealed structural relationships between different conformations (North-anti and South-syn) and enzyme-inhibitory activity. Among the new series of NAD analogues synthesized and tested, the 2′-C-methyl-NAD analogue 1 (Ki = 15 and 21 µM towards NMN and ATP, respectively) emerged as the most potent and selective inhibitor of hNMNAT-2 reported so far. Finally, we rationalized the in vitro bioactivity and selectivity of methylated NAD analogues with in silico studies, helping to lay the groundwork for rational scaffold optimization.