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

Area of Science:

• Biochemistry
• Medicinal Chemistry
• Cancer Biology

Background:

• Nicotinamide adenine dinucleotide (NAD) metabolism is crucial for cancer cell growth and proliferation.
• Targeting NAD biosynthesis enzymes offers a potential anticancer therapeutic strategy.
• Nicotinamide/nicotinic acid adenylyltransferase (NMNAT) is a key enzyme in NAD synthesis.

Purpose of the Study:

• To synthesize novel NAD/NaAD analogues with methyl groups at the ribose 2'-C and 3'-C positions.
• To evaluate these analogues as inhibitors of the three human NMN-adenylyltransferase (hNMNAT) isoforms.
• To understand the structure-activity relationships between analogue conformation and enzyme inhibition.

Main Methods:

• Synthesis of novel NAD/NaAD analogues (1-4).
• NMR-based conformational analysis of the synthesized analogues.
• Biological evaluation of analogues as inhibitors against hNMNAT-1, hNMNAT-2, and hNMNAT-3.
• In silico studies to rationalize bioactivity and selectivity.

Main Results:

• Analogues 1-4 exhibited distinct conformational preferences (North-anti and South-syn).
• Structure-activity relationships were identified between conformation and hNMNAT inhibition.
• The 2'-C-methyl-NAD analogue 1 demonstrated potent and selective inhibition of hNMNAT-2 (Ki = 15 and 21 µM for NMN and ATP, respectively).

Conclusions:

• Novel methylated NAD analogues were successfully synthesized and characterized.
• Analogue 1 represents a potent and selective inhibitor of hNMNAT-2, a potential anticancer target.
• In silico and in vitro studies provide a foundation for rational drug design and scaffold optimization in NAD metabolism-targeted cancer therapy.