SYNTESIS AND BIOLOGICAL EVALUATION NMN-ADENYLYTRANSFERASE INIBITORS FOR DEVELOPEMENT OF NEW CHEMIOTERAPIC AGENTS

The pivotal role of nicotinamide adenine dinucleotide (NAD) in cellular metabolic processes justifies the interest in nicotinamide mononucleotide adenylyltransferase (NMNAT) as an attractive target for the rational design of new drugs. NMNAT is an indispensable enzyme in both de novo and salvage pathways of NAD or deamido-NAD (NaAD) biosynthesis. Significant differences between bacterial and human enzymes in both substrate specificity and active site conformations have been described that might lead to the rational design of highly selective chemotherapic agents. Therefore, the specific aim of my research was the identification of molecular ligands which could act as selective inhibitors of NMNAT from specific species, useful for the biological characterization of the enzymes and the discovery of new drugs. With this purpose in view, two dinucleoside polyphosphate NAD analogs, having a polyphosphate linker between the nicotinamide riboside function and adenosine (Np3AD and Np4AD), that should mimic the structure of the hypothetical transition state of the reaction catalyzed by NMNAT, were prepared. Interestingly, both dinucleosides polyphosphates and, in particular Np4AD, exerted a different inhibitory effect toward the bacterial enzyme with respect to eukaryotic NMNAT. So, Np4AD might represent the first lead compound for the development of novel potent and selective bacterial NMNAT inhibitors. Another part of the research was addressed to the synthesis of ?-nicotinamide and nicotinic acid ribosides (?-NAR and ?-NaR, respectively) in order to investigate the subdomain of NMNAT that binds the substrates nicotinic mononucleotides (NMN/NaMN). Nicotinamide riboside is an intermediate in one biosynthetic pathway by which nicotinamide is converted into NAD. It is known that kinetic and structural studies of both bacterial and human NMNAT require the ?-anomer of nicotinic ribosides as substrates and of monoribotides as products of the NAD degradation pathways. ?-NAR and its nicotinic acid analog (?-NaR) were obtained in high yields by a stereoselective synthesis via glycosylation of the presilylated bases under Vorbruggen's protocol and controlled conditions. In order to obtain novel NAD analogs as substrate/inhibitors of key enzymes involved in the nucleotide biosynthesis, a ?-NAR analog, methylated in 3-position of the ribofuranose moiety, was also synthesised. Novel potential inhibitors of NMNAT from different sources were also developed as product analogs of the enzymatic reaction. Recently, three distinct human NMNAT isoforms, named hNMNAT-1, hNMNAT-2, and hNMNAT-3, have been characterized. The tissue distribution pattern of the human isoenzymes is different, suggesting that their expression is differentially controlled at transcription level. Thus, each isoenzyme may have a distinct cellular and physiological role; however, several questions remain to clarify and characterize the specific roles of these isoenzymes in NAD metabolism and regulation. In this respect, the proposed study combines efforts directed toward the design and synthesis of mechanism-based enzyme-inhibitors, with biochemical studies on targeted enzymes to better understand the biological function of the human NMNAT isoforms and define their suitability as specific targets for drug development. With this in view, a NAD analog and its deamido derivative were synthesized in which the adenylyl part of the dinucleotides was modified at the C2' position of the furanose ring by introduction of a methyl group (N2'-MeAD and Na2'-MeAD, respectively). The effect of the dinucleotides on the enzymatic activity of NMNAT resulted in a selective inhibition of the human enzyme with respect to the archeal enzyme, suggesting that the modification in the ribose moiety of NAD, which induces a stabilization of the North(3T2)-anti conformation, as detected for both N2'-MeAD and Na2'-MeAD, proved to be favourable to the binding at human enzymes. N2'-MeAD showed a significant competitive inhibitory activity toward the hNMNAT-2, but no activity toward the hNMNAT-1 isoform. Surprisingly, the deamido dinucleotide Na2'-MeAD resulted a non-competitive inhibitor of hNMNAT-2 and a selective competitive inhibitor of hNMNAT-3 isoform. A molecular modeling study was also carried out starting from the recently solved structures of NAD/NaAD-bound hNMNAT-1 and hNMNAT-3 complexes. Compared with these complexes, the N2'-MeAD/Na2'-MeAD-bound showed conformational changes in the binding sites of both hNMNAT-1 and hNMNAT-3 induced by the modified adenylyl part of the dinucleotides. The inhibitory effect of the modified NAD analogs toward the human enzymes might be helpful for the rational design of molecules potentially useful as new chemotherapic agents.