Investigator: Karen Almeida, Rhode Island College

Scientific Theme: Cancer

Abstract: NAD+ is essential for life and therefore must be strictly monitored. NAD+ is widely used as the cofactor in cell energy production and metabolism by providing high-energy electrons required for oxidation/reduction reactions. However, NAD+ concentration is diminished by its use as a substrate for enzymes such as poly (ADP-ribose) polymerases (PARPs) and Sirtuins. In each case, the ADP-ribose portion of NAD+ is cleaved from the nicotinamide (NAM) moiety. To restore NAD+, NAM is converted to nicotinamide mononucleotide (NMN) by the rate-limiting enzyme Nicotinamide phosphoribosyltransferase (NAMPT) also called NAmPRTase, Pre-B cell-enhancing factor (PBEF), or Vistafin. NAD+ is regenerated in the second step with concomitant hydrolysis of ATP by the enzyme NMNAT. Within the NAD+ salvage pathway, NAMPT has been shown to modulate the cellular NAD+ levels and therefore is a potential target for regulation by inhibitors. NAMPT is a 55-kDa enzyme that forms a homodimer to generate two active sites. The channel leading into the active site is primarily housed in one monomer with contributions at the active site from the opposing monomer. NAMPT enzymatic activity as a phosphoribosyltransferase has been well established. Little to no information is available regarding the dimerization and thus activation of the NAMPT monomers. We hypothesize that the increased activity shown in our initial screens is the result of small molecule binding to affect a shift in the monomer/dimer equilibrium. In other words, these compounds are binding across the dimerization plane, stabilizing the active dimer and thus increasing enzymatic activity. The overall goal of this proposal is to determine which structural components of the two recently identified activators are required to elicit the increase in NAMPT activity. This information can then direct the screening of drug libraries for novel activators. We propose to 1) screen commercially available derivatives of the two known activators 2) establish a method to monitor the monomer/dimer equilibrium based on a Bimolecular Fluorescence Complementation assay based on Enhanced Yellow Fluorescence Protein (EYFP). We anticipate identifying several new compounds that influence NAMPT activity and/or dimerization.

Human Health Relevance: NAMPT is under investigation for its relevance to human disease states, including cancer, diabetes, and heart disease. NAMPT inhibits apoptosis in cancer cells, necessary for the transition to a cancerous state. Additionally, NAMPT activates the IL-6/STAT3 cell survival pathway by a non-enzymatic mechanism. It is likely that one or more of these pathways are affected by the dimerization state of NAMPT. Taken together, the studies proposed will provide critical information regarding the biological mechanism of NAMPT.