STUDY OF NAMPT ACTIVITY IN MELANOMA CELLS BY LC-ESI-MSn ANALYSES

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in nicotinamide adenine dinucleotide NAD metabolism. It converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN), via 5-phosphoribosyl pyrophosphate (PRPP) and Adenosine 5′-triphosphate (ATP). Afterwards, nicotinamide mononucleotide adenylyltransferase (NMNAT) synthesizes NAD from NMN in presence of ATP. NAMPT is involved in many inflammatory and metabolic diseases, including cancer. Its activity is essential for maintaining the NAD pool especially in tumor cells, in which the activity of NAD degrading enzymes such as poly (ADP-ribose) polymerases (PARPs), mono (ADP-ribose) transferases (ARTs) and sirtuins is increased (Figure 1). Indeed, NAMPT is over-express by most of tumoural cells and its high levels are correlated to prognosis and overall survival. In this work, a new LC-ESI-MSn bioanalytical method was developed to evaluate all the analytes involved in the NAD homeostasis related to NAMPT activity and to deeply understand the mechanism of action of NAMPT in the B16 engineered melanoma cells. The method demonstrated to be sensitive, specific and very accurate for the quantification of six different molecules (AMP, ADP, ATP, NMN, NAD and NAM). The sample preparation was characterized by cells isolation followed by liquid-liquid extraction (LLE); all the analytes studied were separated by isocratic elution on a Luna HILIC column using acetonitrile and ammonium acetate buffer (pH 5,8, 100mM). The ESI MS detection was made either in positive or in negative scan, in MS2, SRM or MRM mode.