Targeting the NAD⁺ salvage pathway: Identification of bifunctional NAMPT inhibitors and SIRT1 activators through...

Nicotinamide phosphoribosyltransferase (NAMPT) and Sirtuin 1 (SIRT1) are key regulators of cellular metabolism and NAD⁺ homeostasis, representing attractive targets in cancer therapy. In this study, we identified novel bifunctional small molecules acting as simultaneous NAMPT inhibitors and SIRT1 activators through pharmacophore-based virtual screening, followed by biochemical validation with computational support. Among the candidates, compound F1 emerged as a leading hit, exhibiting potent NAMPT inhibition (IC₅₀ = 0.89 μM) and selective SIRT1 activation (EC_1.5 = 16.45 μM) in vitro. In cellular assays using the human Hep3B hepatocellular carcinoma cells, F1 reduced cell viability (IC₅₀ = 17.03 μM) through a p53-independent mechanism. Notably, despite its NAMPT inhibitory activity in vitro, F1 treatment resulted in a time-dependent increase in intracellular nicotinamide adenine dinucleotide (NAD⁺) levels (∼1.5-fold) and an elevated NAD⁺/NADH ratio within 24 h, accompanied by upregulation of NAMPT and SIRT1 protein expression, reflecting a compensatory response aimed at maintaining NAD⁺ homeostasis. This elevated NAD⁺ level, coupled with SIRT1 activation, led to a marked reduction in global lysine acetylation. Collectively, our findings demonstrate that F1 disrupted cancer cell homeostasis by inducing a unique metabolic reprogramming of the NAD⁺ salvage pathway, positioning it as a promising scaffold for multi-target metabolic modulation.