Structural Basis for Resistance to Diverse Classes of NAMPT Inhibitors

Inhibiting NAD biosynthesis by blocking the function of nicotinamide phosphoribosyl transferase (NAMPT) is an attractive therapeutic strategy for targeting tumor metabolism. However, the development of drug resistance commonly limits the efficacy of cancer therapeutics. This study identifies mutations in NAMPT that confer resistance to a novel NAMPT inhibitor, GNE-618, in cell culture and in vivo, thus demonstrating that the cytotoxicity of GNE-618 is on target. We determine the crystal structures of six NAMPT mutants in the apo form and in complex with various inhibitors and use cellular, biochemical and structural data to elucidate two resistance mechanisms. One is the surprising finding of allosteric modulation by mutation of residue Ser165, resulting in unwinding of an α-helix that binds the NAMPT substrate 5-phosphoribosyl-1-pyrophosphate (PRPP). The other mechanism is orthosteric blocking of inhibitor binding by mutations of Gly217. Furthermore, by evaluating a panel of diverse small molecule inhibitors, we unravel inhibitor structure activity relationships on the mutant enzymes. These results provide valuable insights into the design of next generation NAMPT inhibitors that offer improved therapeutic potential by evading certain mechanisms of resistance.

通过阻断烟酰胺磷酸核糖转移酶（nicotinamide phosphoribosyl transferase, NAMPT）的功能以抑制烟酰胺腺嘌呤二核苷酸（NAD）生物合成，是靶向肿瘤代谢的极具吸引力的治疗策略。然而，耐药性的产生通常会限制癌症治疗的疗效。本研究在细胞培养体系与体内模型中，鉴定出可使肿瘤细胞对新型NAMPT抑制剂GNE-618产生耐药性的NAMPT突变体，从而证实GNE-618的细胞毒性作用具有靶点特异性。我们解析了6种空载形式以及与多种抑制剂结合状态的NAMPT突变体的晶体结构，并结合细胞实验、生化实验与结构数据，阐明了两种耐药机制：其一为Ser165残基突变引发的别构调控，该突变会导致结合NAMPT底物5-磷酸核糖-1-焦磷酸（PRPP）的α螺旋发生解旋；其二为Gly217突变对抑制剂结合产生的正位阻断作用。此外，通过对一系列多样化的小分子抑制剂进行评估，我们阐明了突变酶上的抑制剂构效关系。上述研究结果为下一代NAMPT抑制剂的设计提供了宝贵见解，这类抑制剂可通过规避特定耐药机制，获得更优的治疗潜力。