Structural Basis for Resistance to Diverse Classes of NAMPT Inhibitors

<div><p>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 <i>in vivo</i>, 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.</p></div

G217 derived resistance.

<p>a) Structure of NAMPT-G217A superimposed onto the wild-type structure in b) (the brown and green ribbons). Only the side chains of residues Asp219, His191, and Ala217 of the G217A mutant are shown (cyan) together with those of wild-type (brown) for comparison. The transparent blue sticks indicate the position of GNE-618 in wild-type structure. Note that Ala217 induced a rotation of the His191 imidazole ring. Similar effects were caused by the G217V mutant (magenta) and G217R mutant (orange) depicted in <b>b</b>) and <b>c</b>). d) The crystal structure of APO866 in complex with G217-NAMPT with similar representations as above. G217R side chain is shown in sticks, and colored in red. <b>e</b>) Complex structure of NAMPT with GNE-618 shown in surface rendering colored by electrostatic potential (blue positive, red negative, white neutral). GNE-618 is shown as sticks (carbon in blue). <b>f</b>) Structure of NAMPT G217R shown in surface rendering colored by electrostatic potential as in g). The transparent black sticks indicate the GNE-618 binding position in wild-type NAMPT.</p

Nampt mutations Identified in Resistant Cell Lines.

<p>Cell lines that were selected to grow in the presence of 100 fold the parental IC₅₀ for GNE-618. NA indicates whether the cell line was selected in the presence or absence of 10 uM nicotinic acid. NAPRT1 status did not change as a result of selection for GNE-618 resistance.</p><p>Nampt mutations Identified in Resistant Cell Lines.</p

Crystal structure of NAMPT with resistant mutations mapped.

<p>The NAMPT protein structure is shown in ribbon diagram displaying an active dimer. The monomers of the NAMPT dimer are colored in brown and green, respectively. Two inhibitor molecules of GNE-618 are bound to this structure and are shown as yellow spheres. Some of the resistant mutations are mapped on the structure and are shown as red spheres.</p

H191 derived resistance.

<p>a) A close-up view of NAMPT inhibitor binding site. GNE-618 is shown in sticks (carbon in blue). NAMPT is shown in ribbons diagram, and colored by monomers, brown and green, respectively. The key residues (Asp219, His191, Gly217, Tyr188) forming hydrogen bond network are shown in sticks (carbon in brown). A water molecule WAT mediating hydrogen bonds is shown as a red sphere, dotted lines are hydrogen bonds. <b>b</b>) The structure of NAMPT in complex with GNE-618. GNE-618 is shown in sticks and colored by atom-type (carbons in blue). NAMPT is shown in both ribbons and surface rendering, with monomers in brown and green, respectively. The interior of the NAMPT protein is blinded in gray. H191 side chain is shown in sticks. Mutation H191R side chain is plotted in sticks and transparent spheres, in magenta. <b>c</b>) Complex structure of APO866 with NAMPT-H191R. APO866 is shown in sticks (blue for carbons). For comparison, APO866 conformation in wild-type NAMPT is shown in transparent sticks, colored in yellow. The protein is shown in both surface rendering and ribbons. The monomers are in brown and green, respectively. The interior of the NAMPT protein is blinded in gray. The His191 side chain is shown in red sticks.</p

Efficacy of GNE-618 in the S165Y NCI-H460 xenograft model.

<p>a–b) Mean tumor volume + standard error of the mean (SEM) in NCI-H460 parental (a) and NCI-H460 S165Y mutant (b) xenograft models after treatment with GNE-618 at the doses indicated. c) Fold change in IC₅₀ of NAMPT inhibitors on purified mutant NAMPT proteins bearing mutations in G217 and H191 compared to wild-type enzyme; d) fold change in IC₅₀ of NAMPT inhibitors on purified mutant NAMPT proteins bearing mutations in S165 compared to wild-type enzyme. Rx  =  treatment period. *p = 0.007 compared to vehicle control.</p

Nampt inhibitors evaluated for potency on mutant NAMPT enzymes.

<p>a) Chemical structures of NAMPT inhibitors, compound 1 is GNE-618, compound 9 is GMX1778, compound 11 is APO866. b) Fold change in IC₅₀ of NAMPT inhibitors on purified mutant NAMPT proteins compared to wild-type enzyme. c) Fold change in IC₅₀ values for S165Y/F mutants compared to wild-type. * fold-change assuming IC₅₀  =  maximum concentration tested (5 µM). Actual fold-change is likely larger than that depicted.</p

Conformational changes in 380-helix induced by resistant mutations at S165.

<p>a) Superimposition of NAMPT-S165F/GNE-618 complex structure (ribbons colored in cyan) onto a wild-type apo-structure (PDB entry 4KFO, ribbons colored in brown). GNE-618 is shown in sticks, and colored by atom type (carbons in light blue). The inhibitor-binding site is drawn in surface representation, two monomers colored in cyan and green, respectively. S165F is shown in red sticks, which imposes a steric clash onto residue L386 (sticks colored in wheat) of the wild-type structure. The normal PRPP binding mode is illustrated in transparent red spheres. For comparison, a structure of GNE-617 is shown in sticks (carbons in yellow). b) Superimposition of NAMPT-S165F/pRib-GNE-617 complex (ribbons colored in orange) onto the structure of NAMPT-S165F/GNE-617 ribbons colored in cyan). The pRib-GNE-617 molecule is shown in sticks (carbons in brown). GNE-617 is also shown in sticks (carbons in yellow). S165F is shown in red sticks. The resulting 380-helix avoided the steric clash between with Phe165. c) NMN production by wild-type (WT) and mutant NAMPT enzymes in the indicated concentrations of PRPP.</p

Biocheµical IC₅₀Values of Structurally Diverse NAMPT inhibitors.

<p>Values are in umol/L, sd =  standard deviation, NA = not applicable.</p><p>Biocheµical IC₅₀Values of Structurally Diverse NAMPT inhibitors.</p

Characterization of GNE-618 resistant cell lines.

<p>a) Example IC₅₀ of RD parent versus the resistant derivative line harboring the S165F NAMPT mutation in the absence (solid lines) or presence (dashed line) of 10 µM NA. b) Fold shifts in absolute IC₅₀ values in resistant versus parental cell lines. Error bars represent the standard deviation of three independent runs. c-e) NAMPT S165F and S165Y were expressed in 293T cells and evaluated for response to c) GNE-618, d) APO866 and e) GMX1778. WT = wild-type NAMPT, UT = untransfected.</p