Crystal structure of checkpoint kinase 2 in complex with NSC 109555, a potent and selective inhibitor

Abstract

Checkpoint kinase 2 (Chk2), a ser/thr kinase involved in the ATM-Chk2 checkpoint pathway, is activated by genomic instability and DNA damage and results in either arrest of the cell cycle to allow DNA repair to occur or apoptosis if the DNA damage is severe. Drugs that specifically target Chk2 could be beneficial when administered in combination with current DNAdamaging agents used in cancer therapy. Recently, a novel inhibitor of Chk2, NSC 109555, was identified that exhibited high potency (IC 50 = 240 nM) and selectivity. This compound represents a new chemotype and lead for the development of novel Chk2 inhibitors that could be used as therapeutic agents for the treatment of cancer. To facilitate the discovery of new analogs of NSC 109555 with even greater potency and selectivity, we have solved the crystal structure of this inhibitor in complex with the catalytic domain of Chk2. The structure confirms that the compound is an ATP-competitive inhibitor, as the electron density clearly reveals that it occupies the ATPbinding pocket. However, the mode of inhibition differs from that of the previously studied structure of Chk2 in complex with debromohymenialdisine, a compound that inhibits both Chk1 and Chk2. A unique hydrophobic pocket in Chk2, located very close to the bound inhibitor, presents an opportunity for the rational design of compounds with higher binding affinity and greater selectivity.

Cellular Inhibition of Checkpoint Kinase 2 (Chk2) and Potentiation of Camptothecins and Radiation by the Novel Chk2 Inhibitor PV1019 [7-Nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide]

“…2D). PV1019 retains many of the same interactions with the ATP-binding pocket that were observed in the complex with NSC 109555 (Lountos et al, 2009), including the hydrogen bond with Glu 273 and the hydrophobic interactions between the aryl and methyl moieties of the phenyl guanidinohydrazone and the ATP-binding pocket. The methyl group of PV1019 also projects toward a hydrophobic pocket in the enzyme as observed previously in the Chk2/NSC 109555 complex.…”

“…We recently identified and characterized a Chk2 inhibitor, NSC 109555, with a novel chemotype (Jobson et al, 2007) and cocrystallized NSC 109555 with the catalytic domain of Chk2 (Lountos et al, 2009). Seeking to improve the cellular activity of NSC 109555 while maintaining selectivity for Chk2, we synthesized a new analog PV1019 (NSC 744039) (Fig.…”

“…The methyl group of PV1019 also projects toward a hydrophobic pocket in the enzyme as observed previously in the Chk2/NSC 109555 complex. This hydrophobic pocket is of considerable interest because it may present an opportunity to modulate the potency and specificity of Chk2 inhibitors; its shape, accessibility, and degree of hydrophobicity vary among kinases (Toledo et al, 1999;Scapin, 2002;Lountos et al, 2009). Accordingly, functional replacements for the methyl group of PV1019, such as branched and cyclic al- and OVCAR-4 (C and E) were incubated with various concentrations of TPT (A and C) and CPT (E) alone or coincubated with various concentrations of TPT or CPT and fixed concentrations of PV1019 for 48 h in 96-well plates.…”

“…kanes, may fill this pocket and further improve potency and specificity. Because of the incorporation of the 7-nitroindole substituent, PV1019 picks up new interactions with the ATPbinding pocket, which probably account for its increased potency over the parent compound NSC 109555 (Lountos et al, 2009). The 7-nitro group incorporates two new interactions, a water-mediated hydrogen bond to the backbone carbonyl oxygen of Glu 302 and a hydrogen bond to the backbone amide of Met 304 in the hinge, and the amide nitrogen of PV1019 also picks up a water-mediated hydrogen bond to the carboxyl side chain oxygen of Glu 308 .…”

“…For this reason we sought to optimize the molecule. Figure 1A shows the structure of PV1019 -an analog of NSC 109555 we designed from the crystal structure (Lountos et al, 2009) and selected for the present study. As part of the optimization process we desymmetrized the lead molecule (NSC 109555), retaining one of the guanidinohydrazone moieties and coupling this via an amide bond to a 7-nitroindole substituent (shown as dotted box for PV1019) (Supplemental Materials and Methods).…”