Chemoproteomic Evaluation of Target Engagement by the Cyclin-Dependent Kinase 4 and 6 Inhibitor Palbociclib Correlates with Cancer Cell Response

Palbociclib is a cyclin-dependent kinase (CDK) 4/CDK6 inhibitor approved for breast cancer that is estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-negative. We profiled palbociclib in cells either sensitive or resistant to the drug using an ATP/ADP probe-based chemoproteomics platform. Palbociclib only engaged CDK4 or CDK6 in sensitive cells. In resistant cells, no inhibition of CDK4 or CDK6 was observed, although the off-target profiles were similar in both cell types. Prolonged incubation of sensitive cells with the compound (24 h) resulted in the downregulation of additional kinases, including kinases critical for cell cycle progression. This downregulation is consistent with cell cycle arrest caused by palbociclib treatment. Both the direct and indirect targets were also observed in a human tumor xenograft study using the COLO-205 cell line in which phosphorylation of the retinoblastoma protein was tracked as the pharmacodyanamic marker. Together, these results suggest that this probe-based approach could be an important strategy toward predicting patient responsiveness to palbociclib

Structure-Based Identification of Ureas as Novel Nicotinamide Phosphoribosyltransferase (Nampt) Inhibitors

Nicotinamide phosphoribosyltransferase (Nampt) is a promising anticancer target. Virtual screening identified a thiourea analogue, compound <b>5</b>, as a novel highly potent Nampt inhibitor. Guided by the cocrystal structure of <b>5</b>, SAR exploration revealed that the corresponding urea compound <b>7</b> exhibited similar potency with an improved solubility profile. These studies also indicated that a 3-pyridyl group was the preferred substituent at one inhibitor terminus and also identified a urea moiety as the optimal linker to the remainder of the inhibitor structure. Further SAR optimization of the other inhibitor terminus ultimately yielded compound <b>50</b> as a urea-containing Nampt inhibitor which exhibited excellent biochemical and cellular potency (enzyme IC₅₀ = 0.007 μM; A2780 IC₅₀ = 0.032 μM). Compound <b>50</b> also showed excellent in vivo antitumor efficacy when dosed orally in an A2780 ovarian tumor xenograft model (TGI of 97% was observed on day 17)

Structure-Based Discovery of Novel Amide-Containing Nicotinamide Phosphoribosyltransferase (Nampt) Inhibitors

Crystal structures of several urea- and thiourea-derived compounds in complex with the nicotinamide phosphoribosyltransferase (Nampt) protein were utilized to design a potent amide-containing inhibitor bearing an aza-indole moiety (<b>7</b>, Nampt BC IC₅₀ = 9.0 nM, A2780 cell proliferation IC₅₀ = 10 nM). The Nampt–<b>7</b> cocrystal structure was subsequently obtained and enabled the design of additional amide-containing inhibitors which incorporated various other fused 6,5-heterocyclic moieties and biaryl sulfone or sulfonamide motifs. Additional modifications of these molecules afforded many potent biaryl sulfone-containing Nampt inhibitors which also exhibited favorable in vitro ADME properties (microsomal and hepatocyte stability, MDCK permeability, plasma protein binding). An optimized compound (<b>58</b>) was a potent inhibitor of multiple cancer cell lines (IC₅₀ <10 nM vs U251, HT1080, PC3, MiaPaCa2, and HCT116 lines), displayed acceptable mouse PK properties (F = 41%, CL = 52.4 mL/min/kg), and exhibited robust efficacy in a U251 mouse xenograft model