John Newcomb

Structural analysis of the lymphocyte-specific kinase Lck in complex with non-selective and Src family selective kinase inhibitors

BACKGROUND The lymphocyte-specific kinase Lck is a member of the Src family of non-receptor tyrosine kinases. Lck catalyzes the initial phosphorylation of T-cell receptor components that is necessary for signal transduction and T-cell activation. On the basis of both biochemical and genetic studies, Lck is considered an attractive cell-specific target for the design of novel T-cell immunosuppressants. To date, the lack of detailed structural information on the mode of inhibitor binding to Lck has limited the discovery of novel Lck inhibitors. RESULTS We report here the high-resolution crystal structures of an activated Lck kinase domain in complex with three structurally distinct ATP-competitive inhibitors: AMP-PNP (a non-selective, non-hydrolyzable ATP analog); staurosporine (a potent but non-selective protein kinase inhibitor); and PP2 (a potent Src family selective protein tyrosine kinase inhibitor). Comparison of these structures reveals subtle but important structural changes at the ATP-binding site. Furthermore, PP2 is found to access a deep, hydrophobic pocket near the ATP-binding cleft of the enzyme; this binding pocket is not occupied by either AMP-PNP or staurosporine. CONCLUSIONS The potency of staurosporine against Lck derives in part from an induced movement of the glycine-rich loop of the enzyme upon binding of this ligand, which maximizes the van der Waals interactions present in the complex. In contrast, PP2 binds tightly and selectively to Lck and other Src family kinases by making additional contacts in a deep, hydrophobic pocket adjacent to the ATP-binding site; the amino acid composition of this pocket is unique to Src family kinases. The structures of these Lck complexes offer useful structural insights as they demonstrate that kinase selectivity can be achieved with small-molecule inhibitors that exploit subtle topological differences among protein kinases.

Discovery of Novel, Induced-Pocket Binding Oxazolidinones as Potent, Selective, and Orally Bioavailable Tankyrase Inhibitors

Tankyrase (TNKS) is a poly-ADP-ribosylating protein (PARP) whose activity suppresses cellular axin protein levels and elevates β-catenin concentrations, resulting in increased oncogene expression. The inhibition of tankyrase (TNKS1 and 2) may reduce the levels of β-catenin-mediated transcription and inhibit tumorigenesis. Compound 1 is a previously described moderately potent tankyrase inhibitor that suffers from poor pharmacokinetic properties. Herein, we describe the utilization of structure-based design and molecular modeling toward novel, potent, and selective tankyrase inhibitors with improved pharmacokinetic properties (39, 40).

Structure-Guided Design of Aminopyrimidine Amides as Potent, Selective Inhibitors of Lymphocyte Specific Kinase: Synthesis, Structure–Activity Relationships, and Inhibition of in Vivo T Cell Activation

The lymphocyte-specific kinase (Lck), a member of the Src family of cytoplasmic tyrosine kinases, is expressed in T cells and natural killer (NK) cells. Genetic evidence, including knockout mice and human mutations, demonstrates that Lck kinase activity is critical for normal T cell development, activation, and signaling. Selective inhibition of Lck is expected to offer a new therapy for the treatment of T-cell-mediated autoimmune and inflammatory disease. With the aid of X-ray structure-based analysis, aminopyrimidine amides 2 and 3 were designed from aminoquinazolines 1, which had previously been demonstrated to exhibit potent inhibition of Lck and T cell proliferation. In this report, we describe the synthesis and structure-activity relationships of a series of novel aminopyrimidine amides 3 possessing improved cellular potency and selectivity profiles relative to their aminoquinazoline predecessors 1. Orally bioavailable compound 13b inhibited the anti-CD3-induced production of interleukin-2 (IL-2) in mice in a dose-dependent manner (ED 50 = 9.4 mg/kg).

Development of Novel Dual Binders as Potent, Selective, and Orally Bioavailable Tankyrase Inhibitors

Tankyrases (TNKS1 and TNKS2) are proteins in the poly ADP-ribose polymerase (PARP) family. They have been shown to directly bind to axin proteins, which negatively regulate the Wnt pathway by promoting β-catenin degradation. Inhibition of tankyrases may offer a novel approach to the treatment of APC-mutant colorectal cancer. Hit compound 8 was identified as an inhibitor of tankyrases through a combination of substructure searching of the Amgen compound collection based on a minimal binding pharmacophore hypothesis and high-throughput screening. Herein we report the structure- and property-based optimization of compound 8 leading to the identification of more potent and selective tankyrase inhibitors 22 and 49 with improved pharmacokinetic properties in rodents, which are well suited as tool compounds for further in vivo validation studies.

Discovery of 2,4-bis-arylamino-1,3-pyrimidines as insulin-like growth factor-1 receptor (IGF-1R) inhibitors

The insulin-like growth factor-1 receptor (IGF-1R) plays an important role in the regulation of cell growth and differentiation, and in protection from apoptosis. IGF-1R has been shown to be an appealing target for the treatment of human cancer. Herein, we report the synthesis, structure-activity relationships (SAR), X-ray cocrystal structure and in vivo tumor study results for a series of 2,4-bis-arylamino-1,3-pyrimidines.

2-Phenylamino-6-cyano-1H-benzimidazole-based isoform selective casein kinase 1 gamma (CK1γ) inhibitors

Screening of the Amgen compound library led to the identification of 2-phenylamino-6-cyano-1H-benzimidazole 1a as a potent CK1 gamma inhibitor with excellent kinase selectivity and unprecedented CK1 isoform selectivity. Further structure-based optimization of this series resulted in the discovery of 1h which possessed good enzymatic and cellular potency, excellent CK1 isoform and kinase selectivity, and acceptable pharmacokinetic properties.

Structure-Based Design of Potent and Selective CK1γ Inhibitors.

Aberrant activation of the Wnt pathway is believed to drive the development and growth of some cancers. The central role of CK1γ in Wnt signal transduction makes it an attractive target for the treatment of Wnt-pathway dependent cancers. We describe a structure-based approach that led to the discovery of a series of pyridyl pyrrolopyridinones as potent and selective CK1γ inhibitors. These compounds exhibited good enzyme and cell potency, as well as selectivity against other CK1 isoforms. A single oral dose of compound 13 resulted in significant inhibition of LRP6 phosphorylation in a mouse tumor PD model.