Discovery of AMG 925, a FLT3 and CDK4 dual kinase inhibitor with preferential affinity for the activated state of FLT3.

We describe the structural optimization of a lead compound 1 that exhibits dual inhibitory activities against FLT3 and CDK4. A series of pyrido[4',3':4,5]pyrrolo[2,3-d]pyrimidine derivatives was synthesized, and SAR analysis, using cell-based assays, led to the discovery of 28 (AMG 925), a potent and orally bioavailable dual inhibitor of CDK4 and FLT3, including many FLT3 mutants reported to date. Compound 28 inhibits the proliferation of a panel of human tumor cell lines including Colo205 (Rb(+)) and U937 (FLT3(WT)) and induced cell death in MOLM13 (FLT3(ITD)) and even in MOLM13 (FLT3(ITD, D835Y)), which exhibits resistance to a number of FLT3 inhibitors currently under clinical development. At well-tolerated doses, compound 28 leads to significant growth inhibition of MOLM13 xenografts in nude mice, and the activity correlates with inhibition of STAT5 and Rb phosphorylation.

Preclinical evaluation of AMG 925, a FLT3/CDK4 dual kinase inhibitor for treating acute myeloid leukemia.

Acute myeloid leukemia (AML) remains a serious unmet medical need. Despite high remission rates with chemotherapy standard-of-care treatment, the disease eventually relapses in a major proportion of patients. Activating Fms-like tyrosine kinase 3 (FLT3) mutations are found in approximately 30% of patients with AML. Targeting FLT3 receptor tyrosine kinase has shown encouraging results in treating FLT3-mutated AML. Responses, however, are not sustained and acquired resistance has been a clinical challenge. Treatment options to overcome resistance are currently the focus of research. We report here the preclinical evaluation of AMG 925, a potent, selective, and bioavailable FLT3/cyclin-dependent kinase 4 (CDK4) dual kinase inhibitor. AMG 925 inhibited AML xenograft tumor growth by 96% to 99% without significant body weight loss. The antitumor activity of AMG 925 correlated with the inhibition of STAT5 and RB phosphorylation, the pharmacodynamic markers for inhibition of FLT3 and CDK4, respectively. In addition, AMG 925 was also found to inhibit FLT3 mutants (e.g., D835Y) that are resistant to the current FLT3 inhibitors (e.g., AC220 and sorafenib). CDK4 is a cyclin D-dependent kinase that plays an essential central role in regulating cell proliferation in response to external growth signals. A critical role of the CDK4-RB pathway in cancer development has been well established. CDK4-specific inhibitors are being developed for treating RB-positive cancer. AMG 925, which combines inhibition of two kinases essential for proliferation and survival of FLT3-mutated AML cells, may improve and prolong clinical responses.

Development of a time-resolved fluorescence resonance energy transfer assay for cyclin-dependent kinase 4 and identification of its ATP-noncompetitive inhibitors.

Protein kinases are recognized as important drug targets due to the pivotal roles they play in human disease. Many kinase inhibitors are ATP competitive, leading to potential problems with poor selectivity and significant loss of potency in vivo due to cellular ATP concentrations being much higher than K(m). Consequently, there has been growing interest in the development of ATP-noncompetitive inhibitors to overcome these problems. There are challenges to identifying ATP-noncompetitive inhibitors from compound library screens because ATP-noncompetitive inhibitors are often weaker and commonly excluded by potency-based hit selection criteria in favor of abundant and highly potent ATP-competitive inhibitors in screening libraries. Here we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for protein kinase cyclin-dependent kinase 4 (CDK4) and the identification of ATP-noncompetitive inhibitors by high-throughput screening after employing a strategy to favor this type of inhibitors. We also present kinetic characterization that is consistent with the proposed mode of inhibition.