Targeting the TGF-β RI kinase with LY2157299: A PK/PD-driven drug discovery and clinical development program
Authors: Jonathan Yingling
Journal: Cancer Research (2005)

SY13-2  Transforming growth factor-β (TGF-β) is a cytokine with diverse biological activities. TGF-β’s role as a tumor suppressor and tumor promoter has been well documented. In most cells, TGF-β mediates a growth inhibitory signal via a heteromeric receptor complex composed of two transmembrane serine/threonine kinase receptors, the type I and type II receptors (TβRI and TβRII). However, once this inhibitory pathway is disrupted in tumor cells, TGF-β becomes a potent tumor promoter that can be secreted at high levels from the resistant tumor cells. Clinically, elevation of TGF-β has been reported in glioblastoma, breast, prostate, non-small cell lung, renal and bladder cancer. Increased TGF-β expression affects the tumor microenvironment by modulating the extracellular matrix, promoting angiogenesis and decreasing immune surveillance. In addition, TGF-β induces epithelial-to-mesenchymal transition of tumor cells, thus creating a more invasive and metastatic phenotype. The tumor microenvironment and tumor cell effects of TGF-β provide strong rationale to target this pathway for therapeutic intervention. The drug discovery effort aimed at inhibiting TGF-β signal transduction initially relied on a cell-based proliferation assay. Taking advantage of the growth suppressive properties of TGF-β in epithelial cells, a high-throughput screen was performed to identify active compounds based on their ability to promote cell growth. Subsequent analysis confirmed that the molecular target of the original pyrazole hit, LY364947, was the TβRI and TβRII kinase domain. Biochemical mechanism of action and crystallography studies confirmed the ATP-competitive nature of this chemical scaffold. Structure-activity relationships were generated in a series of TGF-β dependent cell-based assays that correlated with potency at TβRI including evaluation of phospho-Smad2 inhibition. Structural biology confirmed interaction in the ATP-binding site with the protein backbone at His283 and a water-mediated H-bonding network involving Tyr249 and Glu245. These interactions in the active site of TβRI have been observed with all reported inhibitory scaffolds at this target. Biochemical and cellular selectivity analysis confirmed the highly selective nature of advanced compounds from the dihydropyrrolopyrazole series, including the clinical candidate LY2157299. The critical role of TGF-β in maintaining homeostasis is substantiated by the phenotype of mice where ligands, receptors or SMAD proteins that constitute the primary signal transduction cascade are genetically manipulated. Knock-out and transgenic mouse models suggest that significant and prolonged inhibition of TGF-β signaling may result in disrupted hematopoiesis and vasculogenesis, autoimmunity, multi-focal inflammation as well as renal, pulmonary, musculoskeletal and cardiovascular defects. The potential for significant adverse events with a potent and selective TGF-β RI kinase inhibitor drove our drug discovery effort to focus on a detailed understanding of the in vivo PK/PD profile of target modulation using phospho-Smad2 as a pharmacodynamic endpoint. Our lead optimization strategy focused on identifying a compound with optimal properties balancing target modulation and safety endpoints. Interestingly, LY2157299 was not the most potent compound tested, highlighting the importance of understanding compound PK/PD properties in conjunction with its toxicity profile to select molecules with an optimized therapeutic index. Not unexpectedly, high doses that abrogate TGF-β signaling result in multi-focal inflammation, while lower pharmacological doses exhibit endochondral hypertrophy and increases in trabecular bone. A biomarker strategy is currently being implemented to guide clinical development of LY2157299. This strategy involves identification and validation of biochemical and functional biomarker endpoints to monitor modulation of TGF-β signaling in patients. In collaboration with 3 clinical sites a cohort of 40-50 patients is being evaluated for biological variability in the primary phospho-Smad2 biomarker as well as quantitation of circulating TGF-β levels and several downstream secreted biomarkers. This study characterizing biomarker methodologies is aiding design of future Phase I clinical trials using continuous reassessment based on relevant pharmacodynamic markers. It is anticipated that this approach will guide dose selection for future Phase II clinical investigation. In conclusion, the preclinical drug discovery paradigm that led to the identification of LY2157299 and the translational biomarker strategy will allow a PK/PD focused Phase I trial design to establish the potential clinical benefit of modulating TGF-β signaling in cancer patients.