Bioavailable Dual-Protein Degraders of CK1α and Transcriptional Kinase CDK9 As Potential Therapeutics for Hematological Malignancies
Authors: Fung Leah , Chourasia Aparajita Hoskote , Torres Eduardo , Lam Imelda , Erdman Paul , Hecht David , Sullivan Robert , McElwee Brooke , Richard Normand , Venkatachalam Avanthika , Snir-Alkalay Irit , Chan Kyle WH , Mercurio Frank , Ben-Neriah Yinon , Stirling David I 
Published: November 2019

BioTheryx's small-molecule kinase inhibitor, BTX-A51 (the ditosylated salt of A51), has recently received FDA approval of its IND application to initiate a Phase I clinical trial in relapsed or refractory acute myeloid leukemia (AML). A51 (API of BTX-A51) is a multi-kinase inhibitor that blocks the leukemic stem cell target, Casein Kinase 1α (CK1α), as well as the super-enhancer regulator, Cyclin-Dependent Kinase 9 (CDK9), thus preventing the transcription of key oncogenic genes. This molecule has demonstrated remarkable preclinical animal efficacy inferring the eradication of AML stem cells and its potential for use in treating multiple malignancies. To exploit the unique properties of this multi-kinase inhibitor in the context of kinase protein degradation, Proteolysis-Targeting Chimeras (PROTACs) of A51 were investigated. PROTACs utilize the cell's natural ubiquitin-proteasome system to induce the selective and sustained degradation of unwanted disease-causing proteins. PROTACs are heterobifunctional molecules which are comprised of an E3-ubiquitin-ligase ligand which is covalently linked to a target-protein ligand. Through concomitant binding to an E3 ligase and to a target protein, a PROTAC promotes ubiquitination and ultimately degradation of the target protein via the proteasome. As a reversible kinase inhibitor, A51 binds stoichiometrically to CK1α and CDK9 and requires continuous occupancy of these proteins to sustain its unique inhibitory activity. A PROTAC of A51, however, would act catalytically and ablate the target proteins. In this study, PROTACs were assembled through chemically linking A51 to BioTheryx's proprietary Protein Homeostatic Modulators (PHMs™). PHMs™ are a new class of small molecules which bind to the E3 ubiquitin ligase, Cereblon (CRBN), and promote proteasomal degradation of known as well as unreported clinically-relevant CRBN neo-substrates. For the discovery of PHM®-A51 PROTACs, CDK9 crystal structure (6GZD.pdb) and CRBN crystal structure (5FQD.pdb) were used independently for computational drug design. Upon testing in AML and lymphoma cell lines, the PHM®-A51 PROTACs rapidly induced the degradation of both CK1α and CDK9. Additionally, these PROTACs had diminished effect on the protein levels of other A51 targets while inducing the degradation of other desirable targets unaffected by A51. This distinct selectivity arises from the PROTACs' ability to facilitate formation of a ternary complex that enables subsequent ubiquitination of the bound target protein - a selectivity not inherent to any kinase inhibitor. Further, the PHM®-A51 PROTACs have demonstrated low-nanomolar inhibition of cell proliferation in both AML and lymphoma cell lines yet have significantly less toxicity in fibroblast cells and PBMCs. In addition, this class of PROTACs has good pharmaceutical properties, as demonstrated by pharmacokinetic studies in mice, and will advance into AML and lymphoma in vivo studies. The targeted degradation of CK1α and CDK9 using safe, bioavailable drugs designed using strategically-selected PHMs™ with unique biological properties of their own is a very promising approach to treating hematological malignancies and other devasting cancers.

Disclosures
No relevant conflicts of interest to declare.