Traditional drug discovery is based on sequentially using approaches to optimizing compounds. Cloud computing, machine learning, and automation allow us to use several approaches in parallel to quickly cover a large chemical space in search of the best compound to modulate the activity of the kinase of interest. The talk will show the implementation of these approaches in ORION.

Nearly all aspects of cellular signaling are touched by the kinome, with dysregulation of this network a key driver of disease. In this talk, I'll describe recent work focused on characterizing the architecture of the kinome, along with its response to targeted perturbations. With particular emphasis on poorly studied "dark kinases", this work includes new experimental efforts to quantify kinome behavior in response to targeted drug perturbations, along with newly developed machine learning approaches that greatly enhance our ability to identify novel druggable targets.

HER2-targeted tyrosine kinase inhibitors (TKIs), such as lapatinib, tucatinib and neratinib, have shown excellent clinical activity in HER2-positive breast cancer. However, the effectiveness of these drugs has been limited in other cancer types. Our study examined these HER2-targeted TKIs in a large-scale cell line analysis in order to determine novel biomarkers of response to these drugs, and discover new potential indications.

BRAF kinase is a critical effector of the MAPK/ERK signaling pathway that is hyperactivated in several cancers, including melanoma, colorectal, and thyroid. FDA-approved RAF inhibitors poorly inhibit BRAF dimers, leading to tumor resistance. Through a cell-based screening assay, crystal structures, and structure-based drug design, we identified novel inhibitors that use an allosteric site. We will present a novel class of BRAF dimer selective inhibitors that use this allosteric site.

Pre-mRNA Processing Factor 4 (PRPF4) is a kinase previously associated with splicing regulation and triple-negative breast cancer metastasis formation in vivo, although it remains largely understudied. Using this compound as a starting point, several analogues were synthesized aiming at maintaining high potency while further increasing selectivity and cell permeability. Compounds were tested in the differential scanning fluorimetry, MDCK permeability, and 320-kinase selectivity assays. The best compounds exhibited high potency with relevant gains in cell permeability and selectivity. Further optimization is ongoing to attenuate the activity against some kinase off-targets. The co-crystal structure between one of the analogues and PRPF4 was obtained to rationalize the next cycles of design by exploring unique interactions between the ligand and kinase hinge residues.

Mutations in the epidermal growth factor receptor (EGFR) are a major driver of non-small-cell lung cancer. We developed mutant-selective allosteric inhibitors to combat acquired resistance to ATP-competitive drugs. Select allosteric inhibitors simultaneously and cooperatively bind with a subset of ATP-site inhibitors as confirmed by structural and biochemical studies. Such inhibitor combinations synergize in vivo, overcoming clinically relevant resistance mutations and delaying the emergence of additional mutations.

Targeted protein degradation has rapidly gained traction as a promising therapeutic approach. Despite this, relatively little is understood about the factors governing a target's propensity for ubiquitination and degradation by specific E3-ligases, and the influence of chemical and cell type-specific variables on the effects of a given degrader molecule. We employed a chemo-proteomic approach to systematically characterize the factors that influence endogenous kinase degradation in different cellular contexts, profiling a library of well-characterized, diverse kinase degraders using a quantitative global proteomics approach.

This talk outlines the design, physicochemical optimisation, and biological activity of a set of novel PROTACs targeting the JAK family of proximal membrane-bound proteins. JAK PROTACs from two distinct chemotypes were designed, optimising the physicochemical properties for each template to enhance cell permeation. These PROTACs are capable of inducing significant JAK1/JAK2 degradation, demonstrating an extension of the PROTAC methodology to an unprecedented class of protein targets and their mechanisms of action.

The on-target and dose-limiting thrombocytopenia limits the use of BCL-X_L inhibitors, such as ABT263, as safe and effective anticancer agents. To reduce this toxicity, we converted ABT263 into DT2216, a BCL-X_L PROTAC that targets BCL-X_L to VHL for degradation. DT2216 was more potent against cancer cells, but less toxic to platelets than ABT263, because VHL is poorly expressed in platelets. This demonstrates the use of PROTACs to reduce on-target drug toxicities.

With PROTACs progressing rapidly towards therapeutic applications, it is important to understand whether and how resistance to these novel agents may emerge. Using BET-PROTACs as a model system, we demonstrate that resistance to both VHL- and CRBN-based PROTACs was primarily caused by genomic alterations that compromise core components of the relevant E3 ligase complexes. This study reveals a novel resistance mechanism distinct from current targeted therapies and warrants future investigations.