Discovery and Development of Allosteric Inhibitors of Src-family Kinases for AML

University Of Pittsburgh At Pittsburgh, Pittsburgh PA

Investigators

Abstract

Abstract. In this exploratory project, we propose to discover and develop allosteric kinase inhibitors targeting Hck and Fgr, members of the Src protein-tyrosine kinase family expressed in myeloid hematopoietic cells. Both kinases represent promising drug targets for acute myeloid leukemia (AML), an aggressive blood cancer with substantial unmet clinical need. Hck and Fgr are frequently over-expressed in AML patient bone marrow cells, and ATP-site inhibitors for these kinases suppress AML cell growth in vitro and in mouse models. However, orthosteric inhibitors are subject to acquired resistance through kinase domain active site mutations. Allosteric inhibitors may provide a new path to selectivity, and when combined with existing ATP-site inhibitors, are likely to prevent acquired resistance. Work with the structurally related Bcr-Abl tyrosine kinase associated with CML supports this view, where the combination of allosteric (asciminib) and ATP-site (nilotinib) Abl kinase inhibitors completely suppressed emergence of drug resistant disease in vivo. Allosteric inhibitors exploit structural fea- tures outside of the ATP-binding site that are unique to individual kinases and are thus more likely to exhibit target selectivity. Src-family kinases are dynamic, multi-domain structures which adopt distinct conformations dependent on the spatial arrangement of the regulatory SH2 and SH3 domains relative to the catalytic kinase domain. When these regulatory domains pack against the back of the kinase domain, they stabilize an inactive conformation. Our goal is to develop small molecules that lock Src-family kinases in this natural inactive confor- mation with the following Specific Aims: Aim 1. Synthesize and test analogs of existing allosteric modulators for the AML associated kinase, Hck. Our preliminary and published studies have identified small molecules that bind to an allosteric pocket involving the regulatory SH3-SH2 region of Hck. One of these compounds, a pyrimidine diamine analog known as PDA1, stabilizes a closed, inactive Hck structure. Using structure-based design, we will synthesize second generation analogs of PDA1 to improve binding potency and selectivity, and test efficacy against AML cells both alone and together with ATP-site inhibitors. Aim 2. Identify additional scaffolds for allosteric regulation of AML-associated Src-family kinases. In preliminary studies, we devel- oped a highly sensitive fluorescence complementation assay, based on the ‘SplitFAST’ fluorogenic protein sys- tem, for discovery of small molecules that bind the regulatory regions of Hck and Fgr. We will automate this assay and screen ~100,000 diverse small molecules to identify additional scaffolds for allosteric inhibitor devel- opment. Both Aims will employ a suite of biochemical, biophysical, and cellular assays to assess candidate inhibitor activity. Surface plasmon resonance (SPR) will reveal the kinetics and affinity of small molecule inter- actions with target kinases, while 1H-15N HSQC protein NMR will identify the ligand binding sites. In vitro kinase assays will determine inhibitor potency, efficacy, and selectivity. Promising candidates will be assessed in AML cell lines driven by the respective Src-family kinases to demonstrate on-target activity.