Targeting a Pleckstrin Homology Domain with a Lysine-Reactive Covalent Binder

ABSTRACT Bruton’s Tyrosine Kinase (BTK) is a validated target for haematological malignancies, with numerous FDA approved inhibitors on the market. Current therapies target the highly conserved ATP binding site and hence limit the therapeutic index given the site’s highly conserved nature across the kinome. We explore a novel approach for BTK inhibition, by targeting the PH domain-mediated membrane recruitment and activation of BTK. We have identified a fragment which covalently labels a lysine in the inositol phosphate (PIP3) binding site. Fragment growth and an extensive structure-binding relationship study uncovered 27 crystal structures and a best-in-class analog, 24. Evaluation of pKa values of the targeted lysine in BTK and other PH domains suggests this as a more general approach to PH domain inhibition. Competing Interest Statement The authors have declared no competing interest. ABBREVIATIONS - ATP - Adenosine triphosphate - BTK - Bruton’s Tyrosine Kinase - DCM - dichloromethane - DMF - dimethyl formamide - DMSO - di-methyl sulfoxide - DSF - Differential Scanning Fluorimery - ERBB4 - erythroblastic leukemia viral oncogene 4 - FCC - flash column chromatography - FDA - Food and Drug Administration - HEPES - 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - HPLC - high-performance liquid chromatography - HRMS - high resolution mass spectrometry - IC50 - half maximal inhibitory concentration - IP4 - Inositol 1,3,4,5-tetrakisphosphate - ITC - iso-thermal titration calorimetry - Kd - dissociation constant - LCMS - liquid chromatography-mass spectrometry - LDA - Lithium diisopropylamide - MALDI-TOF - Matrix-assisted laser desorption/ionization Time of Flight - MOE - Molecular Operating Environment - NMR - nuclear magnetic resonance - PDB - Protein Data Bank - PE - petroleum ether - PH - Pleckstrin Homology - PIP3 - Phosphatidylinositol (3,4,5)-trisphosphate - PLC - Phopholipase C - rt - room temperature - SH2 - Src Homology 2 - SH3 - Src Homology 3 - SFC - supercritical fluid chromatography - s-phos - 2-Dicyclohexylphosphino-2′,6′-dimethoxybiphenyl - TCEP - tris(2-carboxyethyl)phosphine - THF - tetrahydrofuran - TLC - thin layer chromatography - WT - wildtype