Figure 8. Crystal structure of both a type II inhibitor, ima7nib, and a type IV inhbitor, GNF-­‐2, bound to the kinase domain of c-­‐ Abl (3K5V.pdb). GNF-­‐2 is a fully allosteric inhibitor of WT Abl and rescues type I/II inhibitor eﬃcacy against T315I both in vitro and in vivo. Gly250 Tyr253 Glu255 Met351 Phe359 Thr315 In addi7on to playing an essen7al role in cellular energe7cs, kinases and their associated signaling pathways regulate a plethora of intracellular processes. When aberrantly expressed or controlled, kinases can cause cellular dysregula7on and contribute to the onset of several diseases, including cancer. Based on the fundamental understanding of kinase dysregula7on in cancer biology, the discovery of small organic molecules to alter kinase func7on has culminated in the development of targeted cancer therapy. However, limited selec7vity and the emergence of drug resistance remain fundamental challenges for current modern medicinal chemistry eﬀorts aimed at the development of kinase inhibitors that are both safe and eﬀec7ve as long-­‐term treatments. Muta7ons resistant to classical ATP-­‐compe77ve (Type I/II) kinase inhibitors emerged at a rapid pace and oTen limit the success of newly available targeted cancer therapies. At present, there are more than 70 individual muta7ons at more than 50 muta7on sites conferring diﬀerent levels of ima7nib resistance found within CML pa7ents. Recently, a number of Type IV kinase inhibitors that func7on via a fully allosteric mechanism of ac7on have demonstrated promise toward addressing muta7on dependent drug resistance. BINDING STUDIES OF TYPE I, II, AND IV KINASE INHIBITORS AGAINST ABL KINASE USING BACK-­‐SCATTERING INTERFEROMETRY MOA FOR ALLOSTERIC INHIBITORS THAT OVERCOME DRUG RESISTANCE Fei Shen, Nathaniel Gilbert, Robert R. Lavieri, Richard J. Isaacs, Scot R. Weinberger Molecular Sensing, Inc. Back-­‐scaNering Interferometry The Back-­‐scaYering device is a micro-­‐scale interferometer (see ﬁgure 4). The BSI device consists of a HeNe laser, a microchip, and a CCD camera. The microchip receives light from the laser and illuminates the sample containing channel. As light passes into the channel, interference fringe paYerns arise and a camera images the fringes (Figure 4). When molecules bind, the resultant complex causes a change in molecular mean polarizability that is measured as a fringe paYern shiT. Monitoring the change in fringe phase as a func7on of ligand concentra7on allows equilibrium dissocia7on constant (Kd) measurements to be performed. SeRng up the BSI assay BSI Kd determina7ons are performed in end-­‐point fashion, with target and ligand pre-­‐incubated to establish equilibrium. Target and ligand concentra7ons are chosen to ini7ate pseudo-­‐ﬁrst order binding condi7ons, for which the target is typically held at a sparing concentra7on and the ligand is present in excess to avoid ligand deple7on during the binding process. Back-­‐scaNering Interferometry Kinase inhibitor binding analysis and BSI data validaTon References Materials and Methods www.molsense.com Sample Reference Figure 11. Prepara7on of kinase Inhibitors. Ima7nib, dasa7nib, nilo7nib and GNF-­‐5 were prepared as 50 mM working stocks in 100% DMSO. Dose response series were created by dilu7ng each working stock with MOPS buﬀer to establish the appropriate target concentra7on range for each binding system using a 12-­‐point doubling dilu7on (range: 50 pM – 125 μM). DasaTnib (nM) NiloTnib (nM) ImaTnib (nM) c-­‐Abl Kinase IC50 KD IC50 KD IC50 KD Wild-­‐type 1.83 1.08 17.69 13.3 527 472 M351T 1.61 0.42 7.8 2.7 926 1086 Q252H 5.6 5.49 46.7 47.6 733 961 H396P 1.95 0.7 42.6 32.6 1280 1228 T315I 137 86.5 696 761 9221 4050 Figure 9. 9A. The Kd of ATP for both WT and T315I Abl is about 4-­‐5 micromolar. 9B. The Kd values for GNF-­‐5 vs. WT and T315I Abl are about 510 nM and 130 nM respec7vely. 9C. Interes7ngly, in the presence of a satura7ng amount of GNF-­‐5 (50 μM) the binding curve for ATP is right-­‐shiTe