Targeting the Myristoyl Binding Pocket in BCR/ABL: A Rational Approach for the Design of Molecular Therapy Against Philadelphia Chromosome-Positive Leukemia
Final Report Abstract
The t(9;22) leads to the formation of the chimaeric bcr/abl fusion gene which encodes for the BCR/ABL fusion protein. In contrast to its physiological counterpart c-ABL, whose kinase activity is finely regulated by growth factors and other stimuli, BCR/ABL is constitutively activated. Thus it aberrantly activates down-stream signaling pathways inducing the leukemic phenotype 6. In the BCR/ABL, the N-terminus part of ABL is replaced by the N-terminus part of BCR. The N-terminus end (Cap region) of c-ABL, absent in BCR/ABL, is implicated in regulating the kinase function of ABL. The N-terminus of ABL is myristoylated, and the myristate residue binds to a hydrophobic pocket in the kinase domain (Myristoyl binding pocket, MBP), a process called “capping” which turns c-ABL into an auto-inhibited conformation. BCR/ABL “escapes” this auto-inhibition, because the myristoylated N-terminus of ABL is lost by the fusion to BCR in the BCR/ABL fusion protein 8. In this project we provided the proof of principle of i.) an efficient drug design for compounds actively targeting the MBP of BCR/ABL; ii.) that allosteric inhibitors alone are able to inhibit the BCR/ABL-T315I, which is resistant against all molecular therapy approaches with the exception of the multitargeted AKI Ponatinib. Further we were able to show that BCR/ABL-T315I can be efficiently targeted by a combination of oligomerization inhibitors with the allosteric inhibitor GNF-2, which binds to the MBP 1. Moreover, we showed that GNF-2 regains activity against T315I mutation when used together with ATP-competitive inhibitors such as Nilotinib and Dasatinib9,12. We further optimized the computational drug design of MBP binders by additional docking strategies, tested more than 150 new compound for their inhibitory effects against the BCR/ABL-kinase and then further investigated in pre-clinical models of Ph+ leukemia. We developed model systems of Ph+ leukemia nearly aspects of clinically relevant aspects in vitro as well as in vivo for investigating mechanisms of mutational and not mutational resistance mechanisms as well as tor establishing novel therapy approaches for the treatment of both Ph+ CML and Ph+ ALL; iii.) we optimized response to allosteric inhibition by the combination with oligomerization inhibition10 using interfering peptides which were further optimized “in silico”; iv.) evidenced a strong allosteric inhibitory activity against ABL of the dual Met/Alk-inhibitor Crizotinib, developed for the treatment of lung cancer; v.) we disclosed the active principle of a natural mushroom extract able to target BCR/ABL-kinase.
Publications
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(2009) Oligomerization inhibition combined with allosteric inhibition abrogates the transformation potential of T315I-positive BCR/ABL. Leukemia 23:2242-7
Mian, A.A., Oancea, C., Zhao, Z., Ottmann, O.G. and Ruthardt, M.
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(2009) The gatekeeper mutation T315I confers resistance against small molecules by increasing or restoring the ABL-kinase activity accompanied by aberrant transphosphorylation of endogenous BCR, even in loss-of-function mutants of BCR/ABL. Leukemia 9:1614-21
Mian, A.A., Schüll, M., Zhao, Z., Oancea, C., Hundertmark, A., Beissert, T., Ottmann, O.G. and Ruthardt, M.
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(2011) Oleic Acid Is the Active Component in the Mushroom Daedalea gibbosa Inhibiting Bcr-Abl Kinase Autophosphorylation Activity. Anticancer Res. (1):177-183
Khamaisie, H., Sussan, S., Tal, M., Najajreh, Y., Ruthardt, M., Mahajna, J.
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(2012) Allosteric inhibition enhances the efficacy of AKIs to inhibit unmutated BCR-ABL and BCR-ABL-T315I. BMC Cancer Sep 17;12:411
Mian, A.A., Metodieva, A., Badura, S., Khateb, M., Ruini, N., Najajreh, Y., Ottmann, O.G., Mahajna, J., and Ruthardt M.
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(2012) Overcoming Bcr-Abl T315I mutation by combination of GNF-2 and ATP competitors in an Ablindependent mechanism. BMC Cancer. Nov 27;12:563
Khateb, M., Ruimi, N., Khamisie, H., Najajreh ,Y., Mian, A., Metodieva, A., Ruthardt. M., and Mahajna, J.
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(2012) p185BCR/ABL and p210BCR/ABL are differentially targeted by the allosteric inhibitor GNF-2 in models of Philadelphia chromosome-positive acute lymphatic leukemia. Haematologica 97: 245-251
Mian, A.A., Metodieva, A., Najajreh, Y., Ottmann, O.G., Mahajna, J., and Ruthardt M.
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(2013) Oleylamine-carbonyl-valinol inhibits auto-phosphorylation activity of native and T315I mutated Bcr-Abl, and exhibits selectivity towards oncogenic Bcr-Abl in SupB15 ALL cell lines. Mol Biol Rep. Dec 5
Najajreh, Y., Khamaisie, H., Ruimi, N., Khatib, S., Katzhendler, J., Ruthardt, M., and Mahajna, J.
