Discovery of Selective Binding Molecules for the Pseudokinase Domain of Janus Kinase 2
Date of Award
Doctor of Philosophy (PhD)
Janus Kinase 2 (JAK2) is a nonreceptor tyrosine kinase that plays an important role in the regulation of hematopoiesis through the JAK-STAT pathway. JAK2 is comprised of seven Janus Homology (JH) domains, including a C-terminal kinase domain (JH1) responsible for the catalytic activity, and an adjacent pseudokinase domain (JH2). The pseudokinase domain adopts a prototypical protein-kinase fold and can bind ATP, but it lacks critical residues for phosphorylation catalysis. Its primary role is to regulate the function of the JH1 domain, and according to recent insights, it also plays critical role in cytokine receptor dimerization. V617F, the most frequently occurring mutation in JAK2, has been associated with the pathogenesis of myeloproliferative neoplasms (MPNs), like polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (MF). Despite that the V617F mutation in JH2 domain results in hyperactivation of the kinase activity, so far only the kinase domain of JAK2 has been targeted for therapeutic treatments of MPNs by type-I ATP-competitive inhibitors. Current therapies for MPNs, like the FDA approved drug ruxolitinib (JAK1/JAK2 inhibitor) for the treatment of myelofibrosis, are non-selective to the site of the mutation and cause hematopoietic toxicities, so alternative therapeutic approaches are desired. Evidence from mutagenesis studies suggest that the hyperactivation caused by the V617F mutant could be attenuated by displacing ATP from the binding site of the JAK2 JH2 domain. Moreover, the studies indicate that the displacement event itself could attenuate the hyperactive V617F variant without affecting wild-type JAK2. Despite these auspicious insights, the value of the pseudokinase domain of JAK2 as a pharmacological target has yet to be demonstrated by small molecules. Along these lines, we aimed at developing small molecules that could selectively bind the JH2 over the JH1 domain and could serve as chemical probes to test this hypothesis in cells. Toward this goal, we utilized de novo design, synthesis, in vitro assaying, and X-ray crystallography. Following the structural evolution of different ligand series during lead optimization and their associated trends in binding, we were able to identify structural features that were required to achieve strong binding to JAK2 JH2 and selectivity over JH1. By growing toward non-conserved residues in the binding site of the JH2 domain, we obtained the first JH2 selective leads that have ever been identified. Following this milestone, further optimization was achieved by appending pharmacophores with rational design. In a final stage of optimization, we improved the drug-like properties of lead compounds to enhance membrane permeation. These studies led to the discovery of the most potent, selective, cell-permeable binding molecules for the JAK2 pseudokinase known to date. Cell assays on these leads provided insights on the feasibility of JAK2 modulation through the pseudokinase domain.
Liosi, Maria-Elena, "Discovery of Selective Binding Molecules for the Pseudokinase Domain of Janus Kinase 2" (2022). Yale Graduate School of Arts and Sciences Dissertations. 494.