Hsp90 inhibition in B cell acute lymphoblastic leukemia overcomes resistance to JAK2 inhibitors.

The incidence of dengue fever epidemics has increased dramatically over the last few decades. However, no vaccine or antiviral therapies are available. Therefore, the need for safe and effective antiviral drugs has become imperative. The entry of dengue virus into a host cell is mediated by its major envelope (E) protein. The crystal structure of the E protein reveals a hydrophobic pocket that is presumably important for low-pHmediated membrane fusion. High-throughput docking with this hydrophobic pocket was performed, and hits were evaluated in cell-based assays. Compound 6 was identified as one of the inhibitors and had an average 50% effective concentration of 119 nM against dengue virus serotype 2 in a human cell line. Mechanism-ofaction studies demonstrated that compound 6 acts at an early stage during dengue virus infection. It arrests dengue virus in vesicles that colocalize with endocytosed dextran and inhibits NS3 expression. The inhibitors described in this report can serve as molecular probes for the study of the entry of flavivirus into host cells.

To assess the potential of protein kinase CK2 as a target for developing new antitumor agents, we have undertaken a search for inhibitors of this enzyme. As part of this effort, we report here the discovery of the potent and selective CK2 inhibitor (5-oxo-5,6-dihydroindolo[1,2-a]quinazolin-7-yl)acetic acid. We identified this inhibitor of a novel structural type by high-throughput docking of our corporate compound collection in the ATP binding site of a homology model of human CK2, using an appropriate protocol. The synthesis of the inhibitor as well as that of related analogues whose CK2 inhibitory activities give support to the binding mode proposed by the docking program is described. The results obtained suggest that virtual screening of a 3D database by molecular docking is a useful approach for lead finding provided that adapted postdocking filtering and reranking procedures are applied to the primary hit list.

JAK inhibitors are being developed for the treatment of rheumatoid arthritis, psoriasis, myeloproliferative neoplasms and leukemias. Most of these drugs target the ATP-binding pocket and stabilize the active conformation of the JAK kinases. This type-I binding mode leads to an increase in JAK activation-loop phosphorylation, despite blockade of kinase function. Here we report that stabilizing the inactive state via type-II inhibition acts in the opposite manner, leading to a loss of activation-loop phosphorylation. We used X-ray crystallography to corroborate the binding mode and report for the first time the crystal structure of the JAK2 kinase domain in an inactive conformation. Importantly, JAK inhibitor-induced activation-loop phosphorylation requires receptor interaction, as well as intact kinase and pseudokinase domains. Hence, depending on the respective conformation stabilized by a JAK inhibitor, hyperphosphorylation of the activation-loop may or may not be elicited.

The recent discovery of an acquired activating point mutation in JAK2, substituting valine at amino acid position 617 for phenylalanine, has greatly improved our understanding of the molecular mechanism underlying chronic myeloproliferative neoplasms. Strikingly, the JAK2 V617F mutation is found in nearly all patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia and primary myelofibrosis. Thus, JAK2 represents a promising target for the treatment of myeloproliferative neoplasms and considerable efforts are ongoing to discover and develop inhibitors of the kinase. Here, we report potent inhibition of JAK2 V617F and JAK2 wild-type enzymes by a novel substituted quinoxaline, NVP-BSK805, which acts in an ATP-competitive manner. Within the JAK family, NVP-BSK805 displays more than 20-fold selectivity towards JAK2 in vitro, as well as excellent selectivity in broader kinase profiling. The compound blunts constitutive STAT5 phosphorylation in JAK2 V617F-bearing cells, with concomitant suppression of cell proliferation and induction of apoptosis. In vivo, NVP-BSK805 exhibited good oral bioavailability and a long half-life. The inhibitor was efficacious in suppressing leukemic cell spreading and splenomegaly in a Ba/F3 JAK2 V617F cell-driven mouse mechanistic model. Furthermore, NVP-BSK805 potently suppressed recombinant human erythropoietin-induced polycythemia and extramedullary erythropoiesis in mice and rats. Mol Cancer Ther; 9(7); 1945-55. ©2010 AACR.

Summary A variety of cancers depend on JAK2 signaling, including the high-risk subset of B-cell acute lymphoblastic leukemias (B-ALLs) with CRLF2 rearrangements. Type I JAK2 inhibitors induce paradoxical JAK2 hyperphosphorylation in these leukemias and have limited activity. To improve the efficacy of JAK2 inhibition in B-ALL, we developed the type II inhibitor CHZ868, which stabilizes JAK2 in an inactive conformation. CHZ868 potently suppressed the growth of CRLF2-rearranged human B-ALL cells, abrogated JAK2 signaling, and improved survival in mice with human or murine B-ALL. CHZ868 and dexamethasone synergistically induced apoptosis in JAK2-dependent B-ALLs and further improved in vivo survival compared to CHZ868 alone. These data support the testing of type II JAK2 inhibition in patients with JAK2-dependent leukemias and other disorders.

Novel 2-benzylidene-benzofuran-3-ones were designed and synthesized to mimic flavopiridol, a well-established inhibitor of cyclin-dependent kinases (CDKs) which is currently undergoing clinical evaluation. The underlying design concepts as well as the synthesis and structure-activity relationships (CDKs 1, 2, and 4 enzyme assays) of these mimics are described. Inhibitors of CDKs 1 and 2 that are more potent and selective than flavopiridol were obtained.

We describe a synthetic approach toward the rapid modification of phenyl-indolyl maleimides and the discovery of potent Jak3 inhibitor 1 with high selectivity within the Jak kinase family. We provide a rationale for this unprecedented selectivity based on the X-ray crystal structure of an analogue of 1 bound to the ATP-binding site of Jak3. While equally potent compared to the Pfizer pan Jak inhibitor CP-690,550 (2) in an enzymatic Jak3 assay, compound 1 was found to be 20-fold less potent in cellular assays measuring cytokine-triggered signaling through cytokine receptors containing the common γ chain (γC). Contrary to compound 1, compound 2 inhibited Jak1 in addition to Jak3. Permeability and cellular concentrations of compounds 1 and 2 were similar. As Jak3 always cooperates with Jak1 for signaling, we speculate that specific inhibition of Jak3 is not sufficient to efficiently block γC cytokine signal transduction required for strong immunosuppression.