Importantly, the hybrids composed of Az and geldanamycin, which have high inhibitory activities towards both Hsp70 and Hsp90, exhibit enhanced anti-leukemia activity relative to the individual inhibitors. a caspase-dependent apoptotic pathway. Taken together, the findings suggest that Hsp70 inhibitors as well as their hybrids can serve as potential anti-leukemia agents. Introduction Leukemia is a class of cancers, which cause the increased number of abnormal white blood cells. Imatinib (or Glivec), a Dapansutrile selective Abl kinase inhibitor (Fig.?1), is a highly efficacious drug to treat early-phase chronic myeloid leukemia which expresses an oncogenic Bcr-Abl fusion protein with a constitutively active Abl kinase1, 2. However, late-phase chronic myeloid leukemia becomes resistant to imatinib by expressing various Abl mutants3. The heterogeneity of leukemia caused by gene mutations and the status of patients with regards to the stage of leukemia reduce the efficacy of imatinib. Therefore, novel anti-leukemia agents that display broad selectivity towards a wide range of patients are urgently needed4. Open in a separate window Figure 1 Chemical structures of substances used in this study. Members of the Hsp70 proteins exhibit ATP-dependent chaperone activities, including protein folding, degradation of misfolded proteins, blocking denatured protein aggregation, and protein translocation5, 6. The two major members of cytosolic Hsp70 proteins are constitutive Hsc70 and inducible Hsp70. It is known that inducible Hsp70 is greatly produced in both solid and hematological tumors, a phenomenon that leads to an enhancement in cancer cell survival7, 8. The increased level of Hsp70 expression also correlates with resistance of cancers to chemotherapeutic agents, including imatinib9, 10. Moreover, simultaneous attenuation of the expression of both inducible Hsp70 and constitutive Hsc70 promotes apoptotic death of cancer cells without affecting normal cells11. As a consequence, Hsp70 proteins are potent targets for cancer diagnosis and prognosis, and their inhibitors are potential chemotherapeutic agents for treatment of various cancers12, 13. Another class of ATP-dependent molecular chaperone, Hsp90, is known to bind and stabilize various cancer-associated client proteins, including Bcr-Abl14, 15. Thus, targeting Hsp90 with small molecule inhibitors represents yet another promising approach to the treatment of tumors16, 17. For example, geldanamycin (Fig.?1), which associates with the ATP Dapansutrile binding site of Hsp90 and blocks its activity, is a candidate for anticancer therapy18, 19. However, the results of previous investigations indicate that inhibition of Hsp90 in itself is insufficient to bring about cancer cell death because exposure to geldanamycin or its synthetic derivatives induces upregulation of Hsp7020. This upregulation leads to a decrease in the anticancer activities of Hsp90 inhibitors. Therefore, dual inhibition of Hsp90 and Hsp70 proteins FGF22 should be an important therapeutic strategy to generate efficacious anticancer agents21, 22. Recently we showed that apoptozole (Az, Fig.?1), a small molecule inhibitor of both Hsc70 and Hsp7023C27, induces death of various solid tumor cells25. Guided Dapansutrile by this finding, we designed an investigation to evaluate the anti-leukemia activity of Az. In addition, based on observations that unimolecular dual inhibitors with dual activities often have enhanced therapeutic efficacies relative to the individual components28, 29, we designed hybrids of Az. Specifically, hybrids in which Az is covalently linked to an inhibitor of either Hsp90 (geldanamycin) or Abl (imatinib) were synthesized and evaluated for their leukemia cell death activities. The results of our effort demonstrate that Az induces leukemic cell death and that its hybrids containing geldanamycin exhibit an improved anti-leukemia efficacy compared to that of Az or geldanamycin. Results NMR studies We previously showed that Az inhibits the ATPase activities of both Hsc70 and Hsp70, with high amino acid sequence and structural similarities, by binding to their ATPase domains24, 25. However, the detailed mode of Az binding to the proteins has not been elucidated. To gain information about the molecular basis of Az binding to Hsp70 proteins, NMR studies were carried out on Dapansutrile a complex of Az with an ATPase domain (1-386 residues) of.