Potent non-benzoquinone ansamycin Hsp90 inhibitors from mutasynthesis

C232

Tanespimycin (KOS-953, 17-allylamino-17-desmethoxygeldanamycin) and alvespimycin (KOS-1022, 17-(dimethylamino)ethylamino-17-desmethoxygeldanamycin) are potent ansamycin Hsp90 inhibitors currently under evaluation in clinical trials. These compounds inhibit the binding of Hsp90 to its “client” proteins, including oncoproteins such as Her2, cKit and BCR-Abl, and protein kinases such as Akt and Raf-1, resulting in proteasomal degradation of these clients and cancer cell growth arrest and/or cell death. We have undertaken a program to discover non-benzoquinone ansamycins (“NBAs”), which we predict will be potent Hsp90 inhibitors and potentially show different pharmaceutical properties. We have developed a powerful mutasynthesis strategy to prepare a diverse set of NBAs by fermentation, and we show here that these compounds can be potent, efficacious Hsp90 inhibitors. Binding measurements to the N-terminal domain of Hsp90 using isothermal titration calorimetry showed high affinity. The most avid analog shows K_d = 0.15 nM, making it the highest affinity Hsp90 inhibitor reported to date. Cell growth inhibition assays show that NBAs are as effective as tanespimycin and alvespimycin against NQO1-expressing cell lines, and retain potency against NQO1-deficient cell lines. NBAs accumulate to high level in cancer cell lines in vitro, and cause the depletion of Hsp90 client proteins such as ErbB2 in SKBr3 cells. A selected analog was examined further. Examination of the inhibitory activity of this NBA against a diversity panel of 60 kinases revealed no significant kinase inhibition at 10 µM, indicating high selectivity of the NBA for Hsp90. A PK/PD study in mice bearing HCT116 xenografts revealed high accumulation and long retention of the NBA in tumor tissue, resulting in a significant induction of Hsp70, an established pharmacodynamic marker of Hsp90 inhibition. Upon intravenous administration to mice bearing HCT116 xenografts, doses up to 30 mg/kg were well-tolerated on a weekly qdx5 dosing schedule, showing no body weight loss. Intravenous dosing of 15 or 30 mg/kg of this compound for two weeks on a weekly qdx5 schedule led to a dose responsive and statistically significant (p<0.001) inhibition of growth of HCT116 xenografts. Mutasynthesis thus can facilitate the discovery of the next generation of Hsp90 inhibitors by providing a diverse set of compounds from fermentation of a single organism.