Small molecules that target specific DNA sequences have the potential to control gene expression. Ligands designed for therapeutic application must bind any predetermined DNA sequence with high affinity and permeate living cells. Synthetic polyamides containing N-methylimidazole and N-methylpyrrole amino acids have an affinity and specificity for DNA comparable to naturally occurring DNA-binding proteins. We report here that an eight-ring polyamide targeted to a specific region of the transcription factor TFIIIA binding site interferes with 5S RNA gene expression in Xenopus kidney cells. Our results indicate that pyrrole-imidazole polyamides are cell-permeable and can inhibit the transcription of specific genes.

Inhibition of heat shock protein 90 (Hsp90) results in the degradation of oncoproteins that drive malignant progression, inducing cell death, making Hsp90 a target of substantial interest for cancer therapy. BIIB021 is a novel, fully synthetic inhibitor of Hsp90 that binds competitively with geldanamycin in the ATP-binding pocket of Hsp90. In tumor cells, BIIB021 induced the degradation of Hsp90 client proteins including HER-2, AKT, and Raf-1 and up-regulated expression of the heat shock proteins Hsp70 and Hsp27. BIIB021 treatment resulted in growth inhibition and cell death in cell lines from a variety of tumor types at nanomolar concentrations. Oral administration of BIIB021 led to the degradation of Hsp90 client proteins measured in tumor tissue and resulted in the inhibition of tumor growth in several human tumor xenograft models. Studies to investigate the antitumor effects of BIIB021 showed activity on both daily and intermittent dosing schedules, providing dose schedule flexibility for clinical studies. Assays measuring the HER-2 protein in tumor tissue and the HER-2 extracellular domain in plasma were used to show interdiction of the Hsp90 pathway and utility as potential biomarkers in clinical trials for BIIB021. Together, these data show that BIIB021 is a promising new oral inhibitor of Hsp90 with antitumor activity in preclinical models. [Mol Cancer Ther 2009;8(4):921-9] IntroductionHeat shock protein 90 (Hsp90) is a widely expressed molecular chaperone that functions in the maturation and stabilization of cellular proteins (1-3). Hsp90, in complex with other cochaperone proteins, catalyzes the conformational changes of client proteins via its ATPase activity (4). The activity of Hsp90 maintains a variety of client proteins in their active conformation (5). Hsp90 also plays an important role in the regulation of several key oncogenic signaling proteins (6-8) and steroid receptors (9). Mutated proteins are particularly dependent on Hsp90 for the maintenance of the active conformation (2, 3).Ansamycin drugs such as geldanamycin bind in the ATPbinding site in the NH 2 terminus of Hsp90 (6, 10). This binding inhibits the chaperone activity of Hsp90 and results in proteasomal degradation of the client proteins (5, 11-13). Because tumor cells rely on the activity of client proteins for cell proliferation and survival, drug-induced client protein degradation leads to cytostasis and/or selective cell killing of tumor cell in vitro and in vivo (14-16).The semisynthetic Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) is currently in clinical trials for cancer (17)(18)(19). However, 17-AAG is expensive to prepare and difficult to formulate. The problematic nature of the formulations may well contribute to the dose-limiting toxicity observed with this compound. 17-AAG is also susceptible to metabolism by NQO1/DT-diaphorase enzymes (20) and to efflux by P-glycoprotein (21). The identification of a synthetic Hsp90 inhibitor would be of great therapeutic interest as it would circumvent t...

The selective heat shock protein 90 (HSP90) inhibitor 17-allyamino-17-demethoxygeldanamycin (17-AAG) is currently in phase I/II clinical studies at numerous institutions. Heretofore, the biomarkers to detect 17-AAG bioactivity (Hsp70, Raf-1, and cyclin-dependent kinase 4) had to be analyzed by Western blot of cellular samples, either from tumor biopsies or peripheral blood leukocytes, a method that is both laborious and invasive. We have identified two new biomarkers [insulin-like growth factor binding protein-2 (IGFBP2) and HER-2 extracellular domain] that can be readily detected in patient sera by ELISA. Both secreted proteins are derived from or regulated by Hsp90 client proteins, raising hopes that they might be sensitive serum markers of HSP90 inhibitor activity. Several structurally unrelated HSP90 inhibitors dose-dependently decreased secretion of both IGFBP-2 and HER-2 extracellular domain into culture medium, and both proteins were more sensitive to HSP90 inhibitors than previously identified biomarkers. In sera from BT474 tumor-bearing mice, both IGFBP-2 and HER-2 extracellular domain were downregulated by 17-AAG in a time-dependent and dosedependent manner, coincident with the degradation of HER-2 and attenuation of AKT activity in the tumors. Furthermore, IGFBP-2 levels at the end of treatment correlated with residual tumor load, suggesting that IGFBP-2 might serve as an early indicator of therapeutic response. In addition, we also found that both IGFBP-2 and HER-2 extracellular domain levels are elevated in patient sera from several cancer types, suggesting that these novel secreted biomarkers could be valuable pharmacodynamic tools in clinical trials of HSP90 inhibitors.

17-AAG, the first-generation clinical Hsp90 inhibitor, exhibits promising antitumor activity in clinical studies, but is limited by poor solubility and hepatotoxicity. To pursue compounds with better biopharmaceutical properties, we have developed a series of fully synthetic orally bioavailable inhibitors of Hsp90. Here, we report that 17-AAG and other ansamycin derivatives are inactive in P-gp and/or MRP-1 expressing cell lines and sensitivity could be restored by coadministration of P-gp or MRP inhibitors. In contrast, the synthetic Hsp90 inhibitor, BIIB021 was active in these models. Accordingly, BIIB021 was considerably more active than 17-AAG against adrenocortical carcinoma, a tumor that naturally expresses P-gp, both in vitro and in vivo. This efflux pump-mediated resistance is manifested in both cytotoxicity assays and measurements of target inhibition, such as client protein degradation. Other than this, the cytotoxic activity of BIIB021 was also not influenced by loss of NQO1 or Bcl-2 overexpression, molecular lesions that do not prevent client loss but are nonetheless associated with reduced cell killing by 17-AAG. Our results indicate that the activity of 17-AAG and other ansamycins may be curtailed in tumors that have upregulated efflux pumps or antiapoptotic proteins or other genetic alterations. These data indicate that the new generation of synthetic anti-Hsp90 drugs, exemplified by BIIB021 that is currently undergoing Phase II testing, may have broader application against tumors with acquired multidrug resistance or tumors located in organs protected by MDR proteins, such as the adrenal glands, brain and testis.Hsp90 is an attractive target in cancer therapy due to its critical role in mediating the maturation and stability of a variety of cancer-associated proteins, including p53, Src, Her-2, IGFR, VEGFR2, steroid receptors, AKT, Raf and Bcr-abl. Many Hsp90 inhibitors in clinical development, including KOS-953 (tanespimycin), IPI-504 (retaspimycin) and KOS-1022 (DMAG), are all semisynthetic geldanamycin derivatives and are structurally closely related to 17-AAG.1,2 Previous studies have shown that the antitumor activity of 17-AAG and ansamycins are significantly curtailed by the expression of multidrug resistance (MDR) proteins, a major contributor to drug resistance commonly observed in heavily pretreated cancer patients. 3,4 This raised the possibility that increased MDR expression in these patients may limit the response of the patients to 17-AAG and its analogues.MDR is a significant obstacle in cancer therapy. It is mediated by a family of ATP-binding cassette (ABC) proteins, which function as efflux pumps. Two major members in this family are P-glycoprotein (P-gp) and MRP1 (MDRrelated protein 1), whose expression are found to be elevated in a variety of cancers.3,5 Expression of MDR proteins confers resistance to a broad range of structurally and functionally unrelated chemotherapeutic agents and molecularly targeted drugs. 4,6,7 P-gp not only elicits drug resistance at the cellular level...

Hsp90 inhibitors are under investigation in multiple human clinical trials for the treatment of cancers, including myeloma, breast cancer, prostate, lung, melanoma, gastrointestinal stromal tumour and acute myeloid leukaemia. The pharmacodynamic activity of Hsp90 inhibitors in the clinic is currently assessed by Hsp70 induction in peripheral blood mononuclear cells using Western blot analysis, a method that is laborious, semiquantitative and difficult to implement in the clinic. Since Hsp70 was reported to be secreted by tumour cells and elevated in sera of cancer patients, serum Hsp70 has been evaluated as a potentially more robust, easily and reproducibly measured biomarker of Hsp90 inhibition as an alternative to cytosolic Hsp70. A highly sensitive and specific electrochemiluminescent ELISA was developed to measure serum Hsp70 and employed to evaluate Hsp70 levels in both ex vivo and xenograft samples. In ex vivo studies, maximal secretion of Hsp70 by tumour cells was observed between 48 and 72 h after exposure to Hsp90 inhibitors. In in vivo studies a 3-4-fold increase in serum Hsp70 was observed following treatment with BIIB021 in tumour-bearing mice. Strikingly, secreted Hsp70 was detectable in mice transplanted with human tumours but not in naive mice indicating a direct origination from the transplanted tumours. Analysis of clinical samples revealed low baseline levels (2 - 15 ng ml(-1)) of Hsp70 in the serum of cancer patients and normal donors. Together these findings in laboratory studies and archived cancer patient sera suggest that serum Hsp70 could be a novel biomarker to assess reliably the pharmacological effects of Hsp90 inhibitors in clinical trials, especially under conditions where collection of tumour biopsies is not feasible.