Inhibitors targeting human HSF1 as a new class of cancer therapeutic agents

A multitude of studies qualified heat shock transcription factor 1 (HSF1) as a new and promising target for cancer therapy (1-9). HSF1 activity is controlled by interactions with a number of other proteins and by modification (10-22). None of these interaction partners or modifying enzymes appear to be specific to HSF1. Targeting any one of the latter proteins or enzymes in an attempt to inhibit HSF1 activity can be expected to have unnecessarily pleiotropic effects that are likely manifested in excessive general toxicity of inhibitor compounds. This situation motivated a search for inhibitors of human HSF1 (IHSFs) that directly target the transcription factor. Several years ago, HSF Pharmaceuticals embarked on such an IHSF discovery program, in spite of the apparently well-deserved reputation of unliganded transcription factors as near impossible targets for pharmacological development.

Several structures for the DNA-binding domains (DBDs) of yeast and Drosophila HSF were published in the Brookhaven Protein Databank at the time work on this project was initiated. A comparative model for the human HSF1 DBD was generated based on Drosophila NMR structures, from which model several possible binding pockets for small molecules were predicted (23). Corresponding 3-point pharmacophores were defined and used to virtually screen a library of 300,000 commercially available drug-like compounds. Based on the results of this screen, a biased sub-library of about 2,000 compounds was assembled. To screen the latter sub-library, a cell-based assay was designed and validated. This assay identified compounds that inhibited the heat-induced activity of HSF1 but not, or less, that of a chimeric transcription factor in which the DNA-binding domain of HSF1 had been replaced with the DNA-binding domain of bacterial protein LEXA (24,25). A screen of the sub-library employing the latter assay identified lead molecule IHSF001 (23). Several rounds of improvement chemistry yielded IHSF115. Surface plasmon resonance assays confirmed that this molecule was capable of interacting with the DNA-binding domain of human HSF1.

Concerning the mechanism of inhibition of HSF1, IHSF115 neither inhibited the heat-induced oligomerization of HSF1 nor the heat-induced binding of the transcription factor to its target sequence or its heat-induced phosphorylation (monitored by EMSA). The inhibitor also did not impair the stability of HSF1. Apparently, IHSF115 inhibited the transcriptional function of HSF1. Consistent with this assignment, the inhibitor was found to interfere with the interaction between HSF1 and ATF1.  ATF1-BRG1 is known to promote the establishment of an active chromatin state and HSP gene expression (22).

An analysis of mRNA levels in human HeLa cells by hybridization to Affymetrix microarrays revealed 667 heat-induced and 406 heat-repressed genes. Over 64% of the heat-induced genes, including HSP genes, were inhibited by IHSF115; the inhibitor also caused partial or complete de-repression of over 20% of heat-repressed genes (23). Inhibitory effects of IHSF115 on a small group of heat-induced genes were quantified by RT-qPCR. IC50 values were found to range from about 2 µM to about 10 µM (about 1 µM for the HSPA7-RLUC reporter gene). As would have been expected, IHSF115 not only inhibited the heat-induced expression of HSP genes but also expression induced by other stress stimuli (e.g., proteasome inhibitor MG132).

IHSF115 significantly but variably affected the viability of human cancer cell lines (23). Most sensitive were multiple myeloma and certain triple-negative breast cancer lines. EC50 values for multiple myeloma cells that were shown to be killed largely by an apoptotic mechanism ranged from 1.7 to 3.9 µM. The EC50 values for the two most sensitive triple-negative breast cancer lines were 2.2 µM and 4.2 µM, respectively.

Most recently, it was discovered that IHSF115 synergistically enhances the activity of proteasome inhibitor bortezomib against multiple myeloma cells. Bortezomib is approved in the U.S. and in Europe for the treatment of multiple myeloma. It is noted that additional improvement chemistry uncovered analogs of IHSF115 with several-fold greater potency. These analogs killed multiple myeloma cells with IC50 values in the submicromolar range.

Based on the results obtained to date, the company believes that further work on IHSF as anti-cancer agents is warranted. While IHSF may be considered for monotherapy, they may be particularly attractive for use in combination with other active agents such as inhibitors of HSP90, HDAC6 or the proteasome, as the effectiveness of the latter inhibitors is expected to be limited by their coincident ability to induce a cytoprotective response through the activation of HSF1.


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