Chemical Inhibitors of Hsp70 for Breast Cancer

Institution: The Burnham Institute for Medical Research
Investigator(s): Chung-Wai Shiau, Ph.D. -
Award Cycle: 2006 (Cycle 12) Grant #: 12FB-0098 Award: $103,232
Award Type: Postdoctoral Fellowship
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure



Initial Award Abstract (2006)

It is a critical problem that chemotherapy drugs are often ineffective for treatment of advanced breast cancer. Endogenous protection mechanisms within breast cancer cells often blocks effective chemotherapy treatment. Heat shock protein 70 (Hsp70) represents one of critical cell protection proteins in breast cancer. Normal cells contain few Hsp70 molecules. However, the amount of Hsp70 molecules in breast cancer is increased hundreds of times over that of normal cells. Therefore, breast cancer cells can escape the toxicity of chemotherapy drugs, in part, because they contain too much protective Hsp70. The purpose of this research is to generate chemical inhibitors of Hsp70 that might eventually be used as new drugs for treating breast cancer.

By generating chemicals that selectively inhibit Hsp70, we will be able to directly test this hypothesis and begin the process of creating new drugs for breast cancer therapy. To identify chemicals that inhibit Hsp70, we will use robotic instrumentation to screen a large collection (i.e., library) of chemicals, which each have a different shape (structure). This empirical search is expected to yield a small number of molecules that have just the right shape to bind to surface features of the Hsp70 protein. Then, the active chemicals will be modified by to adjust their shape, so they bind more avidly to the surface of Hsp70, thus increasing the chances of inhibition. In addition, the structural optimization of Hsp70 inhibitors will be assisted by modern techniques of structure-based drug discovery, especially using high-field nuclear magnetic resonance (NMR) and computer modeling. This allows a state-of-the-art visualization of the 3-dimensional structure of Hsp70 with chemical inhibitors bound to it. Finally, the chemical inhibitors of Hsp70 will be tested for their ability to kill breast cancer cells in culture, when used alone or in combination with anti-cancer drugs, such as Taxotere and Adriamycin. Ultimately, we will be poised to advance the best of our Hsp70 inhibitors into animal models of breast cancer, to more definitively test their anti-tumor activity as well as toxicity.

To date, potent Hsp70 inhibitors have never been produced or tested as cancer therapies. However, recently it was shown that chemicals that inhibit a molecule with similar functions, called Hsp90, have anti-tumor activity and an acceptable safety profile. Hsp90 inhibitors are now in Phase II clinical trials for refractory cancers. These recent advances make it more likely that this project will succeed and advance a new breast cancer target and drug strategy closer to eventual translation.




Final Report (2010)

Note: the PI resigned the grant in the third year of the grant.

In this project we have completed
1) screen a chemical library using the HSP70 Fluorescence Polarization Assay (FPA),
2) employ NMR-based lead optimization techniques and computer modeling to increase the potency of qualified compounds discovered by the screen,
3) test the optimized compounds for anti-HSP70 activity in biochemical and cell –based assays,
4) determine the cytotoxic activity of the optimized compounds against cultured breast cancer cell lines.

To summarize our previous results, we screened 60,000 compounds with the HSP70 FPA and confirmed their effects by experimental repetition and in biochemical assays designed to test the ability of the compound to interfere with the chaperone functions of HSC70 and its co-chaperone as it acts on an unfolded ß-galactosidase polypeptide.

We used NMR to measure Inter-Ligand Nuclear Overhauser Effects (ILOE) to discriminate compound fragments that exchange Nuclear Overhauser Effects (NOE) in the presence but not the absence of the HSP70 protein and thus assign these peaks in an effort to synthesize these fragments into a single incorporation. To this effect, 32 compounds elicit NOE signatures and 25 fragment pairs derived from 19 fragments have been confirmed to give ILOEs with HSC70

Additionally we have solved the crystal structures for ADP, phosphate, betaine and glycerol as in complex with HSC70 as they are found to occupy the active site when nucleotides are removed by activated charcoal. Hence we have generated a set of 17 fragments that are phosphate and glycerol mimics in order to further explore the active sites and elucidate pharmacophores with a high affinity for HSC70. This is where we have concluded this study, since the PI was unable to continue his work due to circumstances in his home country (Taiwan) to which he has returned.




Symposium Abstract (2007)
It is a critical problem that chemotherapy drugs are often ineffective for treatment of advanced breast cancer. Endogenous cell protection mechanisms in breast cancer cells prevent effective chemotherapy treatment. Heat shock protein 70 (Hsp70) represents one of critical cell protection proteins in breast cancer. Normal cells contain few Hsp70 molecules. However, the amount of Hsp70 molecules in breast cancer is increased hundreds of times. Therefore, breast cancer cells can escape toxicity of chemotherapy drugs, in part, because they contain too much protective Hsp70. The purpose of this research is to generate chemical inhibitors of Hsp70 that might eventually be used as new drugs for treating breast cancer.

Our central hypothesis is that Hsp70 when neutralized will suppress breast cancer growth and make it easier to kill breast cancer cells using conventional anti-cancer drugs. To identify chemicals that inhibit Hsp70, we used robotic instrumentation to screen a large library of chemicals, which each have a different shape (structure) to bind to surface features of the Hsp70 protein. We found 10 compounds could bind to the pocket of Hsp70 where its cofactor ATP binds, displacing ATP. These compounds also show an ability to inhibit activity of Hsp70 in protein re-folding assays. Further, the active chemicals were combined with fragment chemical library to adjust their shape so they fit perfectly on the surface of Hsp70, thus increasing the strength of their binding and hence their potency. This optimization of the shapes of our chemicals is assisted by modern techniques of structure-based drug discovery, especially using high-field nuclear magnetic resonance (NMR) and computer modeling to view the 3-dimensional structure of Hsp70 with our chemical inhibitors bound to it. We are using organic synthesis methods to connect the active compounds with fragment chemicals to confirm the NMR and computer modeling data. Finally, the chemical inhibitors of Hsp70 will be tested for their ability to kill breast cancer cells in the test tube, when used alone or in combination with currently used anti-cancer drugs, such as Taxotere and Adriamycin. If all goes well, then we will be poised beyond the requested funding period to advance the best of our Hsp70 inhibitors into animal models of breast cancer, to more definitively test their anti-tumor activity while also assessing their safety in animals. If those animal studies produce acceptable results, then we will be ready to advance the Hsp70 inhibitors into human clinical trials.

To date, potent Hsp70 inhibitors have never been produced or tested as cancer therapies. However, recently it was shown that chemicals that inhibit a molecule with similar functions, called Hsp90, have anti-tumor activity and an acceptable safety profile. Hsp90 inhibitors are now in Phase II clinical trials for refractory cancers. Our project is innovative for tackling Hsp70 as a new possible cancer target. Our project is also innovative because it uses advanced methods of drug discovery and optimization that normally exist only at pharmaceutical companies. Most companies are risk adverse, and will not pursue targets such as Hsp70 because they are insufficiently validated. Our goal will determine whether Hsp70 and related proteins are valid drug discovery targets for breast cancer, and our project will produce chemical inhibitors to understand more about the role of Hsp70 in breast cancer biology and the underlying mechanisms of chemoresistance and radioresistance.