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SMALL MOLECULE SCREEN FOR THE STRESS RESPONSE

Small Molecules that Regulate the Heat Shock Response and the Activities of Molecular Chaperones

Regulation of the heat shock response by pharmacologically active small molecules has proven invaluable as a tool to elucidate steps in the regulation of HSF1 activity. We are interested in the further identification of novel small molecules for mechanistic studies on HSF1 regulation. Moreover, with the potential applications of HSF1 and molecular chaperones to diseases of protein conformation, the identification of novel small molecule regulators and their structure/function analysis has taken on additional interest for therapeutic purposes (Morimoto and Santoro, 1998; Westerheide and Morimoto, 2005).

1. Small Molecules Inducers of the Heat Shock Response

a. NSAIDS

We have, for some time, been interested in non-steroidal anti-inflammatory drugs (NSAIDS), including sodium salicylate and indomethacin, as potent regulators of HSF1 activity. Sodium salicylate treatment induces HSF1 trimer formation and in vivo occupancy of heat shock elements (HSEs) in the promoters of target genes. However, sodium salicylate does not induce the hyperphosphorylation of HSF1 that is required for transcriptional activity. Studies with sodium salicylate were thus central to the initial dissection of the HSF1 activation pathway: trimerization and DNA binding of HSF1 occur prior to hyperphosphorylation and transcriptional activity (Cotto et al., 1996; Jurivich et al., 1992). Indomethacin, on the other hand, leads to full induction of a heat shock-like response and cytoprotection (Lee et al., 1995). The protective effects of HSF1 and molecular chaperones could, in part, be responsible for the many and diverse positive actions of NSAIDS.

b. Prostaglandins

Many molecules that regulate inflammation can activate the heat shock response by induction of HSF1, providing a potential link between the heat shock response and inflammation. The inflammatory response is induced by a signaling cascade involving arachidonic acid release and metabolism. Phospholipase A2, which stimulates arachidonic acid release, and arachidonate itself both activate the heat shock response (Jurivich et al., 1996; Jurivich et al., 1994). The cyclopentenone prostaglandins PGA1, PGA2 and PGJ2, which are produced downstream of arachidonic acid release, also lead to activation of HSF1 (Amici et al., 1992; Ohno et al., 1988).

c. Celastrol

The expression of molecular chaperones has been shown to suppress protein misfolding/aggregation and cellular toxicity phenotypes in model systems associated with Huntington’s disease, Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis (ALS). Therefore, we hypothesized that the identification of small molecules that activate HSF1 may lead to the development of novel therapies beneficial to the arrest, retardation, or prevention of neurodegenerative diseases. On the basis of this hypothesis, our laboratory recently took part in a multi-lab screening program organized by the National Institute of Neurological Disorders and Stroke (NINDS), Huntington Disease Society of America (HDSA), Hereditary Disease Foundation (HDF), and the Amyotrophic Lateral Sclerosis Association (ALSA) to identify new drugs for treating neurodegenerative diseases. In our screen, we searched for drugs that activated the heat shock response and identified a common set of compounds, the most effective being the natural product celastrol.

Celastrol, a quinone methide triterpene and an active component from Chinese herbal medicine, strongly activates the human heat shock response (Westerheide et al., 2004). From a structure/function examination, the celastrol structure is remarkably specific and activates the heat shock transcription factor HSF1 with kinetics similar to those of heat stress, as determined by the induction of HSF1 DNA binding, hyperphosphorylation of HSF1, and expression of chaperone genes. Celastrol can activate heat shock gene transcription synergistically with other stresses and exhibits cytoprotection against subsequent exposures to other forms of lethal cell stress. These results suggest that celastrols exhibit promise as a new class of pharmacologically active regulators of the heat shock response.

2. Small Molecule Inhibitors of the Heat Shock Response

Triptolide

Inhibitors of HSF1 activity represent another potentially interesting class of small molecules. The expression of molecular chaperones is elevated in most tumors and transformed cell lines with constitutive activation of HSF1 implicated in tumor formation. We have identified triptolide, a diterpene triepoxide from the plant Triptergium wilfordii, as an inhibitor of the human heat shock response (Westerheide et al., 2006). Triptolide treatment of human tissue culture cells inhibits the heat shock-induced expression of an hsp70 promoter-reporter construct and suppresses the stress-inducible expression of endogenous hsp70 gene expression. Upon examining each of the steps in the HSF1 activation pathway, we have found that triptolide abrogates the transactivation function of HSF1 without interfering in the early events of trimer formation, hyperphosphorylation, and HSF1 DNA binding. The ability of triptolide to inhibit the cellular heat shock response renders these cells hyper-sensitive to stress-induced cell death. This suggests that triptolide may potentiate the efficacy of chemotherapeutic drugs in cancer treatments.

Future screens

Additional major efforts are ongoing for screening small molecule libraries and characterization of a number of promising small molecule candidates, the identification of the intracellular targets, and complete structure/function analyses to identify an optimal chemistry (in collaboration with Prof. Rick Silverman, Department of Chemistry).

References

  1. Trott A., J.D. West, L. Klaic, S.D. Westerheide, R.B. Silverman, R.I. Morimoto, and K.A. Morano. Activation of Heat Shock and Antioxidant Responses by the Natural Product Celastrol: Transcriptional Signatures of a Thiol-targeted Molecule. Molecular Biology of the Cell 19: 1104-1112 (2008).
  2. Westerheide, S., T. Kawahara, K. Orton, and R.I. Morimoto, Triptolide, an Inhibitor of the Human Heat Shock Response that Enhances Stress-Induced Cell Death. J. Biol. Chem. 281: 9616-9622. (2006).
  3. Westerheide, S., and R.I. Morimoto. Heat Shock Response Modulators as Therapeutic Tools for Diseases of Protein Misfolding. J. Biol. Chem. 380: 33097-33100 (2005).
  4. Westerheide, S., J. Bosman, B. Mbadugha, T. Kawahara, G. Matsumoto, W. Gu, S. Kim, J. Devlin, R. Silverman, and R.I. Morimoto. Celastrols as Inducers of the Human Heat Shock Response and Cytoprotection J. Biol. Chem. 279: 56053-60 (2004).
  5. Morimoto RI, Santoro MG Stress-inducible responses and heat shock proteins: new pharmacologic targets for cytoprotection Nat Biotechnol 1998 Sep;16(9):833-8.
  6. Cotto J, Kline MJ, Morimoto RI. Activation of Heat Shock Factor 1 DNA Binding Precedes Stress-Induced Serine Phosphorylation: Evidence for Multi-step Pathway of Regulation. J. Biological Chemistry 271: 3355-3358 (1996).
  7. Lee B, Chen J, Jurivich D, Angelidis C, Morimoto RI. Pharns/lee/pnas/macological Modulation of the Heat Shock Factor Activity by Anti-Inflammatory Drugs Protects Against Stress-Induced Cellular Damage. Proc. Natl. Acad. Sci. USA 92: 7207-7211 (1995).
  8. Jurivich D, Sistonen L, Sarge KD, Morimoto RI. Arachidonic Acid is a Potent Modulator of Human Heat Shock Gene Transcription. Proc. Natl. Acad. Sci. USA 91: 2280-2284 (1994).
  9. Jurivich D, Sistonen L, Kroes R, Morimoto RI. Effects of Sodium Salicylate on the Human Heat Shock Response. Science 255, 1243-1245 (1992).
  10. Amici C, Sistonen L, Santoro MG, Morimoto RI. Anti-Proliferative Prostaglandins Activate Heat Shock Transcription Factor. Proc. Natl. Acad. Sci. 89, 6277-6231 (1992).
Transcriptional regulation of heat shock response
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Small molecule screen for the stress response
Systems Approach to Stress Biology