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Small Molecule Proteostasis Regulators

Pharmacologically active small molecules that restore or enhance the heat shock response (HSR) should counterbalance the effects of aging on the proteostasis network and the proteome imbalance in protein misfolding diseases.  By promoting HSF-1 activity and enhancing the levels of molecular chaperones, we propose to compensate for the age-associated decline in cellular stress resilience by restoring the robustness of the proteostasis network and suppressing protein misfolding and aggregation.  The identification of novel small molecule regulators takes on additional relevance as a therapeutic strategy for diverse protein misfolding diseases including Alzheimer’s disease, Parkinson’s disease, ALS, Huntington’s disease, cancer, metabolic diseases and muscle wasting diseases (Morimoto and Santoro, 1998; Westerheide and Morimoto, 2005; Brandvold and Morimoto, 2015).

 

Barbara Calamini led a tour-de-force ~1mil molecule cell-based screen for small molecule proteostasis regulators using a high throughput approach employing HeLa cells expressing the human Hsp70 luciferase reporter (Calamini et al., 2011).  We identified 263 novel small molecule proteostasis regulators that chemically induced the HSR by activation of HSF-1 resulting in the elevated expression of multiple chaperones. These compounds do not appear to cause protein misfolding directly or inhibit the ubiquitin-proteasome or Hsp90, however the underlying mechanism of action of many of these compounds remains to be determined.  A notable observation is that many of these proteostasis regulators appear to have complex cell stress response signatures. In addition to inducing both HSF-1 and the expression of multiple cytoplasmic chaperones, for a subset of these molecules we also observed the induction of the UPR and the antioxidant response.  The ability of these proteostasis regulators to activate multiple cell stress response pathways was shown to have potential therapeutic effects by suppressing polyQ and mutant Huntingtin aggregation (Calamini et al., 2011) and to restore aggregation-induced inhibition of clathrin mediated endocytosis (Yu et al., 2019).  Our approach employs the cell’s biological response to damaged proteins to protect cells against chronic disease.  This supports a systems and network approach strategy for drug discovery as we harness the protective abilities of cellular stress responses to protect the cell against the multitude of deficiencies that occur during chronic proteotoxicity and stress (Calamini and Morimoto, 2012).

 

Another approach toward small molecules for ALS was initiated when Gen Matsumoto established a mammalian cell-based system to examine the properties of wild type and mutant (G85R and G93A) SOD1-CFP/YPF reporters expressed in PC12 and HeLa cells using live cell imaging to monitor the structure and properties of protein aggregates (Matsumoto et al., 2005; 2006).  Whereas Hsp70 associates transiently with mutant SOD1 aggregates in a chaperone activity-dependent manner, the proteasome was shown to be irreversibly sequestered.  Nearly all (90%) of aggregate-containing cells expressing mutant SOD1 died within 48 h, whereas 70% of cells expressing a soluble mutant SOD1 survived. We further developed this cell-based assay in collaboration with Rick Silverman (Northwestern Chemistry) and Don Kirsch (Cambia Biosciences) to identify small molecules that suppressed proteotoxicity.  This led to the identification of the arylsulfanyl pyrazolone scaffold that had potent and favorable ADME properties but required additional rounds of medicinal chemistry to increase in vitro potency and pharmacological properties in an ALS mouse model (Chen et al., 2012).  To establish a mechanism-of-action, pyrazolone based affinity probes were synthesized to identify high affinity binding partners.  These probes retained their neuroprotective properties in PC12-SOD1(G93A) cells and were used to identify by protein pull-down, affinity purification and proteomic analysis using LC-MS/MS specific subunits of the 26S proteasome and the CCT chaperonin as putative protein targets (Trippier et al., 2014).

 

Another intriguing class of bioactive molecules with established human safety that induce the HSR are the non-steroidal anti-inflammatory drugs (NSAIDS) including sodium salicylate and indomethacin.  Don Jurivich showed that sodium salicylate treatment of HeLa cells induces HSF1 trimer formation and in vivo binding to the heat shock elements (HSEs) in the promoters of the human Hsp70 gene. However, unlike the effects of heat shock, sodium salicylate did not induce hyperphosphorylation of HSF1 that is required for HSF-1 transcriptional activity (Jurivich et al., 1992). From a mechanistic perspective, the effects of sodium salicylate provided a tool to dissect the HSF-1 cycle by demonstrating that HSF-1 trimerization and acquisition of DNA binding in vivo is insufficient for inducible transcription, and that these events precede HSF-1 hyperphosphorylation and transcriptional activity. Indomethacin, on the other hand, studied by Betty Lee lead to full induction of a heat shock response and at the cellular level conferred stress resilience (Lee et al., 1995). The protective effects of HSF-1 and molecular chaperones could, in part, be related to the many and diverse beneficial effects of NSAIDS.

 

In further support of the relationship between inflammation and the HSR is the regulation of the inflammatory response by a signaling cascade involving arachidonic acid release and metabolism. Don Jurivich further examined the effects of Phospholipase A2, which stimulates arachidonic acid release, and showed that arachidonate itself activates the HSR (Jurivich et al., 1996; Jurivich et al., 1994). These results were consistent with observations made by Carla Amici that cyclopentenone prostaglandins PGA1, PGA2 and PGJ2, which are downstream products of arachidonic acid, also activate HSF-1 and the HSR (Amici et al., 1992).

 

A parallel large-scale study initiated 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) brought together ~30 laboratories to join forces on a multi-lab screening program using repurposed drugs for neurodegenerative diseases.  Using HeLa cells expressing the human Hsp70-luciferase reporter (Calamini et al., 2011), we identified a small number of compounds including the natural product Celastrol that was independently identified by other research teams using different assays for suppression of neurodegenerative disease phenotypes.  Celastrol is a quinone methide triterpene and an active component from Chinese herbal medicine that strongly induces the human HSR.  Sandy Westerheide subsequently demonstrated that Celastrol activates HSF-1 in human tissue culture cells with kinetics similar to those of heat shock, as determined by the induction of HSF-1 DNA binding, hyperphosphorylation of HSF-1, and transcription induction of heat shock genes (Westerheide et al., 2004).  Celastrol activates heat shock gene transcription synergistically with other types of cell stress conditions to confer resilience against subsequent exposures to other forms of lethal cell stress.  Together with Lada Klaic in the Silverman laboratory, medical chemistry was used to increase the specificity of Celastrol analogues on induction of the human HSR (Klaic et al., 2012).

 

Another class of interesting regulators of the HSR are inhibitors of HSF-1. The expression of molecular chaperones is elevated in most tumors and transformed cell lines with constitutive activation of HSF1 implicated in tumor formation. Sandy Westerheide identified triptolide, a diterpene triepoxide from the plant Triptergium wilfordii, as an inhibitor of the HSR (Westerheide et al., 2006). Triptolide treatment of human tissue culture cells inhibited 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 HSF-1 activation pathway, we have found that triptolide abrogates the transactivation function of HSF-1 without interfering in the early events of trimer formation, hyperphosphorylation, and HSF1 DNA binding. The ability of triptolide to inhibit the cellular HSR renders these cells hyper-sensitive to stress-induced cell death. This suggests that triptolide or other compounds that inhibit HSF-1 could be used to potentiate the efficacy of chemotherapeutic drugs in cancer treatments.

References

Amici C, Sistonen L, Santoro MG, Morimoto RI. Anti-Proliferative Prostaglandins Activate Heat Shock Transcription Factor. Proc. Natl. Acad. Sci. 89: 6277-6231. (1992).

 

Benmohamed, R., A. C. Arvanites, K. Cormier, D. J. Amante, R. J. Ferrante, R. B. Silverman, R.I. Morimoto, and D. R. Kirsch. Identification of Compounds Protective Against G93A SOD1 Toxicity for the Potential Treatment of Amyotrophic Lateral Sclerosis.  Amyotrophic Lateral Sclerosis 12(2): 87-96.  DOI: 10.3109/17482968.2010.522586 (2011).

 

Brandvold, K.R., and R.I. Morimoto. Chemical Biology of Proteostasis – Targeting Molecular Chaperones. In Molecular Chaperones and Protein Quality Control, editor – J. Buchner. J. Mol. Biology 427: 2931-2947. DOI:10.1016/j.jmb.2015.05.010 PMID: 26003923 (2015).

 

Calamini, B., and R.I. Morimoto.  Protein Homeostasis as a Therapeutic Target for Diseases of Protein Conformation. Current Topics in Medicinal Chemistry 12: 2623-2640. PMID: 23339312 (2012).

 

Calamini, B., C. Silva, F. Madoux, D. M. Hutt, S. Khanna, M. Chalfant, P. Hodder, B. Tait, D. Garza, W. Balch, and R.I. Morimoto. Small Molecule Proteostasis Regulators for Protein Conformational Disease. Nature Chemical Biology 8(2): 185-96. DOI. 10.1038/nchembio.763 (2011).

 

Chen, T, R. Benmohamed, A.C. Arvanites, H.R. Ranaivo, R.I. Morimoto, R.J. Ferrante, D.M. Watterson, D.R. Kirsch, and R.B. Silverman. Arylsulfanyl pyrazolones block mutant SOD1-G93A aggregation. Potential application for the treatment of amyotrophic lateral sclerosis. Bioorganic and Medicinal Chemistry 19: 613-622. PMID: 21095130 (2011).

 

Chen, T. R. Benmohamed, J. Kim, K. Smith, D. Amante, R.I. Morimoto, D. Kirsch, R. Ferrante, and R.B. Silverman.  ADME-Guided Design and Synthesis of Aryloxanyl Pyrazolone Derivatives to Block Mutant Superoxide Dismutase 1 (SOD1) Cytotoxicity and Protein Aggregation: Potential Application for the Treatment of Amyotrophic Lateral Sclerosis. J. Med. Chem. PMID: 22191331 (2011).

 

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).

 

Jurivich D, Sistonen L, Kroes R, Morimoto RI. Effects of Sodium Salicylate on the Human Heat Shock Response. Science 255: 1243-1245. (1992).

 

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).

 

Klaic, L., R.I. Morimoto, and R.B. Silverman. Celastrol Analogs as Inducers of the Heat Shock Response: Design and Synthesis of Affinity Probes for the Identification of Protein Targets. American Chemical Society Chemical Biology 7: 928-37. PMID: 22380712 (2012).

 

Klaic, L., P. Trippier, R. Mishra, R.I. Morimoto, and R. B. Silverman. Remarkable Stereospecific Conjugate Additions to the Hsp90 Inhibitor Celastrol. Journal of the American Chemical Society 133: 19634-7. (2011). 

 

 

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).

 

Matsumoto, G., A. Stojanovic, C. Holmberg, S. Kim, and R.I. Morimoto.  Structural Properties and Neuronal Toxicity of ALS-Associated SOD1 Aggregates.  J. Cell Biology 171: 75-85. (2005).

 

Morimoto RI, Santoro MG Stress-inducible responses and heat shock proteins: new pharmacologic targets for cytoprotection. Nat Biotechnol Sep;16(9): 833-8. (1998).

 

Trippier, P., K.T. Zhao, S.G. Fox, I. Schiefer, R. Benmohamed, J. Moran, D. Kirsch, R.I. Morimoto, and R. Silverman.  Proteasome Activation is a Mechanism for Pyrazolone Small Molecules Displaying Therapeutic Potential in Amyotrophic Lateral Sclerosis.  ACS Chemical Neuroscience 5: 823-829. DOI: 10.1021/cn500147v, PMID: 25001311 (2014).

 

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).

 

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).

 

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).

 

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).

 

Xia, G., R. Benmohamed, J. Kim, A. C. Arvanites, R. I. Morimoto, R. J. Ferrante, D. R. Kirsch, and R. B. Silverman. Pyrimidine-2,4,6-trione Derivatives and Their Inhibition of Mutant SOD1-Dependent Protein Aggregation. Towards a Treatment for Amyotrophic Lateral Sclerosis. J. Med. Chem. 54: 2409-2421 (2011).

 

Yu, A., S.G. Fox, A. Cavallini, C. Kerridge, M. J. O’Neill, J. Wolak, S. Bose, and R. I. Morimoto. Tau Protein Aggregates Inhibit the Protein-Folding and Vesicular Trafficking Arms of the Cellular Proteostasis Network. J. Biological Chemistry 294(19): 7917-7930 DOI:10.1074/jbc.RA119.007527 (2019).

 

Zhang, W., R. Benmohamed, A.C. Arvanites, R.I. Morimoto, R.J. Ferrante, D.R. Kirsch, and R.B. Silverman.  Cyclohexane 1,3-diones and Their Inhibition of Mutant SOD1-dependent Protein Aggregation and Toxicity in PC12 Cells. Bioorganic and Medicinal Chemistry 20: 1029-1045. PMID: 22189272 (2012).

 

Zhang, Y., K. Zhao, S. Fox, J. Kim, D. Kirsch, D. Ferrante, R.I. Morimoto, and R. Silverman. Tertiary Amine Pyrazolones and Their Salts as Inhibitors of Mutant Superoxide Dismutase 1-Dependent Protein Aggregation for the Treatment of Amyotrophic Lateral Sclerosis. Journal of Medicinal Chemistry 58: 5942-5949. PMID: 26186011 (2015).