Daniel Gewirth, PhD
Humans owe their existence to the proteins that reside in, on, and around every cell in the body. Proteins are useless, however, unless they are folded correctly into their active form. Proteins don’t last forever; they also get old and need to be disposed of properly. Failure at either end of the protein life cycle – folding at the beginning or disposal at the end – is a cellular catastrophe that leads inevitably to misfunction or death.
Recognizing the absolute importance of protein quality control, organisms have evolved a specialized class of proteins called chaperones. Chaperones help other proteins achieve their folded, active form or partition into the disposal system when they are old or damaged. Virtually every protein in the body at one time or another will interact with a member of the chaperone machinery, an indication of the importance placed on these mediators for cellular quality control. Diseases such as cancer, neurodegeneration, and infection, to name a few, are characterized by potentially lethal alterations to the amount and overall composition of the proteins in the cell. Paradoxically, the same chaperone systems that help maintain cellular order in normal cells are often co-opted by diseases to perpetuate the survival of diseased cells with altered protein burdens.
Drugs that selectively target individual members of the chaperone machinery offer great potential in the treatment of disease. The goal of the Gewirth lab is to understand these chaperone systems and to exploit these insights in the development of novel therapeutics.
A Deeper Understanding
Laboratory Research Interests:
- Structural studies of Hsp90 chaperones, drug design, protein folding.
- Steroid hormone receptors.
1. Structure-Function Studies of hsp90 chaperones. The hsp90 family of molecular chaperones are key players in the conformational maturation and folding of an extensive array of cellular client proteins ranging from steroid receptors to regulatory kinases, Toll-like receptors, G-proteins, telomerases and many others. Inhibitors of hsp90s, such as geldanamycin, are potent anti-tumor compounds because of their inhibitory effect on the maturation of clients that are key players in cellular transformation and malignancy, and are the subject of intense pharmacological interest. The mechanism by which hsp90 chaperones act to mature client proteins is still poorly understood. Over the past decade, our group has determined a series of structures of Grp94, the endoplasmic recticulum hsp90 paralog. Grp94 has a specialized but important portfolio of client proteins that include cell surface receptors, integrins, and growth factors. The structures we have determined have been of both the intact protein as well as its regulatory N-terminal domain, and have been solved in complex with a variety of inhibitory and naturally-occuring ligands. These structures and the associated biochemical and functional studies have revealed, among other highlights, a ligand-dependent switch in the N-terminal domain, unique conformational and quaternary states of the intact chaperone, a regulatory role for the extensive pre-N terminal portion of the molecule, and the mechanism by which Grp94 selectively binds to certain types of inhibitory ligands. These last insights are currently being exploited in the design and synthesis of novel Grp94 inhibitors, early versions of which have been shown to target HER2+ breast cancer cell lines. Grp94-selective compounds not only have therapeutic potential but should also prove useful for dissecting the cellular roles of the individual hsp90s. Recent structures of intact Grp94 have now begun to shed light on potential client binding interactions, and ongoing studies aim to deepen our understanding of this important mechanistic question.
2. Studies of the Androgen Receptor and characterization of novel AR inhbitors. The androgen receptor (AR) is the key cellular mediator of prostate cancer, the most common form of cancer to afflict western men after lung cancer. Late stage prostate cancers are often associated with androgen insensitivity, which renders standard castration and anti-androgen therapies ineffective. The androgen receptor is a transcription factor that binds to specific DNA targets. Our group has determined the structure of the AR DBD bound to a selective DNA target. The structure explains how AR forms the dimeric interactions that allow it to bind to these elements. Compounds that specifically interfere with the protein-protein or protein-DNA interactions constitute a new approach to anti-androgen therapy. Ongoing studies are also now examining the N-terminal activation domain of the receptor, which may form tertiary interactions with the other domains of the receptor and allow constituitive gene activation. This analysis should explain the hormone independent activity of the AR and may lead to the development of novel anti-androgens that do not target the mutagenically sensitive hormone binding domain.
Molecular Stressors Engender Protein Connectivity Dysfunction through Aberrant N-Glycosylation of a Chaperone. Yan P, Patel HJ, Sharma S, Corben A, Wang T, Panchal P, Yang C, Sun W, Araujo TL, Rodina A, Joshi S, Robzyk K, Gandu S, White JR, de Stanchina E, Modi S, Janjigian YY, Hill EG, Liu B, Erdjument-Bromage H, Neubert TA, Que NLS, Li Z, Gewirth DT, Taldone T, Chiosis G. Cell Rep. 2020 Jun 30;31(13):107840. doi: 10.1016/j.celrep.2020.107840. PMID: 32610141
Thrombin contributes to cancer immune evasion via proteolysis of platelet-bound GARP to activate LTGF-β. Metelli A, Wu BX, Riesenberg B, Guglietta S, Huck JD, Mills C, Li A, Rachidi S, Krieg C, Rubinstein MP, Gewirth DT, Sun S, Lilly MB, Wahlquist AH, Carbone DP, Yang Y, Liu B, Li Z. Sci Transl Med. 2020 Jan 8;12(525):eaay4860. doi: 10.1126/scitranslmed.aay4860. PMID: 31915300
NECA derivatives exploit the paralog-specific properties of the site 3 side pocket of Grp94, the endoplasmic reticulum Hsp90. Huck JD, Que NLS, Immormino RM, Shrestha L, Taldone T, Chiosis G, Gewirth DT. J Biol Chem. 2019 Nov 1;294(44):16010-16019. doi: 10.1074/jbc.RA119.009960. Epub 2019 Sep 9. PMID: 31501246
Structures of Hsp90α and Hsp90β bound to a purine-scaffold inhibitor reveal an exploitable residue for drug selectivity. Huck JD, Que NLS, Sharma S, Taldone T, Chiosis G, Gewirth DT. Proteins. 2019 Oct;87(10):869-877. doi: 10.1002/prot.25750. Epub 2019 Jun 12. PMID: 31141217
Chaperome heterogeneity and its implications for cancer study and treatment. Wang T, Rodina A, Dunphy MP, Corben A, Modi S, Guzman ML, Gewirth DT, Chiosis G. J Biol Chem. 2019 Feb 8;294(6):2162-2179. doi: 10.1074/jbc.REV118.002811. Epub 2018 Nov 8. PMID: 30409908
Potential impact of combined inhibition of 3α-oxidoreductases and 5α-reductases on prostate cancer. Asian J Urol. 2019 Jan;6(1):50-56. doi: 10.1016/j.ajur.2018.09.002. Epub 2018 Sep 26.PMID: 30775248
Structure Based Design of a Grp94-Selective Inhibitor: Exploiting a Key Residue in Grp94 To Optimize Paralog-Selective Binding.
Que NLS, Crowley VM, Duerfeldt AS, Zhao J, Kent CN, Blagg BSJ, Gewirth DT. J Med Chem. 2018 Apr 12;61(7):2793-2805. doi: 10.1021/acs.jmedchem.7b01608. Epub 2018 Mar 20. PMID: 29528635
Bypassing Drug Resistance Mechanisms of Prostate Cancer with Small Molecules that Target Androgen Receptor-Chromatin Interactions.
Dalal K, Che M, Que NS, Sharma A, Yang R, Lallous N, Borgmann H, Ozistanbullu D, Tse R, Ban F, Li H, Tam KJ, Roshan-Moniri M, LeBlanc E, Gleave ME, Gewirth DT, Dehm SM, Cherkasov A, Rennie PS. Mol Cancer Ther. 2017 Oct;16(10):2281-2291. doi: 10.1158/1535-7163.MCT-17-0259. Epub 2017 Aug 3. PMID: 28775145
Structural and Functional Analysis of GRP94 in the Closed State Reveals an Essential Role for the Pre-N Domain and a Potential Client-Binding Site. Huck JD, Que NL, Hong F, Li Z, Gewirth DT. Cell Rep. 2017 Sep 19;20(12):2800-2809. doi: 10.1016/j.celrep.2017.08.079.
Exploring the Functional Complementation between Grp94 and Hsp90. Maharaj, K.A., Que, N.L.S., Gill, S., Wu, S., Li, Z., and Gewirth, D.T. (2016) PloS ONE. 11: e0166271. PMID: 27824935.
Paralog Specific Hsp90 Inhibitors – A Brief History and a Bright Future. Gewirth DT. Curr Top Med Chem. 2016;16(25):2779-91. Review.
Clients and Oncogenic Roles of Molecular Chaperone gp96/grp94. Ansa-Addo EA, Thaxton J, Hong F, Wu BX, Zhang Y, Fugle CW, Metelli A, Riesenberg B, Williams K, Gewirth DT, Chiosis G, Liu B, Li Z. Curr Top Med Chem. 2016;16(25):2765-78. Review.
Structure-activity relationship in a purine-scaffold compound series with selectivity for the endoplasmic reticulum Hsp90 paralog Grp94. Patel HJ, Patel PD, Ochiana SO, Yan P, Sun W, Patel MR, Shah SK, Tramentozzi E, Brooks J, Bolaender A, Shrestha L, Stephani R, Finotti P, Leifer C, Li Z, Gewirth DT, Taldone T, Chiosis G. J Med Chem. 2015 May 14;58(9):3922-43. doi: 10.1021/acs.jmedchem.5b00197. Epub 2015 Apr 22.
Characterization of the Grp94/OS-9 chaperone-lectin complex. Seidler PM, Shinsky SA, Hong F, Li Z, Cosgrove MS, Gewirth DT. J Mol Biol. 2014 Oct 23;426(21):3590-605. doi: 10.1016/j.jmb.2014.08.024. Epub 2014 Sep 3.
Paralog-selective Hsp90 inhibitors define tumor-specific regulation of HER2. Patel PD, Yan P, Seidler PM, Patel HJ, Sun W, Yang C, Que NS, Taldone T, Finotti P, Stephani RA, Gewirth DT, Chiosis G. Nat Chem Biol. 2013 Nov;9(11):677-84. doi: 10.1038/nchembio.1335. Epub 2013 Sep 1.
Experimental and structural testing module to analyze paralogue-specificity and affinity in the Hsp90 inhibitors series. Taldone T, Patel PD, Patel M, Patel HJ, Evans CE, Rodina A, Ochiana S, Shah SK, Uddin M, Gewirth D, Chiosis G. J Med Chem. 2013 Sep 12;56(17):6803-18. doi: 10.1021/jm400619b. Epub 2013 Aug 21.
α7 helix region of αI domain is crucial for integrin binding to endoplasmic reticulum chaperone gp96: a potential therapeutic target for cancer metastasis. Hong F, Liu B, Chiosis G, Gewirth DT, Li Z. J Biol Chem. 2013 Jun 21;288(25):18243-8. doi: 10.1074/jbc.M113.468850. Epub 2013 May 13.
The molecular chaperone gp96/GRP94 interacts with Toll-like receptors and integrins via its C-terminal hydrophobic domain. Wu S, Hong F, Gewirth D, Guo B, Liu B, Li Z. J Biol Chem. 2012 Feb 24;287(9):6735-42. doi: 10.1074/jbc.M111.309526. Epub 2012 Jan 5.
Different poses for ligand and chaperone in inhibitor-bound Hsp90 and GRP94: implications for paralog-specific drug design. Immormino RM, Metzger LE 4th, Reardon PN, Dollins DE, Blagg BS, Gewirth DT. J Mol Biol. 2009 May 22;388(5):1033-42. doi: 10.1016/j.jmb.2009.03.071. Epub 2009 Apr 8.
Structures of GRP94-nucleotide complexes reveal mechanistic differences between the hsp90 chaperones. Dollins DE, Warren JJ, Immormino RM, Gewirth DT. Mol Cell. 2007 Oct 12;28(1):41-56.
The role of antibody polyspecificity and lipid reactivity in binding of broadly neutralizing anti-HIV-1 envelope human monoclonal antibodies 2F5 and 4E10 to glycoprotein 41 membrane proximal envelope epitopes. Alam SM, McAdams M, Boren D, Rak M, Scearce RM, Gao F, Camacho ZT, Gewirth D, Kelsoe G, Chen P, Haynes BF. J Immunol. 2007 Apr 1;178(7):4424-35.
Structural and quantum chemical studies of 8-aryl-sulfanyl adenine class Hsp90 inhibitors. Immormino RM, Kang Y, Chiosis G, Gewirth DT. J Med Chem. 2006 Aug 10;49(16):4953-60.
Structure of UDP-N-acetylglucosamine acyltransferase with a bound antibacterial pentadecapeptide. Williams AH, Immormino RM, Gewirth DT, Raetz CR. Proc Natl Acad Sci U S A. 2006 Jul 18;103(29):10877-82. Epub 2006 Jul 11.
Identification of potent water soluble purine-scaffold inhibitors of the heat shock protein 90. He H, Zatorska D, Kim J, Aguirre J, Llauger L, She Y, Wu N, Immormino RM, Gewirth DT, Chiosis G. J Med Chem. 2006 Jan 12;49(1):381-90.
Structure of unliganded GRP94, the endoplasmic reticulum Hsp90. Basis for nucleotide-induced conformational change. Dollins DE, Immormino RM, Gewirth DT. J Biol Chem. 2005 Aug 26;280(34):30438-47. Epub 2005 Jun 11.
Characterization of transcriptional activation and DNA-binding functions in the hinge region of the vitamin D receptor. Shaffer PL, McDonnell DP, Gewirth DT. Biochemistry. 2005 Feb 22;44(7):2678-85.
Ligand-induced conformational shift in the N-terminal domain of GRP94, an Hsp90 chaperone. Immormino RM, Dollins DE, Shaffer PL, Soldano KL, Walker MA, Gewirth DT. J Biol Chem. 2004 Oct 29;279(44):46162-71. Epub 2004 Aug 2.
DNA recognition by nuclear receptors. Claessens F, Gewirth DT. Essays Biochem. 2004;40:59-72. Review.
Structural analysis of RXR-VDR interactions on DR3 DNA. Shaffer PL, Gewirth DT. J Steroid Biochem Mol Biol. 2004 May;89-90(1-5):215-9.
Vitamin D receptor-DNA interactions. Shaffer PL, Gewirth DT. Vitam Horm. 2004;68:257-73. Review.
Structure of the N-terminal domain of GRP94. Basis for ligand specificity and regulation. Soldano KL, Jivan A, Nicchitta CV, Gewirth DT. J Biol Chem. 2003 Nov 28;278(48):48330-8. doi: 10.1074/jbc.M308661200. Epub 2003 Sep 11. PMID: 12970348
Structural basis of VDR-DNA interactions on direct repeat response elements. Shaffer PL, Gewirth DT. EMBO J. 2002 May 1;21(9):2242-52. doi: 10.1093/emboj/21.9.2242.
Structural studies of a yeast quaternary transcription-initiation complex. Wang SM, Kim GJ, Gewirth DT. Acta Crystallogr D Biol Crystallogr. 2001 Mar;57(Pt 3):441-4. doi: 10.1107/s0907444901000683. PMID: 11223526
The basis for half-site specificity explored through a non-cognate steroid receptor-DNA complex. Gewirth DT, Sigler PB. Nat Struct Biol. 1995 May;2(5):386-94. doi: 10.1038/nsb0595-386. PMID: 7664096
On the use of T7 RNA polymerase transcripts for physical investigation. Szewczak AA, White SA, Gewirth DT, Moore PB. Nucleic Acids Res. 1990 Jul 25;18(14):4139-42. doi: 10.1093/nar/18.14.4139. PMID: 1696001
Exploration of the L18 binding site on 5S RNA by deletion mutagenesis. Gewirth DT, Moore PB. Nucleic Acids Res. 1988 Nov 25;16(22):10717-32. doi: 10.1093/nar/16.22.10717. PMID: 3060848
Preparation of 5S RNA-related materials for nuclear magnetic resonance and crystallography studies. Moore PB, Abo S, Freeborn B, Gewirth DT, Leontis NB, Sun G. Methods Enzymol. 1988;164:158-74. doi: 10.1016/s0076-6879(88)64041-9. PMID: 3071660.
Effects of mutation on the downfield proton nuclear magnetic resonance spectrum of the 5S RNA of Escherichia coli. Gewirth DT, Moore PB.
Biochemistry. 1987 Sep 8;26(18):5657-65. doi: 10.1021/bi00392a012. PMID: 3314994
Secondary structure of 5S RNA: NMR experiments on RNA molecules partially labeled with nitrogen-15. Gewirth DT, Abo SR, Leontis NB, Moore PB. Biochemistry. 1987 Aug 11;26(16):5213-20. doi: 10.1021/bi00390a047. PMID: 2444255
Assignment of resonances in the downfield proton spectrum of Escherichia coli 5S RNA and its nucleoprotein complexes using components of a ribonuclease-resistant fragment. Kime MJ, Gewirth DT, Moore PB. Biochemistry. 1984 Jul 17;23(15):3559-68. doi: 10.1021/bi00310a027. PMID: 6380589
Daniel T. Gewirth, PhD
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