Restoration of testis function in hypogonadotropic hypogonadal mice harboring a misfolded GnRHR mutant by pharmacoperone drug therapy

Jo Ann Janovick, M. David Stewart, Darla Jacob, L. D. Martin, Jian Min Deng, C. Allison Stewart, Ying Wang, Anda Cornea, Lakshmi Chavali, Suhujey Lopez, Shoukhrat Mitalipov, Eunju Kang, Hyo-Sang Lee, Pulak R. Manna, Douglas M. Stocco, Richard R. Behringer, and P. Michael Conn
PNAS published ahead of print December 9, 2013 https://doi.org/10.1073/pnas.1315194110

  1. Edited by William F. Crowley, Massachusetts General Hospital, Boston, MA, and accepted by the Editorial Board November 4, 2013 (received for review September 10, 2013)

Significance

Many diseases result from genetic mutations that cause protein misfolding. Medical treatments often address the symptoms, but do not correct the underlying etiology. This study illustrates proof of principle that a disease caused by a misfolded cell surface receptor can be corrected with a pharmacoperone, a unique class of target-specific drugs that assist protein folding.

Abstract

Mutations in receptors, ion channels, and enzymes are frequently recognized by the cellular quality control system as misfolded and retained in the endoplasmic reticulum (ER) or otherwise misrouted. Retention results in loss of function at the normal site of biological activity and disease. Pharmacoperones are target-specific small molecules that diffuse into cells and serve as folding templates that enable mutant proteins to pass the criteria of the quality control system and route to their physiologic site of action. Pharmacoperones of the gonadotropin releasing hormone receptor (GnRHR) have efficacy in cell culture systems, and their cellular and biochemical mechanisms of action are known. Here, we show the efficacy of a pharmacoperone drug in a small animal model, a knock-in mouse, expressing a mutant GnRHR. This recessive mutation (GnRHR E90K) causes hypogonadotropic hypogonadism (failed puberty associated with low or apulsatile luteinizing hormone) in both humans and in the mouse model described. We find that pulsatile pharmacoperone therapy restores E90K from ER retention to the plasma membrane, concurrently with responsiveness to the endogenous natural ligand, gonadotropin releasing hormone, and an agonist that is specific for the mutant. Spermatogenesis, proteins associated with steroid transport and steroidogenesis, and androgen levels were restored in mutant male mice following pharmacoperone therapy. These results show the efficacy of pharmacoperone therapy in vivo by using physiological, molecular, genetic, endocrine and biochemical markers and optimization of pulsatile administration. We expect that this newly appreciated approach of protein rescue will benefit other disorders sharing pathologies based on misrouting of misfolded protein mutants.

Footnotes

  • ↵1J.A.J. and M.D.S. contributed equally to this work.

  • ↵2Present address: Laboratory Animal Center, Osong Medical Innovation Foundation, Chungbuk 363-951, Republic of Korea.

  • ↵3To whom correspondence should be addressed. E-mail: michael.conn{at}ttuhsc.edu.

  • Author contributions: J.A.J., M.D.S., R.R.B., and P.M.C. designed research; J.A.J., M.D.S., D.J., L.D.M., J.M.D., C.A.S., Y.W., A.C., L.C., S.L., S.M., E.K., H.-S.L., P.R.M., D.M.S., R.R.B., and P.M.C. performed research; J.A.J., M.D.S., R.R.B., and P.M.C. contributed new reagents/analytic tools; J.A.J., M.D.S., D.J., L.D.M., J.M.D., C.A.S., Y.W., A.C., L.C., S.L., S.M., E.K., H.-S.L., P.R.M., D.M.S., R.R.B., and P.M.C. analyzed data; and J.A.J., M.D.S., R.R.B., and P.M.C. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission. W.C. is a guest editor invited by the Editorial Board.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1315194110/-/DCSupplemental.

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Pharmacoperone therapy for misfolded GPCR diseases
Jo Ann Janovick, M. David Stewart, Darla Jacob, L. D. Martin, Jian Min Deng, C. Allison Stewart, Ying Wang, Anda Cornea, Lakshmi Chavali, Suhujey Lopez, Shoukhrat Mitalipov, Eunju Kang, Hyo-Sang Lee, Pulak R. Manna, Douglas M. Stocco, Richard R. Behringer, P. Michael Conn
Proceedings of the National Academy of Sciences Dec 2013, 201315194; DOI: 10.1073/pnas.1315194110
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