Green mamba peptide targets type-2 vasopressin receptor against polycystic kidney disease Justyna Cioleka,1, Helen Reinfrankb,1,2, Lo¨ıc Quintonc, Say Viengchareund, Enrico A. Sturaa, Laura Veraa,3, Sabrina Sigismeaua, Bernard Mouillace,Hel´ ene` Orcele, Steve Peigneurf, Jan Tytgatf, Laura Droctove´ a, Fabrice Beaua, Jerome Nevouxd, Marc Lombes` d, Gilles Mouriera, Edwin De Pauwc, Denis Serventa, Christiane Mendree,4, Ralph Witzgallb,4, and Nicolas Gillesa,4 aService d’Ingenierie´ Moleculaire´ des Proteines,´ Institut des Sciences du Vivant Fred´ eric´ Joliot, Commissariat a` l’Energie Atomique, Universite´ Paris-Saclay, F-91191 Gif sur Yvette, France; bInstitute for Molecular and Cellular Anatomy, University of Regensburg, 93053 Regensburg, Germany; cLaboratoire de Spectrometrie´ de Masse, Unite´ de Recherche Molecular Systems, Universite´ de Liege,` Liege` 4000, Belgium; dINSERM U1185, Universite´ Paris Sud, Universite´ Paris-Saclay, F-94276, Le Kremlin-Bicetre,ˆ France; eInstitut de Genomique´ Fonctionnelle, CNRS, INSERM, Universite´ Montpellier, F-34094 Montpellier, France; and fLaboratory of Toxicology, University of Leuven, Leuven B-3000, Belgium Edited by David W. Russell, University of Texas Southwestern Medical Center, Dallas, TX, and approved April 13, 2017 (received for review December 17, 2016) Polycystic kidney diseases (PKDs) are genetic disorders that can therapeutic approach, the use of V2R antagonists has certain cause renal failure and death in children and adults. Lowering advantages (7, 8). Most renal ADPKD cysts develop within the cAMP in cystic tissues through the inhibition of the type-2 vaso- vasopressin-sensitive tubular parts of the nephron, where vaso- pressin receptor (V2R) constitutes a validated strategy to reduce pressin is also the main hormonal regulator of adenylyl cyclase disease progression. We identified a peptide from green mamba and thus a stimulator of cAMP production. Moreover, increased venom that exhibits nanomolar affinity for the V2R without any levels of circulating vasopressin and elevated expression of V2R activity on 155 other G-protein–coupled receptors or on 15 ionic have been observed in human ADPKD and in animal models. channels. Mambaquaretin-1 is a full antagonist of the V2R acti- Finally, in contrast to somatostatin receptors, V2R has kidney- vation pathways studied: cAMP production, beta-arrestin interac- specific expression. tion, and MAP kinase activity. This peptide adopts the Kunitz fold Among the orally bio-available vasopressin antagonists, orig- known to mostly act on potassium channels and serine proteases. inally licensed for short-term treatment of heart failure and Mambaquaretin-1 interacts selectively with the V2R through its hyponatraemia, tolvaptan has been proved to be beneficial for first loop, in the same manner that aprotinin inhibits trypsin. the long-term treatment of ADPKD in adult patients (9). Tolvap- Injected in mice, mambaquaretin-1 increases in a dose-dependent tan is an aquaretic agent that inhibits V2R and reduces cAMP manner urine outflow with concomitant reduction of urine osmo- production in principal cells of the collecting tubules (10). In lality, indicating a purely aquaretic effect associated with the in vivo blockade of V2R. CD1-pcy/pcy mice, a juvenile model of Significance PKD, daily treated with 13 µg of mambaquaretin-1 for 99 d, developed less abundant (by 33%) and smaller (by 47%) cysts Polycystic kidney diseases (PKDs) are genetic disorders in than control mice. Neither tachyphylaxis nor apparent toxicity has which multiple cysts grow in kidneys, leading to end-stage been noted. Mambaquaretin-1 represents a promising therapeutic renal failure. Vasopressin antagonists (vaptans) currently used agent against PKDs. to treat PKDs have side effects due to liver toxicity. We report the characterization of Mambaquaretin-1, a Kunitz-fold j j polycystic kidney disease Kunitz peptide snake toxin polypeptide isolated from mamba venom that selectively and fully inhibits three major signaling pathways of the vaso- olycystic kidney diseases (PKDs) are potentially life-threat- pressin type-2 receptor. Mambaquaretin-1 induces a purely Pening genetic disorders that are characterized by the pres- aquaretic effect on mice and reduces cyst development in a ence of multiple fluid-filled cysts in the kidney. In PKDs, cyst for- mouse model. We produced mambaquaretin-1 by peptide syn- mation and enlargement progressively compromise normal renal thesis and determined its X-ray structure, its binding mode, parenchyma functions and with time severely distort the entire and functional properties. With high selectivity and without kidney, leading to end-stage renal failure (1). With an estimated toxic metabolic byproducts associated with its peptidic nature, prevalence of 1 in 1,000, autosomal-dominant PKD (ADPKD), mambaquaretin-1 could become the preferential treatment caused by mutations in either the polycystin-1 or polycystin-2 for these disorders. gene, is one of the most common monogenetic disorders affect- ing millions of people worldwide (2, 3). Autosomal-recessive Author contributions: E.A.S., B.M., C.M., R.W., and N.G. designed research; J.C., H.R., L.Q., PKD caused by mutations in the fibrocystin gene is 20 times less S.V., E.A.S., L.V., S.S., B.M., H.O., S.P., J.T., L.D., F.B., J.N., M.L., G.M., C.M., R.W., and N.G. performed research; J.C., H.R., L.Q., S.V., E.A.S., L.V., S.S., B.M., H.O., S.P., J.T., L.D., F.B., common than ADPKD and affects children in the first year of life J.N., M.L., G.M., C.M., R.W., and N.G. analyzed data; and J.C., L.Q., E.A.S., B.M., S.P., J.T., with a high mortality rate. No satisfactory therapy for PKDs has M.L., E.D.P., D.S., C.M., R.W., and N.G. wrote the paper. yet been developed despite huge efforts for more than 30 y. All of The authors declare no conflict of interest. the strategies, from microtubule stabilization with the antitumor This article is a PNAS Direct Submission. drug paclitaxel to the inhibition of either epidermal growth factor Data deposition: The atomic coordinates and structure factors have been deposited in receptor or B-Raf, MAPK/ERK, and mTOR kinases have failed the Protein Data Bank, www.pdb.org (PDB ID code 5M4V). to significantly improve the pathological state (4). A key advance 1 in ADPKD treatment has been the observation that in the poly- J.C. and H.R. contributed equally to this work. 2 cystic kidneys a pathogenic cAMP accumulation stimulates cell Present address: Department of Nephrology, University Hospital of Regensburg, 93053 Regensburg, Germany. proliferation and chloride-driven fluid secretion into cyst lumen 3Present address: Laboratory of Biomolecular Research, Division of Biology and Chem- (5, 6). The cAMP level can be reduced in two ways: through istry, Paul Scherrer Institute, 5232 Villigen, Switzerland. Gi-protein activation by the somatostatin SSTR2 receptor or, 4To whom correspondence may be addressed. Email: [email protected], christiane. alternatively, through type-2 vasopressin receptor (V2R) inhibi- [email protected], or [email protected]. tion that decreases Gs-protein signaling. Although both strate- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. gies appear to be effective in humans and constitute an efficient 1073/pnas.1620454114/-/DCSupplemental. 7154–7159 j PNAS j July 3, 2017 j vol. 114 j no. 27 www.pnas.org/cgi/doi/10.1073/pnas.1620454114 Downloaded by guest on September 24, 2021 a large randomized phase III clinical trial, tolvaptan was found A B 0,7 3 to slow down cyst development and renal insufficiency (8). B Since 2014, marketing authorization has been granted in Japan, 0,6 [AU] 0,5 2 NaCl [M] Canada, and four European countries (France, Germany, the 0,4 A C DEF United Kingdom, and Switzerland) and only more recently in the nm 280 0,3 J K 1 0,2 G H I United States, delayed because of concerns regarding its harm- Abs 0,1 ful effects on liver function. Indeed, tolvaptan degradation by 0 0 cytochrome P450 generates hepatotoxic metabolites (9). Addi- 0 2 4 6 8 10 12 14 16 tionally, because vaptans bind at a highly conserved orthosteric Time [min] site, common to all vasopressin-sensitive receptors, they have C D weak selectivity. 0,6 100 0,3 B 100 C Acetonitrile [%] 0,5 80 Acetonitrile [%] Venoms constitute a reservoir of millions of disulfide-rich D 80 [AU] 0,4 60 0,2 peptides, which are biochemically stable with particular phar- 0,3 60 [AU] 280 nm 280 B E 40 A nm 280 40 macological properties. Due to their high affinity and selectiv- 0,2 0,1 F 20 ity, polypeptides derived from venom of different animals are Abs 0,1 G A 20 Abs interesting alternatives to small nonpeptidic compounds for drug 0 0 0 0 development (11). Mamba snake venom contains toxins, such as 0 10203040506070 25 30 35 40 45 MT7, ρ-Da1b, and ρ-Da1a specific for GPCRs, able to modulate E Time [h] F Time [min] their functions in unique ways. MT7 acts as a negative allosteric 0,6 50 120 modulator of the M1 muscarinic receptor (12), ρ-Da1b is a hV1aR 0,5 46 Acetonitrile [%] 100 hOTR noncompetitive antagonist of the α2A-adrenoreceptor (13), and coinjecon hV1bR [AU] 0,4 42 80 ρ-Da1a is able to antagonize in an insurmountable way the α1A- 280 nm 280 0,3 synthec 60 38 hV2R adrenoreceptor (14). Marine cone snails are currently the only 0,2 40 Abs nave species known to secrete toxins active on vasopressin receptors. 0,1 34 20 These toxins mimic vasopressin with weak activity against the 0 30 0 V1a and V1b receptors (15, 16). Here we report the identifica- 17 18 19 20 21 -11 -10 -9 -8 -7 -6 -5 H-AVP bound (% of control) of (% bound H-AVP tion, 3D structure, and properties of mambaquaretin-1 discov- Time [min] 3 Log (ligand), M ered by screening green mamba venom.