Pharmacological Chaperones Restore Function to …jpet.aspetjournals.org/content/jpet/335/3/520.full.pdf · Pharmacological Chaperones Restore Function to MC4R ... using 2-adrenergic

Pharmacological Chaperones Restore Function to MC4RMutants Responsible for Severe Early-Onset Obesity□S

Patricia Rene, Christian Le Gouill, Irina D. Pogozheva, Gary Lee, Henry I. Mosberg,I. Sadaf Farooqi, Kenneth J. Valenzano, and Michel BouvierDepartment of Biochemistry, Institute for Research in Immunology and Cancer, and University Drug Research Group,University of Montreal, Montreal, Quebec, Canada (P.R., C.L.G., M.B.); Department of Medicinal Chemistry, College ofPharmacy, University of Michigan, Ann Arbor, Michigan (I.D.P., H.I.M.); Amicus Therapeutics, Cranbury, New Jersey (G.L.,K.J.V.); and University of Cambridge Metabolic Research Laboratories, Institute of Metabolic Science, Addenbrooke’s Hospital,Cambridge, United Kingdom (I.S.F.)

Received June 30, 2010; accepted September 7, 2010

ABSTRACTHeterozygous null mutations in the melanocortin-4 receptor(MC4R) cause early-onset obesity in humans, indicating thatmetabolic homeostasis is sensitive to quantitative variation inMC4R function. Most of the obesity-causing MC4R mutationsfunctionally characterized so far lead to intracellular retention ofreceptors by the cell’s quality control system. Thus, recoveringcell surface expression of mutant MC4Rs could have a bene-ficial therapeutic value. We tested a pharmacological chaper-one approach to restore cell surface expression and function of10 different mutant forms of human melanocortin-4 receptorfound in obese patients. Five cell-permeant MC4R-selectiveligands were tested and displayed pharmacological chaperone

activities, restoring cell surface targeting and function of thereceptors with distinct efficacy profiles for the different muta-tions. Such mutation-specific efficacies suggested a structure-activity relationship between compounds and mutant receptorconformations that may open a path toward personalized ther-apy. In addition, one of the five pharmacological chaperonesrestored function to most of the mutant receptors tested. Com-bined with its ability to reach the central nervous system and itsselectivity for the MC4R, this pharmacological chaperone mayrepresent a candidate for the development of a targeted ther-apy suitable for a large subset of patients with MC4R-deficientobesity.

IntroductionDisease-causing mutations in G protein-coupled receptors

(GPCRs) often lead to decreased cell surface expression andconcomitant loss of function as a result of improper folding(Schoneberg et al., 2004; Thompson et al., 2008). These mu-tant receptors, generally recognized by the cell’s quality con-trol system within the endoplasmic reticulum (ER) and Golgi

apparatus, are retained intracellularly and targeted for deg-radation. In many of these conformational diseases, the mu-tation occurs in receptor domains that do not directly affectligand binding or G protein coupling, opening the possibilityfor interventions that could restore receptor function by res-cuing folding and cell surface expression (Bernier et al., 2004;Conn et al., 2007). Such functional rescue has been achievedfor several GPCRs, indicating that pharmacologically selec-tive compounds, termed pharmacological chaperones (PCs),can stabilize the misfolded receptors to facilitate their exportfrom the ER to the plasma membrane where they are active(Morello et al., 2000; Petaja-Repo et al., 2002; Noorwez et al.,2004; Bernier et al., 2006; Robben et al., 2007; Conn andJanovick, 2009). The clinical effectiveness of a PC approachhas been tested for one such disease, nephrogenic diabetes

This work was supported by Amicus Therapeutics (to M.B.); the CanadianInstitute for Health Research [Grant 10501] (to M.B.); and a Canada ResearchChair in Cell Signaling and Molecular Pharmacology (to M.B.).

Article, publication date, and citation information can be found athttp://jpet.aspetjournals.org.

doi:10.1124/jpet.110.172098.□S The online version of this article (available at http://jpet.aspetjournals.org)

contains supplemental material.

ABBREVIATIONS: GPCR, G protein-coupled receptor; ER, endoplasmic reticulum; PC, pharmacological chaperone; MC4R, melanocortin-4receptor; hMC4R, human melanocortin-4 receptor; HA, hemagglutinin; MTHP, 2-(2-(2-methoxy-5-nitrobenzylthio)phenyl)-1,4,5,6-tetrahydropyri-midine; PPPone, 3-(4-(2-(4-fluorophenyl)-2-(4-methylpiperazin-1-yl)ethyl)piperazin-1-yl)-2-methyl-1-phenylpropan-1-one; MPCI, 2-(5-bromo-2-methoxyphenethyl)-N-(N-((1-ethylpiperidin-4-yl)methyl)carbamimi doyl)-3-fluorobenzamide; DCPMP, N-((2R)-3(2,4-dichlorophenyl)-1-(4-(2-((1-methoxypropan-2-ylamino)methyl)phenyl)piperazin-1-yl)-1-oxopropan-2-yl)propionamide; NBP, 1-(1-(4-fluorophenyl)-2-(4-(4-(naphthalene-1-yl)butyl)piperazin-1-yl)ethyl)-4-methylpiperazine; PBS, phosphate-buffered saline; BSA, bovine serum albumin; NDP, [Nle4,D-Phe7]; �-MSH, �-melanocyte stimulating hormone; HEK,human embryonic kidney; D-PBS, Dulbecco’s PBS; WT, wild type; vYFP, venus yellow fluorescent protein; SAR, structure-activity relationship; THIQ,tetrahydroisoquinoline; TM, transmembrane domain; EXL, extracellular loop.

0022-3565/10/3353-520–532$20.00THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 335, No. 3Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 172098/3641728JPET 335:520–532, 2010 Printed in U.S.A.

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insipidus, where a vasopressin antagonist, acting as PC,rescued the function of ER-retained V2-vasopressin receptormutants and significantly improved the kidney function ofpatients with nephrogenic diabetes insipidus (Bernier et al.,2006).

PCs may present an attractive therapeutic option for se-vere early-onset morbid obesity that results from mutationsin the melanocortin-4 receptor (MC4R), a receptor that playsa pivotal role in energy homeostasis (Cone, 2005). In humans,MC4R mutations lead to an obese phenotype similar to thehomozygous null-mouse model (Huszar et al., 1997; Chen etal., 2000) and represent the most common monogenic causeof severe early-onset obesity (Farooqi and O’Rahilly, 2006).To date, �80 distinct mutations of MC4R have been reportedin the obese human population. One MC4R antagonist waspreviously shown to have pharmacological chaperone actionon two MC4R mutants (Fan and Tao, 2009). However, thelarge diversity of obesity-related trafficking-defective muta-tions in MC4R calls into question the ability of a singlecompound to restore cell surface expression and function toall mutant forms. This idea is reinforced by the fact thatmutations, which are broadly distributed throughout the re-ceptor structure, might lead to different conformationalchanges. To address this question and determine whetherdifferent PC candidates show different efficacies toward dis-tinct mutants, we tested five cell-permeant MC4R antago-nists that belong to three structurally distinct chemicalclasses. The compounds were studied for their ability to res-cue cell surface expression and signaling activity of 10 nat-urally occurring mutant forms of MC4R that cause obesity(Tao, 2005; Tan et al., 2009). Clear differences were found inthe efficacies and potencies between compounds on each mu-tant, revealing unique rescue profiles for individual PCs. It isnoteworthy that one compound emerged as the most univer-sal PC, rescuing nearly all mutations with the highest po-tency and efficacy. Furthermore, its bioavailability in brain,its clearance properties, and its receptor subtype selectivitymake it a good candidate for the development of a clinicallyuseful drug to treat genetic obesity caused by distinct MC4Rmutations in humans.

Materials and MethodsGeneration of Mutant Human Melanocortin-4 Receptor

Constructs. Ten mutant forms of human MC4R (hMC4R) [S58C,E61K, N62S, I69T, I125K, T162I, R165Q, R165W, C271Y, andP299H] were double-tagged with a 3�HA tag at the N terminusand a venus yellow fluorescent protein (vYFP) tag at the C termi-nus (see Supplemental Materials and Methods).

Compound Synthesis. Compounds selected in the study weresynthesized at Amicus Therapeutics (Cranbury, NJ) according toprocedures described previously: 2-(2-(2-methoxy-5-nitrobenzylthio)phenyl)-1,4,5,6-tetrahydropyrimidine (MTHP) (Maguire et al., 2002);3-(4-(2-(4-fluorophenyl)-2-(4-methylpiperazin-1-yl)ethyl)piperazin-1-yl)-2-methyl-1-phenylpropan-1-one (PPPone) (Arasasingham et al.,2003); 2-(5-bromo-2-methoxyphenethyl)-N-(N-((1-ethylpiperidin-4-yl)methyl)carbamimidoyl)-3-fluorobenzamide (MPCI) (Chaki et al.,2005); N-((2R)-3(2,4-dichlorophenyl)-1-(4-(2-((1-methoxypropan-2-ylamino)methyl)phenyl)piperazin-1-yl)-1-oxopropan-2-yl)propionamide(DCPMP) (Pontillo et al., 2005a); 1-(1-(4-fluorophenyl)-2-(4-(4-(naphthalene-1-yl)butyl)piperazin-1-yl)ethyl)-4-methylpiperazine(NBP) (Vos et al., 2006).

Structural Modeling. The homology modeling of the inactiveconformation of human MC4R was done as described previously (Tan

et al., 2009) using �2-adrenergic receptor fused with T4 lysozyme(Protein Data Bank ID 2rh1) as a structural template. Three-dimen-sional structures of five small-molecule antagonists (including fourstereoisomers of PPPone and two stereoisomers of both DCPMP andNBP) were generated by QUANTA (Accelrys, San Diego, CA). pKa

values for charged groups were calculated by Marvin software (http://www.chemaxon.com/marvin/sketch/index.html). Conformational anal-ysis of the ligands was performed using grid scan search for torsionangles of all rotatable bonds. The lowest-energy conformations of allstereoisomers of all ligands (except one stereoisomer of NBP with �E �2.2 kcal/mol) were used for docking (see Supplemental Materials andMethods).

Transfection and Cell Culture. HEK293T cells were tran-siently transfected with plasmids encoding chimeric WT or mutanthMC4Rs using FuGENE 6 (Roche Applied Science, Laval, QC, Can-ada) as the transfection agent. Cells were maintained in Dulbecco’smodified Eagle’s medium supplemented with 10% fetal bovine se-rum, and 100 U/ml penicillin/streptomycin for 24 h. Transientlyexpressing cells were incubated in the presence or absence of antag-onist for 12 h before flow cytometry or cAMP detection assays.

Quantitative Assessment of WT and Obesity-AssociatedMutant hMC4R Membrane Expression by Flow Cytometry.Cells were harvested 48 h after transfection, rinsed once in 1�Dulbecco’s PBS (D-PBS), and transferred into 1� Tyrode’s solution(140 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, 12 mM NaHCO3, 5.6 mMD-Glucose, 0.49 mM MgCl2, 0.37 mM NaHPO4, and 25 mM HEPES,pH 7.4) supplemented with 1% BSA (Sigma-Aldrich, Oakville, ON,Canada) (Tyrode/BSA) containing mouse monoclonal anti-HA anti-body (1:1000; HA.11; Covance, Burlington, ON, Canada) to label cellsurface receptors. After 1-h incubation at room temperature, cellswere washed once and resuspended in Tyrode/BSA containing anti-mouse Alexa Fluor 647 secondary antibody (1:1000; Invitrogen Can-ada Inc., Burlington, ON, Canada). After 1-h incubation at roomtemperature, cells were washed with Tyrode/BSA, resuspended inTyrode’s solution, and kept on ice. Just before analysis, propidiumiodide was added to each sample to exclude labeled nonviable cells.Cells were analyzed through a LSR II flow cytometer (BD Bio-sciences, Mississauga, ON, Canada) set to detect yellow fluorescentprotein, propidium iodide, and Alexa Fluor 647 nm in distinctchannels.

For agonist-promoted endocytosis experiments, transiently trans-fected cells were incubated with 100 nM NDP-�-MSH, and cell sur-face expression was monitored after different times of agonist expo-sure (30 min and 1, 2, 4, 6, 8, and 22 h).

cAMP Assay. Intracellular cAMP accumulation was measuredusing a competitive immunoassay based on homogeneous time-resolved fluorescence technology (cAMP dynamic-2; Cis-Bio, Bed-ford, MA).

Each double-tagged construct was transiently transfected intoHEK293T cells. Thirty-six hours after transfection, cells weretreated in the presence or absence of 10 �M antagonist for 12 h. Cellswere then collected and washed (1� D-PBS, pH 7.4, and 0.1% glu-cose). Then 4 � 104 cells/well were dispensed in 96-well plates incAMP buffer [1� D-PBS, 1% BSA, 0.1% glucose, 0.75 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich, Oakville, ON, Canada)] and incu-bated for 15 min at 37°C in the presence of 100 nM NDP-�-MSH(Sigma-Aldricht, Oakville, ON, Canada). Next, 104 cells were trans-ferred in 384-well plates, lysed, and incubated with cAMP labeledwith the dye d2 and anti-cAMP M-Antibody labeled with Cryptateaccording to the manufacturer’s protocol. Reading of the homoge-neous time-resolved fluorescence signal was performed on an Arte-mis time-resolved fluorescence resonance energy transfer platereader (Cosmo Bio USA, Carlsbad, CA).

Affinity for Melanocortin Receptors. The affinities (IC50 val-ues) of DCPMP for recombinant human MC1R, MC3R, MC4R, andMC5R were determined at MDS Pharma Services using traditionalradioligand displacement assays at the tracer concentrations indi-cated in Table 6.

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Plasma and Brain Quantitation of DCPMP in C57BL/6Mice. All in vivo procedures were conducted at Eurofins ProductSafety Laboratories (Des Moines, IA) under protocols approved bytheir Institutional Animal Care and Use Committee and followed allrelevant guidance and regulation, including that set out by theInstitute of Laboratory Animal Resources (1996). One of two doses (3or 30 mg/kg) of DCPMP, formulated in 90% cottonseed oil/10%EtOH, was administered to 8-week-old C57BL/6 mice by a singleintraperitoneal injection. Conventional liquid chromatography tan-dem mass spectrometry was used to achieve separation and detec-tion of analytes in plasma and brain tissue (see Supplemental Ex-perimental Procedures for description).

The elimination rate constant (kel) was calculated from the for-mula Ln[(Cp/2) � (1/Cp)] � �kel � t1/2 3 kel � (0.693/t1/2), wheret1/2 � [t2 � t1]. The half-life (t1/2) is the time taken for the plasma orbrain concentration of DCPMP to fall to half its highest value. Cp isthe highest concentration of DCPMP found in plasma or brain at t1

and Cp/2 is the concentration one half-life later at t2.Statistical Analyses. All curve fitting was conducted by nonlin-

ear regression analyses using Prism (ver. 4.0c; GraphPad Software,San Diego, CA). Data are presented as mean S.E.M., and statis-tical significance of the differences were assessed by one-way anal-

ysis of variance. Pair-wise comparisons were made by post hoc Bon-ferroni’s multiple comparison test. Differences with P 0.05 wereconsidered statistically significant.

ResultsSelection and Characterization of hMC4R Mutants

for PC Rescue. Ten naturally occurring MC4R point muta-tions (S58C, E61K, N62S I69T, I125K, T162I, R165Q,R165W, C271Y, and P299H) that result in severe early-onsetobesity in humans were used to investigate the ability of fiveMC4R antagonists to act as PCs. The selected mutationswere chosen based on previous reports suggesting their in-tracellular retention (Farooqi et al., 2000, 2003; Dubern etal., 2001; Nijenhuis et al., 2003; Lubrano-Berthelier et al.,2006; Tan et al., 2009), prevalence in patient populations,and distribution throughout the receptor structural domains(Fig. 1 and Supplemental Table 1). Wild type (WT) and eachof the 10 mutant receptors was tagged with a 3�HA epitopeat the N terminus and a vYFP at the C terminus. Relative

Fig. 1. Naturally occurring mutations inhMC4R selected in this study. Schematicrepresentation of hMC4R with the loca-tion of the 10 naturally occurring muta-tions selected in the study (A) and cartoonrepresentation of the homology model ofMC4R based on the �2-adrenoreceptorstructural template: top view (B) and sideview (C). Mutated residues are coloredred, residues in the ligand-binding pocketare colored blue, and receptor helices arecolored gray. Position of hydrophobicmembrane boundaries is shown in accor-dance with the OPM database (http://opm.phar.umich.edu). Figure was pre-pared using PyMOL (http://www.pymol.org).

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cell surface expression was then assessed by differentiallymonitoring cell surface HA immunoreactivity and total cel-lular vYFP fluorescence with the use of dual-flow cytometry.For the WT receptor, a generally linear relationship was seenbetween the total and the cell surface expression (Supple-mental Fig. 1A, left). In contrast, cell surface expression ofmutant receptors increased only minimally as a result oftheir intracellular retention until total receptor expressionreached very high levels (Supplemental Fig. 1A, right). Then,to limit artifacts caused by high receptor expression (bypass-ing the quality control system), our analysis was limited tocells expressing low levels of receptor .The ratio between HAimmunoreactivity and the vYFP signal for each individualcell was used as an index for cell surface trafficking effi-ciency, which has been arbitrarily fixed at 100% for WT-hMC4R and used as a reference value.

Under basal conditions, nine mutant receptors (S58C,E61K, N62S, I69T, T162I, R165Q, R165W, C271Y, andP299H) showed high levels of intracellular retention, dis-playing trafficking efficiencies ranging from 20 to 40% ofthose measured for WT receptor (Fig. 2, top). Contrary toprevious reports (Yeo et al., 2003; Xiang et al., 2006), I125K-hMC4R was expressed at the cell surface to the same extent

as the WT receptor (Fig. 2, top). The reason for the differencebetween the two studies is unclear. It should be noted, how-ever, that despite the apparent lack of cell surface expressionreported by Xiang et al. (2006) using fluorescence-activatedcell sorting analysis, some binding of the cell impermeable125I-NDP-�-MSH was observed by those authors, as well asby Yeo et al. (2003).

It is noteworthy that the reduced cell surface trafficking ofthe nine other mutants was not accompanied by significantchanges in their total expression levels, as illustrated bytheir equivalent average vYFP signals (Fig. 2, top, inset). Toinvestigate the signaling capacity of the mutant receptors,cAMP accumulation was determined after agonist stimula-tion with 100 nM NDP-�-MSH. In contrast to the WT recep-tor, NDP-�-MSH did not significantly stimulate cAMP pro-duction for any of the hMC4R mutants (Fig. 2, bottom).

Selection of MC4R Compounds and Docking in thehMC4R Model. Five MC4R antagonists with distinct struc-tural features (Table 1) were selected as potential PCs basedon published high affinity and selectivity for MC4R and theirpredicted lipophilic properties, allowing penetration into in-tracellular compartments. The putative binding modes ofthese compounds on WT-hMC4R were assessed by virtualdocking and are presented in Supplemental Fig. 2, and themajor contact points are listed in Table 2.

Low-energy conformations of all five MC4R-selective an-tagonists were docked into the binding pocket of the hMC4Rinactive state model, based on structure-activity relationship(SAR) studies (Arasasingham et al., 2003; Pontillo et al.,2005a,b; Vos et al., 2006), knowledge of functionally impor-tant receptor residues (Pogozheva et al., 2005; Yang et al.,2009), and in accordance with geometric and polarity match-ing of ligand and receptor (Table 2 and Supplemental Fig. 2).

Three of the antagonists (PPPone, DCPMP, and NBP)could be docked similarly to the well characterized hMC4R-selective small-molecule agonist THIQ (Sebhat et al., 2002)(Table 2 and Supplemental Fig. 2A). DCPMP is structurallyrelated to THIQ, harboring a central halogen-substituted2,4-Cl-D-Phe aromatic ring (“A”) and a phenylpiperazine moi-ety (“B”) that mimics the cyclohexylpiperazine of THIQ (Ta-ble 2 and Supplemental Fig. 2E). The basic benzylaminegroup of DCPMP replaces the N-terminal basic nitrogen ofTHIQ. The two dipiperazine-based ligands, PPPone andNBP, also have central halogen-substituted phenyl rings(“A”), and a second aromatic function (“B”) that are essentialfor high binding affinity (Arasasingham et al., 2003), as wellas basic nitrogens in the piperazinyl rings. The central halo-gen-substituted aromatic ring of these three ligands (“A”)represents the key pharmacophore and can occupy the sameposition at the bottom of the binding cavity as the p-chlorophe-nyl ring of THIQ, forming multiple contacts with aromatic,aliphatic, and sulfur-containing residues from TM3 (Ile129,Cys130, Leu133), TM5 (Cys196, Met200), TM6 (Phe261,Leu265), and TM7 (Phe284, Leu288). At physiological pH, thebenzylamine group in DCPMP (pKa � 8.97) and the centralpiperazinyl group in NBP (pKa � 8.92) are positively chargedand can form ionic pairs with Asp126 in TM3 (Table 2 andSupplemental Fig. 2, E and F). Furthermore, the nitrogens inthe terminal piperazinyl groups of PPPone (pKa � 8.21) andNBP (pKa � 7.91) are also positively charged and may beinvolved in ionic interactions with both Asp126 (TM3) andGlu100 (TM2) (Table 2 and Supplemental Fig. 2, C and F). The

Fig. 2. Characterization of cell surface expression, total expression levels,and signaling capacity of 10 mutant forms of hMC4R. Top, cell surfaceand total expression of 3HA-MC4R-vYFP mutants measured by fluores-cence-activated cell sorting. vYFP emission represents MC4R total ex-pression, and HA-Alexa Fluor 647 emission represents MC4R plasmamembrane expression. The relative cell surface expression (ratio Alexa/vYFP emission) is calculated for each individual cell in the selected gate(see Supplemental Fig. 1) and is expressed as a percentage of the valueobtained in the same experiment for the WT-hMC4R in the untreatedcondition. The total expression (inset) is calculated as the percentage ofthe mean of vYFP signal emission of each individual cell in the gate ofinterest (see dot-plot graphs in Supplemental Fig. 1) and is expressed asa percentage of the value obtained in the same experiment for the WT-hMC4R in untreated condition. Bottom, NDP-�-MSH-induced cAMP ac-cumulation expressed as the percentage of agonist stimulated cAMPproduction of WT-hMC4R. WT-hMC4R absolute values are basal, 40 8fmol/104 cells; NDP-�-MSH-stimulated cAMP production, 300 20 fmol/104 cells. Each bar represents the mean S.E.M. of at least threeindependent experiments performed in triplicate.

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nitrogen from the central piperazinyl group of PPPone(pKa � 7.48) would be charged only at a more acidic pH. Addi-tional hydrophobic interactions may form between the benzylrings of DCPMP, PPPone, or the naphthyl ring of NBP andresidues from TM4 (Phe184, Ile185), TM5 (Cys196, Met200),and TM6 (Leu265), similar to interactions of the cyclohexylgroup and the triazol ring of THIQ. The benzyl ring of DCPMPextends toward the extracellular surface, where it may interactwith residues from TM4 (Phe184, Ile185) as well as with resi-dues from extracellular loop (EXL) 2 and the N terminus.

Docking of the structurally distinct ligands MPCI andMTHP was different from that proposed for PPPone,DCPMP, and NBP. SAR studies of MPCI indicate the func-tional importance of the basic group in the arylguanidinesubstituent in combination with the central fluorobenzyl ring(“A”) and the second aromatic ring (“B”) with lower lipophi-licity. Therefore, we propose that the low-energy conforma-tions of MPCI may be docked such that the N� of the piper-idinyl group forms an ion pair with Glu100 (TM2) andAsp126 (TM3), whereas the fluoro-benzyl ring occupies a

position similar to the 4-chlorophenyl ring of THIQ (Table 2and Supplemental Fig. 2D). The docking of MTHP is morechallenging. A folded conformation of the ligand was chosen,because this is energetically preferred compared with anextended conformation (�E � 3.5 kcal/mol). Because of thesmall size, two molecules of MTHP can be easily accommo-dated within the relatively large receptor binding pocket: onemolecule occupying the area between TMs 3 and 6 and an-other molecule filling the space between TMs 2, 3, and 7. Thepositively charged nitrogen of the ligand can form an ionicpair with Asp126 (TM3) if the ligand is in the first dockingpose or with Glu100 (TM2) if the ligand occupies the seconddocking pose (Table 2 and Supplemental Fig. 2B).

Cell Surface Rescue of Mutant hMC4Rs. The ability ofthe selected MC4R antagonists to act as PCs was first as-sessed by monitoring receptor cell surface expression after12-h incubation with the compounds at 1 and 10 �M. Quan-titative analysis of the flow cytometry dot plots (Supplemen-tal Fig. 1 for WT and I69T hMC4R) revealed that cell surfaceexpression of all mutant receptors, except for P299H, was

TABLE 1Antagonists selected in the studyValues were obtained from radioligand displacement assays by competition with 0.02 nM 125I-NDP-�-MSH on HEK293 cells expressing hMC4R (Kd,, 0.5 nM; Bmax, 3900fmol/mg protein).

Structure Nomenclature Code Ki Inhibition @ 10 �M

�M %

S

NH

N

N+–O O

O2-(2-(2-Methoxy-5-nitrobenzylthio)phenyl)-1,4,5,6-

tetrahydropyrimidine MTHP 0.094 88

N N

N

N

O

F

3-(4-(2-(4-Fluorophenyl)-2-(4-methylpiperazin-1-yl)ethyl)piperazin-1-yl)-2-methyl-1-phenylpropan-1-one

PPPone 0.691 88

FO

Br

NH

O

NH

NH

N2-(5-Bromo-2-methoxyphenethyl)-N-(N-((1-

ethylpiperidin-4-yl)methyl)carbamimidoyl)-3-fluorobenzamide

MPCI 0.218 94

NN

ONH

Cl Cl

O

HNO

N-((2R)-3(2,4-Dichlorophenyl)-1-(4-(2-((1-methoxypropan-2-ylamino)methyl)phenyl)piperazin-1-yl)-1-oxopropan-2-yl)propionamide

DCPMP 0.0237 88

N

NN

F

N 1-(1-(4-Fluorophenyl)-2-(4-(4-(naphthalene-1-yl)butyl)piperazin-1-yl)ethyl)-4-methylpiperazin NBP 0.0024 89

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significantly increased after incubation with at least one ofthe compounds (Fig. 3, left). It is noteworthy that the effi-ciency of the individual compounds to promote cell surfaceexpression was mutation-dependent.

At the highest concentration tested, DCPMP and NBPshowed the broadest activity, significantly increasing the cellsurface targeting of all nine responsive mutants (Fig. 3, leftand Table 3). PPPone increased cell surface expression of thesame subset of mutant receptors, except for I125K. MTHPsignificantly increased cell surface levels of S58C, I125K,R165Q, and R165W, whereas MPCI enhanced cell surfaceexpression of T162I, R165Q, R165W, and C271Y. In somecases, PCs increased cell surface expression at higher levelsthan that of untreated WT receptor (e.g., DCPMP and NBPfor S58C, I125K, R165Q, and R165W; MTHP for I125K andR165Q; PPPone for I69T and R165Q).

The relative apparent potency of the compounds to pro-mote cell surface targeting also varied among the differentmutants. This is evident based on the efficiency ratios of cellsurface rescue promoted by incubation with the differentcompounds at 1 and 10 �M (Table 3). DCPMP and NBPshowed high relative potencies for promoting cell surfacetargeting (ratios �0.6) of eight of nine and six of nine mutantreceptors, respectively. In contrast, MTHP and PPPoneshowed lower relative potencies (ratios 0.6) for four of fourand eight of eight of the rescued mutants, respectively. Fi-nally, MPCI showed an intermediary relative potency profile

with half of the four rescuable mutants below and half abovethe 0.6 ratio. All compounds had potency ratios below 0.6 torescue N62S, as well as low potencies to rescue R165W andR165Q, except DCPMP. In contrast, T162I and C271Y wererescued with potency ratios above 0.6 by three of the fivecompounds: MPCI, DCPMP, and NBP. It should be notedthat although the relative potency value allows a comparisonof the potencies when the ratio is below 1, it does not allow aconclusion that compounds that have ratios of 1 have iden-tical potencies, because such a ratio simply indicates that thepotency of the compound is below 1 �M.

Although the compounds varied significantly in their abil-ity to rescue the cell surface expression of different mutants,some generalities can be drawn: two mutants (R165Q andR165W) are rescued by all compounds, two compounds(DCPMP and NBP) rescue all mutants, and the subset ofmutations that could be partially or fully rescued is differentfor each compound (Table 3). It is noteworthy that MTHP,DCPMP, and NBP also significantly increased the relativecell surface expression of the WT receptor with high potency(relative potency ratios of 0.74, 0.91, and 0.96, respectively),indicating that trafficking efficiency of WT-hMC4R could alsobe influenced by PCs.

Functional Rescue of hMC4R Mutants with PCs. Wenext investigated whether PC-mediated increases in cell sur-face expression could also restore signaling activity of themutant MC4Rs (Fig. 3, right). In the case of I125K, none of

TABLE 2Description of binding residues for THIQ and antagonists of the studyResidues of MC4R located at 4.5 or 4.0 Å from PCs docked in the models of MC4R receptor in the inactive conformation. Residues that are important for THIQ binding (ProteinData Bank code 2IQU) are in bold. Residues presented in Supplemental Fig. 2 are underlined.

PC THIQ MTHPa MTHPb PPPone MCPI DCPMP NBP

Radius, Å 4.5/4.0 4.5/4.0 4.5/4.0 4.5/4.0 4.5/4.0 4.5/4.0 4.5/4.0NT

Leu44 �/� �/� �/� �/� �/� �/� �/�TM2

Glu100 �/� �/� �/� �/� �/� �/� �/�Val103 �/� �/� �/� �/� �/� �/� �/�Ile104 �/� �/� �/� �/� �/� �/� �/�

TM3Asp122 �/� �/� �/� �/� �/� �/� �/�Asp126 �/� �/� �/� �/� �/� �/� �/�Ile129 �/� �/� �/� �/� �/� �/� �/�Cys130 �/� �/� �/� �/� �/� �/� �/�Leu133 �/� �/� �/� �/� �/� �/� �/�

TM4Phe184 �/� �/� �/� �/� �/� �/� �/�Ile185 �/� �/� �/� �/� �/� �/� �/�

EXL2Ser190 �/� �/� �/� �/� �/� �/� �/�Ser191 �/� �/� �/� �/� �/� �/� �/�Ala192 �/� �/� �/� �/� �/� �/� �/�Val193 �/� �/� �/� �/� �/� �/� �/�

TM5Cys196 �/� �/� �/� �/� �/� �/� �/�Cys200 �/� �/� �/� �/� �/� �/� �/�

TM6Phe261 �/� �/� �/� �/� �/� �/� �/�Phe262 �/� �/� �/� �/� �/� �/� �/�Leu265 �/� �/� �/� �/� �/� �/� �/�His264 �/� �/� �/� �/� �/� �/� �/�Tyr268 �/� �/� �/� �/� �/� �/� �/�

TM7Asn285 �/� �/� �/� �/� �/� �/� �/�Leu288 �/� �/� �/� �/� �/� �/� �/�Phe284 �/� �/� �/� �/� �/� �/� �/�Met292 �/� �/� �/� �/� �/� �/� �/�

NT, N terminus; MTHPa, binds between TM2, TM3, and TM7; MTHPb, binds between TM3 and TM6.

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Fig. 3. Effects of MC4R antagonists on cell surface expression and stimulated cAMP production of mutant and WT hMC4Rs. HEK293T cellstransiently expressing the indicated double-tagged hMC4R were incubated for 12 h in the absence (untreated) or presence of antagonist compounds.Left, cell surface expression of each receptor was measured by flow cytometry. Cells were incubated in the absence (gray bar, untreated) or presenceof 1 �M (open bar) or 10 �M (filled bar) antagonist compounds. The relative membrane expression (ratio of Alexa/vYFP emission) was calculated foreach individual cell in the gate of interest (see dot-plot graphs in Supplemental Fig. 1) and expressed as a percentage of the value obtained in the sameexperiment for the WT-hMC4R in the absence of antagonist. Right, HEK293T cells transiently expressing the indicated double-tagged hMC4R wereincubated for 12 h in the absence (gray bar, untreated) or presence (filled bar) of 10 �M antagonist compounds. cAMP accumulation was measuredafter 100 nM NDP-�-MSH stimulation. The results are expressed as the percentage of NDP-MSH-stimulated cAMP production by WT-hMC4R. Eachbar represents the mean S.E.M. of at least three independent experiments. Dashed lines represent WT-hMC4R cell surface expression (left) or cAMPaccumulation (right) in untreated condition. This level is considered as reference for a full recovery for mutant receptors. The symbol (�) indicates asignificant difference from untreated condition: �, P 0.05; ��, P 0.01; ���, P 0.001.

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the compounds could restore NDP-�-MSH-stimulated cAMPresponse, despite the fact that its cell surface expressioncould be further increased by three compounds (MTHP,DCPMP, and NBP), indicating that this mutant form of thereceptor is unable to respond to agonist. This exception aside,compounds that increased cell surface expression also re-stored NDP-�-MSH-stimulated cAMP production (Fig. 3,right) for all mutants, confirming that rescued mutant recep-tors are in a conformation that can bind agonist and trans-duce signal. However, differences in the extent of cell surfaceand functional rescue were observed. For instance, althoughNBP very efficiently rescued the cell surface expression of sixmutants, it only marginally restored significant signaling fortwo of them. This most likely results from persistent NBPbinding because of its high affinity (2 nM), which preventssubsequent stimulation with NDP-�-MSH. This hypothesisis supported by the observation that pretreatment of WT-

hMC4R with NBP led to a �50% inhibition of NDP-�-MSH-stimulated cAMP production (Fig. 3, right).

Even for compounds that have lower affinity for the recep-tor, discrepancies between rescued cell surface expressionand signaling activity were observed. This is illustrated bythe signaling/cell surface ratio, which is taken as an indica-tion of the signaling efficacy of the rescued receptor and is setat one for the WT-hMC4R (Table 4). For four of the mutants(I69T, T162I, R165Q, and R165W), at least one of thecompounds restored signaling efficacy ratios to valuesabove 0.8. For other mutations (S58C, E61K, N62S, andC271Y), none of the tested compounds promoted signalingratios above 0.8, indicating that the conformation stabi-lized for these mutants may be less efficient for signaling.It is noteworthy that when considering each mutant recep-tor the compounds that favored the highest signaling effi-cacy ratios were not always the same. For example,

Fig. 3. Continued.

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DCPMP promoted the highest signaling ratios for R165Q,T162I, and E61K, whereas MTHP and PPPone did so forR165W and I69T.

In several cases, incubation with specific compounds re-stored cAMP responses that were equivalent or superior tothose observed for untreated WT receptor (Fig. 3, right). Such

complete rescue was seen for different compound/mutationcombinations (MTHP, R165Q and R165W; PPPone, I69T,R165Q, and R165W; DCPMP, S58C, E61K, N62S, I69T,T162I, R165Q, and R165W). Overall, DCPMP emerged as thecompound that restored function to the largest subset ofmutants. As was the case for cell surface expression, incuba-

TABLE 3Overview of efficacy and relative potency of each compound on cell surface rescueUntreated indicates relative membrane expression reported as a percentage of the value obtained in the same experiment for the hMC4R (WT) in the absence of antagonist.Efficacy refers to relative surface expression level attained after overnight incubation with 10 �M antagonist. Relative potency is the ratio between the mean of the relativesurface expression level obtained at 1 and 10 �M. Data shown are the mean S.E.M. of at least three independent experiments and are derived from Fig. 3.

Construct UntreatedEfficacy at 10 �M Relative Potency

MTHP PPPone MPCI DCPMP NBP MTHP PPPone MPCI DCPMP NBP

WT 100 172 7a 135 6a 144 10a 189 34b 192 38b 0.74 0.99 0.75 0.91 0.96S58C 40 5* 102 4a 108 4a 74 17 184 31b 229 20b 0.53 0.45 0.41 0.81 0.73E61K 26 3* 37 3 73 16b 42 7 111 2b 73 9b N.A. 0.47 N.A. 0.64 0.65N62S 18 8** 47 9 130 32b 37 9 165 19b 138 23b N.A. 0.19 N.A. 0.52 0.23I69T 34 1* 63 16 171 39c 63 7 115 17b 97 8a N.A. 0.37 N.A. 0.86 0.76I125K 105 8 204 9c 114 16 89 13 186 11b 217 4c 0.93 0.99 1.08 0.82 0.83T162I 21 2* 61 17a 132 23c 74 10a 134 8b 113 17b 0.38 0.23 0.81 0.82 0.63R165Q 16 4** 208 32c 344 31c 88 15a 278 29c 277 34c 0.19 0.25 0.30 0.86 0.56R165W 16 5** 86 5a 104 10b 116 12b 283 20c 294 16c 0.46 0.37 0.26 1.04 0.41C271Y 24 3* 26 0.5 59 10a 69 15a 105 9b 68 13a N.A. 0.44 0.75 0.67 0.96P299H 18 5** 20 3 27 5 17 4 20 4 20 3 N.A. N.A. N.A. N.A. N.A.

N.A., not applicable (no significant rescue was observed at high concentration).a Significantly different from untreated condition, P 0.05.b Significantly different from untreated condition, P 0.01.c Significantly different from untreated condition, P 0.001.* Significantly different from untreated WT hMC4R, P 0.05.** Significantly different from untreated WT hMC4R, P 0.01.

TABLE 4Comparison of cell surface expression and signaling efficacy for each compoundCells expressing WT or mutant hMC4Rs were preincubated in the absence or presence of 10 �M concentrations of the indicated compound for 12 h. After washes, cellsexpressing WT or mutant hMC4Rs were stimulated for 15 min at 37°C with 100 nM NDP-�-MSH.

Parameters and Mutations Untreated MTHP PPPone MPCI DCPMP NBP

Surface expressionWT 100 172 7 135 6 144 10 189 34 192 38S58C 40 5 102 4 108 4 74 17 184 31 229 20E61K 26 3 37 3 73 16 42 7 111 2 73 9N62S 18 8 47 9 130 32 37 9 165 19 138 23I69T 34 11 63 16 171 39 63 7 115 17 97 8I125K 105 8 204 9 114 16 89 13 186 11 217 4T162I 21 2 61 17 132 23 74 10 134 8 113 17R165Q 16 4 208 32 344 31 88 15 278 29 277 34R165W 16 5 86 5 104 10 116 12 283 20 294 16C271Y 24 3 26 0.5 59 10 69 15 105 9 68 13

SignalingWT 100 383 41a 330 24a 98 9 219 23a 44 6S58C 16 2** 34 9 42 9 24 3 60 12a 10 2E61K 14 2** 4 2 17 3 13 0 81 5a 12 2N62S 10 3** 13 2 28 3b 14 4 68 4a 10 2I69T 5 1** 42 13b 164 25a 18 6 76 14a 25 8I125K 7 1** 6 0 8 0 7 2 8 2 12 1T162I 6 1** 16 0 6 1 33 14 102 17a 6 1R165Q 6 0** 97 9a 127 32a 56 6a 249 54a 36 8b

R165W 3 1** 94 21a 115 16a 17 1b 205 11a 17 1b

C271Y 4 1** 4 1 10 1 7 3 32 8a 9 4Ratio

WT 1 2.23 2.44 0.68 1.16 0.23S58C 0.40 0.33 0.39 0.32 0.33 0.04E61K 0.54 0.11 0.23 0.31 0.73 0.16N62S 0.56 0.28 0.22 0.38 0.41 0.07I69T 0.15 0.67 0.96 0.29 0.66 0.26I125K 0.07 0.03 0.07 0.08 0.04 0.06T162I 0.29 0.26 0.05 0.45 0.76 0.05R165Q 0.38 0.47 0.37 0.64 0.90 0.13R165W 0.19 1.09 1.11 0.15 0.72 0.06C271Y 0.17 0.15 0.17 0.1 0.3 0.13

a Significantly different from untreated condition, P 0.01.b Significantly different from untreated condition, P 0.05.** Significantly different from untreated WT hMC4R, P 0.01.

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tion of WT receptor with MTHP, PPPone, or DCPMP alsoincreased NDP-�-MSH-stimulated cAMP production.

Pharmacological Characterization of Rescued Re-ceptors. To further explore the pharmacological propertiesof the rescued receptor, we generated concentration-responsecurves for NDP-�-MSH- and �-MSH-stimulated cAMP pro-duction in cells expressing WT or three MC4R mutants se-lected for their different rescue profiles (R165W, N62S, andC271Y; Table 5 and Supplemental Fig. 3). Treatment of cellsexpressing the WT receptor with DCPMP did not affect theEC50 of either hormone to stimulate cAMP production. ForDCPMP-rescued R165W, the potency of NDP-�MSH and�-MSH was almost identical or slightly reduced comparedwith WT (1.2- and 4-fold, respectively) (Table 5). The potencyof both agonists for the rescued N62S was reduced by approx-imately 5-fold compared with WT receptor. These data indi-cate that the DCPMP-rescued R165W and N62S have signal-ing potencies that are not drastically different from those ofthe WT receptor. In contrast, the EC50 values for bothNDP-�-MSH- and �-MSH-stimulated cAMP production forthe DCPMP-rescued C271Y were much higher (44- and32-fold, respectively) than for WT receptor, indicating asignificant loss of potency for this receptor mutant(Table 5).

Pharmacological Characterization of DCPMP. Be-cause DCPMP efficiently rescued the largest subset of MC4Rmutants, it emerges as a potential lead for the developmentof a therapeutic PC to treat MC4R-linked obesity. Thus, itspotency, selectivity, and in vivo pharmacokinetic profile wereassessed. The potency of DCPMP to functionally rescue theNDP-�-MSH-stimulated cAMP production by N62S, E61K,and R165W is illustrated in Fig. 4. In agreement with theweaker relative potencies of DCPMP to restore cell surfaceexpression of N62S (0.52) and E61K (0.61) versus R165W(1.04) (see Table 3), the dose-response curve of DCPMP torestore signaling activity was right-shifted for N62S andE61K compared with R165W. The lack of saturation of theresponses for N62S and E61K prevented the determinationof an accurate EC50, but an EC50 of 1.5 �M was calculated forR165W. This value is 10-fold higher than that obtained for

the WT receptor to enhance the signaling response aboveuntreated condition level (data not shown). Although no di-rect information regarding the affinity of DCPMP for themutant form of the receptor is available [i.e., its relativepotency close to 1 for both WT and R165W (Table 3) indicatesthat its potency is better than 1 �M but not necessarily equalfor the two receptor forms], the difference in its potency topromote R165W versus WT signaling could indicate that themutation affects the affinity of the receptor for DCPMP.Alternatively, the persistence of the rescued mutant receptorfunctionality could be reduced compared with the WT recep-tor as a consequence of an accelerated loss of cell surfacereceptor upon activation. Thus, a higher concentration of thePC may be needed to reach an equivalent functional steadystate than with the WT receptor. To compare the kinetics ofcell surface residency of the rescued R165W with that of theWT receptor, we assessed the kinetics of agonist-promotedendocytosis of the R165W and WT receptors after a 12-hpretreatment with DCPMP and compared it with that of theuntreated WT receptor. As shown in Fig. 5, stimulation of thereceptors with the agonist NDP-�-MSH led to a loss of cellsurface receptors with comparable kinetics for the rescuedR165W and the WT receptor (pretreated or not withDCPMP), indicating that the dynamics of cell-rescuedR165W once at the cell surface are identical to those of theWT receptor. It is therefore unlikely that the difference in theapparent potency of DCPMP may result from distinct kinet-ics of cell surface residency of the rescued receptor.

The selectivity of DCPMP for MC4R was assessed by itsability to inhibit 125I-NDP-�-MSH binding to human MC1R,MC3R, MC4R, and MC5R. DCPMP displayed high affinityfor MC4R with an IC50 value of 25 nM, showing a selectivityof 100-fold or more over the three other receptor subtypes(Table 6).

To determine whether DCPMP could reach its intendedtarget in the central nervous system, one of two doses ofDCPMP (3 and 30 mg/kg) were administered to 8-week-oldC57BL/6 mice by a single intraperitoneal injection. The pres-ence of compound was then monitored over a 24-h period inblood and brain (Fig. 6). DCPMP showed similar kinetics in

TABLE 5Efficacy and potency of melanocortin agonists to stimulate cAMP production of WT and mutant hMC4Rs rescued by DCPMP pretreatmentData shown are the mean S.E.M. of the number of experiments indicated in parentheses. EC50 is the concentration of ligand that results in 50% stimulation of the maximalresponse (Rmax).

Agonist and Mutation LocationEC50 Rmax

Untreated 10 �M DCPMP Untreated 10 �M DCPMP

nM fmol/104 cells

NDP-�-MSHWT 4.01 0.82 (7) 3.9 0.9 (9) 170.2 31.7 (7) 393.2 40.0 (9)**N62S TM 1 N.A. 18.5 0.8 (3)* N.A. 275.4 57.1 (3)R165W TM 4 N.A. 4.8 1.3 (3) N.A. 297.0 23.4 (3)C271Y EXL3 N.A. 175 82.1 (4)** N.A. 152.9 50.2 (4)a

�-MSHWT 300 70 (9) 370 60 (9) 196.7 45.3 (9) 405.2 37.3 (9)*N62S TM 1 N.A. 2020 700 (3)**b N.A. 367.3 82.5 (3)R165W TM 4 N.A. 1500 400 (5)*b N.A. 213.5 64.8 (5)C271Y EXL3 N.A. 12,300 6500 (3)***c N.A. 106.7 8.4 (3)b

N.A., not applicable.a Significantly different from treated WT hMC4R, P 0.01.b Significantly different from treated WT hMC4R, P 0.05.c Significantly different from treated WT hMC4R, P 0.001.* Significantly different from untreated WT hMC4R, P 0.05.** Significantly different from untreated WT hMC4R, P 0.01.*** Significantly different from untreated WT hMC4R, P 0.001.

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these two tissues, reaching maximal concentrations at 30and 60 min after injection for the 3 and 30 mg/kg dose groups,respectively. Although the maximal concentrations reachedin the brain were 16- and 8-fold lower than those in theplasma, the level reached after 30 mg/kg administration (0.5�M) is very close to the EC50 for PC activity determined incells (see Fig. 4). The calculated elimination rate constantswere similar for brain and plasma (3 mg/kg: 0.69 ng � ml�1 �h�1 for plasma and brain; 30 mg/kg: 0.35 ng � ml�1 � h�1 forplasma and 0.23 ng � ml�1 � h�1 for brain). It is noteworthy

that more than 80% of the drug was cleared from brain 4 and8 h after injection for the 3 and 30 mg/kg dose groups,respectively.

DiscussionOur study revealed important mutation-specific differ-

ences in the PC action of MC4R antagonists that have dis-tinct chemical structures. Nevertheless, at least one PC res-cued almost every MC4R mutant tested. Indeed, only 2 of the10 MC4R mutants were resistant to PC treatment: P299H,the cell surface expression of which could not be restored byany PC, and I125K, which had normal cell surface expressionbut could not be functionally rescued by any compound de-spite a facilitation of its cell surface targeting by some PCs.The P299H mutation affects the proline from the conservedN/DPxxY motif and is predicted to eliminate the proline-induced kink in TM7, affecting the packing of TM1, TM2, andTM7 (Tan et al., 2009). This conformational change mightresult in an unstable receptor that is most likely unable tobind PCs. The lack of functional response observed for I125Kis consistent with the large decrease in binding affinity ofNDP-�-MSH that was reported previously (Haskell-Luevanoet al., 2001; Yeo et al., 2003; Chen et al., 2007). However, thismutation did not prevent the binding of all ligands; MTHP,DCPMP, and NBP potentiated its cell surface expression.Despite this increased cell surface expression, no signalingactivity was restored by these three PCs, indicating that theconformations stabilized by the PCs are unable to bind NDP-�-MSH with high affinity and/or are unable to transduce thebinding into signaling.

Among the five PCs tested, four were able to rescue sig-naling activity of at least two of the eight rescuable mutants.The only compound that could not restore significant NDP-�-MSH responsiveness was NBP, despite a very potent andefficacious rescue of the cell surface trafficking, most likely asa consequence of prolonged antagonist action resulting fromits very high affinity. The presence of the bulky naphthyl ringas well as two positively charged N� from both piperazinylgroups that form multiple interactions with receptor residues

Fig. 4. Concentration-response curve of DCPMP treatment on mutantforms of hMC4R. Signaling capacity of E61K-, N62S- and R165W-MC4Rupon stimulation with 100 nM NDP-�-MSH, after 12-h incubation withincreasing concentrations of DCPMP. The data are expressed as thepercentage of maximal level of cAMP accumulation of mutant forms ofhMC4R upon agonist stimulation in the presence of DCPMP preincuba-tion. Each point represents the mean S.D. of two to three independentexperiments performed in triplicate.

Fig. 5. Kinetic of agonist-promoted endocytosis of rescued R165W- andWT-MC4R. HEK293T cells transiently expressing R165W and WT dou-ble-tagged hMC4R were incubated for 12 h in the absence (only forWT-MC4R) or presence of 10 �M DCPMP. Agonist-promoted endocytosiswas induced by 100 nM NDP-�-MSH maintained over time. The cellsurface expression level was measured by flow cytometry after incubationwith agonist for 30 min and 1, 2, 4, 6, 8, and 22 h. The cell surfaceexpression level is expressed as the percentage of the value measured atT0 (no agonist stimulation) in each condition. Each point represents themean S.D. of three independent experiments.

Fig. 6. Pharmacokinetic profile of the MC4R antagonist DCPMP. One oftwo doses of DCPMP [3 mg/kg (square) and 30 mg/kg (triangle)] wereadministered to 8-week-old C57BL/6 mice by a single intraperitonealinjection. DCPMP concentrations in blood and brain were measured atthe indicated time. The dashed and solid lines correspond to the concen-tration measured in brain and blood, respectively. To derive approximatemolar concentrations of DCPMP in the brain, we assumed that 1 g oftissue corresponds to a volume of 1 ml.

TABLE 6Melanocortin receptor selectivity of DCPMPThe affinities (IC50 values) of DCPMP for recombinant human MC1R, MC3R, MC4R,and MC5R in HEK293 cells were determined using traditional radioligand displace-ment assays in the presence of 0.04 nM 125I-NDP-�-MSH for MC1R (Kd, 0.037 nM);0.035 nM 125I-NDP-�-MSH for MC3R (Kd, 0.24 nM); 0.02 nM 125I-NDP-�-MSH forMC4R (Kd, 0.5 nM); 0.035 nM 125I-NDP-�-MSH for MC5R (Kd, 0.53 nM).

Ligand DCPMP Inhibition at 10 �M IC50 MC4R Selectivity

% �M

MC4R 88 0.025 1MC1R 15 �10 �400MC3R 70 3.07 123MC5R 68 4.04 162

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(Table 2 and Supplemental Fig. 2) may explain the higherbinding affinity and slow dissociation rate of this ligand.

When considering the four compounds that rescued func-tion, distinct mutation-dependent PC efficacy was observed.Whereas DCPMP significantly restored function to all rescu-able mutants, the signaling activity of only some were sig-nificantly rescued by PPPone (N62S, I69T, R165Q, andR165W), MTHP (I69T, R165Q, and R165W), and MPCI(R165Q and R165W). The broader PC action of DCPMP can-not be predicted simply on the basis of the affinity of theligand for the WT-hMC4R. Indeed, PPPone, which rescuesthe second largest number of mutant receptor forms, has thelowest affinity. This suggests that structural effects of themutation as well as the binding modes of the compoundsmust contribute to the mutant-specific profiles of the PCs.For example, DCPMP restored the highest efficacy ratios forT162I (0.76) and R165Q (0.9), whereas PPPone promotedefficacy ratios of only 0.05 and 0.37 for the same mutants. Incontrast, PPPone restored a higher signaling efficacy thanDCPMP for I69T (0.96 versus 0.66). This mutant-dependentaction of the PCs may be explained by the fact that Thr162and Arg165 are at the bottom of TM4, a TM that is predictedby our docking data (Table 2 and Supplemental Fig. 2, C andE) to have more contacts with DCPMP than with PPPone.Thus, the interactions of DCPMP with Phe184 and Ile185 atthe top of TM4 may compensate for the destabilization of theH-bond network between TM4 and the bottom of TM3 andintracellular loop 2 by maintaining the packing of TM4 in thereceptor bundle. On the other hand, unlike DCPMP, PPPoneinteracts with residues of TM2 (Glu100, Ile104), interactionsthat may stabilize the helix packing between TM2 and TM1,which could have been disrupted by the substitution ofthe hydrophobic Ile69 with the smaller polar threonine. Itis noteworthy that the most broadly active compounds(DCPMP, NBP, and PPPone) share a common general bind-ing mode that involves a greater number of receptor residues(Table 2 and Supplemental Fig. 2), indicating that the mul-tiple interactions formed by these three piperazine-contain-ing compounds can stabilize receptors harboring conforma-tional defects originating from mutations in differentreceptor regions. Despite this general rule, the cell surfaceexpression of two mutants, E61K and C271Y, could be re-stored to WT levels by only one PC (DCPMP). This may beexplained by the more dramatic conformational changes pre-dicted to result from these mutations, making them moreresistant than others to stabilization by most PCs. Indeed,substitution of the TM1 Glu61 by a lysine residue has beenproposed to promote the formation of an aberrant hydrogenbond with the free carbonyl group of Ile296 in TM7 that maychange packing interactions of TM1 and TM7 (Tan et al.,2009). The C271Y substitution prevents the formation of adisulfide bond (Cys271–Cys277) between the end of TM6 andEXL3 (Tarnow et al., 2003).

When considering signaling efficacy in light of the cellsurface rescue, two general scenarios were observed: thesignaling efficacy was either proportional or significantly lessthan predicted from the cell surface targeting. The first sce-nario is exemplified by I69T, T162I, R165Q, and R165W, forwhich some PCs restored the intrinsic signaling efficacy tolevels similar to or above those of untreated WT, indicatingthat PC treatments promoted conformations that are fullycompetent for signaling. This is somewhat surprising for

R165Q and R165W, because this arginine has been proposedto interact with Asp146 of the DRY motif and thus to beimportant for ligand-promoted MC4R activation (Xiang etal., 2006). Our data indicate that, although the Arg165 resi-due may be important in promoting a specific conformation,it is not essential for receptor activation, as confirmed by therecovery of the same agonist’s potency as the WT receptor infunctional assay after rescue with DCPMP (Table 5). Thesecond scenario is exemplified by S58C, E61K, N62S, andC271Y, for which none of the PCs could restore signalingefficacies above 0.75 (see Table 4). Mutations S58C, E61K,and N62S are predicted to alter the H-bonding interactionsbetween polar residues from TM1 (Ser58 and Asn62), TM2(Asp90, Ser94, and Asn97), and TM7 (Asn294, Ser295, andAsp298), which play a central role in receptor activation(Govaerts et al., 2005; Tan et al., 2009). For C271Y, the lossof the disulfide bond in EXL3 most likely results in a majorconformational change that could alter NDP-�-MSH bindingor the translation of ligand binding into receptor activation.Consistent with this notion, the potencies of NDP-�-MSHand �-MSH to stimulate cAMP production for the DCPMP-rescued C271Y-MC4R were decreased by 44- and 32-fold,respectively, compared with WT receptor (Table 5).

The different compounds also had different relative poten-cies and efficacies to promote cell surface expression andenhance cell signaling of WT-hMC4R (Table 4). This obser-vation suggests that a fraction of native receptors fails toachieve proper folding and that the presence of a PC favorssuch folding. Similar effects of PCs have been reported pre-viously for the -opioid (Petaja-Repo et al., 2002) and GnRH(Janovick et al., 2002; Conn et al., 2007) receptors. Therefore,in addition to representing a therapeutic avenue for severelyobese patients carrying MC4R mutations, PC treatmentcould possibly be useful for the treatment of obesity in pa-tients with normal MC4R.

It should be noted that the ability of the PCs to restorefunction was assessed only for the canonical Gs-adenylylcyclase signaling pathway. Given that it has been proposedthat different ligand-promoted GPCR conformations may beresponsible for the activation of distinct signaling pathways(Galandrin et al., 2007; Rajagopal et al., 2010), it will beinteresting to monitor the action of various PCs on the abilityof mutant MC4Rs to activate other signaling effectors, suchas the recruitment of �-arrestin and the activation of MAPK.

To be clinically useful, antagonist PCs need to 1) selec-tively bind the targeted receptor; 2) have a sufficiently highaffinity for the receptor but a rapid dissociation rate thatallows washout and competition by the endogenous ligand;3) be sufficiently lipophilic to penetrate cell membranes; and4) be as potent and efficacious as possible on the largestsubset of receptor mutant forms. DCPMP possesses many ofthese characteristics (e.g., high selectivity toward MC4R,broad efficacy toward many mutants, ability to reach thebrain in sufficient concentration, and relatively rapid clear-ance), making it an interesting lead compound for the devel-opment of a therapeutically useful PC. Nevertheless, ourdata clearly indicate that different compounds could betterrescue some mutations, suggesting that pharmacogeneticsmay play an important role in the establishment of PCs astherapeutics. Because the current strategies to control early-onset obesity are either modestly effective or very invasive

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(bariatric surgery), the development of clinically active PCrepresents an attractive avenue.

Acknowledgments

We thank Dr. Monique Lagace for useful discussion and assistancein the preparation of the manuscript.

ReferencesArasasingham PN, Fotsch C, Ouyang X, Norman MH, Kelly MG, Stark KL, Karbon

B, Hale C, Baumgartner JW, Zambrano M, et al. (2003) Structure-activity rela-tionship of (1-aryl-2-piperazinylethyl)piperazines: antagonists for the AGRP/melanocortin receptor binding. J Med Chem 46:9–11.

Bernier V, Bichet DG, and Bouvier M (2004) Pharmacological chaperone action onG-protein-coupled receptors. Curr Opin Pharmacol 4:528–533.

Bernier V, Morello JP, Zarruk A, Debrand N, Salahpour A, Lonergan M, Arthus MF,Laperriere A, Brouard R, Bouvier M, et al. (2006) Pharmacologic chaperones as apotential treatment for X-linked nephrogenic diabetes insipidus. J Am Soc Neph-rol 17:232–243.

Chaki S, Oshida Y, Ogawa S, Funakoshi T, Shimazaki T, Okubo T, Nakazato A, andOkuyama S (2005) MCL0042: a nonpeptidic MC4 receptor antagonist and seroto-nin reuptake inhibitor with anxiolytic- and antidepressant-like activity. Pharma-col Biochem Behav 82:621–626.

Chen AS, Metzger JM, Trumbauer ME, Guan XM, Yu H, Frazier EG, Marsh DJ,Forrest MJ, Gopal-Truter S, Fisher J, et al. (2000) Role of the melanocortin-4receptor in metabolic rate and food intake in mice. Transgenic Res 9:145–154.

Chen M, Cai M, Aprahamian CJ, Georgeson KE, Hruby V, Harmon CM, and Yang Y(2007) Contribution of the conserved amino acids of the melanocortin-4 receptor in[Nle4,D-Phe7]-alpha-melanocyte-stimulating hormone binding and signaling.J Biol Chem 282:21712–21719.

Cone RD (2005) Anatomy and regulation of the central melanocortin system. NatNeurosci 8:571–578.

Conn PM and Janovick JA (2009) Trafficking and quality control of the gonadotropinreleasing hormone receptor in health and disease. Mol Cell Endocrinol 299:137–145.

Conn PM, Ulloa-Aguirre A, Ito J, and Janovick JA (2007) G protein-coupled receptortrafficking in health and disease: lessons learned to prepare for therapeutic mu-tant rescue in vivo. Pharmacol Rev 59:225–250.

Dubern B, Clement K, Pelloux V, Froguel P, Girardet JP, Guy-Grand B, and TounianP (2001) Mutational analysis of melanocortin-4 receptor, agouti-related protein,and alpha-melanocyte-stimulating hormone genes in severely obese children. J Pe-diatr 139:204–209.

Fan ZC and Tao YX (2009) Functional characterization and pharmacological rescueof melanocortin-4 receptor mutations identified from obese patients. J Cell MolMed 13:3268–3282.

Farooqi IS, Keogh JM, Yeo GS, Lank EJ, Cheetham T, and O’Rahilly S (2003)Clinical spectrum of obesity and mutations in the melanocortin 4 receptor gene.N Engl J Med 348:1085–1095.

Farooqi IS, Yeo GS, Keogh JM, Aminian S, Jebb SA, Butler G, Cheetham T, andO’Rahilly S (2000) Dominant and recessive inheritance of morbid obesity associ-ated with melanocortin 4 receptor deficiency. J Clin Invest 106:271–279.

Farooqi S and O’Rahilly S (2006) Genetics of obesity in humans. Endocr Rev 27:710–718.

Galandrin S, Oligny-Longpre G, and Bouvier M (2007) The evasive nature of drugefficacy: implications for drug discovery. Trends Pharmacol Sci 28:423–430.

Govaerts C, Srinivasan S, Shapiro A, Zhang S, Picard F, Clement K, Lubrano-Berthelier C, and Vaisse C (2005) Obesity-associated mutations in the melanocor-tin 4 receptor provide novel insights into its function. Peptides 26:1909–1919.

Haskell-Luevano C, Cone RD, Monck EK, and Wan YP (2001) Structure activitystudies of the melanocortin-4 receptor by in vitro mutagenesis: identification ofagouti-related protein (AGRP), melanocortin agonist and synthetic peptide antag-onist interaction determinants. Biochemistry 40:6164–6179.

Huszar D, Lynch CA, Fairchild-Huntress V, Dunmore JH, Fang Q, Berkemeier LR,Gu W, Kesterson RA, Boston BA, Cone RD, et al. (1997) Targeted disruption of themelanocortin-4 receptor results in obesity in mice. Cell 88:131–141.

Institute of Laboratory Animal Resources (1996) Guide for the Care and Use ofLaboratory Animals 7th ed. Institute of Laboratory Animal Resources, Commis-sion on Life Sciences, National Research Council, Washington DC.

Janovick JA, Maya-Nunez G, and Conn PM (2002) Rescue of hypogonadotropichypogonadism-causing and manufactured GnRH receptor mutants by a specificprotein-folding template: misrouted proteins as a novel disease etiology and ther-apeutic target. J Clin Endocrinol Metab 87:3255–3262.

Lubrano-Berthelier C, Dubern B, Lacorte JM, Picard F, Shapiro A, Zhang S, BertraisS, Hercberg S, Basdevant A, Clement K, et al. (2006) Melanocortin 4 receptor

mutations in a large cohort of severely obese adults: prevalence, functional clas-sification, genotype-phenotype relationship, and lack of association with bingeeating. J Clin Endocrinol Metab 91:1811–1818.

Maguire MP, Dai M, and Vos TJ (2002), inventors; Millennium Pharmaceuticals,Inc., Maguire MP, Dai M, and Vos TJ, assignees. Melanocortin-4 receptor bindingcompounds and methods of use thereof. World patent WO02062766, 2002 Aug 15.

Morello JP, Salahpour A, Laperriere A, Bernier V, Arthus MF, Lonergan M, Petaja-Repo U, Angers S, Morin D, Bichet DG, et al. (2000) Pharmacological chaperonesrescue cell-surface expression and function of misfolded V2 vasopressin receptormutants. J Clin Invest 105:887–895.

Nijenhuis WA, Garner KM, van Rozen RJ, and Adan RA (2003) Poor cell surfaceexpression of human melanocortin-4 receptor mutations associated with obesity.J Biol Chem 278:22939–22945.

Noorwez SM, Malhotra R, McDowell JH, Smith KA, Krebs MP, and Kaushal S (2004)Retinoids assist the cellular folding of the autosomal dominant retinitis pigmen-tosa opsin mutant P23H. J Biol Chem 279:16278–16284.

Petaja-Repo UE, Hogue M, Bhalla S, Laperriere A, Morello JP, and Bouvier M (2002)Ligands act as pharmacological chaperones and increase the efficiency of deltaopioid receptor maturation. EMBO J 21:1628–1637.

Pogozheva ID, Chai BX, Lomize AL, Fong TM, Weinberg DH, Nargund RP, Mulhol-land MW, Gantz I, and Mosberg HI (2005) Interactions of human melanocortin 4receptor with nonpeptide and peptide agonists. Biochemistry 44:11329–11341.

Pontillo J, Marinkovic D, Tran JA, Arellano M, Fleck BA, Wen J, Tucci FC, NelsonJ, Saunders J, Foster AC, et al. (2005a) Optimization of piperazinebenzylamineswith a N-(1-methoxy-2-propyl) side chain as potent and selective antagonists of thehuman melanocortin-4 receptor. Bioorg Med Chem Lett 15:4615–4618.

Pontillo J, Tran JA, White NS, Arellano M, Fleck BA, Marinkovic D, Tucci FC,Saunders J, Foster AC, and Chen C (2005b) Structure-activity relationship studieson a series of cyclohexylpiperazines bearing a phanylacetamide as ligands of thehuman melanocortin-4 receptor. Bioorg Med Chem Lett 15:5237–5240.

Rajagopal S, Rajagopal K, and Lefkowitz RJ (2010) Teaching old receptors newtricks: biasing seven-transmembrane receptors. Nat Rev Drug Discov 9:373–386.

Robben JH, Sze M, Knoers NV, and Deen PM (2007) Functional rescue of vasopressinV2 receptor mutants in MDCK cells by pharmacochaperones: relevance to therapyof nephrogenic diabetes insipidus. Am J Physiol Renal Physiol 292:F253–F260.

Schoneberg T, Schulz A, Biebermann H, Hermsdorf T, Rompler H, and Sangkuhl K(2004) Mutant G-protein-coupled receptors as a cause of human diseases. Phar-macol Ther 104:173–206.

Sebhat IK, Martin WJ, Ye Z, Barakat K, Mosley RT, Johnston DB, Bakshi R, Palucki B,Weinberg DH, MacNeil T, et al. (2002) Design and pharmacology of N-[(3R)-1,2,3,4-tetrahydroisoquinolinium- 3-ylcarbonyl]-(1R)-1-(4-chlorobenzyl)- 2-[4-cyclohexyl-4-(1H-1,2,4-triazol- 1-ylmethyl)piperidin-1-yl]-2-oxoethylamine (1), a potent, selective,melanocortin subtype-4 receptor agonist. J Med Chem 45:4589–4593.

Tan K, Pogozheva ID, Yeo GS, Hadaschik D, Keogh JM, Haskell-Leuvano C,O’Rahilly S, Mosberg HI, and Farooqi IS (2009) Functional characterization andstructural modeling of obesity associated mutations in the melanocortin 4 recep-tor. Endocrinology 150:114–125.

Tao YX (2005) Molecular mechanisms of the neural melanocortin receptor dysfunc-tion in severe early onset obesity. Mol Cell Endocrinol 239:1–14.

Tarnow P, Schoneberg T, Krude H, Gruters A, and Biebermann H (2003) Mutationallyinduced disulfide bond formation within the third extracellular loop causes melano-cortin 4 receptor inactivation in patients with obesity. J Biol Chem 278:48666–48673.

Thompson MD, Percy ME, McIntyre Burnham W, and Cole DE (2008) G protein-coupledreceptors disrupted in human genetic disease. Methods Mol Biol 448:109–137.

Vos TJ, Balani S, Blackburn C, Chau RW, Danca MD, Drabic SV, Farrer CA, Patane MA,Stroud SG, Yowe DL, et al. (2006) Identification and structure-activity relationships of anew series of Melanocortin-4 receptor antagonists. Bioorg Med Chem Lett 16:2302–2305.

Xiang Z, Litherland SA, Sorensen NB, Proneth B, Wood MS, Shaw AM, Millard WJ, andHaskell-Luevano C (2006) Pharmacological characterization of 40 human melanocortin-4receptor polymorphisms with the endogenous proopiomelanocortin-derived agonistsand the agouti-related protein (AGRP) antagonist. Biochemistry 45:7277–7288.

Yang Y, Cai M, Chen M, Qu H, McPherson D, Hruby V, and Harmon CM (2009) Keyamino acid residues in the melanocortin-4 receptor for nonpeptide THIQ specificbinding and signaling. Regul Pept 155:46–54.

Yeo GS, Lank EJ, Farooqi IS, Keogh J, Challis BG, and O’Rahilly S (2003) Mutationsin the human melanocortin-4 receptor gene associated with severe familial obesitydisrupts receptor function through multiple molecular mechanisms. Hum MolGenet 12:561–574.

Address correspondence to: Dr. Michel Bouvier, Institute for Researchin Immunology and Cancer, Universite de Montreal, C.P. 6128 SuccursaleCentre-Ville, Montreal, Quebec H3C 3J7, Canada. E-mail: [email protected]

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