Patent Application
20180360972
G protein-coupled receptors (GPCRs) are highly sought after drug targets in the pharmaceutical industry with approximately 30-40% of drugs targeting them (Rask-Andersen, M. et al. Nature Reviews Drug Discovery 2011, 10, 579-590 and Santos, R. et al. Nature Reviews Drug Discovery 2017, 16, 19-34). Classically, medicinal chemists targeted GPCRs as monomeric units; however increasing evidence has shown GPCRs form dimers with themselves (homodimers) and with other GPCRs (heterodimers) (Ferre, S. et al. Pharmacol. Rev. 2014, 66, 413-434 and Ferre, S. et al. Trends Pharmacol. Sci. 2015, 36, 145-152). Targeting GPCR homodimers' and heterodimers' distinct and exploitable functions may yield a revolution in GPCR targeting therapeutics. Although ligands targeting heterodimers have shown much promise in both in vitro and in vivo preclinical studies (Daniels, D. J. et al. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 19208-19213; Smeester, B. A. et al. Eur. I Pharmacol. 2014, 743, 48-52; Le Naour, M. et al. J. Med. Chem. 2013, 56, 5505-5513; Akgün, E. et al. J. Med. Chem. 2015, 58, 8647-8657 and Portoghese, P. S. et. al. ACS Chem. Neurosci. 2017, 8, 426-428) there has been limited development of ligands targeting the allosterism that can occur within homodimers.
Pharmacologically targeting homodimers possess a unique conundrum: How to target and detect a homodimer when the two receptors comprising it are structurally similar, usually respond to the same ligands, and appear to have the same propensity to signal in standard cell culture assays? Various groups have devised clever strategies around these problems to demonstrate the functional consequences of asymmetric homodimers (Han, Y. et al. Nat. Chem. Biol. 2009, 5, 688-695; Pellissier, L. P. et al. J. Biol. Chem. 2011, 286, 9985-9997; Teitler, M. et al. Pharmacol. Ther. 2012, 133, 205-217; Comps-Agrar, L. et al. EMBO J. 2011, 30, 2336-2349; Pin, J. P. et al. Febs J. 1 2005, 272, 2947-2955; Hlavackova, V. et al. EMBO J. 2005, 24, 499-509; Prezeau, L. et al. Neuropharmacology 2005, 49, 267-267; Kniazeff, J. et al. Nat. Struct. Mol. Biol. 2004, 11, 706-713; Kniazeff, J. et al. J. Neurosci. 2004, 24, 370-377; Zylbergold, P. et al. Nat. Chem. Biol. 2009, 5, 608-609; Szalai, B. et al. Biochem. Pharmacol. 2012, 84, 477-485; Damian, M. et al. EMBO J. 2006, 25, 5693-5702; Brock, C. et al. J. Biol. Chem. 2007, 282, 33000-33008; Sartania, N et al. Cell. Signal. 2007, 19, 1928-1938; Gracia, E. et al. Neuropharmacology 2013, 71, 56-69; Chapman, K. L. et al. Biochim. Biophys. Acta. 2013, 1828, 535-542; Orcel, H. et al. Mol. Pharmacol. 2009, 75, 637-647 and Iglesias, A. et al. Eur. J. Pharmacol. 2017, 800, 63-69). Some of these groups focus on demonstrating subtle changes in pharmacology utilizing strategically designed in vitro experiments and other groups exploited receptor mutation strategies in order to differentiate between the two protomers making up the dimer (Han, Y. et al. Nat. Chem. Biol. 2009, 5, 688-695; Pellissier, L. P. et al. J. Biol. Chem. 2011, 286, 9985-9997; Teitler, M. et al. Pharmacol. Ther. 2012, 133, 205-217; Comps-Agrar, L. et al. EMBO J. 2011, 30, 2336-2349; Pin, J. P. et al. Febs J. 2005, 272, 2947-2955; Hlavackova, V. et al. EMBO J. 2005, 24, 499-509; Prezeau, L. et al. Neuropharmacology 2005, 49, 267-267; Kniazeff, J. et al. Nat. Struct. Mol. Biol. 2004, 11, 706-713; Kniazeff, J. et al. J. Neurosci. 2004, 24, 370-377; Zylbergold, P. et al. Nat. Chem. Biol. 2009, 5, 608-609; Szalai, B. et al. Biochem. Pharmacol. 2012, 84, 477-485; Damian, M. et al. EMBO J. 2006, 25, 5693-5702; Brock, C. et al. J. Biol. Chem. 2007, 282, 33000-33008; Sartania, N et al. Cell. Signal. 2007, 19, 1928-1938; Gracia, E. et al. Neuropharmacology 2013, 71, 56-69; Chapman, K. L. et al. Biochim. Biophys. Acta. 2013, 1828, 535-542 and Orcel, H. et al. Mol. Pharmacol. 2009, 75, 637-647). For example, Han and coworkers in 2009 combined different receptor-G protein fusions and various mutant receptors to demonstrate allosteric modulation within a dopamine homodimer (Han, Y. et al. Nat. Chem. Biol. 2009, 5, 688-695). They reported that the D2 dopamine receptor homodimers are maximally activated upon a single agonist binding a single protomer in the dimer pair. When a second agonist binds the second protomer, it blunts the signal. If an inverse agonist binds the second protomer, it enhances the signal beyond agonist alone (Han, Y. et al. Nat. Chem. Biol. 2009, 5, 688-695).
In a different strategy, Teitler and coworkers developed pseudo-irreversible inactivators and reactivators that can be used to block only one of the protomers within the dimer pair in order to demonstrate the crosstalk within wild type serotonin homodimers (Teitler, M. et al. Pharmacol. Ther. 2012, 133, 205-217). This approach can and has been used to demonstrate the allosteric regulation within homodimers in native tissue samples. Application of this technique in vivo would be difficult given the multiple dosing regimen necessary and, therefore, would have very limited therapeutic applications (Teitler, M. et al. Pharmacol. Ther. 2012, 133, 205-217). Although these reports provide critical proof of the relevancy and functional significance of asymmetric signaling homodimers, the techniques employed are limited by their use of receptor mutations or subtle pharmacological differences that make adaption of the approaches to in vivo applications difficult and therapeutic applications inexecutable. Ideally, a pharmacological approach is needed to target and exploit allosteric communication between homodimers with a single chemical entity that could be used to examine the in vivo effects of asymmetric GPCR homodimers to study their potential as therapeutic targets.
One approach to pharmacologically targeting GPCR dimers is utilizing bivalent ligands. This approach was pioneered Portoghese and coworkers targeting the opioid receptors (Portoghese, P. S. et al. Life Sci. 1982, 31, 1283-1286 and Erez, M. et al. J. Med. Chem. 1982, 25, 847-849). Heterobivalent ligands featuring pharmacophores for two different receptor types have been utilized to exploit allosteric interactions within heterodimers to develop ligands with novel pharmacological profiles, tissue selectivity, and different functional effects (Daniels, D. J. et al. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 19208-19213; Smeester, B. A. et al. Eur. J. Pharmacol. 2014, 743, 48-52; Le Naour, M. et al. J. Med. Chem. 2013, 56, 5505-5513; Akgün, E. et al. J. Med. Chem. 2015, 58, 8647-8657, Le Naour, M. et al. J. Med. Chem. 2014, 57, 6383-6392 and Hiller, C. et al. J. Med. Chem. 2013, 56, 6542-6559). However, no one has exploited the allosteric communication that may occur between homodimers with bivalent ligands to produce novel pharmacologies. Unmatched bivalent ligands (UmBLs) have an agonist pharmacophore on one side of the bivalent ligand connected to an antagonist pharmacophore through an inert linker. The term UmBLs is used to separate this class of ligands from heterobivalent ligands that also have different pharmacophores on each side of the bivalent ligand, but are usually used to target different receptor types. This UmBL design has been proposed and reported previously, however, it has not been used to successfully exploit asymmetric signaling of GPCR homodimers (Kuhhorn, J. et al. J. Med. Chem. 2011, 54, 7911-7919; Fernandes, S. M. et al. Bioorg. Med. Chem. 2014, 22, 6360-6365 and Smith, N. J. et al. Pharmacol. Rev. 2010, 62, 701-725).
Both agonist and antagonist homobivalent ligands targeting the melanocortin receptor system have been previously reported (Lensing, C. J. et al. J. Med. Chem. 2016, 59, 3112-3128 and Lensing, C. J. et al. ACS Chem. Neurosci. 2017, 8, 1262-1278). Ligands targeting the melanocortin system have been implicated as potential therapeutics or used as pharmacological probes for a wide range of diseases states including cancer (Xu, L. P. et al. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 21295-21300; Josan, J. S. et al. Bioconjugate Chem. 2011, 22, 1270-1278; Barkey, N. M. et al. J. Med. Chem. 2011, 54, 8078-8084; Brabez, N. et al. ACS Med. Chem. Lett. 2013, 4, 98-102 and Brabez, N. et al. J. Med. Chem. 2011, 54, 7375-7384), skin pigmentation disorders (Langendonk, J. G. et al. N. Engl. J. Med. 2015, 373, 48-59), social disorders (Penagarikano, O. et al. Sci. Transl. Med. 2015, 7, 271 and Barrett, C. E. et al. Neuropharmacology 2014, 85, 357-366), sexual function disorders (Uckert, S. et al. Expert Opin. Invest. Drugs 2014, 23, 1477-1483; Clayton, A. H. et al. Women's Health 2016, 12, 325-337 and Kingsberg, S. et al. J. Sex. Med. 2015, 12, 389-389), Alzheimer's disease (Giuliani, D. et al. Mol. Cell. Neurosci. 2015, 67, 13-21 and Giuliani, D. et al. Neurobiol. Aging 2014, 35, 537-547), cachexia (Joppa, M. A. et al. Peptides 2005, 26, 2294-2301; Deboer, M. D. et al. Trends Endocrinol. Metab. 2006, 17, 199-204; Doering, S. R. et al. ACS Med. Chem. Lett. 2015, 6, 123-127 and Ericson, M. D. et al. J. Med. Chem. 2015, 58, 4638-4647), and obesity (Lensing, C. J. et al. J. Med. Chem. 2016, 59, 3112-3128; Irani, B. G. et al. Eur. J. Pharmacol. 2011, 660, 80-87; Marsh, D. J. et al. Nat. Genet. 1999, 21, 119-122 and Fan, W. et al. Nature 1997, 385, 165-168). All five melanocortin receptor subtypes (MC1-5R) signal through the Gas protein signaling pathway. In this pathway, agonist binding to the GPCR activates cAMP signal transduction pathways and also results in the recruitment of β-arrestin (Shinyama, H. et al. Endocrinology 2003, 144, 1301-1314). The melanocortin-3 receptor (MC3R) and melanocortin-4 receptor (MC4R) in particular have been elucidated to play a roles in energy homeostasis (Irani, B. G. et al. Eur. J. Pharmacol. 2011, 660, 80-87; Marsh, D. J. et al. Nat. Genet. 1999, 21, 119-122; Fan, W. et al. Nature 1997, 385, 165-168; Huszar, D. et al. Cell 1997, 88, 131-141 and Chen, A. S. et al. Nat. Genet. 2000, 26, 97-102). Ligands for the MC4R were under intense clinical development to treat obesity and related metabolic disorders; however these ligands were reported to have undesirable effects such as increasing blood pressure (Greenfield, J. R. et al. N. Engl. J. Med. 2009, 360, 44-52) or inducing male erections (Van der Ploeg, L. H. et al. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 11381-11386). It is hypothesized that ligands that target melanocortin homodimers may have unique effects from the current monovalent approaches, and may, therefore circumvent some side effects.
It is previously shown that an agonist homobivalent ligand produces a distinct in vivo pharmacological profile compared to monovalent counterpart suggesting that targeting putative melanocortin dimers may have physiological relevancy (Lensing, C. J. et al. ACS Chem. Neurosci. 2017, 8, 1262-1278). Furthermore, biased ligands would be valuable pharmacological probes to elucidate which signaling pathway is responsible for the various melanocortin dependent effects (i.e. lowered food intake vs increased blood pressure).
Currently, there is a need for new ligands that can asymmetrically signal the melanocortin receptor homodimers.
This invention provides new ligands that are capable of signaling a melanocortin receptor homodimer. Accordingly, the invention provides a compound of formula I:
Y—X—Z I
or a salt thereof, wherein:
X is a linking group; and
Y is a melanocortin receptor agonist and Z is a melanocortin receptor antagonist; or Y is a melanocortin receptor antagonist and Z is a melanocortin agonist.
The invention also provides a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
The invention also provides a method for treating obesity or a disease associated with obesity in an animal (e.g., a mammal, such as a human) comprising administering a compound of formula I or a pharmaceutically acceptable salt thereof to the animal.
The invention also provides a compound of formula I or a pharmaceutically acceptable salt thereof for use in medical therapy.
The invention also provides a compound of formula I or a pharmaceutically acceptable salt thereof for the prophylactic or therapeutic treatment of obesity or a disease associated with obesity.
The invention also provides the use of a compound of formula I or a pharmaceutically acceptable salt thereof to prepare a medicament for treating obesity or a disease associated with obesity.
The invention also provides processes and intermediates disclosed herein that are useful for preparing a compound of formula I or a salt thereof.
The term “alkyl”, by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain hydrocarbon radical, having the number of carbon atoms designated (i.e., C1-4 means one to four carbons). Non limiting examples of “alkyl” include methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl.
The term “halo” means fluoro, chloro, bromo, or iodo.
The term “haloalkyl” means an alkyl that is optionally substituted with one or more (e.g., 1, 2, 3, 4, or 5) halo. Non limiting examples of “haloalkyl” include iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl 2,2-difluoroethyl and pentafluoroethyl.
The term “alkoxy” refers to an alkyl groups attached to the remainder of the molecule via an oxygen atom (“oxy”).
The term “alkylthio” refers to an alkyl groups attached to the remainder of the molecule via a thio group.
The term “cycloalkyl” refers to a saturated all carbon ring having 3 to 8 carbon atoms (i.e., (C3-C8)carbocycle). The term also includes multiple condensed, saturated all carbon ring systems (e.g., ring systems comprising 2, 3 or 4 carbocyclic rings). Accordingly, carbocycle includes multicyclic carbocyles such as a bicyclic carbocycles (e.g., bicyclic carbocycles having about 3 to 15 carbon atoms, about 6 to 15 carbon atoms, or 6 to 12 carbon atoms such as bicyclo[3.1.0]hexane and bicyclo[2.1.1]hexane), and polycyclic carbocycles (e.g tricyclic and tetracyclic carbocycles with up to about 20 carbon atoms). The rings of the multiple condensed ring system can be connected to each other via fused, spiro and bridged bonds when allowed by valency requirements. For example, multicyclic carbocyles can be connected to each other via a single carbon atom to form a spiro connection (e.g., spiropentane, spiro[4,5]decane, etc), via two adjacent carbon atoms to form a fused connection (e.g., carbocycles such as decahydronaphthalene, norsabinane, norcarane) or via two non-adjacent carbon atoms to form a bridged connection (e.g., norbornane, bicyclo[2.2.2]octane, etc). Non-limiting examples of cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptane, pinane, and adamantane.
The term “heterocycle” refers to a single saturated or partially unsaturated ring that has at least one atom other than carbon in the ring, wherein the atom is selected from the group consisting of oxygen, nitrogen and sulfur; the term also includes multiple condensed ring systems that have at least one such saturated or partially unsaturated ring, which multiple condensed ring systems are further described below. Thus, the term includes single saturated or partially unsaturated rings (e.g., 3, 4, 5, 6 or 7-membered rings) from about 1 to 6 carbon atoms and from about 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur in the ring. The ring may be substituted with one or more (e.g., 1, 2 or 3) oxo groups and the sulfur and nitrogen atoms may also be present in their oxidized forms. Exemplary heterocycles include but are not limited to azetidinyl, tetrahydrofuranyl and piperidinyl. The term “heterocycle” also includes multiple condensed ring systems (e.g., ring systems comprising 2, 3 or 4 rings) wherein a single heterocycle ring (as defined above) can be condensed with one or more groups selected from cycloalkyl, aryl, and heterocycle to form the multiple condensed ring system. The rings of the multiple condensed ring system can be connected to each other via fused, spiro and bridged bonds when allowed by valency requirements. It is to be understood that the individual rings of the multiple condensed ring system may be connected in any order relative to one another. It is also to be understood that the point of attachment of a multiple condensed ring system (as defined above for a heterocycle) can be at any position of the multiple condensed ring system including a heterocycle, aryl and carbocycle portion of the ring. In one embodiment the term heterocycle includes a 3-15 membered heterocycle. In one embodiment the term heterocycle includes a 3-10 membered heterocycle. In one embodiment the term heterocycle includes a 3-8 membered heterocycle. In one embodiment the term heterocycle includes a 3-7 membered heterocycle. In one embodiment the term heterocycle includes a 3-6 membered heterocycle. In one embodiment the term heterocycle includes a 4-6 membered heterocycle. In one embodiment the term heterocycle includes a 3-10 membered monocyclic or bicyclic heterocycle comprising 1 to 4 heteroatoms. In one embodiment the term heterocycle includes a 3-8 membered monocyclic or bicyclic heterocycle heterocycle comprising 1 to 3 heteroatoms. In one embodiment the term heterocycle includes a 3-6 membered monocyclic heterocycle comprising 1 to 2 heteroatoms. In one embodiment the term heterocycle includes a 4-6 membered monocyclic heterocycle comprising 1 to 2 heteroatoms. Exemplary heterocycles include, but are not limited to aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, tetrahydrofuranyl, dihydrooxazolyl, tetrahydropyranyl, tetrahydrothiopyranyl, 1,2,3,4-tetrahydroquinolyl, benzoxazinyl, dihydrooxazolyl, chromanyl, 1,2-dihydropyridinyl, 2,3-dihydrobenzofuranyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl, spiro[cyclopropane-1,1′-isoindolinyl]-3′-one, isoindolinyl-1-one, 2-oxa-6-azaspiro[3.3]heptanyl, imidazolidin-2-one imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, and 1,4-dioxane.
The term “alkoxycarbonyl” as used herein refers to a group (alkyl)-O—C(═O)—, wherein the term alkyl has the meaning defined herein.
The term “alkanoyloxy” as used herein refers to a group (alkyl)-C(═O)—O—, wherein the term alkyl has the meaning defined herein.
As used herein, the term “heteroatom” is meant to include oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).
Specifically, (C1-C6)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl; (C3-C6)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; (C1-C6)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy; (C1-C6)alkanoyl can be acetyl, propanoyl or butanoyl; (C1-C6)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, or hexyloxycarbonyl; (C1-C6)alkylthio can be methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, pentylthio, or hexylthio; and (C2-C6)alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy.
As used herein a wavy line “” that intersects a bond in a chemical structure indicates the point of attachment of the bond that the wavy bond intersects in the chemical structure to the remainder of a molecule.
The compounds disclosed herein can also exist as tautomeric isomers in certain cases. Although only one delocalized resonance structure may be depicted, all such forms are contemplated within the scope of the invention.
It is understood by one skilled in the art that this invention also includes any compound claimed that may be enriched at any or all atoms above naturally occurring isotopic ratios with one or more isotopes such as, but not limited to, deuterium (2H or D). As a non-limiting example, a —CH3 group may be substituted with —CD3.
The term “amino acid,” comprises the residues of the natural amino acids (e.g. Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as well as unnatural amino acids (e.g. Dap, PyrAla, ThiAla, (pCl)Phe, (pNO2)Phe, ε-Aminocaproic acid, Met[O2], dehydPro, (31)Tyr, norleucine (Nle), para-I-phenylalanine ((pI)Phe), 2-napthylalanine (2-Nal), β-cyclohexylalanine (Cha), β-alanine β-Ala), phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid (Tic), penicillamine, ornithine, citruline, a-methyl-alanine, para-b enzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine) in D or L form. The term also comprises natural and unnatural amino acids bearing a conventional amino protecting group (e.g. acetyl or benzyloxycarbonyl), as well as natural and unnatural amino acids protected at the carboxy terminus (e.g. as a (C1-C6)alkyl, phenyl or benzyl ester or amide; or as an a-methylbenzyl amide). Other suitable amino and carboxy protecting groups are known to those skilled in the art (See for example, T. W. Greene, Protecting Groups In Organic Synthesis; Wiley: New York, 1981, and references cited therein). An amino acid can be linked to the remainder of a compound of formula I through the carboxy terminus, the amino terminus, or through any other convenient point of attachment, such as, for example, through the sulfur of cysteine.
As used herein, the term “residue of an amino acid” means a portion of an amino acid. Non-limiting examples include a residue of L-histidine, D-histidine, L-phenylalanine, D-phenylalanine, L-arginine, D-arginine, L-tryptophan, D-tryptophan, L-2-naphthyl-alanine, and D-2-naphthyl-alanine, wherein certain atoms (e.g., H or OH) may have been removed to link the amino acids via a peptide bond.
It is understood that Y and Z can be linked to X at any synthetically feasible position on Y or Z.
It will be appreciated by those skilled in the art that compounds of the invention having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase.
When a bond in a compound of formula I herein is drawn in a non-stereochemical manner (e.g. flat), the atom to which the bond is attached includes all stereochemical possibilities. When a bond in a compound formula herein is drawn in a defined stereochemical manner (e.g. bold, bold-wedge, dashed or dashed-wedge), it is to be understood that the atom to which the stereochemical bond is attached is enriched in the absolute stereoisomer depicted unless otherwise noted. In one embodiment, the compound may be at least 51% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 60% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 80% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 90% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 95% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 99% the absolute stereoisomer depicted.
As described herein, the one portion of a compound can be bonded (connected) to the remainder of the compound through an optional linker. In one embodiment the linker is absent. The linker can vary in length and atom composition and for example can be branched or non-branched or cyclic or a combination thereof. The linker may also modulate the properties of the compound such as but not limited to solubility, stability and aggregation.
In one embodiment the linker comprises about 3-100 atoms. In one embodiment the linker comprises about 3-50 atoms. In one embodiment the linker comprises about 3-25 atoms.
In one embodiment the linker comprises atoms selected from H, C, N, S and O.
In one embodiment the linker comprises atoms selected from H, C, N, S, P and O.
In one embodiment the linker comprises a branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from about 1 to 100 (or 1-50, 1-25, 1-10, 1-5, 5-100, 5-50, 5-25, 5-10 or 2-5 carbon atoms) wherein one or more of the carbon atoms is optionally replaced independently by —O—, —S, —N(Ra)—, 3-7 membered heterocycle, 5-6-membered heteroaryl or carbocycle and wherein each chain, 3-7 membered heterocycle, 5-6-membered heteroaryl or carbocycle is optionally and independently substituted with one or more (e.g. 1, 2, 3, 4, 5 or more) substituents selected from (C1-C6)alkyl, (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, —N(Ra)2, hydroxy, oxo (═O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, wherein each Ra is independently H or (C1-C6)alkyl. In one embodiment the linker comprises a branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from about 1 to 100 (or 1-50, 1-25, 1-10, 1-5, 5-100, 5-50, 5-25, 5-10 or 2-5 carbon atoms) wherein one or more of the carbon atoms is optionally replaced independently by —O—, —S, —N(Ra)—, wherein each Ra is independently H or (C1-C6)alkyl.
In one embodiment the linker comprises a polyethylene glycol. In one embodiment the linker comprises a polyethylene glycol linked to the remainder of the targeted conjugate by a carbonyl group. In one embodiment the polyethylene glycol comprises about 1 to about 10 (e.g., —CH2CH2O—) units (Greenwald, R. B., et al., Poly (ethylene glycol) Prodrugs: Altered Pharmacokinetics and Pharmacodynamics, Chapter, 2.3.1., 283-338; Filpula, D., et al., Releasable PEGylation of proteins with customized linkers, Advanced Drug Delivery, 60, 2008, 29-49; Zhao, H., et al., Drug Conjugates with Poly(Ethylene Glycol), Drug Delivery in Oncology, 2012, 627-656).
In one embodiment the linker is —NH(CH2CH2O)4CH2CH2C(═O)—. In one embodiment the linker is —NH(CH2CH2O)nCH2CH2C(═O)— wherein n is 1-10, 1-5, 2-10, 2-5, 3-10, 3-5, 4-10, 4-5. In one embodiment the linker is —(CH2CH2O)4CH2CH2C(═O)—.
The terms “treat” and “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or decrease an undesired physiological change or disorder, such as a metabolic disorder (e.g., obesity) or a disease associated with the metabolic disorder. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
The phrase “effective amount” means an amount of a compound of the present invention that (i) treats or prevents the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.
In one embodiment, the melanocortin receptor agonist comprises an amino acid sequence of His-DPhe-Arg-Trp (SEQ ID NO:1).
In one embodiment, the melanocortin receptor antagonist comprises an amino acid sequence of His-DNal(2′)-Arg-Trp (SEQ ID NO:2).
In one embodiment, the compound of invention has the following formula II:
CH3C(═O)-A-X—B—NH2 II
or a salt thereof, wherein:
A is -His-DPhe-Arg-Trp-, -His-DNal(2′)-Arg-Trp-, -DTrp-DArg-Phe-DHis-, or -DTrp-DArg-Nal(2′)-DHis-;
B is -His-DPhe-Arg-Trp-, -His-DNal(2′)-Arg-Trp-, -DTrp-DArg-Phe-DHis-, or -DTrp-DArg-Nal(2′)-DHis-;
X is a linking group;
His is a residue of L-histidine;
DHis is a residue of D-histidine;
Phe is a residue of L-phenylalanine, wherein the phenyl ring is optionally substituted with one or more groups selected from halo, (C1-C4)alkyl, —O(C1-C4)alkyl, (C1-C4)haloalkyl, or —O(C1-C4)haloalkyl;
DPhe is a residue of D-phenylalanine, wherein the phenyl ring is optionally substituted with one or more groups selected from halo, (C1-C4)alkyl, —O(C1-C4)alkyl, (C1-C4)haloalkyl, or —O(C1-C4)haloalkyl;
Arg is a residue of L-arginine;
DArg is a residue of D-arginine;
Trp is a residue of L-tryptophan, wherein the indolyl ring is optionally substituted with one or more groups selected from halo, (C1-C4)alkyl, —O(C1-C4)alkyl, (C1-C4)haloalkyl, or —O(C1-C4)haloalkyl;
DTrp is a residue of D-tryptophan, wherein the indolyl ring is optionally substituted with one or more groups selected from halo, (C1-C4)alkyl, —O(C1-C4)alkyl, (C1-C4)haloalkyl, or —O(C1-C4)haloalkyl;
Nal(2′) is a residue of L-2-naphthyl-alanine, wherein the phenyl ring is optionally substituted with one or more groups selected from halo, (C1-C4)alkyl, —O(C1-C4)alkyl, (C1-C4)haloalkyl, or —O(C1-C4)haloalkyl; and
DNal(2′) is a residue of D-2-naphthyl-alanine, wherein the phenyl ring is optionally substituted with one or more groups selected from halo, (C1-C4)alkyl, —O(C1-C4)alkyl, (C1-C4)haloalkyl, or —O(C1-C4)haloalkyl;
provided if A is -His-DPhe-Arg-Trp-, B is not -His-DPhe-Arg-Trp-, wherein the phenyl ring and the indolyl ring are not substituted;
and provided if A is -His-DNal(2′)-Arg-Trp-, B is not -His-DNal(2′)-Arg-Trp-, wherein the naphthyl ring and the indolyl ring are not substituted.
In one embodiment, A is -His-DPhe-Arg-Trp- or -His-DNal(2′)-Arg-Trp-.
In one embodiment, A is:
In one embodiment, B is -His-DPhe-Arg-Trp- or -His-DNal(2′)-Arg-Trp-.
In one embodiment, B is:
In one embodiment, the compound of invention is a compound of formula Ia:
or a salt thereof.
In one embodiment, the compound of invention is a compound of formula Ib:
or a salt thereof.
In one embodiment, X is a branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from about 10-100 carbon atoms, wherein one or more of the carbon atoms is optionally replaced independently by —O—, —S, —N(Ra)—, or 3-7 membered heterocycle, wherein the hydrocarbon chain is optionally substituted with one or more substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, —N(Ra)2, hydroxy, oxo (═O), or carboxy, wherein each Ra is independently H or (C1-C6)alkyl.
In one embodiment, X is a branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from about 10-50 carbon atoms, wherein one or more of the carbon atoms is optionally replaced independently by —O—, —S, —N(Ra)—, or 3-7 membered heterocycle, wherein the hydrocarbon chain is optionally substituted with one or more substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, —N(Ra)2, hydroxy, oxo (═O), or carboxy, wherein each Ra is independently H or (C1-C6)alkyl.
In one embodiment, X comprises at least one unit of —CH2CH2O—.
In one embodiment, X comprises about 3 to 10 units of —CH2CH2O—.
In one embodiment, X is —NH(CH2CH2O)nCH2CH2C(═O)—, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
In one embodiment, X is —NH(CH2CH2O)nCH2CH2C(═O)—, wherein n is 3, 4, 5, or 6.
In one embodiment, X is:
In one embodiment, X is:
In one embodiment, the compound of invention is selected from the group consisting of:
Ac-His-DPhe(p-I)-Arg-Trp-(PEDG20)-His-DPhe(p-I)-Arg-Trp-NH2;
Ac-His-DNal(2′)-Arg-Trp-(Pro-Gly)6-His-DPhe-Arg-Trp-NH2;
Ac-His-DPhe-Arg-Trp-(PEDG20)-His-DNal(2′)-Arg-Trp-NH2;
Ac-His-DNal(2′)-Arg-Trp-(PEDG20)-His-DPhe-Arg-Trp-NH2;
Ac-His-DNal(2′)-Arg-Trp-(PEDG20)-(PEDG20)-His-DPhe-Arg-Trp-NH2;
Ac-His-DPhe(p-I)-Arg-Trp-(Pro-Gly)6-His-DPhe-Arg-Trp-NH2;
Ac-His-DPhe(p-I)-Arg-Trp-(PEDG20)-His-DPhe-Arg-Trp-NH2;
Ac-His-DPhe-Arg-Trp-(PEDG20)-His-DPhe(p-I)-Arg-Trp-NH2;
Ac-His-DNal(2′)-Arg-Trp-(PEDG20)-His-DPhe(p-I)-Arg-Trp-NH2;
Ac-His-DPhe(p-I)-Arg-Trp-(PEDG20)-His-DNal(2′)-Arg-Trp-NH2;
Ac-His-DPhe-Arg-Trp-(PEG)2(22atoms)-His-DNal(2′)-Arg-Trp-NH2;
Ac-His-DPhe-Arg-Trp-(PEDG20)-His-DNal(2′)-Arg-Trp-NH2;
Ac-His-DPhe-Arg-Trp-(PEG)(19atoms)-His-DNal(2′)-Arg-Trp-NH2;
Ac-DTrp-DArg-Phe-DHis-(PEG)(22 atoms)-His-DPhe-Arg-Trp-NH2;
Ac-DTrp-DArg-Phe-DHis-(PEDG20)-His-DPhe-Arg-Trp-NH2;
Ac-DTrp-DArg-Phe-DHis-(PEG)(19 atoms)-His-DPhe-Arg-Trp-NH2;
Ac-His-DPhe-Arg-Trp-(PEG)(22 atoms)-DTrp-DArg-Phe-DHis-NH2;
Ac-His-DPhe-Arg-Trp-(PEDG20)-DTrp-DArg-Phe-DHis-NH2;
Ac-His-DPhe-Arg-Trp-(PEG)(19 atoms)-DTrp-DArg-Phe-DHis-NH2;
Ac-DTrp-DArg-Phe-DHis-(PEG)(22 atoms)-DTrp-DArg-Phe-DHis-NH2;
Ac-DTrp-DArg-Phe-DHis-(PEDG20)-DTrp-DArg-Phe-DHis-NH2; and
Ac-DTrp-DArg-Phe-DHis-(PEG)(19 atoms)-DTrp-DArg-Phe-DHis-NH2;
and salts thereof, wherein:
Ac is CH3C(═O)—;
His is a residue of L-histidine;
DHis is a residue of D-histidine;
Phe is a residue of L-phenylalanine;
DPhe is a residue of D-phenylalanine;
Arg is a residue of L-arginine;
DArg is a residue of D-arginine;
Trp is a residue of L-tryptophan;
DTrp is a residue of D-tryptophan;
DNal(2′) is a residue of D-2-naphthyl-alanine;
In one embodiment, the compound of invention is:
Ac-His-DNal(2′)-Arg-Trp-(Pro-Gly)6-His-DPhe-Arg-Trp-NH2;
Ac-His-DPhe-Arg-Trp-(PEDG20)-His-DNal(2′)-Arg-Trp-NH2;
Ac-His-DNal(2′)-Arg-Trp-(PEDG20)-His-DPhe-Arg-Trp-NH2; or
Ac-His-DNal(2′)-Arg-Trp-(PEDG20)-(PEDG20)-His-DPhe-Arg-Trp-NH2;
or a salt thereof, wherein:
Ac is CH3C(═)—;
His is a residue of L-histidine;
DPhe is a residue of D-phenylalanine;
Arg is a residue of L-arginine;
Trp is a residue of L-tryptophan;
DNal(2′) is a residue of D-2-naphthyl-alanine;
In one embodiment the comnound of invention is:
or a salt thereof.
In one embodiment, the compound of invention comprises first amino acid sequence having at least 80% sequence identity to His-DPhe-Arg-Trp (SEQ ID NO:1), and second amino acid sequence at least 80% identity to His-DNal(2′)-Arg-Trp (SEQ ID NO:2), or a salt thereof.
In one embodiment, the compound of invention comprises first amino acid sequence having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:1, and second amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:2, or a salt thereof.
In one embodiment, the compound of invention comprises first amino acid sequence having at least 90% sequence identity to SEQ ID NO:1, and second amino acid sequence at least 90% identity to SEQ ID NO:2, or a salt thereof.
In one embodiment, the compound of invention comprises first amino acid sequence having at least 99% sequence identity to SEQ ID NO:1, and second amino acid sequence at least 99% identity to SEQ ID NO:2, or a salt thereof.
In one embodiment, the compound of invention comprises first amino acid sequence of SEQ ID NO:1, and second amino acid sequence of SEQ ID NO:2, or a salt thereof.
In one embodiment, the compound of invention is an agonist for MC1R, MC3R, MC4R or MC5R.
In one embodiment, the compound of invention is a selective agonist for MC1R, MC3R, MC4R or MC5R.
One embodiment of the invention provides a dietary supplement comprising a compound of formula I, or a salt thereof.
Another embodiment of the invention provides a prodrug of a compound of formula I or a salt thereof. As used herein the term “prodrug” refers to a biologically inactive compound that can be metabolized in the body to produce a biologically active form of the compound.
In one embodiment, the disease associated with obesity is diabetes, cardiovascular disease or hypertension.
One embodiment of the invention provides a method of modulating (e.g., increasing or decreasing) the activity of a melanocortin receptor in vitro or in vivo comprising contacting the receptor with an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof.
One embodiment of the invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof for use in modulating (e.g., increasing or decreasing) the activity of a melanocortin receptor in vitro or in vivo.
One embodiment of the invention provides the use of a compound of formula I, or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for modulating (e.g., increasing or decreasing) the activity of a melanocortin receptor in vitro or in vivo.
One embodiment of the invention provides a method of modulating (e.g. increasing or decreasing) the activity of a melanocortin receptor homodimer in vitro or in vivo comprising contacting the homodimer with an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof.
One embodiment of the invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof for use in modulating (e.g., increasing or decreasing) the activity of a melanocortin receptor homodimer in vitro or in vivo.
One embodiment of the invention provides the use of a compound of formula I, or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for modulating (e.g., increasing or decreasing) the activity of a melanocortin receptor homodimer in vitro or in vivo.
One embodiment of the invention provides a method of activing cAMP signaling and simultaneously blocking β-arrestin recruitment in vitro or in vivo comprising contacting a melanocortin receptor homodimer with an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof.
One embodiment of the invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof, for use in activing cAMP signaling and simultaneously blocking β-arrestin recruitment in vitro or in vivo.
One embodiment of the invention provides the use of a compound of formula I, or a pharmaceutically acceptable salt thereof for manufacture of a medicament for activing cAMP signaling and simultaneously blocking β-arrestin recruitment in vitro or in vivo.
In one embodiment, the melanocortin receptor or the melanocortin receptor homodimer is MC1R, MC3R, MC4R or MC5R.
In one embodiment, the melanocortin receptor or the melanocortin receptor homodimer is MC3R.
Another embodiment of the invention provides a method of modulating (e.g., increasing or decreasing) metabolic activity in an animal in need thereof, comprising administering an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, to the animal.
Another embodiment of the invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof for use in modulating (e.g., increasing or decreasing) metabolic activity.
Another embodiment of the invention provides the use of a compound of formula I, or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for modulating (e.g., increasing or decreasing) metabolic activity in an animal in need thereof.
Another embodiment of the invention provides a method of modulating (e.g., increasing or decreasing) appetite in an animal in need thereof, comprising administering an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, to the animal.
Another embodiment of the invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof for use in modulating (e.g., increasing or decreasing) appetite.
Another embodiment of the invention provides the use of a compound of formula I, or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for modulating (e.g., increasing or decreasing) appetite in an animal in need thereof.
Another embodiment of the invention provides a method of decreasing food intake, reducing body fat percentage, and/or increasing fat consumption in an animal in need thereof, comprising administering an effective amount of compound of formula I, or a pharmaceutically acceptable salt thereof, to the animal.
Another embodiment of the invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof, for use in decreasing food intake, reducing body fat percentage, and/or increasing fat consumption in an animal in need thereof.
Another embodiment of the invention provides the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for decreasing food intake, reducing body fat percentage, and/or increasing fat consumption in an animal in need thereof.
Another embodiment of the invention provides a method of activating one downstream signaling event and simultaneously blocking a different downstream signaling event of a G protein-couple receptor (GPCR) homodimer comprising contacting the GPCR homodimer a ligand that comprises an agonist pharmacophore and an antagonist pharmacophore, wherein the agonist pharmacophore occupies and activates one receptor within the GPCR homodimer and the antagonist pharmacophore occupies and deactivates the other receptor within the GPCR homodimer.
In one embodiment, the GPCR homodimer is a melanocortin receptor homodimer.
In one embodiment, the agonist pharmacophore activates cAMP signaling and the antagonist pharmacophore deactivates β-arrestin recruitment.
In one embodiment, the agonist pharmacophore is linked to the antagonist pharmacophore through a linking group.
In one embodiment, the agonist pharmacophore comprises an amino acid sequence of SEQ ID NO:1.
In one embodiment, the antagonist pharmacophore comprises an amino acid sequence of SEQ ID NO:2.
Compounds of the invention can also be administered in combination with other therapeutic agents, for example, other agents that are useful for the obesity. Accordingly, in one embodiment the invention also provides a composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, at least one other therapeutic agent, and a pharmaceutically acceptable diluent or carrier. The invention also provides a kit comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, at least one other therapeutic agent, packaging material, and instructions for administering the compound of formula I or the pharmaceutically acceptable salt thereof and the other therapeutic agent or agents to an animal to treat obesity.
In cases where compounds are sufficiently basic or acidic, a salt of a compound of formula (I) can be useful as an intermediate for isolating or purifying a compound of formula (I). Additionally, administration of a compound of formula (I) as a pharmaceutically acceptable acid or base salt may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.
Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.
Compounds of formula (I) (including salts and prodrugs thereof) can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical, nasal, inhalation, suppository, sub dermal osmotic pump, or subcutaneous routes.
Thus, the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.
The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.
The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
For topical administration, the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
Examples of useful dermatological compositions which can be used to deliver the compounds of formula Ito the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).
Useful dosages of the compound of formula (I) can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.
The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.
In general, however, a suitable dose will be in the range of from about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day.
The compound is conveniently formulated in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form. In one embodiment, the invention provides a composition comprising a compound of the invention formulated in such a unit dosage form.
The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.
Compounds of the invention can also be administered in combination with other therapeutic agents. For example, compounds of formula (I), or salts thereof, may be administered with other agents that are useful for modulating appetite (i.e., increasing or decreasing), modulating metabolic activity, treating obesity or diseases associated with obesity (e.g., diabetes, cardiovascular disease or hypertension), inducing weight loss, increasing or decreasing weight gain. Accordingly, in one embodiment the invention also provides a composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, at least one other therapeutic agent, and a pharmaceutically acceptable diluent or carrier. The invention also provides a kit comprising compound of formula (I), or a pharmaceutically acceptable salt thereof, at least one other therapeutic agent, packaging material, and instructions for administering the compound of formula (I) or the pharmaceutically acceptable salt thereof and the other therapeutic agent or agents to an animal to modulate appetite, modulate metabolic activity, treat obesity or diseases associated with obesity (e.g., diabetes, cardiovascular disease or hypertension), induce weight loss, increase weight gain, or decrease weight gain.
The invention will now be illustrated by the following non-limiting Examples.
Homobivalent ligands targeting melanocortin receptors have previously resulted in increased binding affinity (˜14 to 25-fold) consistent with a synergistic binding mode resulting from receptor dimer binding (Fernandes, S. M. et al. Bioorg. Med. Chem. 2014, 22, 6360-6365; Lensing, C. J. et al. J. Med. Chem. 2016, 59, 3112-3128; Lensing, C. J. et al. ACS Chem. Neurosci. 2017, 8, 1262-1278; Brabez, N. et al. J. Med. Chem. 2011, 54, 7375-7384; Carrithers, M. D. et al. Chem. Biol. 1996, 3, 537-542; Elshan, N. G. R. D. et al. Org. Biomol. Chem. 2015, 13, 1778-1791; Handl, H. L. et al. Bioconjugate Chem. 2007, 18, 1101-1109; Vagner, J. et al. Bioorg. Med. Chem. Lett. 2004, 14, 211-215; Bowen, M. E. et al. J. Org. Chem. 2007, 72, 1675-1680; Jagadish, B. et al. Bioorg. Med. Chem. Lett. 2007, 17, 3310-3313; Dehigaspitiya, D. C. et al. Tetrahedron Lett. 2015, 56, 3060-3065 and Dehigaspitiya, D. C. et al. Org. Biomol. Chem. 2015, 13, 11507-11517). In spite of an increased binding affinity, much smaller fold increases in cAMP based functional activity have been observed (3 to 5-fold) (Lensing, C. J. et al. J. Med. Chem. 2016, 59, 3112-3128). Hruby and coworkers noted similar effects with melanocortin bivalent ligands in which cAMP accumulation was not as dramatically increased with synergistic multivalent binding (Brabez, N. et al. J. Med. Chem. 2011, 54, 7375-7384). One possibility for the incongruity between binding affinity increases and functional signaling increases with bivalent ligands may be due to allosterism between the melanocortin receptors within homodimers (Lensing, C. J. et al. J. Med. Chem. 2016, 59, 3112-3128). Such asymmetric signaling with GPCR homodimers has previously been reported for a variety of systems including the vasopressin (Orcel, H. et al. Mol. Pharmacol. 2009, 75, 637-647), dopamine (Han, Y. et al. Nat. Chem. Biol. 2009, 5, 688-695), adenosine (Gracia, E. et al. Neuropharmacology 2013, 71, 56-69), metabotropic glutamate (Kniazeff, J. et al. Nat. Struct. Mol. Biol. 2004, 11, 706-713), and serotonin receptors (Pellissier, L. P. et al. J. Biol. Chem. 2011, 286, 9985-9997).
A new paradigm can be hypothesized in which one receptor within the melanocortin homodimer might be responsible for cAMP signaling and the other receptor might be responsible for signaling through a different cellular pathway (e.g. β-arrestin recruitment pathway) (
Because bivalent ligands presumably occupy both sites in a receptor dimer due to synergistic binding, a MUmBL is postulated to occupy one receptor within a dimer pair with an agonist pharmacophore and the other receptor within the same dimer with an antagonist pharmacophore (
Ligands CJL-1-124, CJL-5-74, and CJL-1-63 feature the His-DPhe-Arg-Trp scaffold on the C-terminus and the His-DNa!(2′)-Arg-Trp scaffold on the N-terminus (
Peptides were synthesized utilizing standard solid phase peptide synthesis and fluorenyl-9-methoxycarbonyl (Fmoc) methodologies to protect the elongating peptide chain (Stewart, J. M. et al. Solid Phase Peptide Synthesis. 2nd edition (Pierce Chemical Co., 1984) and Carpino, L. A. et al. J. Org. Chem. 1972, 37, 3404-3409). A CEM Discover SPS microwave peptide synthesizer was used to expedite couplings and deprotections. A split resin technique was used to synthesize common sequences as previously described (Lensing, C. J. et al. J. Med. Chem. 2016, 59, 3112-3128). The O-(N-Fmoc-3-aminopropyl)-O′-(N-diglycolyl-3-aminopropyl)-diethyleneglycol [Fmoc-NH-(PEG)2-COOH (20atoms) or Fmoc-NH2-PEDG20-COOH] was purchased from Novobiochem® EMD Millipore Corp (Billerica, Mass., USA). The N,N-diisopropylethylamine (DIEA), triisopropylsilane (TIS), 1,2-ethanedithiol (EDT), piperidine, pyridine, and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (St. Louis, Mo.). The 4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetyl-MBHA resin [Rink-amide-MBHA (200-400 mesh), 0.35-0.37 meq/g substitution], 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), and Fmoc-protected amino acids [Fmoc-Pro, Fmoc-Gly, Fmoc-His(Trt), Fmoc-DPhe, Fmoc-Arg(Pbf), Fmoc-Trp(Boc), and Fmoc-DNal(2′)] were purchased from Peptides International (Louisville, Ky., USA). Acetonitrile (MeCN), N,N-dimethylformamide (DMF), acetic anhydride, dichloromethane (DCM), and methanol (MeOH) were purchased from FischerScientific. All reagents were ACS grade or better and were used without further purification.
Peptides were assembled in a fritted polypropylene reaction vessel (25 mL CEM reaction vessel) on the Rink-amide-MBHA resin. A repeated two-step cycle of deprotection with 20% piperidine in DMF, then amide coupling with the Fmoc-amino acid, HBTU, and DIEA was employed until the final peptide was synthesized on resin. Excess reagents were removed between all deprotection or coupling by 3-5 washes of DMF between steps. A Kaiser/ninhydrin test was used after each deprotection or coupling step (except with Pro residues) to indicated the presence or lack of a free primary amine (Kaiser, E. et al. Anal. Biochem. 1970, 34, 595-598). For Pro residues, the presence or lack of a free secondary amine was indicated by a chloranil test (Stewart, J. M. et al. Solid Phase Peptide Synthesis. 2nd edn, (Pierce Chemical Co., 1984) and Christensen, T. Acta Chem. Scand. 1979, 33, 763-766). Removal of the Fmoc group was achieved in a twostep process. First an initial two minute deprotection was performed outside of the microwave. Then a second aliquot of 20% piperidine was added and further deprotection was assisted by microwave heating (75° C., 30 W, 4 min).
Amide coupling was achieved by addition of 3.1-fold excess Fmoc-protected amino acids (5.1-fold for Arg) and 3-fold excess of HBTU (5-fold for Arg) in DMF added to the free amine on the elongating peptide on the resin. After which the 5-fold excess of DIEA (7-fold for Arg) was added, and the reaction was heated in the microwave synthesizer (75° C., 30 W or 50° C., 30 W for His) for five minutes (10 min for Arg). The Fmoc-NH-(PEDG20)-COOH was incorporated into the peptide using the same protocol except it was allowed to cool for at least one hour after microwave heating to ensure the reaction went to completion.
Acetylation was achieved on resin after the final Fmoc deprotection by addition of 3:1 mixture of acetic anhydride to pyridine and were mixed at room temperature with bubbling nitrogen for 30 minutes. Before cleavage, all peptides were washed with DCM at least 3 times and dried in a desiccator. Side chain deprotection and resin cleavage was simultaneously accomplished via addition of 8 mL of a cleavage cocktail (91% TFA, 3% EDT, 3% TIS, 3% water) for 1.5-3 hours. Peptides were precipitated from cleavage solution using cold (4° C.) anhydrous diethyl ether. The cloudy mixture of peptides was vortexed and centrifuged at 4° C. and 4000 RPMs for 4 minutes (Sorval Super T21 high-speed centrifuge swinging bucket rotor). The supernatant was discarded. The crude peptide pellet was then washed with cold (4° C.) diethyl ether and centrifuged. This process was repeated until no thiol aroma was present (usually 3 times) and the peptides were dried overnight in a desiccator.
A Shimadzu chromatography system with a photodiode array detector and a semipreparative RP-HPLC C18 bonded silica column (Vydac 218TP1010, 1 cm×25 cm) were used to purify 5-20 mg sample of crude peptide by RP-HPLC. The solvent system for purification was either MeCN or MeOH in 0.1% aqueous TFA. Purified fractions were collected and peptides were concentrated in vacuo and lyophilized. A purity of 95% or greater was confirmed by RP-HPLC in two diverse solvent systems (10% MeCN in 0.1% TFA/water and a gradient to 90% MeCN over 35 min; and 10% MeOH in 0.1% TFA/water and a gradient to 90% MeOH over 35 minutes). ESI-MS was used to confirm the correct molecular mass (University of Minnesota Department of Chemistry Mass Spectrometry Laboratory) (
Additional ligands were synthesized and purified in an analogous fashion (
Upon agonist stimulation, melanocortin receptors are known to signal through a Gαs-protein mediated signaling pathway that results in intracellular cAMP accumulation. Agonist stimulation of the melanocortin receptors also results in β-arrestin recruitment and receptor desensitization (Shinyama, H. et al. Endocrinology 2003, 144, 1301-1314; Cai, M. Y. et al. Chem. Biol. Drug Des. 2006, 68, 183-193 and Gao, Z. H. et al. J. Pharmacol. Exp. Ther. 2003, 307, 870-877). In order to evaluate the ligands efficacy and potency to stimulate cAMP signaling, ALPHAScreen™ cAMP Assay Technology was utilized to assess live HEK293 cells stably expressing human (h)MC4R (Xiang, Z. et al. Biochemistry 2010, 49, 4583-4600 and Xiang, Z. et al. Biochemistry 2006, 45, 7277-7288). All ligands that contained the His-DPhe-Arg-Trp pharmacophore, including the MUmBLs, were single-digit or sub-nanomolar agonists in the cAMP assay (
The MUmBLs (i.e. CJL-1-63, CJL-5-58, CJL-1-124, and CJL-5-74) were all single digit nanomolar agonists at the hMC4R. For comparison with the MUmBLs and as a control, an equal mixture of tetrapeptides CJL-1-14+CJL-1-80 was assayed. In order to give the best comparison to the MUmBLs, 1 nM of the tetrapeptide mixture contained 1 nM CJL-1-14 and 1 nM CJL-1-80 (for a final concentration of 2 nM total peptide) was tested. This would be directly comparable to 1 nM of a MUmBL when looking at final pharmacophore concertation. This tetrapeptide mixture resulted in an agonist dose response curve with an EC50 of 1.9±0.2 nM. From this data, it appears that antagonist scaffold His-DNal(2′)-Arg-Trp is not capable of effecting the cAMP agonist pharmacology of His-DPhe-Arg-Trp agonist scaffold when mixed in equal portions.
Theoretically, if both the agonist scaffold and antagonist scaffold compete equally for binding, then at 100% receptor occupancy 50% of the receptors would be occupied by agonist tetrapeptide scaffold and 50% would be occupied by the antagonist tetrapeptide scaffold. This likelihood of 50:50 binding should be amplified by the synergistic bivalent binding mode (Lensing, C. J. et al. J. Med. Chem. 2016, 59, 3112-3128). Based on this assumption of 50:50 binding, the MUmBLs full cAMP agonist pharmacology would be achieved by only 50% receptor occupancy by the agonist scaffold at the receptors, since the antagonist scaffold would be occupying approximately 50% of the receptors. This is consistent with both the spare receptor theory (Stephenson, R. P. Br. J. Pharmacol. Chemother. 1956, 11, 379-393 and Takeyasu, K. et al. Life Sci. 1979, 25, 1761-1771), and the hypothesis presented above for asymmetric signaling homodimers in which ˜50% of the receptors are responsible for β-arrestin recruitment and 50% are responsible for cAMP signaling (
It was, therefore, hypothesized that the second binding event within the GPCR dimer may be responsible for a different functional response not detected in the cAMP functional assays. It has previously been observed that β-arrestin recruitment of one protomer within the AT1 angiotensin receptor homodimer can be allosterically regulated by selective stimulation of the other protomer (Szalai, B. et al. Biochem. Pharmacol. 2012, 84, 477-485). In order to examine if β-arrestin recruitment to the hMC4R was regulated differently by MUmBLs versus agonist or antagonist homobivalent ligands, we utilized the PRESTO-Tango assay developed by Roth and colleagues (Kroeze, W. K. et al. Nat. Struct. Mol. Biol. 2015, 22, 362-369 and Barnea, G. et al. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 64-69). The PRESTO-Tango technology is an open-source resource that has been utilized to identify ligands for orphan receptors based on β-arrestin recruitment. This assay has previously been validated at the hMC4R that agonist stimulation results in β-arrestin recruitment (Kroeze, W. K. et al. Nat. Struct. Mol. Biol. 2015, 22, 362-369). In agreement with these results, classic monovalent agonist ligands result in the recruitment of β-arrestin and high signal (
The ligands containing only the antagonist His-DNal(2′)-Arg-Trp pharmacophore resulted in minimal β-arrestin recruitment consistent with a classical antagonist pharmacology. The tetrapeptide Ac-His-DNal(2′)-Arg-Trp-NH2 resulted in 30% response at 10 μM compared to the maximal efficacy of NDP-MSH. The other linker control ligands and the bivalent ligand CJL-1-140 resulted in equal or lower β-arrestin recruitment. This result was not surprising given the antagonist nature of these compounds and that antagonist have previously been reported to result in minimal β-arrestin recruitment and receptor internalization (Shinyama, H. et al. Endocrinology 2003, 144, 1301-1314; Cai, M. Y. et al. Chem. Biol. Drug Des. 2006, 68, 183-193 and Gao, Z. H. et al. J. Pharmacol. Exp. Ther. 2003, 307, 870-877).
The MUmBLs resulted in minimal β-arrestin recruitment. The most potent MUmBL was CJL-1-63 that resulted in 55% maximal efficacy at 10 μM compared to NDP-MSH. All other MUmBLs resulted in less β-arrestin recruitment. Because these ligands still potently stimulate cAMP signaling but result in minimal β-arrestin recruitment, it supports the current hypothesis that one pharmacophore is responsible for the activation of the cAMP pathway, but the other pharmacophore is responsible for the β-arrestin recruitment. When a bivalent ligand is comprised of an agonist scaffold and an antagonist scaffold, it should favor a binding mode in which equal portions of agonist scaffold and antagonist scaffold bind to a GPCR dimer as discussed above (i.e. one MUmBL to two receptors or one dimer). The agonist pharmacophore would then signal effectively through the cAMP pathway, but the antagonist pharmacophore would block the β-arrestin recruitment pathway (
An explanation of the biased agonism is through a model for allosterically interacting receptor dimers (
There is an assumption above that the agonist tetrapeptide scaffold of the MUmBLs binds first before the antagonist tetrapeptide scaffold, but in practice the order of binding is not determined (
Functional activity data and competitive binding data of the ligands at the mouse melanocortin receptor subtypes are summarized in
HEK293 cells for the ALPHAScreen assay, competitive binding assay, and PRESTO-Tango assay were maintained in humidified atmosphere of 95% air and 5% CO2 at 37° C. in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% newborn calf serum (NCS), and 1% penicillin/streptomycin. Stable cell lines were generated with wild type mMC1R, mMC4R, mMC5R, hMC4R-Flag, and mMC3R-Flag DNA in pCDNA3 expression vector (20 μg) using the calcium phosphate transfection method (Chen, C. A. et al. Biotechniques 1988, 6, 632-638). Stable populations were selected for using G418 selection (0.7-1.0 mg/mL) and used in bioassays unless indicated otherwise. In vitro experimental ligands were dissolved to a 10−2 M stock in DMSO and stored at −20° C. Subsequent dilutions were performed in each assay's specific buffer to achieve the final concentration in the well. The ligands were assayed as TFA salts.
AlphaScreen cAMP Functional Bioassay
The AlphaScreen® cAMP technology (PerkinElmer Life Sciences, Cat #6760625M) was utilized to measure cAMP signaling after ligand stimulation in HEK293 cells stably expressing the mMC1R, mMC3R, mMC4R, hMC4R and mMC5R. The AlphaScreen® assay was performed as described by manufacturer. This method has been previously utilized by our lab (Lensing, C. J. et al. J. Med. Chem. 2016, 59, 3112-3128 and Singh, A. et al. ACS Med. Chem. Lett. 2015, 6, 568-572), and it is described briefly below.
On the day of the assay, cells were 70-95% confluent in 10 cm plates. Cells were removed from plates using Gibco® Versene solution and pelleted by centrifugation (Sorvall Super T21 high speed centrifuge, swinging bucket rotor) at 800 rpm for five minutes. Media was gently aspirated and cells were resuspended in Dulbecco's phosphate buffered saline solution (DPBS 1× [−] without calcium and magnesium chloride, Gibco ® Cat # 14190-144). A 10 μL aliquot of cell suspension was counted manually using a hemocytometer after addition of Trypan blue dye (BioRad). Cells were again pelleted by centrifugation, and DPBS was gently aspirated. The pelleted cells were then resuspended in a solution of freshly made stimulation buffer (Hank's Balanced Salt Solution [HBSS 10× [−] sodium bicarbonate] and [−] phenol red, Gibco®], 0.5 mM isobutylmethylxanthine [IBMX], 5 mM HEPES buffer solution [1M, Gibco®], 0.1% bovine serum albumin [BSA] in Milli-Q water, pH=7.4) and anti-cAMP acceptor beads (1.0 unit per well, AlphaScreen®). A cell/acceptor bead solution was added manually to each well of a 384 well microplate (OptiPlate-384; PerkinElmer) for final concentrations of 10,000 cells/well and 1.0 Unit anti-cAMP acceptor beads/well. The cells were then stimulated with ligand diluted in stimulation buffer to achieve their final concentrations in the well ranging from 10−13 to 10−4 M. The stimulated plates were incubated in a dark laboratory drawer at room temperature for two hours.
Meanwhile, a biotinylated cAMP/streptavidin donor bead working solution was made by adding biotinylated cAMP (1 Unit/well, AlphaScreen®) and streptavidin donor beads (1 Unit/well, AlphaScreen®) to a lysis buffer (10% Tween-20, 5 mM HEPES buffer solution [1M, Gibco®], 0.1% bovine serum albumin [BSA] in Milli-Q water, pH=7.4). After the two hour stimulation, the biotinylated cAMP/Streptavidin donor bead working solution was added to each well under green light and mixed well by pipetting up and down. The cells were incubated for another two hours at room temperature in a dark drawer at room temperature. The plate was then read on an EnSpire™ Alpha plate reader using a pre-normalized assay protocol set by the manufacturer. Assays were performed with duplicate data points on each plate and repeated in at least three independent experiments. Each plate contained a control ligand dose response (NDP-MSH, α-MSH, or γ2-MSH), a 10−4M forskolin positive control, and a no ligand assay buffer negative control.
Dose response curves were analyzed using the PRISM program (v4.0; GraphPad Inc.). Potency EC50 values (concentration that caused 50% maximal signal) were calculated by a nonlinear regression method. To be consistent with functional data being represented as an increasing response with increasing concentration and because the AlphaScreen® assay is a competition assay, a transformation was carried out for illustration purposes to normalize data to control compounds and flip dose response curves as previously described (Lensing, C. J. et al. J. Med. Chem. 2016, 59, 3112-3128 and Singh, A. et al. ACS Med. Chem. Lett. 2015, 6, 568-572).
The PRESTO-Tango assay was developed by Kroeze and coworkers for identifying biologically activate compounds by the rapid screening for most of the entire druggable GPCRome (Kroeze, W. K. et al. Nat. Struct. Mol. Biol. 2015, 22, 362-369 and Barnea, G. et al. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 64-69). The plasmids and assay technology was kindly provided by the Bryan Roth laboratory (University of North Carolina at Chapel Hill) and are now available through ADDGENE (Kit # 1000000068). Briefly, HTLA cells (HEK293 cells that stably express a tTA-dependent luciferase reporter and a β-arrestin 2—TEV fusion gene and were kindly provided by Richard Axel) (Barnea, G. et al. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 64-69) were maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 μg/mL puromycin, and 100 μg/mL hygromycin B in humidified atmosphere of 95% air and 5% CO2 at 37° C.
The first day of the assay, HTLA cells were plated at approximately 1×106 cells per 10 cm plate and grown to 20-40% confluency. The second day cells were transiently transfected using the calcium phosphate method with 4 μg/plate of hMC4R PRESTO-Tango plasmid construct and incubated 15-24 hours in humidified atmosphere of 97% air and 3% CO2 at 35° C. (Kroeze, W. K. et al. Nat. Struct. Mol. Biol. 2015, 22, 362-369 and Chen, C. A. et al. Biotechniques 1988, 6, 632-638). The third day, cells were removed from 10 cm plates using Gibco® Versene solution and pelleted by centrifugation (Sorvall Super T21 high speed centrifuge, swinging bucket rotor) at 800 rpm for five minutes at room temperature. Cells were manually counted on a hemocytometer and resuspended in 1% dialyzed FBS and 1% penicillin/streptomycin in DMEM to a final concentration of 400,000 cells/mL. Cells were plated into 384-well white wall and clear bottom microplate (ViewPlate-384 TC, PerkinElmer Cat # 6007480) for a final concentration of 20,000 cells/well and incubated in 5% CO2 at 37° C. On the fourth day, cells were stimulated by ligands diluted to the appropriate in well concentrations (i.e. 10−12 to 10−5 M) in filter-sterilized assay buffer (20 mM HEPES, 1× HBSS, water, titrated to pH 7.4 with 1 N NaOH). Stimulated cells were incubated for 18 hours in 5% CO2 at 37° C. On the fifth day, the assay buffer and cell medium was removed by aspiration. Then 20 μL of Bright-Glo (Promega, Cat # N1661) diluted 20-fold in assay buffer was added to each well and incubated to 15-20 minutes. After incubation, luminescence was then read on an EnSpire™ Alpha plate reader using a pre-normalized assay protocol for luminescence set by the manufacturer. Dose response curves were analyzed using the PRISM program (v4.0; GraphPad Inc.). Potency EC50 values (concentration that caused 50% maximal signal) were calculated by a nonlinear regression method.
NDP-MSH was radioiodinated with Na125I utilizing the chloramine T procedure (26). Monoradioiodinated NDP-MSH (specific activity: 2175 Ci/mmol) was separated from uniodinated and diradioiodinated peptide by HPLC using a C18 column eluted isocratically with 24% acetonitrile: 76% trimethylamine phosphate (pH 3.0) mobile phase. HEK293 cells stably expressing wildtype mMC1R or mMC4R were maintained as described above. Transiently transfected HEK293 cells were used for binding experiments on the mMC3R. Transfection was performed in 10 cm plates using FuGene6 transfection reagent (15 μL/plate; Promega), Opti-Mem medium (1.7 mL/plate; Invitrogen), and mMC3R-Flag DNA (3.33 μg/plate) two days prior to binding experiment. One or two days preceding the competition experiments, cell were plated into 12-well tissue culture plates (Corning Life Sciences, Cat. # 353043) and grown to 90-100% confluency. On the day of the assay, media was removed gently. The cells were treated with a freshly diluted aliquot of non-labeled compound at the in well concentration being tested (ranging from 10−12 to 10−4 M as appropriate) in assay buffer (DMEM and 0.1% BSA) and a constant amount of 1251-NDP-MSH (100,000 cpm/well) and were incubated at 37° C. for one hour. Media was gently aspirated and cells were washed gently once with assay buffer. The assay buffer was gently aspirated, and then cells were lysed with NaOH (500 μL; 0.1 M) and Triton X-100 (500 μL; 1%) for a minimum of 10 minutes. The cell lysate was transferred to 12×75 mm polystyrene tubes. The radioactivity was quantified on WIZARD2 Automatic Gamma Counter (PerkinElmer). All experiments included unlabeled NDP-MSH as a positive control. All experiments were performed with duplicate data points and repeated in at least two independent experiments. The non-specific values used for calculations was radioactivity of the 10−6 M unlabeled NDP-MSH. Data was analyzed by a nonlinear regression method using the PRISM program (v4.0; GraphPad Inc.) to generate and calculate dose-response curves and IC50 values. The standard error of the mean (SEM) was derived from the IC50 values from at least two independent experiments.
Bioluminescence resonance energy transfer (BRET) has been routinely used to assess GPCR dimerization (Pfleger, K. D. et al. Nat. Protoc. 2006, 1, 337-345). Specifically, the MC3R and MC4R have been reported to result in high basal BRET signal supporting the formation of homodimers (Lensing, C. J. et al. ACS Chem. Neurosci. 2017, 8, 1262-1278; Kopanchuk, S. et al. Neurochem. Int. 2006, 49, 533-542; Mandrika, I. et al. Biochem. Biophys. Res. Commun. 2005, 326, 349-354 and Nickolls, S. A. et al. Peptides 2006, 27, 380-387). Furthermore, BRET has been utilized to support the existence of hMC1R-hMC3R and mMC3R-mMC4R heterodimers (Lensing, C. J. et al. ACS Chem. Neurosci. 2017, 8, 1262-1278 and Mandrika, I. et al. Biochem. Biophys. Res. Commun. 2005, 326, 349-354). It has also been suggested that ligand treatment can increase or decrease dimerization which should be detectable with changes in BRET signal (Tabor, A. et al. Sci. Rep. 2016, 6, 33233; Cottet, M. et al. Front. Endocrinol. (Lausanne) 2012, 3, 92; Grant, M. et al. J. Biol. Chem. 2010, 279, 36179-36183; Albizu, L. et al. Nat. Chem. Biol. 2010, 6, 587-594; Zheng, Y. et al. J. Med. Chem. 2009, 52, 247-258; Journe, A. S. et al. Medchemcomm 2014, 5, 792-796 and Russo, O. et al. J. Med. Chem. 50, 4482-4492). However, these reports vary depending on the receptor system and ligands used (Cottet, M. et al. Front. Endocrinol. (Lausanne) 2012, 3, 92). For example, agonist treatment at the somatostatin receptor 2 has been reported to cause the homodimers to dissociate into monomers (Grant, M. et al. J. Biol. Chem. 2010, 279, 36179-36183). Whereas at the vasopressin V1a receptor, agonist ligand had no observable effect on dimerization ratio (Albizu, L. et al. Nat. Chem. Biol. 2010, 6, 587-594). Also there are several examples of bivalent ligand treatment resulting increased BRET signal suggesting they are inducing or increasing dimerization (Tabor, A. et al. Sci. Rep. 2016, 6, 33233; Zheng, Y. et al. J. Med. Chem. 2009, 52, 247-258; Journe, A. S. et al. Medchemcomm 2014, 5, 792-796 and Russo, O. et al. J. Med. Chem. 50, 4482-4492). In previous reports focused melanocortin receptors, no significant effect of agonist or antagonist ligand was reported for the hMC1R, hMC3R, or hMC4R homodimerization (Mandrika, I. et al. Biochem. Biophys. Res. Commun. 2005, 326, 349-354 and Nickolls, S. A. et al. Peptides 2006, 27, 380-387). However, in the BRET study involving the hMC4R, there does appear to be a trend towards decreasing BRET signal after agonist dosing, albeit not significant. After dosing α-MSH at 1 μM the mean BRET signal decreased by approximately 20% compared to basal BRET signal of the hMC4R (Nickolls, S. A. et al. Peptides 2006, 27, 380-387). Because of the potential of the compounds to be modulating the dimer or oligomer state or changing the dimer conformational state, we investigated the response of BRET signal from mMC4R in response to ligand treatment (
Ligands α-MSH, CJL-1-14, and CJL-1-87, that have full agonist activity in both the cAMP signaling assay and the β-arrestin recruitment assay, resulted in a dose dependent decrease in BRET signal (
The reduction of BRET signal observed with agonist containing ligands could be the result of two different mechanisms: 1) The dimerization or oligomerization is being disrupted and moving towards a lower oligomer state (e.g. dimers to monomers). 2) A conformational change is occurring within the intact dimer or higher-order oligomer in which the NanoLuc®-donor and the HaloTag®-acceptor are being orientated such that the BRET signal is being reduced (e.g. moving further away or dipole orientation is incorrect) (Broussard, J. A. et al. Nat. Protoc. 2013, 8, 265-281). It is currently difficult to determine which of these two possibilities are the driving force for the BRET signal reduction observed in our studies. Regardless, it is apparent that some sort of conformational change is occurring that effects the BRET signal that relates with ligands agonist activity both for cAMP and for β-arrestin recruitment.
These changes match the proposed asymmetric signaling of MC4R homodimers. It follows from the proposed model that at basal levels in which only assay buffer is added (
The current studies support the hypothesis that the bias agonism observed currently with CJL-5-58 is the result of a conformational change of the dimeric state as correlated with the changes observed in the BRET signal. These conformational changes could be changes in the oligomeric number (e.g. dimers to monomers), orientation of the receptors within a dimer pair (e.g. which transmembrane helixes are interacting), or changes in the cellular location of the receptors (e.g. receptor internalization) (Akgün, E. et al. J. Med. Chem. 2015, 58, 8647-8657; Chapman, K. L. et al. Biochim. Biophys. Acta. 2013, 1828, 535-542 and Piechowski, C. L. et al. J. Mol. Endocrinol. 2013, 51, 109-118).
The NanoBRET™ Protein:Protein Interaction System was utilized according to manufacturer's instructions to examine the association and proximity of the melanocortin receptors as previously reported (Lensing, C. J. et al. ACS Chem. Neurosci. 2017, 8, 1262-1278). Briefly, the plasmids were constructed to incorporate the NanoLuc® fusion protein and the HaloTag® fusion protein onto the C-terminus the mMC4R of the plasmids described above. Proper cell membrane expression and ligand binding have previously been supported by competitive binding experiments (Lensing, C. J. et al. ACS Chem. Neurosci. 2017, 8, 1262-1278). The specificity of signal has also previously been shown (Lensing, C. J. et al. ACS Chem. Neurosci. 2017, 8, 1262-1278). On the first day, cells were plated into 6 well plates in the morning. In the afternoon of the same day, cells were transiently transfected with mMC4R-NanoLuc® fusion protein and the MC4R-HaloTag® fusion protein by adding FuGene6 Transfection (8 μL/well, Promega), DNA (2 μg/well) in OptiMem medium (Invitrogen) at a total volume of 100 μL/well. The ratio of donor NanoLuc® to acceptor HaloTag® DNA has previously been optimized and a ratio of 1 Receptor-NanoLuc® plasmid: 4 Receptor-HaloTag® plasmid was utilized for all experiments (Lensing, C. J. et al. ACS Chem. Neurosci. 2017, 8, 1262-1278). Cells were incubated with transfection reagent overnight at 5% CO2 at 37° C. One day after the transfection, cells were re-plated into 96-well black clear bottom plates (Cat # 3603, Corning Life Sciences) at 30,000 cells in 90 μL of assay buffer (4% FBS in OptiMem).
To each well, 1 μL of 0.1 mM HaloTag® NanoBRET™ 618 ligand was added and incubated 18-24 h at 5% CO2 at 37° C. As a negative control, each assay also included; “no acceptor controls” in which 1 μL of DMSO was added instead of 618 ligand rendering the BRET relay system incomplete. This provides the background signal and was subtracted from the final experimental signal. Plates were then developed 48 to 60 hours after transfection. Two hours before the plates were developed, 10 μL of a 10× aliquot of the ligand diluted in assay buffer was added to each well to yield the final in well concentration (10−5, 10−7, or 10−9M) of each compound. For the assay buffer control, 10 μL of assay buffer was added instead of compound. To develop plates, 25 μL of 5× solution of NanoBRET™ Nano-Glo® Substrate in Opti-MEM® was added to each well. Plates were then read within 10 min on a FlexStation® 3 plate reader (Molecular Devices) at the donor emission wavelength (460 nm) and acceptor emission wavelength (618 nm). The milli BRET Units (mBUs) were calculated by dividing the acceptor emission of 618 nm by the donor emission at 460 nm and multiplying it by 1000. The standard error of the mean (SEM) was derived from at least three independent experiments.
The novel in vitro pharmacological profile of the MUmBLs warranted further evaluation in vivo to study their effects on energy homeostasis and physiological consequences. In particular, compound CJL-5-58 was selected due to its biased agonism at the hMC4R, consistent pharmacology in cAMP accumulation assays between the mouse and human isoforms, and the increased serum stability of a PEDG20 linker compared to a Pro-Gly linker (Lensing, C. J. et al. ACS Chem. Neurosci. 2017, 8, 1262-1278). The compound was administered intracerebroventricularly (ICV) directly into the lateral ventricle of the brain in order to avoid the confounding effects of metabolism and brain delivery and to be consistent with previous work in the field (Lensing, C. J. et al. J. Med. Chem. 2016, 59, 3112-3128; Lensing, C. J. et al. ACS Chem. Neurosci. 2017, 8, 1262-1278; Irani, B. G. et al. Eur. J. Pharmacol. 2011, 660, 80-87; Marsh, D. J. et al. Nat. Genet. 1999, 21, 119-122; Ericson, M. D. et al. Biochim. Biophys. Acta, Mol. Basis Dis. 2017, 1863, 2414-2435 and Lensing, C. J. et al. ACS Chem. Neurosci. 2016, 7, 1283-1291).
Dose response studies were performed in “conventional” standard mouse cages in which all measurements were taken manually. Compound CJLS-58 resulted in no signs of adverse effects at doses of 2.5 nmols and 5.0 nmols in the conventional cage experiments. Compound CJL-5-58 resulted in a dose dependent decrease in food intake when refeeding was measured after a 22 hour fast. Significant decreased food intake was observed at 2, 4, 6, and 8 hours after compound administration in male mice (
No statistically significant effect on either food intake or body weight was observed with CJL-5-58 at a 5.0 nmol dose in a nocturnal free-feeding paradigm. In the nocturnal feeding paradigm, mice have free access to food the entire course of the experiments, and compound is administered 2 hours before lights out (Lensing, C. J. et al. J. Med. Chem. 2016, 59, 3112-3128; Lensing, C. J. et al. ACS Chem. Neurosci. 2017, 8, 1262-1278 and Lensing, C. J. et al. ACS Chem. Neurosci. 2016, 7, 1283-1291). Since mice consume approximately 70% of their food during the dark cycle with their biggest meal being soon after lights out, this paradigm should measure the effect on food intake with minimal disruption of homeostasis (Ellacott, K. L. et al. Cell Metab. 2010, 12, 10-17). However, in the fasting-refeeding paradigm, mice are fasted from the start of the previous dark cycle until 2 hours before lights out. At which point mice were administered the compound and food was returned. This disrupts the normal homeostasis of the mice by putting them in a fasting state, but the fast creates a robust feeding response that can help to detect significant effects (Lensing, C. J. et al. ACS Chem. Neurosci. 2017, 8, 1262-1278 and Ellacott, K. L. et al. Cell Metab. 2010, 12, 10-17). Specific to the melanocortin system, expression of endogenous antagonist AGRP is upregulated (Haskell-Luevano, C. et al. Endocrinology 1999, 140, 1408-1415; Marsh, D. J. et al. Brain Res. 1999, 848, 66-77 and Hahn, T. M. et al. Nat. Neurosci. 1998, 1, 271-272).
CJL-5-58's potent binding affinity (IC50=14 nM) may allow it to compete more effectively against endogenous ligands for binding. In the fasting paradigm, compound CJL-5-58 is directly competing with agouti-related peptide (AGRP) which is an endogenous MC3R/MC4R antagonist whose expression levels are upregulated during fasting (Haskell-Luevano, C. et al. Endocrinology 1999, 140, 1408-1415; Marsh, D. J. et al. Brain Res. 1999, 848, 66-77 and Hahn, T. M. et al. Nat. Neurosci. 1998, 1, 271-272). It, therefore, may be hypothesized that CJL-5-58 achieves its effects in the fasting state by blocking the orexigenic effects of AGRP. Regardless, the agonist pharmacophore is overriding the antagonist pharmacophore in the regulation of food intake behavior in vivo.
In order to better characterize the effects of CJL-5-58 on energy homeostasis, it was decided to perform compound administration in TSE Phenotypic metabolic cages that are configured to automatically measure water intake, food intake, changes in the CO2 and O2 levels within the cages, and beam break activity in wild type mice, MC3RKO mice, and MC4RKO mice. In these experiments, a similar trend of CJL-5-58 acting as an agonist by reducing food intake was observed. In must also be noted for full disclosure and good scientific practices that some adverse behaviors were observed during the metabolic cage experiments. These adverse reactions did not appear to affect the parameters measured (i.e. activity, food intake, water intake, RER, and energy expenditure). Also, the behaviors were different depending on the housing conditions and experimental paradigm utilized suggesting that they may not necessarily be compound specific.
All experiments were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) at the University of Minnesota. Female and male littermates with mixed background from C57BL/6J and 129/Sv inbred strains were 8 weeks old at the time of surgeries. Mice were individually housed after surgeries and for the remainder of the experiment. Mice were maintained on a 12 hr light/dark cycle (Lights off was at 11:59 AM) in a temperature controlled room (23-25° C.). In the nocturnal feeding paradigm, mice had free access to normal chow (Harlan Teklad 2018 Diet: 18.6% crude protein, 6.2% crude fat, 3.5% crude fiber, with energy density of 3.1 kcal/g). In the fasting-refeeding paradigm, mice were fasted from lights out on the previous day until compound administration 2 hours prior to lights out for a total fasting time of 22 h (Lensing, C. J. et al. ACS Chem. Neurosci. 2017, 8, 1262-1278 and Ellacott, K. L. et al. Cell Metab. 2010, 12, 10-17). Mice had free access to tap water throughout all the experiments. The cannula placement validation studies and conventional cage studies were performed in standard mouse polycarbonate conventional cages provided by University of Minnesota's Research Animal Resources (RAR). Weekly cage changes were conducted by lab research staff.
A cannula was surgically placed into the lateral cerebral ventricle as previously reported (Lensing, C. J. et al. J. Med. Chem. 2016, 59, 3112-3128; Lensing, C. J. et al. ACS Chem. Neurosci. 2017, 8, 1262-1278; Irani, B. G. et al. Eur. J. Pharmacol. 2011, 660, 80-87 and Lensing, C. J. et al. ACS Chem. Neurosci. 2016, 7, 1283-1291). Mice were anesthetize with a mixture of ketamine (100 mg/kg) and xylazine (5 mg/kg) and were positioned in a stereotaxic apparatus (David Kopf Instruments). A 26-gauge cannula (Cat# 81C315GS4SPC; PlasticsOne, Roanoke, Va.) was inserted in the lateral cerebral ventricle at the coordinates 1.0 mm lateral and 0.46 mm posterior to bregma and 2.3 mm ventral to the skull (Franklin, K. B. J. & Paxinos, G. The Mouse Brain in Stereotaxic Coordinates. (Academic Press, 1997)). The cannula was secured to the skull using dental cement (C&B-Metabond Adhesive cement (Kit # S380) followed by Lang's Jet™ Denture Repair Kit (Jet Denture Repair Powder Ref #1220; Jet Liquid Ref # 1403). A post-surgery dose of flunixin meglumine (FluMegluine, Clipper Distribution Company) and 0.5 mL of 0.9% saline (Hospira, Lake Forrest, Ill.) was given subcutaneously to aid in surgery recovery. All mice were given at least seven days to recover from surgery before any treatment.
Cannula placement was verified by evaluating the feeding response after ICV administration of 2.5 μg of human (h)PYY3-36 (Bachem Cat # H-8585) as described previously (Lensing, C. J. et al. J. Med. Chem. 2016, 59, 3112-3128; Lensing, C. J. et al. ACS Chem. Neurosci. 2017, 8, 1262-1278; Irani, B. G. et al. Eur. J. Pharmacol. 2011, 660, 80-87; Marsh, D. J. et al. Nat. Genet. 1999, 21, 119-122 and Lensing, C. J. et al. ACS Chem. Neurosci. 2016, 7, 1283-1291). Each mouse was administered hPYY and saline on different days following a crossover design in the nocturnal feeding paradigm. In the nocturnal paradigm, compound is administered 2 hours prior to lights out with free access to food and water throughout the entire experiment. There was at least a 4 day washout period between administration to ensure that normal feeding patterns and body weight returned. Food intake and body weight were manually measured 2, 4, and 6 hours after hPYY or saline administration. A mouse with a validated cannula placement for the TSE cage experiments consumed at least 1.0 g more after hPYY administration compared to saline administration 4 hours post-administration.
As stated above, conventional cage experiments were performed in standard mouse polycarbonate conventional cages. The indicated amount (nmols) of compound in 3 μL of saline was delivered two hours prior to lights out (t=0 hr) through the implanted cannula using an infusion internal cannula (Cat# 8IC315IS4SPC; PlasticsOne, Roanoke, Va.) in either a satiated nocturnal feeding paradigm or a fasting refeeding paradigm as described above (Lensing, C. J. et al. ACS Chem. Neurosci. 2017, 8, 1262-1278 and Ellacott, K. L. et al. Cell Metab. 2010, 12, 10-17). All experiments followed a cross-over paradigm in which the mouse received saline and compound on different days with a washout period in between each treatment. Statistical analysis was performed using SPSS V23 software (IBM) using a multivariate general linear model followed by a Bonferroni's post hoc test. Results are presented as Mean±SEM. Statistically significant was considered p<0.05.
There is a growing amount of evidence that GPCR homodimers are functionally relevant and are pharmaceutical targets. A broadly applicable drug design strategy that targets homodimers, as opposed to monomeric receptors, would theoretically double the amount GPCR drug targets. Although various labs have presented different techniques and proof of concepts for methods to target asymmetrically signaling GPCR homodimers (Han, Y. et al. Nat. Chem. Biol. 2009, 5, 688-695; Pellissier, L. P. et al. J. Biol. Chem. 2011, 286, 9985-9997; Teitler, M. et al. Pharmacol. Ther. 2012, 133, 205-217; Comps-Agrar, L. et al. EMBO J. 2011, 30, 2336-2349; Pin, J. P. et al. Febs J. 1 2005, 272, 2947-2955; Hlavackova, V. et al. EMBO J. 2005, 24, 499-509; Prezeau, L. et al. Neuropharmacology 2005, 49, 267-267; Kniazeff, J. et al. Nat. Struct. Mol. Biol. 2004, 11, 706-713; Kniazeff, J. et al. J. Neurosci. 2004, 24, 370-377; Zylbergold, P. et al. Nat. Chem. Biol. 2009, 5, 608-609; Szalai, B. et al. Biochem. Pharmacol. 2012, 84, 477-485; Damian, M. et al. EMBO J. 2006, 25, 5693-5702; Brock, C. et al. J. Biol. Chem. 2007, 282, 33000-33008; Sartania, N et al. Cell. Signal. 2007, 19, 1928-1938 and Gracia, E. et al. Neuropharmacology 2013, 71, 56-69), these techniques would be difficult to adapt to therapeutic design and in vivo applications. The instant invention presents a design strategy that targets asymmetric homodimers that should be easily amendable to various GPCR systems and in vivo targeting applications.
The MUmBL design strategy aims at occupying each of the two receptors within the homodimer with a different pharmacophore such that an agonist pharmacophore and an antagonist pharmacophore each occupy one of the two receptors within each homodimer. This design strategy produced biased ligands at the hMC4R in which the cAMP signaling pathway was robustly activated at nanomolar concentrations (EC50˜2 to 6 nM) but the β-arrestin pathway was only partially activated at a concentration of 10 μM. These are the first melanocortin biased ligands favoring cAMP signaling over β-arrestin recruitment and will be valuable chemical probes to study melanocortin signaling in the disease states and disorders in which the melanocortin receptors are implicated including: cancer (Xu, L. P. et al. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 21295-21300; Josan, J. S. et al. Bioconjugate Chem. 2011, 22, 1270-1278; Barkey, N. M. et al. J. Med. Chem. 2011, 54, 8078-8084; Brabez, N. et al. ACS Med. Chem. Lett. 2013, 4, 98-102 and Brabez, N. et al. J. Med. Chem. 2011, 54, 7375-7384), skin pigmentation disorders (Langendonk, J. G. et al. N. Engl. J. Med. 2015, 373, 48-59), social disorders (Penagarikano, O. et al. Sci. Transl. Med. 2015, 7, 271 and Barrett, C. E. et al. Neuropharmacology 2014, 85, 357-366), sexual function disorders (Uckert, S. et al. Expert Opin. Invest. Drugs 2014, 23, 1477-1483; Clayton, A. H. et al. Women's Health 2016, 12, 325-337 and Kingsberg, S. et al. J. Sex. Med. 2015, 12, 389-389), Alzheimer's disease (Giuliani, D. et al. Mol. Cell. Neurosci. 2015, 67, 13-21 and Giuliani, D. et al. Neurobiol. Aging 2014, 35, 537-547), cachexia (Joppa, M. A. et al. Peptides 2005, 26, 2294-2301; Deboer, M. D. et al. Trends Endocrinol. Metab. 2006, 17, 199-204; Doering, S. R. et al.ACS Med. Chem. Lett. 2015, 6, 123-127 and Ericson, M. D. et al. J. Med. Chem. 2015, 58, 4638-4647), and obesity (Lensing, C. J. et al. J. Med. Chem. 2016, 59, 3112-3128; Irani, B. G. et al. Eur. J. Pharmacol. 2011, 660, 80-87; Marsh, D. J. et al. Nat. Genet. 1999, 21, 119-122 and Fan, W. et al. Nature 1997, 385, 165-168).
Two of the compounds showed species difference in which a partial agonist dose response curve was observed at the mMC4R. This identified CJL-5-58 as the lead ligand for in vivo evaluation due to its biased agonism at the hMC4R, consistent pharmacology in cAMP signaling assays between the mouse and human receptors, and the increased serum stability of a PEDG20 linker compared to a Pro-Gly linker (Lensing, C. J. et al. ACS Chem. Neurosci. 2017, 8, 1262-1278). Evaluation in vivo showed that CJL-5-58 reduce food intake after administration in a fasting-refeeding paradigm consistent with its cAMP agonist function.
The UmBL methodology presented currently should be applicable to various other GPCRs and can easily accommodate the plethora of well-studied and developed selective agonists and antagonists for various GPCR systems. This bivalent ligand targeting method should allow for biased ligands or unique pharmacologies at various receptors by combining known agonists and antagonists with an effective linker. Considering the wide array of GPCRs that are already reported to exist as allosterically modulated or asymmetric homodimers (including the vasopressin (Orcel, H. et al. Mol. Pharmacol. 2009, 75, 637-647), dopamine (Han, Y. et al. Nat. Chem. Biol. 2009, 5, 688-695), adenosine (Gracia, E. et al. Neuropharmacology 2013, 71, 56-69), metabotropic glutamate (Kniazeff, J. et al. Nat. Struct. Mol. Biol. 2004, 11, 706-713), and serotonin receptors (Pellissier, L. P. et al. J. Biol. Chem. 2011, 286, 9985-9997)) this strategy should be broadly applicable. In order to effectively synthesize UmBLs for other receptor systems, it will be necessary to perform some standard medicinal chemistry to optimize the connection points of the linker to the pharmacophores, optimize the linker properties, and optimize the orientation of the pharmacophores. Based on studies at the mouse melanocortin receptors, it was desirable if the agonist scaffold and the antagonist scaffold had approximately equal binding affinities.
The exact pharmacology that may be achieved through the UmBLs design strategy will be as diverse as the allosteric mechanisms between different GPCR homodimers. For example, based on the results of Han and coworkers it can be hypothesized that UmBLs targeting the dopamine D2 receptor would result in increased receptor activation beyond just monovalent agonist alone. This is because allosteric cross-talk of a second agonist protomer was shown to blunt activation, so the occupation of the second protomer with an antagonist scaffold instead of an agonist scaffold should increase signal activation (Han, Y. et al. Nat. Chem. Biol. 2009, 5, 688-695). In contrast if the UmBL approach was applied to the metabotropic glutamate receptor, it would be hypothesized to result in lower than full receptor activation of agonist alone as Kniazeff and coworkers observed that one agonist can partially activate a dimeric unit but two agonists are required for full activation (Kniazeff, J. et al. Nat. Struct. Mol. Biol. 2004, 11, 706-713). Finally, it has been reported that the vasopressin Vib receptor signals through both the Gq/11-inositol phosphate (IP) and the cAMP pathways (Orcel, H. et al. Mol. Pharmacol. 2009, 75, 637-647). It was hypothesized by Orcel and coworkers, that “the IP pathway could be activated by the binding of either one or two AVP molecules to a single receptor dimer. . . By contrast, cAMP production could only be turned on upon the binding of two ligands to a dimer.” Their observations and hypothesis is consistent with asymmetric homodimers such that the IP pathway is activated by the first agonist binding event and the cAMP pathway is activated second (Orcel, H. et al. Mol. Pharmacol. 2009, 75, 637-647). Therefore, if the UmBL design strategy was applied to ligands targeting the vasopressin V1b receptor, it would be predicted to result in biased ligands in which the agonist pharmacophore would activate the IP pathway, and the antagonist pharmacophore would block the cAMP pathway activation within the homodimer. The UmBL design approach could also be applied to GPCR systems in which asymmetry between homodimers has not been identified, or even systems in which homodimerization has not yet been observed. In these situations, designed UmBLs could be evaluated for their ability to induce signaling in multiple signaling pathways (e.g. cAMP, Ca+, kinase signaling, β-arrestin signaling, ect.) to identify asymmetrically signaling GPCR homodimers.
The initial dose response studies were performed in “conventional” standard mouse cages in which all measurements were taken manually. Compound CJL-5-58 resulted in no signs of adverse effects at doses of 2.5 nmols and 5.0 nmols in the conventional cage experiments. Compound CJL-5-58 resulted in a dose dependent decrease in food intake when refeeding was measured after a 22 hour fast. Significant decreases in food intake were observed at 2, 4, 6, and 8 hours after compound administration in male mice (
Interestingly, no statistically significant effect on either food intake or body weight was observed with CJL-5-58 at a 5.0 nmol dose in a nocturnal free-feeding paradigm (
It is possible that CJL-5-58 achieves its effects in the fasting state by blocking the orexigenic effects of AGRP, and not from melanocortin agonist action. Indeed, food intake after CJL-5-58 is consistent between the nocturnal paradigm and the fasting paradigm suggesting that it maintains consistent feeding patterns regardless of endogenous homeostasis regulation.
In order to better characterize the effects of CJL-5-58 on energy homeostasis, it was decided to perform compound administration in TSE Phenotypic metabolic cages that are configured to automatically measure water intake, food intake, changes in the CO2 and O2 levels within the cages, and beam break activity. A new cohort of littermate age match male mice was cannulated and acclimated to the TSE metabolic cages for one week. Consistent with the conventional cage data, the administration of 5 nmols of CJL-5-58 resulted in a decrease in food intake up to 12 hours after administration in the fasting paradigm (
Melanocortin ligands have previously been reported to effect the respiratory exchange ratio (RER), with agonist compounds decreasing the RER, and antagonist compounds increasing the RER. The RER can be measured indirectly utilizing TSE metabolic cage system by measuring the amount of CO2 and O2 entering and exiting the sealed cages. A RER value of c.a. 0.7 gives an indication thatfats are the primary fuel source that the animal is utilizing. A RER value of c.a. 1.0 gives an indication that carbohydrates are the primary fuel source the animal is utilizing. During the fast a baseline RER value of slightly above 0.7 was observed (
Melanocortin ligands have been reported to effect the energy expenditure such that agonists increase the amount of calories burned, and antagonist decrease the amount of calories burned. In the fasting paradigm, the energy expenditure decreases rapidly during fasting which is consistent with the mice conserving energy (
Another hypothesis for the lowered energy expenditure may be the biased signaling of CJL-5-58 for the cAMP signaling pathway over the β-arrestin recruitment pathway. It could be hypothesized that the β-arrestin pathway is responsible for classic agonist effect to increase energy expenditure. Therefore, CJL-5-58, with minimal β-arrestin recruitment, results in a more gradual change in energy expenditure from baseline. However, further experimentation is necessary before hypothesis could be validated. In the nocturnal paradigm, an increase in energy expenditure was observed 5, 13, 15, and 16 hours after treatment which is consistent with agonist function (
Of note some adverse reactions were observed during the TSE cage experiments that were not observed during the conventional cage experiments (
During the fasting paradigm, four adverse reactions were observed within 30 minutes of injection, and one mouse died about 2 hours post-injection. During the nocturnal paradigm there was a total of 2 adverse reactions and one mouse died 30 minutes post-administration. Mice experiencing adverse reactions recovered rapidly (<1 hr). Due to the lack of significant effects observed in the nocturnal paradigm group as well as in the ambulatory activity measurements in both paradigms (
In order to help elucidate if the in vivo effects were due to the MUmBL design or were due to the co-treatment of an agonist and antagonist, co-administration experiments were performed in the same mice as the CJL-5-58 experiment with 5 nmols of CJL-1-14, Ac-His-DPhe-Arg-Trp-NH2, and 5 nmols of CJL-1-80, Ac-His-DNal(2′)-Arg-Trp-NH2, that would reconstitute the 10 nmols of tetrapeptide scaffolds administered with CJL-5-58 at the 5 nmol dose. In the fasting paradigm and the nocturnal paradigm, no statistically significant effect on food intake or water intake were observed compared to saline within 24 hours of administration expect for in the 2 hours timepoint in the nocturnal paradigm (
A direct comparison of CJL-5-58 to the co-administration of CJL-1-14 and CJL-1-80 reveals some differences. There was significant differences in the food intake at 2 and 4 hour time point in the fasting paradigm comparing CJL-5-58 to the tetrapeptide combination. There was significant differences in RER at the 2, 3, 6, and 7 hour time points in the fasting paradigm. Energy expenditure was also significantly higher for CJL-5-58 at the 13 and 17 hour time points in the fasting paradigm. In the nocturnal paradigm, no significant differences were observed for any parameter between the co-administration of the tetrapeptide combination and CJL-5-58.
The combination of CJL-1-14 and CJL-1-80 resulted more adverse observations than CJL-5-58. In the fasting paradigm, three mice died after compound administration. In the nocturnal paradigm, one mouse died. As with CJL-5-58, in the energy homeostasis parameters measured compound toxicity was not observed. If compound toxicity was suspect for the in vivo effects on energy homeostasis, it would be expected that decreases in food intake and water intake would be observed in both the fasting paradigm and the nocturnal paradigm. Furthermore, ambulatory activity resulted in very little significant changes compared to saline. The only significant changes observed in ambulatory activity were at hours 18 and 19 post-administration. It would be expected in the case of compound toxicity, a more significant effect on activity would be observed.
In order to more clearly understand the effects of the biased agonism at the MC4R, the effects of administration of CJL-5-58 was explored in MC3R knockout (KO) mice and MC4RKO mice. In male MC3RKO mice, a significant decrease in food intake was observed in the nocturnal feeding paradigm (
The effects of CJL-5-58 on male MC3RKO mice in a fasting-refeeding paradigm were evaluated for comparison with wild type mice. However due to the adverse reactions observed with higher dosing in the nocturnal paradigm, the effects were only evaluated at 0.5 and 1 nmols (
In male MC4RKO mice, CJL-5-58 was administered at 5 nmols in both a nocturnal paradigm and a fasting-refeeding paradigm in standard conventional cages (
Due to the observed adverse physiological effects that were observed in the different housing and different genotypes (
In order to study the effects of the orientation of the tetrapeptide scaffolds at the N-terminus and the C-terminus in vivo, CJL-1-124 was administered to wild type, MC3RKO, and MC4RKO mice and parameters about their energy homeostasis was recorded using TSE phenotypic metabolic cages. In preliminary conventional cages experiments, strong adverse effects as described above were observed after compound administration in the fasting paradigm, therefore only the nocturnal paradigm was performed. No significant effect on male wild type mice was observed in the nocturnal paradigm at either the 2.5 nmol or 5 nmol dose of CJL-1-124 compared to saline on food intake, water intake, or activity (
The administration of 2.5 nmols or 5.0 nmols CJL-1-124 to male MC3RKO mice resulted in no significant changes in food intake or water intake (
All publications, patents, and patent documents (including Lensing, C. J. et al. J Med. Chem. 2018, Just Accepted, DOI: 10.1021/acs.jmedchem.8b00238) are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.
This application claims the benefit of priority of U.S. Provisional Application Ser. No. 62/500,445 filed on May 2, 2017, which application is incorporated by reference herein.
This invention was made with government support under R01 DK091906 and R01 DK108893 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62500445 | May 2017 | US |
