ABSTRACT
The α-hemoglobin-derived dodecapeptide RVD-hemopressin (RVDPVNFKLLSH) has been proposed to be an endogenous agonist for the cannabinoid receptor type 1 (CB(1)). To study this peptide, we have raised mAbs against its C-terminal part. Using an immunoaffinity mass spectrometry approach, a whole family of N-terminally extended peptides in addition to RVD-Hpα were identified in rodent brain extracts and human and mouse plasma. We designated these peptides Pepcan-12 (RVDPVNFKLLSH) to Pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH), referring to peptide length. The most abundant Pepcans found in the brain were tested for CB(1) receptor binding. In the classical radioligand displacement assay, Pepcan-12 was the most efficacious ligand but only partially displaced both [(3)H]CP55,940 and [(3)H]WIN55,212-2. The data were fitted with the allosteric ternary complex model, revealing a cooperativity factor value α < 1, thus indicating a negative allosteric modulation. Dissociation kinetic studies of [(3)H]CP55,940 in the absence and presence of Pepcan-12 confirmed these results by showing increased dissociation rate constants induced by Pepcan-12. A fluorescently labeled Pepcan-12 analog was synthesized to investigate the binding to CB(1) receptors. Competition binding studies revealed K(i) values of several Pepcans in the nanomolar range. Accordingly, using competitive ELISA, we found low nanomolar concentrations of Pepcans in human plasma and â¼100 pmol/g in mouse brain. Surprisingly, Pepcan-12 exhibited potent negative allosteric modulation of the orthosteric agonist-induced cAMP accumulation, [(35)S]GTPγS binding, and CB(1) receptor internalization. Pepcans are the first endogenous allosteric modulators identified for CB(1) receptors. Given their abundance in the brain, Pepcans could play an important physiological role in modulating endocannabinoid signaling.
Subject(s)
Cannabinoid Receptor Modulators/metabolism , Hemoglobins/metabolism , Peptide Fragments/metabolism , Receptor, Cannabinoid, CB1/metabolism , Allosteric Regulation , Amino Acid Sequence , Animals , Antibodies, Monoclonal, Murine-Derived/biosynthesis , Binding, Competitive , Brain/metabolism , CHO Cells , Cannabinoid Receptor Modulators/blood , Cannabinoid Receptor Modulators/chemical synthesis , Cannabinoid Receptor Modulators/immunology , Cricetinae , Cyclohexanols/metabolism , Epitope Mapping , Female , HL-60 Cells , Hemoglobins/biosynthesis , Hemoglobins/chemical synthesis , Hemoglobins/chemistry , Hemoglobins/immunology , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Inbred NZB , Molecular Sequence Data , Peptide Fragments/biosynthesis , Peptide Fragments/blood , Peptide Fragments/chemical synthesis , Peptide Fragments/immunology , Protein Binding , Protein Transport , Rats , Receptor, Cannabinoid, CB1/agonists , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Signal Transduction , Sus scrofa , Tandem Mass SpectrometryABSTRACT
CB1 receptor antagonists that are peripherally restricted were targeted. Compounds with permanent charge as well as compounds that have increased polar surface area were made and tested against CB1 for binding and activity. Sulfonamide and sulfamide with high polar surface area and good activity at CB1 were rationally designed and pharmacologically tested. Further optimization of these compounds and testing could lead to the development of a new class of therapeutics to treat disorders where the CB1 receptor system has been implicated.
Subject(s)
Cannabinoid Receptor Modulators/chemical synthesis , Cannabinoid Receptor Modulators/pharmacology , Drug Design , Drug Discovery , Receptor, Cannabinoid, CB1/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , CHO Cells , Cannabinoid Receptor Modulators/chemistry , Cannabinoid Receptor Modulators/metabolism , Cell Line , Cricetinae , Dogs , Ligands , Molecular Structure , Piperidines/metabolism , Protein Binding , Pyrazoles/metabolism , Radioligand Assay , Receptor, Cannabinoid, CB1/chemistry , Rimonabant , Stereoisomerism , Structure-Activity Relationship , Sulfonamides/chemistryABSTRACT
We report the synthesis of new chemical probes (1a,b, 2a-c, 3a-c) based on the structure of the main endocannabinoids for their use in biological systems directly or via click chemistry. As proof of concept, 2-arachidonyl glyceryl ether based biotinylated 3b enables direct visualization of CB(1) receptor in cells. These results represent the starting point for the development of advanced small molecule chemical probes able to generate valuable information about the cannabinoid receptors.
Subject(s)
Cannabinoid Receptor Modulators/chemical synthesis , Endocannabinoids , Molecular Probes/chemical synthesis , Receptor, Cannabinoid, CB1/metabolism , Receptor, Cannabinoid, CB2/metabolism , Alkenes/chemical synthesis , Alkenes/chemistry , Alkenes/pharmacology , Arachidonic Acids/chemical synthesis , Arachidonic Acids/chemistry , Arachidonic Acids/pharmacology , Benzophenones/chemical synthesis , Benzophenones/chemistry , Benzophenones/pharmacology , Binding, Competitive , Biotin/chemical synthesis , Biotin/chemistry , Biotin/pharmacology , Cannabinoid Receptor Modulators/chemistry , Cannabinoid Receptor Modulators/pharmacology , Cell Line , Click Chemistry , Glycerides/chemical synthesis , Glycerides/chemistry , Glycerides/pharmacology , Humans , Ligands , Molecular Probes/chemistry , Molecular Probes/pharmacology , Polyunsaturated Alkamides/chemical synthesis , Polyunsaturated Alkamides/chemistry , Polyunsaturated Alkamides/pharmacology , Radioligand Assay , Structure-Activity RelationshipABSTRACT
Arachidonyl and linoleyl sulfamide derivatives have been synthesized and their potential cannabimimetic properties evaluated in in vitro functional and binding assays. Replacement of the ethanolamide moiety of anandamide by -CH(2)NHSO(2)NH-R considerably reduces the CB1 receptor activity and only some of the compounds showed modest cannabinoid properties in binding assays. The new compounds were also tested as inhibitors of the FAAH enzyme but were inactive.
Subject(s)
Arachidonic Acids/chemical synthesis , Arachidonic Acids/pharmacology , Cannabinoid Receptor Modulators/chemical synthesis , Cannabinoid Receptor Modulators/pharmacology , Polyunsaturated Alkamides/chemical synthesis , Polyunsaturated Alkamides/pharmacology , Sulfonamides , Amidohydrolases/antagonists & inhibitors , Animals , Arachidonic Acids/chemistry , Cannabinoid Receptor Modulators/chemistry , Cells, Cultured , Endocannabinoids , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Male , Molecular Structure , Polyunsaturated Alkamides/chemistry , Rats , Vas Deferens/drug effectsABSTRACT
A series of 21 analogues of tetrahydrolipstatin (THL, 1) were synthesized and tested as inhibitors of the formation or hydrolysis of the endocannabinoid 2-arachidonoylglycerol (2-AG). Three of the novel compounds, i.e., 11, 13, and 15, inhibited 2-AG formation via the diacylglycerol lipase alpha (DAGLalpha) with IC 50 values lower than 50 nM (IC 50 of THL = 1 microM) and were between 23- and 375-fold selective vs 2-AG hydrolysis by monoacylglycerol lipase (MAGL) as well as vs cannabinoid CB 1 and CB 2 receptors and anandamide hydrolysis by fatty acid amide hydrolase (FAAH). Three other THL analogues, i.e., 14, 16, and 18, were slightly more potent than THL against DAGLalpha and appreciably selective vs MAGL, CB receptors, and FAAH (15-26-fold). One compound, i.e., 8, was a potent inhibitor of MAGL-like activity (IC 50 = 0.41 microM), and relatively ( approximately 7-fold) selective vs the other targets tested.
Subject(s)
Arachidonic Acids/metabolism , Cannabinoid Receptor Modulators/chemical synthesis , Cannabinoid Receptor Modulators/pharmacology , Endocannabinoids , Glycerides/metabolism , Lactones/chemical synthesis , Lactones/pharmacology , Animals , Arachidonic Acids/chemistry , Cannabinoid Receptor Modulators/chemistry , Cell Line, Tumor , Glycerides/chemistry , Inhibitory Concentration 50 , Lactones/chemistry , Mice , Molecular Structure , Orlistat , Structure-Activity RelationshipABSTRACT
Anandamide (AEA) presents the four double bonds in the cis configuration, deriving from the arachidonic acid moiety. In the context of an antisense strategy based on the double bond configuration, all-trans AEA (t-AEA) was synthesized in high yield starting from all-trans methyl arachidonate and ethanolamine in the presence of KCN. t-AEA was assayed on rabbit platelet aggregation, obtaining effect only at high concentrations (>10(-4) M) after an also concentration-dependent lag phase. At lower concentrations it inhibited PAF-induced rabbit platelet aggregation with an IC(50)=4.6 x 10(-6) M. In contrast to anandamide, the activation of platelets was not due to the conversion of t-AEA to trans arachidonic acid, as ascertained by negative results with FAAH inhibitors. However, t-AEA was found to be a substrate for fatty acid amide hydrolase (FAAH), the enzyme that cleaves anandamide and regulates in vivo the magnitude and duration of the signaling induced by this lipid messenger.
Subject(s)
Amidohydrolases/metabolism , Arachidonic Acids/pharmacology , Calcium Channel Blockers/pharmacology , Cannabinoid Receptor Modulators/pharmacology , Platelet Activation/drug effects , Polyunsaturated Alkamides/pharmacology , Animals , Arachidonic Acid/metabolism , Arachidonic Acids/chemical synthesis , Calcium Channel Blockers/chemical synthesis , Cannabinoid Receptor Modulators/chemical synthesis , Endocannabinoids , Ethanolamine/metabolism , Lipid Metabolism , Polyunsaturated Alkamides/chemical synthesis , Rabbits , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Sulfonamide analogues of the potent CB1R inverse agonist taranabant were prepared and optimized for potency and selectivity for CB1R. They were variably more potent than the corresponding amide analogues. The most potent representative 22 had good pharmacokinetic and brain levels, but was modestly active in blocking CB1R agonist-mediated hypothermia.
Subject(s)
Cannabinoid Receptor Modulators/chemical synthesis , Receptor, Cannabinoid, CB1/drug effects , Sulfonamides/chemical synthesis , Animals , Anti-Obesity Agents/chemical synthesis , Anti-Obesity Agents/pharmacology , Brain Chemistry , Cannabinoid Receptor Modulators/pharmacology , Humans , Hypothermia/drug therapy , Inhibitory Concentration 50 , Pharmacokinetics , Rats , Receptor, Cannabinoid, CB1/agonists , Structure-Activity Relationship , Sulfonamides/pharmacologyABSTRACT
We have designed, synthesized and evaluated the CB(1) binding affinity of a number of new conformationally restricted lipopeptides (1-17). All of them present some of the AEA key structural elements incorporated in a hairpinlike peptide framework. Among them, compounds 1-3 and 8 showed CB(1) affinities in competitive binding assays with K(i) values in the micromolar range (K(i) of AEA = 0.8 microM in the same assay). The remaining pseudopeptides showed little binding to the CB(1) receptor (with K(i) values >or= 50 microM). Conformational analysis on two representative compounds, performed by a combination of NMR studies, restrained molecular dynamics and QM calculations, allowed us to shed light on the structure-activity relationships (SAR), pointing to a correlation between the predominance of the hairpin-like structural motif and the CB(1) binding affinity. In a more general context, the present study may also prove useful in gaining additional insight into the biological relevance of the various AEA conformations.
Subject(s)
Arachidonic Acids/chemistry , Arachidonic Acids/metabolism , Cannabinoid Receptor Modulators/chemistry , Cannabinoid Receptor Modulators/metabolism , Polyunsaturated Alkamides/chemistry , Polyunsaturated Alkamides/metabolism , Receptor, Cannabinoid, CB1/chemistry , Receptor, Cannabinoid, CB1/metabolism , Amino Acid Sequence , Animals , Arachidonic Acids/chemical synthesis , Binding Sites , Cannabinoid Receptor Modulators/chemical synthesis , Endocannabinoids , Magnetic Resonance Spectroscopy , Male , Models, Molecular , Molecular Mimicry , Molecular Sequence Data , Polyunsaturated Alkamides/chemical synthesis , Rats , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Structure-Activity RelationshipABSTRACT
Recent advances in the synthesis of endocannabinoid-related ligands for the period 2001-2004 are covered in this review. During this period the first solid phase synthesis of anandamide (AEA) analogs was developed, which allows modification at both the head group and the end pentyl chain. Synthesis of water-soluble prodrugs of noladin ether was reported, which are chemically stable, rapidly release noladin ether under enzymatic conditions and are shown to reduce intraocular pressure. The structure-activity relationships (SAR) of alkylcarbamic acid aryl esters and the discovery of potent archidonylsulfonyl derivatives as fatty acid amide hydrolase (FAAH) inhibitors are summarized. Recent synthetic developments in the controversial area of anandamide membrane transporter (AMT) inhibitors are also discussed.
Subject(s)
Cannabinoid Receptor Modulators/chemical synthesis , Endocannabinoids , Technology, Pharmaceutical/methods , Animals , Cannabinoid Receptor Modulators/metabolism , Humans , Ligands , Receptors, Cannabinoid/metabolism , Technology, Pharmaceutical/trendsSubject(s)
Cannabinoid Receptor Modulators/physiology , Endocannabinoids , Signal Transduction/physiology , Animals , Cannabinoid Receptor Modulators/chemical synthesis , Cannabinoid Receptor Modulators/metabolism , Cannabis/chemistry , Dronabinol/pharmacology , Glutamic Acid/metabolism , Glutamic Acid/physiology , Hallucinogens/pharmacology , Humans , Neurotransmitter Agents/metabolism , Neurotransmitter Agents/physiology , Receptor, Cannabinoid, CB1/drug effects , Receptor, Cannabinoid, CB1/physiology , Signal Transduction/drug effects , Synaptic Transmission/drug effects , gamma-Aminobutyric Acid/physiologyABSTRACT
Three new endocannabinoid analogues in which amide moiety was replaced either by oxomethylene group or ester moiety with simultaneous substitution of both alpha-hydrogens with methyl groups were synthesized and their abilities to interact with CB1-receptor and FAAH were investigated.