Evaluation of the human prediction of clearance from hepatocyte and microsome intrinsic clearance for 52 drug compounds.

We compare three different approaches to scale clearance (CL) from human hepatocyte and microsome CL(int) (intrinsic CL) for 52 drug compounds. By using the well-stirred model with protein binding included only 11% and 30% of the compounds were predicted within 2-fold and the average absolute fold errors (AAFE) for the predictions were 5.9 and 4.1 for hepatocytes and microsomes, respectively. When predictions were performed without protein binding, 59% of the compounds were predicted within 2-fold using either hepatocytes or microsomes and the AAFE was 2.2 and 2.3, respectively. For hepatocytes and microsomes there were significant correlations (P = 8.7 x 10(-13), R(2) = 0.72; P = 2.8 x 10(-9), R(2) = 0.61) between predicted CL(int in vivo) (obtained from in vitro CL(int)) and measured CL(int in vivo) (obtained using the well-stirred model). When CL was calculated from the regression, 76% and 70% of the compounds were predicted within 2-fold and the AAFE was 1.6 and 1.8 for hepatocytes and microsomes, respectively. We demonstrate that microsomes and hepatocytes are in many cases comparable when scaling of CL is performed from regression. By using the hepatocyte regression, CL for 82% of the compounds in an independent test set (n = 11) were predicted within 2-fold (AAFE 1.4). We suggest that a regression line that adjusts for systematic under-predictions should be the first-hand choice for scaling of CL.

Melanocortin receptors: ligands and proteochemometrics modeling.

The melanocortin receptors exist in five subtypes, MC(1-5)R. These receptors participate in important regulations of the immune system, central behavior, and endocrine and exocrine glands. Here we provide a short review on MCR subtype selective peptides and organic compounds with activity on the MCRs, developed in our laboratory. Also provided is an overview of our new proteochemometric modeling technology, which has been applied to model the interaction of MSH peptides with the MCRs.

Detection of regions in the MC1 receptor of importance for the selectivity of the MC1 receptor super-selective MS04/MS05 peptides.

We have investigated the ability of our earlier identified MS04-MS05 MSH-peptide analogues to bind to chimeric MC1-MC3 receptors. While the MS04 and MS05 peptides bind with nanomolar and sub-nanomolar affinities to the wild type MC1 receptor, they bind only with micromolar affinities for the wild type MC3 receptor, thus being the hitherto most MC1 receptor selective ligands. Upon exchanging portions involving transmembrane regions TM1, TM2-3, and TM6-7 of the MC1 receptor with corresponding portions of the MC3 receptor both of these peptides showed major losses of affinities. By contrast exchanges involving TM4-5 did not appreciably affect the affinity of either MS04 or MS05. Our data suggest that the binding pocket for the MS04-MS05 MSH-peptides is located between TM1-3 and TM6-7 of the melanocortin receptors.

PLS modeling of chimeric MS04/MSH-peptide and MC1/MC3-receptor interactions reveals a novel method for the analysis of ligand-receptor interactions.

A novel method has been developed for the analysis of ligand-receptor interactions. The method utilizes binding data generated from the analysis of chimeric proteins with chimeric peptides. To each chimeric part of the peptide and receptor are assigned descriptors, thus creating a matrix of X descriptors. These descriptors are then correlated with the experimentally determined interaction binding affinities for each chimeric receptor/peptide pair by use of partial least-squares projection to latent structures (PLS). The method was applied to analyze the interactions of chimeric MSH-peptides with wild-type MC1 and MC3 receptors, and MC1/MC3 receptor chimeras (in total 40 peptide-receptor combinations). Two types of PLS models could be created, one that revealed the relationships between receptor and peptide structure and peptide binding pK(i) values (i.e., affinity) (R2 and Q2 being 0.71 and 0.62, respectively), and another that revealed the relationships between peptide and receptor structure and peptide-receptor selectivity (R2 and Q2 being 0.64 and 0.57, respectively). After addition of cross-terms these models improved significantly; the R2 and Q2 being 0.93 and 0.75 for affinity, and 0.92 and 0.72 for selectivity, respectively. The analysis shows that the high affinity of the MSH-peptides is primarily achieved by interactions of the peptides' C-terminal amino acids with TM2 and TM3 of the receptor, and, to a lesser extent, by the interaction of the N-terminus with TM1, TM2 and TM3 of the receptor. However, in contrast, the MC1 receptor selectivity is primarily determined by an interaction of the peptides' N-termini with TM2/3 of the receptor. Moreover, the cross-terms of the PLS model revealed the existence of a strong interaction between TM6/7 and TM2/3 of the receptors.

Development of proteo-chemometrics: a novel technology for the analysis of drug-receptor interactions.

A novel method for the analysis of drug receptor interactions has been developed and used to explore mechanisms involved in the binding of 4-piperidyl oxazole antagonists to alpha1a-, alpha1b- and alpha1d-adrenoceptors. The method exploits affinity data for a series of organic chemical compounds binding to wild-type and artificially mutated receptors. The receptor sequences and compounds are assigned predictor variables that are correlated to the measured pharmacological activities using partial least-squares projections to latent structures. The predictor variables consist of one descriptor block derived from the chemical properties of the receptors' primary amino acid sequences and another descriptor block derived from the chemical properties of the organic compounds. The cross-terms generated from the two descriptor blocks are also derived. Using this approach, very sturdy models were generated describing the interactions of the chemical compounds with the receptors. Models are useful to predict binding affinity and receptor subtype selectivity of compounds prior to their synthesis, and may find use in rational drug design. Moreover, models also give quantitative information about the interactions of the amino acids of the receptors with the ligands, thereby giving an insight into the molecular mechanisms involved in ligand binding.

Design of new small cyclic melanocortin receptor-binding peptides using molecular modelling: Role of the His residue in the melanocortin peptide core.

The conserved core of melanocyte stimulating hormones (MSH), His-Phe-Arg-Trp, was probed by comparing a cyclic pentapeptide containing His-DPhe-Arg-Trp, with three structurally similar cyclic peptides, that lacked the His residue. All three peptides bound to the MC(1), MC(3), MC(4) and MC(5) receptors with similar affinities. Molecular modelling indicated that the 3D structure of the DPhe-Arg-Trp of all three peptides were closely similar. The data indicate that the His residue of the small rigid cyclic MSH core peptides does not participate in binding with the melanocortin receptors.

New aspects on the melanocortins and their receptors.

Knowledge of melanocortins and their receptors has increased tremendously over the last few years. The cloning of five melanocortin receptors, and the discovery of two endogenous antagonists for these receptors, agouti and agouti-related peptide, have sparked intense interest in the field. Here we give a comprehensive review of the pharmacology, physiology and molecular biology of the melanocortins and their receptors. In particular, we review the roles of the melanocortins in the immune system, behaviour, feeding, the cardiovascular system and melanoma. Moreover, evidence is discussed suggesting that while many of the actions of the melanocortins are mediated via melanocortin receptors, some appear to be mediated via mechanisms distinct from melanocortin receptors.

Thyrotropin releasing hormone (TRH) selectively binds and activates the melanocortin 1 receptor.

We tested the endogenous tripeptide TRH (thyrotropin releasing hormone) ability to bind to MC (melanocortin) receptor subtypes. We discovered that TRH binds to the human MCI receptor expressed in COS cells and to murine B16 melanoma cells with 5790+/-1010 nM and 6370+/-1260 nM Ki's, respectively. TRH did not bind to the human MC3, MC4 or MC5 receptor subtypes. Moreover, TRH also stimulated cAMP production in murine B16 melanoma cells reaching the same maximum level of cAMP as found for alpha-MSH. However, several analogues of TRH, including TRH-OH, TRH-Gly-NH2 and other analogues, where each of the three amino acid residues in TRH had been exchanged by a related residue, did not bind to any of the MC receptors tested in this study. C(alpha) atoms of molecular models of TRH and the core of a MSH peptide were aligned with r.m.s. of 0.01 A. Moreover, TRH could be docked into a binding pocket of a molecular model of the MC1 receptor at only a little higher energy than a short cyclic MSH peptide. The data indicate a similarity in the mode of TRH and MSH activation of the MCI receptor.

Long term orexigenic effect of a novel melanocortin 4 receptor selective antagonist.

1. We designed and synthesized several novel cyclic MSH analogues and tested their affinities for cells expressing the MC1, MC3, MC4 and MC5 receptors. 2. One of the substances HS028 (cyclic [AcCys11, dichloro-D-phenylalanine14, Cys18, Asp-NH2(22)]-beta-MSH11-22) showed high affinity (Ki of 0.95nM) and high (80 fold) MC4 receptor selectivity over the MC3 receptor. HS028 thus shows both higher affinity and higher selectivity for the MC4 receptor compared to the earlier first described MC4 receptor selective substance HS014. 3. HS028 antagonised a alpha-MSH induced increase in cyclic AMP production in transfected cells expressing the MC3 and MC4 receptors, whereas it seemed to be a partial agonist for the MC1 and MC5 receptors. 4. Chronic intracerebroventricularly (i.c.v.) administration of HS028 by osmotic minipumps significantly increased both food intake and body weight in a dose dependent manner without tachyphylaxis for a period of 7 days. 5. This is the first report demonstrating that an MC4 receptor antagonist can increase food intake and body weight during chronic administration providing further evidence that the MC4 receptor is an important mediator of long term weight homeostasis.

Characterization of the enzymatic activity for biphasic competition by guanoxabenz (1-(2,6-dichlorobenzylidene-amino)-3-hydroxyguanidine) at alpha2-adrenoceptors. II. Description of a xanthine-dependent enzymatic activity in spleen cytosol.

The mechanism for formation of high affinity binding of guanoxabenz (1-(2,6-dichlorobenzylidene-amino)-3-hydroxyguanidine) to alpha2-adrenoceptors by the rat spleen cytosol was studied. We report here that the spleen cytosolic fraction mediated the reduction of guanoxabenz to guanabenz (1-(2,6-dichlorobenzylidene-amino)-3-guanidine), the latter having an almost 100-fold higher affinity for rat alpha2A-adrenoceptors than guanoxabenz itself. The reaction product could be separated by high-performance liquid chromatography and its identity as guanabenz confirmed by nuclear magnetic resonance. The spleen cytosolic activity could be separated into high and low molecular weight components, the high molecular weight component requiring low molecular weight factors for maximal activity. Xanthine oxidase seems to be the most likely candidate responsible for the activity, as the guanoxabenz-reducing activity of the high molecular weight component could be sustained by exogenously applied xanthine, while it was potently blocked by allopurinol. The conversion of guanoxabenz by the cytosolic activity was also quite potently blocked by DWO1, 1-(3,4-dimethoxybenzylideneamino)3-hydroxyguanidine, a hydroxyguanidine analogue to guanoxabenz.

Conditions for biphasic competition curves in radioligand binding for ligands subjected to metabolic transformation.

The conditions affording biphasic competition curves in radioligand binding for ligands subjected to metabolic transformation was analyzed theoretically. It was shown that when a competing ligand was subjected to transformation to a ligand that showed higher affinity than the parent compound, biphasic competition curves, which might wrongly be interpreted as indicating the presence of two receptor sites, could be observed in binding assays containing a homogenous receptor population. Biphasic competition curves were seen if the conversion of the competitor occurred according to zero and second order kinetics, as well as by enzymatic catalytic processes. However, when the conversion occurred according to a first order kinetics, the competition curves were uniphasic and resolved only into one-site fits, with the apparent affinity of the competitor reflecting the degree of conversion of the competitor to its metabolite. When the metabolic conversion resulted in a metabolite that showed lower affinity for the receptor than the parent compound, the competition curves became supersteep for conversions according to zero and second order kinetics, as well as for conversion by enzymatic catalytic processes.

Selective properties of C- and N-terminals and core residues of the melanocyte-stimulating hormone on binding to the human melanocortin receptor subtypes.

We synthesised nine analogues of [Nle4,D-Phe7]alpha-MSH (melanocyte-stimulating hormone) (NDP) where (1) the N- or C-terminals were deleted or exchanged by those of beta- or gamma-MSH and (2) the core residues His6, Phe7, Arg8 and Trp9 were individually substituted by Glu6, beta-(2-naphthyl)-D-alanine (D-Nal7), Lys8 and His9, respectively. We tested these analogues in ligand binding assays with cells transiently expressing the human melanocortin MC1, MC3, MC4 and MC5 receptors. The results show that the N-terminal segment (Ser1-Tyr2-Ser3) of NDP was not important for binding to melanocortin MC1 and MC4 receptors whereas it affects binding to melanocortin MC3 and MC5 receptors. The C-terminal segment (Gly10-Lys11-Pro12-Val13) of NDP was clearly important for binding to all the four melanocortin receptor subtypes. The data indicate that the low affinity of gamma-MSH for the melanocortin MC4 receptor is due to its C-terminal (Asp10)-Arg11-Phe12). Substitution of D-Phe7 by D-Nal7 increased the affinity for the melanocortin MC4 receptor but not for the other melanocortin receptor subtypes. The other core residue substitutions lowered the affinity in a differentiated manner for each of the melanocortin receptors. These results are valuable for the molecular modelling and design of selective drugs for the melanocortin receptors.

Discovery of novel melanocortin4 receptor selective MSH analogues.

1. We synthesized a novel series of cyclic melanocyte stimulating hormone (MSH) analogues and tested their binding properties on cells transiently expressing the human melanocortin1 (MC1), MC3, MC4 and MC5 receptors. 2. We discovered that compounds with 26 membered rings of [Cys4,D-Nal7,Cys11]alpha-MSH(4-11) displayed specific MC4 receptor selectivity. The preference order of the different MC receptor subtypes for the novel [Cys4D-Nal7Cys11]alpha-MSH(4-11) analogues are distinct from all other known MSH analogues, particularly as they bind the MC4 receptor with high and the MC1 receptor with low relative affinities. 3. HS964 and HS014 have 12 and 17 fold MC4/MC3 receptor selectivity, respectively, which is much higher than for the previously described cyclic lactam and [Cys4,Cys10]alpha-MSH analogues SHU9119 and HS9510. 4. HS964 is the first substance showing higher affinity for the MC5 receptor than the MC1 receptor. 5. HS014, which was the most potent and selective MC4 receptor ligand (Ki 3.2 nM, which is approximately 300 fold higher affinity than for alpha-MSH), was also demonstrated to antagonize alpha-MSH stimulation of cyclic AMP in MC4 receptor transfected cells. 6. We found that a compound with a 29 membered ring of [Cys3,Nle10,D-Nal7,Cys11]alpha-MSH(3-11) (HS010) had the highest affinity for the MC3 receptor. 7. This is the first study to describe ligands that are truly MC4 selective and a ligand having a high affinity for the MC3 receptor. The novel compounds may be of use in clarifying the physiological roles of the MC3, MC4 and MC5 receptors.

Characterisation of D117A and H260A mutations in the melanocortin 1 receptor.

Recent site directed mutagenesis studies on the melanocortin 1 (MC1) receptor have indicated the importance of D117 and H260 amino acid residues for the binding of alpha-MSH (melanocyte stimulating hormone). Here, we report the testing of 12 cyclic and linear MSH peptides on the D117A and H260A mutant receptors. Moreover, we constructed a double mutant which displayed a major loss in affinity for [Nle4, D-Phe7]alpha-MSH. Our new data of His6 and Phe7 substituted MSH peptides are compared with previous results and the hypothesis of putative interactions of D117 and H260 with single amino acids in the MSH peptide. Our conclusions are that the D117A and the H260A mutations may cause conformational changes in the receptor which can not be linked to any specific amino acid in the MSH-peptides.

Binding of cyclic and linear MSH core peptides to the melanocortin receptor subtypes.

We report here the binding of 5-, 6- and 7-amino-acid-long linear and cyclic core peptides of MSH (melanocyte-stimulating hormone) to cells transiently expressing the human melanocortin MC1, MC3, MC4 and MC5 receptors. The results show that, in contrast to the natural peptides, the core peptides did not differentiate between the melanocortin MC3 and MC4 receptors. All tested cyclic peptides had much lower affinities than their corresponding linear homologues. Interestingly, the relative loss of binding due to the cyclisation did not change as the ring size decreased. Therefore, decreasing the ring size does not seem to force the peptide into a more unfavourable conformation.

Selectivity of cyclic [D-Nal7] and [D-Phe7] substituted MSH analogues for the melanocortin receptor subtypes.

The binding of the 2 cyclic lactam MSH (4-10) analogues (MTII, SHU9119), and 5 cyclic [Cys4, Cys10] alpha-MSH analogues were tested on cells transiently expressing the human MC1, MC3, MC4 and MC5 receptors. The results indicate a differential importance of the C-terminal (Lys-Pro-Val) and N-terminal (Ser-Tyr-Ser) of cyclic [Cys4, Cys10] alpha-MSH analogues in binding to the MC receptor subtypes. Substitution of D-Phe7 by D-Nal(2')7 in both the cyclic lactam MSH (4-10) and the cyclic disulphide MSH (4-10) analogues resulted in a shift in favour of selectivity for the MC4 receptor; the disulphide analogue, [Cys4, D-Nal(2')7 Cys10] alpha-MSH (4-10) (HS9510), showing the highest selectivity for the MC4 receptor among all the substances tested. However, the cyclic lactams displayed an over all higher affinity for the MC receptors, than any of the cyclic disulphide MSH (4-10) analogues.

Modeling of the three-dimensional structure of the human melanocortin 1 receptor, using an automated method and docking of a rigid cyclic melanocyte-stimulating hormone core peptide.

A model is presented of the melanocortin 1 receptor (MC1R), constructed by use of an unbiased, objective method. The model is created directly from data derived from multiple sequence analysis, a low-resolution EM-projection map of rhodopsin, and the approximate membrane thickness. The model agrees well with available data concerning natural mutations of MC1Rs occurring in different species. A model is also presented of the most rigid ligand for this receptor, the cyclic pentapeptide cHFRWG, shown docked in the receptor model. The receptor-ligand complex model agrees well with available experimental data. The ligand is located between transmembrane region 1 (TM1), TM2, TM3, TM6, and TM7 of the receptor. Multiple interactions occur between ligand and receptor, including interactions with Leu-48 (TM1), Ser-52 (TM1), Glu-55 (TM1), Asn-91 (TM2), Glu-94 (TM2), Thr-95 (TM2) Ile-98 (TM2), Asp-121 (TM3), Thr-124 (TM3), Phe-257 (TM6), Phe-283 (TM7), Asn-290 (TM7), and Asp-294 (TM7) of the receptor.

Evidence indicating that the TM4, EL2, and TM5 of the melanocortin 3 receptor Do not participate in ligand binding.

The TM4, EL2 and TM5 show low amino acid homology within the MC receptor family. Three mutants of the human MC3 receptor were created in order to investigate the participation of these regions in ligand binding. The TM4, EL2 and TM5 were separately changed by multiple mutagenesis so that their amino acid sequences became identical with the human MC1 receptor. The mutants were expressed in COS cells and they bound peptide ligands in the same fashion as the wild type MC3 receptor clone. Our results indicate that the amino acids that were mutated in the MC3 receptor do not affect the binding of MSH peptides. The data provide further evidence, that the mutated regions may not participate at all in ligand binding, as indicated by modelling experiments and homology comparison.

A three dimensional model for the interaction of MSH with the melanocortin-1 receptor.

A model for the interaction of the melanocortin-1 (MC1) receptor with MSH peptide has been constructed. The model was built by homology modelling using bacteriorhodopsin as template. A cyclic analogue of MSH could be docked into a binding pocket located between transmembrane (TM) domains 2, 3 and 6 of the receptor. The most significant receptor to ligand interactions occur between D117 in TM3 of receptor with histidine in cyclic MSH-peptide, H260 in TM6 with glutamic in peptide and D121 in TM3 with arginine in peptide. The model finds support from earlier mutagenesis data.

Evidence for alternate points of attachment for alpha-MSH and its stereoisomer [Nle4, D-Phe7]-alpha-MSH at the melanocortin-1 receptor.

The molecular basis for the alpha-melanocyte stimulating hormone (alpha-MSH) stereoselectivity was studied by examining ligand binding to site specific mutants of the melanocortin 1 receptor (MC1R). The amino acids Asp117, Phe179, His209 and His260 were targeted for mutation to alanine as they are conserved in the melanocortin receptor family. Expression of the wild type and the MC1R mutants in COS-7 cells revealed that the binding affinities for the alpha-MSH (L-isomer) was reduced by 267 fold for the D117-->A mutant and 132 fold for the H260-->A mutant. In contrast, the binding affinity for the [Nle4, D-Phe7]-alpha-MSH (NDP-MSH; D-isomer) remain unchanged between the wild type and the mutants. Moreover, the mutants also displayed reduction in affinity to L-isomers of all the other melanocortic peptides examined. Thus, the mutation of single amino acids in the third and sixth transmembrane segments results in the display of ligand stereoselectivity of the MC1R. In addition, these data represent the first evidence of the different binding epitopes on a G-protein coupled receptor for a peptide ligand stereoisomers, both of which are shown to be potent agonists.