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Background:Cardiovascular disease (CVD) is a leading cause of death worldwide. It is predicted that approximately 23.6 million people will die from CVDs annually by 2030. Therefore, there is a great need for an effective therapeutic approach to combat this disease. The European Cardiovascular Target Discovery (CarTarDis) consortium identified Oncostatin M (OSM) as a po-tential therapeutic target for atherosclerosis. The benefits of modulating OSM - an interleukin (IL)-6 family cytokine - have since been studied for multiple indications. However, as decades of high at-trition rates have stressed, the success of a drug target is determined by the fine balance between benefits and the risk of adverse events. Safety issues should therefore not be overlooked.Objective:In this review, a risk/benefit analysis is performed on OSM inhibition in the context of atherosclerosis treatment. First, OSM signaling characteristics and its role in atherosclerosis are de-scribed. Next, an overview of in vitro, in vivo, and clinical findings relating to both the benefits and risks of modulating OSM in major organ systems is provided. Based on OSM’s biological function and expression profile as well as drug intervention studies, safety concerns of inhibiting this target have been identified, assessed, and ranked for the target population.Conclusion:While OSM may be of therapeutic value in atherosclerosis, drug development should also focus on de-risking the herein identified major safety concerns: tissue remodeling, angiogene-sis, bleeding, anemia, and NMDA- and glutamate-induced neurotoxicity. Close monitoring and/or exclusion of patients with various comorbidities may be required for optimal therapeutic benefit.

In the present study, our aim was to investigate whether the novel highly selective 5-hydroxytryptamine(6) (5-HT6) receptor antagonist SLV can ameliorate impairments in cognition and social interaction with potential relevance for both schizophrenia and Alzheimer's disease (AD). SLV sub-chronically- treated Wistar rats reared in isolation showed significantly enhanced prepulse inhibition (PPI) and object recognition performance when compared to vehicle - treated rats. In the isolated rats, also a significant reduction in expression of hippocampal neural cell adhesion molecule polysialylation (NCAM-PSA) was found which was ameliorated following treatment with SLV (30 mg/kg). The social engagement deficit in rats exposed in utero (on gestational day 12.5) to valproic acid (VPA) was reversed by treatment with SLV (30 mg/kg). SLV (20 and 30 mg/kg, p.o.) fully reversed MK-801 - induced deficits in the ORT and also scopolamine - induced deficits in both the Object Recognition Task (ORT) and Object Location Task (OLT) in Wistar rats. In addition, a combination of sub-optimal doses of SLV and donepezil attenuated scopolamine-induced ORT deficits. Furthermore, SLV (10 mg/kg, p.o.) reversed spontaneous alternation deficits in the T-maze induced by MK-801 administration in Swiss mice and in aged C57131/6J mice. SLV additionally improved T-Maze spatial learning and passive avoidance learning in Sprague-Dawley rats with amyoid-beta (A beta) injections into the hippocampus. In contrast, no benefits were found with SLV or the tested reference compounds (donepezil and RVT-101) on cognitive performance of 12 months old Tg2576 mice. Also, in the social recognition task, an absence of cognitive enhancing properties was observed with SLV on "normal forgetting" in Wistar rats. Finally, analysis of spontaneous inhibitory postsynaptic currents (sIPSCs) frequency recorded from pyramidal cells revealed a reduction in the presence of 1 mu M of SLV. In conclusion, SLV was investigated in several rodent animal models and found to be effective at a least effective dose (LED) of 20 mg/kg and 10 mg/kg (p.o.) in the rat and the mouse, respectively. (C) 2016 Elsevier Inc. All rights reserved.

The discovery of non-basic N'-(arylsulfonyl)pyrazoline-1-carboxamidines as 5-HT6 antagonists with unique structural features was recently disclosed. Here we describe how this structural class was further developed by addressing an unexplored interaction site of the 5-HT6 receptor. Compound 13 resulting from this effort is a highly potent and selective 5-HT6 antagonist with improved metabolic stability. It is furthermore devoid of hERG affinity. Despite its modest CNS/plasma ratio, a high brain free fraction ensured substantial exposure to allow for rodent cognition studies.

The 5-HT(6) receptor (5-HT(6)R) has been in the spotlight for several years regarding CNS-related diseases. We set out to discover novel, neutral 5-HT(6)R antagonists to improve off-target selectivity compared to basic amine-containing scaffolds dominating the field. High-throughput screening identified the N'-(sulfonyl)pyrazoline-1-carboxamidine scaffold as a promising neutral core for starting hit-to-lead. Medicinal chemistry, molecular modeling, small molecule NMR and X-ray crystallography were subsequently applied to optimize the leads into antagonists (compounds 1-49) displaying high 5-HT(6)R affinity with optimal off-target selectivity. Unique structural features include a pseudoaromatic system and an internal hydrogen bond freezing the bioactive conformation. While physicochemical properties and CNS availability were generally favorable, significant efforts had to be made to improve metabolic stability. The optimized structure 42 is an extremely selective, hERG-free, high-affinity 5-HT(6)R antagonist showing good human in vitro metabolic stability. Rat pharmacokinetic data were sufficiently good to enable further in vivo profiling.

The 5-hydroxytryptamine(6) (5-HT(6)) receptor has been suggested to play an important role in the regulation of memory and cognition. In the present study, our aim was to investigate whether the novel, selective 5-HT(6) antagonists compound (CMP) X and CMP Y and the reference 5-HT(6) antagonist GSK-742457 could ameliorate impairments in episodic memory in 3-months-old male Wistar rats. The acetylcholinesterase inhibitor (AChEI) donepezil (Aricept®, approved for symptomatic treatment of Alzheimer's disease, AD) was used as a positive reference compound. First, effects of the 5-HT(6) antagonists CMP X, CMP Y and GSK-742457 were investigated on object recognition task (ORT) performance in rats treated with the muscarinic antagonist scopolamine (0.1mg/kg, administered intraperitoneally, i.p., 30 min before trial 1). Second, effects of the combination of suboptimal doses of 5-HT(6) antagonists CMP X and CMP Y with the AChEI donepezil were studied, to determine whether the 5-HT(6) antagonists show additive synergism with donepezil in the ORT. Finally, effects of CMP Y, GSK-742457 and donepezil were investigated on object location task (OLT) performance in rats treated with scopolamine. Donepezil (1mg/kg, oral administration, p.o.), GSK-742457 (3mg/kg, i.p.), CMP X (3mg/kg, i.p.) and CMP Y (30 mg/kg, p.o.), all ameliorated the scopolamine-induced deficits in object recognition. In the ORT, we have found that combined administration of subthreshold doses of CMP X (1mg/kg, i.p.) and CMP Y (10mg/kg, p.o.) with the AChEI donepezil (0.1mg/kg, p.o.), enhanced memory performance in Wistar rats with deficits induced by scopolamine. Donepezil (0.1mg/kg, p.o.) alone had no discernable effects on performance. This suggests additive synergistic effects of the 5-HT(6) antagonists (CMP X and CMP Y) with donepezil on cognitive impairment. Finally, donepezil (1mg/kg, p.o.), GSK-742457 (10mg/kg, p.o.) and CMP Y (30 mg/kg, p.o.) also reduced scopolamine-induced deficits in the OLT. In conclusion, the 5-HT(6) antagonists were found to clearly improve episodic memory deficits induced by scopolamine. In addition, co-administration of the 5-HT(6) receptor antagonists CMP X and CMP Y with the AChEI donepezil to cognitively impaired rats also resulted in potentially additive enhancing effects on cognition. This suggests that these compounds could have potential as monotherapy, but also as adjunctive therapy in patients with AD treated with common treatments such as donepezil.

Molecular graph theory was used to design a unique and diverse, high-efficiency fragment screening collection. A data set retrieved from the annotated database AurSCOPE GPS was used as the reference set, and the GDB-13 database, a virtual library of enumerated organic molecules, was used as a source for the fragment selection. The data graph collection of Discngine as implemented in PipelinePilot was applied to perform the graph pharmacophore similarity matching between the reference and the GDB-13 data sets, leading to the ultimate fragment screening library. The relevance of this unique fragment collection was demonstrated by means of a virtual screening exercise using human trypsin as a test case. Several novel entities with high similarity to known trypsin inhibitors were identified in the in silico exercise. The application of this unique, high fragment efficiency collection to other protein targets in the framework of fragment-based drug discovery is warranted.

1. To investigate whether cytochrome P450 (P450) inhibition can contribute to the chemopreventive activity of selenocysteine Se-conjugates (SeCys conjugates), 21 SeCys conjugates were screened for their inhibitory potency towards seven of the most important human P450s. 2. The majority of the SeCys conjugates produced near complete inhibition of CYP1A1 at a concentration of 250 microm. The most potent inhibitor, Se-benzyl-L-selenocysteine, displayed an IC50 of 12.8 +/- 1.2 microm. CYP2C9, -2C19 and -2D6 were moderately (50-60%) inhibited by the SeCys conjugates. CYP1A2, -2E1 and -3A4 were least inhibited. 3. Studies on the susceptibility of CYP1A1 to SeCys conjugates implicated a thiol-reactive intermediate, as evidenced by reduced inhibition levels in the presence of glutathione and N-acetyl cysteine. Uncoupling of the P450-catalytic cycle was of no importance as ROS scavengers did not influence inhibition levels. 4. P450 inhibition by two physiologically relevant metabolite classes of SeCys conjugates was also studied. N-acetylation of SeCys conjugates consistently increased the inhibitory potency towards CYP1A2, -2C19, -2E1 and -3A4. Beta-lyase catalysed bioactivation of alkyl-substituted SeCys conjugates or Se-benzyl-L-selenocysteine produced little or no additional inhibition of P450 activity. For Se-phenyl-L-selenocysteine, however, significant increases in P450 inhibition were obtained by beta-lyase pre-incubation. 5. It is concluded that the potent and relatively selective CYP1A1 inhibition exerted by SeCys conjugates may contribute to their chemopreventive activity.

The granulocyte colony-stimulating factor receptor (G-CSF-R) forms a tetrameric complex with G-CSF containing two ligand and two receptor molecules. The N-terminal Ig-like domain of the G-CSF-R is required for receptor dimerization, but it is not known whether it binds G-CSF or interacts elsewhere in the complex. Alanine scanning mutagenesis was used to show that residues in the Ig-like domain of the G-CSF-R (Phe(75), Gln(87), and Gln(91)) interact with G-CSF. This binding site for G-CSF overlapped with the binding site of a neutralizing anti-G-CSF-R antibody. A model of the Ig-like domain showed that the binding site is very similar to the viral interleukin-6 binding site (site III) on the Ig-like domain of gp130, a related receptor. To further characterize the G-CSF-R complex, exposed and inaccessible regions of monomeric and dimeric ligand-receptor complexes were mapped with monoclonal antibodies. The results showed that the E helix of G-CSF was inaccessible in the dimeric but exposed in the monomeric complex, suggesting that this region binds to the Ig-like domain of the G-CSF-R. In addition, the N terminus of G-CSF was exposed to antibody binding in both complexes. These data establish that the dimerization interface of the complete receptor complex is different from that in the x-ray structure of a partial complex. A model of the tetrameric G-CSF.G-CSF-R complex was prepared, based on the viral interleukin-6.gp130 complex, which explains these and previously published data.

In this study, a selective substrate for cytochrome P450 2D6 was designed using a small molecule model developed by M. J. De Groot et al. [(1997) Chem. Res. Toxicol. 10, 41-48]. The substrate, 7-methoxy-4-(aminomethyl)coumarin (MAMC), and its putative O-demethylated metabolite 7-hydroxy-4-(aminomethyl)coumarin (HAMC) were synthesized, and their respective fluorescence properties were characterized. The selectivity of MAMC for P450 2D6 was characterized using microsomes containing single human P450 isoenzymes and human liver microsomes. Formation of the metabolic product HAMC was easily assessed in real time with fluorescence spectroscopy, since MAMC and HAMC excitation and emission wavelengths differed significantly. HPLC analysis confirmed that HAMC was the single metabolic product of MAMC and that HAMC formation accounts for the total increase in fluorescence. It was found that, in microsomes from yeast or lymphoblastoid cells selectively expressing P450 isoenzymes, MAMC was selective for P450 2D6 at a concentration of 25 microM with only P450 1A2 contributing significantly to the formation of HAMC. P450s 2A6, 2B6, 2C8, 2C9, 2C19, 2E1, 3A4, and 3A5 were shown not to metabolize MAMC at a concentration of 25 microM. K(m) and v(max) values of MAMC for P450 2D6 were found to be 26.2 +/- 2.8 microM and 2.9 +/- 0.07 min(-)(1), respectively. For P450 1A2, MAMC was found to have a K(m) value of 29.7 +/- 6.2 microM and a v(max) of 0.57 +/- 0.07 min(-)(1). Formation of HAMC in human liver microsomes could be completely inhibited by quinidine, at a concentration of 0.5 microM selective for P450 2D6, and furafylline, at a concentration of 30 microM selective for P450 1A2. In conclusion, O-demethylation of 7-methoxy-4-(aminomethyl)coumarin is a rapid and easily determined parameter for P450 2D6 activity and, due to the fluorescent properties of the metabolite formed, may be a valuable new tool for high-throughput screening purposes.

One of the major processes that occur as a result of radical-induced oxidative stress is lipid peroxidation (LPO). Degradation of lipid peroxides results in various products, including a variety of carbonyl compounds. In the present study eight different lipid degradation products, i.e., formaldehyde, acetaldehyde, acetone, propanal, butanal, pentanal, hexanal and malondialdehyde were identified and measured simultaneously and quantitatively in rat urine after derivatization with O-(2,3,4,5,6-pentafluorbenzyl)hydroxylamine hydrochloride, extraction with heptane and using gas chromatography-electron-capture detection (GC-ECD). The identity of the respective oximes in urine was confirmed by gas chromatography-negative ion chemical ionization mass spectrometry (GC-NCI-MS). Simultaneously measured standard curves were linear for all oxime-products and the detection limits were between 39.0 +/- 5.3 (n=9) and 500 +/- 23 (n=9) fmol per microl injected sample. Recoveries of all products from urine or water were 73.0 +/- 5.2% and higher. In urine of CCl4-treated rats an increase in all eight lipid degradation products in urine was found 24 h following exposure. ACON showed the most distinct increase, followed by PROPA, BUTA and MDA. It is concluded that the rapid, selective and sensitive analytical method based on GC-ECD presented here is well suited for routine measurement of eight different lipid degradation products. These products appear to be useful as non-invasive biomarkers for in vivo oxidative stress induced in rats by CCl4.

Recent studies suggest a potential role for 5-hydroxytryptamine(6) (5-HT(6)) receptors in the regulation of addictive behavior. In the present study, our aim was to investigate whether the novel highly selective 5-HT(6) receptor antagonist compound (CMP) 42 affected nicotine and ethanol seeking behavior in Wistar rats. We have also studied whether CMP 42 had beneficial effects in a model of impulse control, as measured in the 5-choice serial reaction time task (5-CSRTT). Rats were trained to nose poke to receive intravenous infusions of nicotine or an ethanol drop. CMP 42 (3-30 mg/kg intraperitoneally, i.p.) was administered to investigate the effects on nicotine self-administration. Rats were also tested for cue-induced reinstatement of nicotine and ethanol seeking. In addition, the effects of CMP 42 were studied on the number of anticipatory responses in the 5-CSRTT. CMP 42 was effective in reducing nicotine self-administration and reinstatement of nicotine seeking at a dose of 30 mg/kg (i.p.). CMP 42 was also effective in reducing reinstatement of ethanol seeking (30 mg/kg i.p.). In contrast, CMP 42 did not affect anticipatory responding at doses tested, indicating no effects on impulse control. These results add to a body of evidence implicating the 5-HT(6) receptor as a viable target for the control of drug abuse. Specifically, we demonstrated for the first time effects on nicotine self-administration and on nicotine and ethanol reinstatement. Further, these effects are probably not mediated by effects on impulse control.

The 5-HT(6) receptor (5-HT(6)R) has been in the spotlight for several years regarding CNS-related diseases. We set out to discover novel, neutral 5-HT(6)R antagonists to improve off-target selectivity compared to basic amine-containing scaffolds dominating the field. High-throughput screening identified the N'-(sulfonyl)pyrazoline-1-carboxamidine scaffold as a promising neutral core for starting hit-to-lead. Medicinal chemistry, molecular modeling, small molecule NMR and X-ray crystallography were subsequently applied to optimize the leads into antagonists (compounds 1-49) displaying high 5-HT(6)R affinity with optimal off-target selectivity. Unique structural features include a pseudoaromatic system and an internal hydrogen bond freezing the bioactive conformation. While physicochemical properties and CNS availability were generally favorable, significant efforts had to be made to improve metabolic stability. The optimized structure 42 is an extremely selective, hERG-free, high-affinity 5-HT(6)R antagonist showing good human in vitro metabolic stability. Rat pharmacokinetic data were sufficiently good to enable further in vivo profiling.

In this review, we begin by introducing the basic principles of kinetics and thermodynamics of target-drug binding within the context of drug discovery. In addition, we present a meta-analysis of the recent literature describing the kinetic and thermodynamic resolution of successful clinical candidates with diverse mechanisms of action. We finish by discussing the best practices in the triage and chemical optimization towards clinical candidates with maximal in vivo efficacy devoid of adverse events.

Many early drug research efforts are too reductionist thereby not delivering key parameters such as kinetics and thermodynamics of target–ligand binding. A set of human D-Amino Acid Oxidase (DAAO) inhibitors 1–6 was applied to demonstrate the impact of key biophysical techniques and physicochemical methods in the differentiation of chemical entities that cannot be adequately distinguished on the basis of their normalized potency (ligand efficiency) values. The resulting biophysical and physicochemical data were related to relevant pharmacodynamic and pharmacokinetic properties. Surface Plasmon Resonance data indicated prolonged target–ligand residence times for 5 and 6 as compared to 1–4, based on the observed koff values. The Isothermal Titration Calorimetry-derived thermodynamic binding profiles of 1–6 to the DAAO enzyme revealed favorable contributions of both ΔH and ΔS to their ΔG values. Surprisingly, the thermodynamic binding profile of 3 elicited a substantially higher favorable contribution of ΔH to ΔG in comparison with the structurally closely related fused bicyclic acid 4. Molecular dynamics simulations and free energy calculations of 1, 3, and 4 led to novel insights into the thermodynamic properties of the binding process at an atomic level and in the different thermodynamic signatures of 3 and 4. The presented holistic approach is anticipated to facilitate the identification of compounds with best-in-class properties at an early research stage.

A novel scoring algorithm based on molecular interaction fingerprints (IFPs) was comparatively evaluated in its scaffold hopping efficiency against four virtual screening standards (GlideXP, Gold, ROCS, and a Bayesian classifier). Decoy databases for the two targets under examination, adenosine deaminase and retinoid X receptor alpha, were obtained from the Directory of Useful Decoys and were further enriched with approximately 5% of active ligands. Structure and ligand-based methods were used to generate the ligand poses, and a Tanimoto metric was chosen for the calculation of the similarity interaction fingerprint between the reference ligand and the screening database. Database enrichments were found to strongly depend on the pose generator algorithm. In spite of these dependencies, enrichments using molecular IFPs were comparable to those obtained with GlideXP, Gold, ROCS, and the Bayesian classifier. More interestingly, the molecular IFP scoring algorithm outperformed these methods at scaffold hopping enrichment, regardless of the pose generator algorithm.

The ligand-binding characteristics of rat and human CYP2D isoforms, i.e., rat CYP2D1-4 and human CYP2D6, were investigated by measuring IC(50) values of 11 known CYP2D6 ligands using 7-methoxy-4-(aminomethyl)coumarin (MAMC) as substrate. Like CYP2D6, all rat CYP2D isozymes catalyzed the O-demethylation of MAMC with K(m) and V(max) values ranging between 78 and 145 microM and 0.048 and 1.122 min(-1), respectively. To rationalize observed differences in the experimentally determined IC(50) values, homology models of the CYP2D isoforms were constructed. A homology model of CYP2D6 was generated on the basis of crystallized rabbit CYP2C5 and was validated on its ability to reproduce binding orientations corresponding to metabolic profiles of the substrates and to remain stable during unrestrained molecular dynamics simulations at 300 K. Twenty-two active site residues, sharing up to 59% sequence identity, were identified in the CYP2D binding pockets and included CYP2D6 residues Phe120, Glu216, and Asp301. Electrostatic potential calculations displayed large differences in the negative charge of the CYP2D active sites, which was consistent with observed differences in absolute IC(50) values. MD studies on the binding mode of sparteine, quinidine, and quinine in CYP2D2 and CYP2D6 furthermore concurred well with experimentally determined IC(50) values and metabolic profiles. The current study thus provides new insights into differences in the active site topology of the investigated CYP2D isoforms.

The homodimeric, pyridoxal 5'-phosphate (PLP)-dependent enzyme glutamine transaminase K/cysteine conjugate beta-lyase (GTK/beta-lyase) has been implicated in the bioactivation of chemopreventive compounds. This paper describes the first homology model of rat renal GTK/beta-lyase and its active site residues, deduced from molecular dynamics (MD) simulations of the binding mode of 13 structurally diverse cysteine S-conjugates and amino acids after Amber-parametrization of PLP. Comparison with Thermus thermophilus aspartate aminotransferase (tAAT) and Trypanosoma cruzi tyrosine aminotransferase (tTAT), used as templates for modeling GTK/beta-lyase, showed that the PLP-binding site of GTK/beta-lyase is highly conserved. Binding of the ligand alpha-carboxylate-group occurred via the conserved residues Arg(432) and Asn(219), and Asn(50) and Gly(70). Two pockets accommodated the various ligand side chains. A small pocket, located directly above PLP, was of a highly hydrophobic and aromatic character. A larger pocket, formed partly by the substrate access channel, was more hydrophilic and notably involved the salt bridge partners Glu(54) and Arg(99*) (* denotes the other subunit). Ligand-binding residues included Leu(51), Phe(71), Tyr(135), Phe(373) and Phe(312*), and pi-stacking interactions were often observed. Tyr(135) and Asn(50) were prominent in hydrogen bonding with the sulfur-atom of cysteine S-conjugates. The observed binding mode of the ligands corresponded well with their experimentally determined inhibitory potency toward GTK/beta-lyase. The current homology model thus provides a starting point for further validation of the role of active site residues in ligand-binding by means of mutagenesis studies. Ultimately, insight in the binding of ligands to GTK/beta-lyase may result in the rational design of new ligands and selective inhibitors.

We recently reported on the design, synthesis and characterisation of a novel and selective substrate of human cytochrome P450 2D6 (CYP2D6), 7-methoxy-4-(aminomethyl)-coumarin (MAMC). Here, we describe a high-throughput microplate reader assay, which makes use of MAMC as a fluorescent probe for determining the inhibition and activity of CYP2D6 in heterologously expressed systems and human liver microsomes. The high-throughput screening (HTS) assay can be used both in an end-point and real-time configuration, and is easy to use, rapid and sensitive. In addition, end-point measurements by means of flow injection analysis have also successfully been performed. The HTS-assay was validated by performing inhibition experiments for several low- and high-affinity ligands (n=6) of CYP2D6, and comparing the findings to those obtained with the standard O-demethylation assay of dextromethorphan. The results indicate that all compounds tested display competitive inhibition in both the MAMC and dextromethorphan assay, and the K(i) values reveal a very good correlation (R(2)=0.984) between the two assays. To further demonstrate the usefulness of the HTS-assay, IC(50) values of a series of five N-substituted analogs of 3, 4-methylenedioxyamphetamine for CYP2D6 have been determined. The results obtained demonstrate that the current HTS-assay represents a significant improvement over previous assays, with a higher turnover of MAMC and a higher selectivity for CYP2D6.

A new pharmacophoric model for the H1-antagonist binding site is derived which reveals that a simple atom to atom matching of compounds is not sufficient; in this model, interacting residues from the receptor need to be included. To obtain this model, the bioactive conformations of several (semi-)rigid classical histamine H1-receptor antagonists have been investigated (cyproheptadine, phenindamine, triprolidine, epinastine, mequitazine, IBF28145, and mianserine). In general, these antihistamines contain two aromatic rings and a basic nitrogen atom. A previously derived pharmacophoric model with the nitrogen position fixed relative to the two aromatic rings is now found not to be suitable for describing the H1-antagonist binding site. A procedure is described which allows for significant freedom in the position of the basic nitrogen of the histamine H1-antagonist. The area accessible to the basic nitrogen is confined to the region accessible to its counterion on the histamine H1-receptor, i.e., the carboxylate group of Asp116. The basic nitrogen is assumed to form an ionic hydrogen bond with this aspartic acid which C alpha- and C beta-carbons are fixed with respect to the protein backbone. Via this hydrogen bond, the direction of the acidic proton of the antagonist is taken into account. Within these computational procedures, an aspartic acid is coupled to the basic nitrogen of each H1-antagonist considered; the carboxylate group is connected to the positively charged nitrogen via geometric H-bonding restraints obtained from a thorough database search (CSD). Also to the basic nitrogen of the pharmacophore is coupled an aspartic acid (to yield our new template). In order to derive a model for the H1-antagonist binding site, the aromatic ring systems of the antagonists and template are matched according to a previously described procedure. Subsequently, the C alpha- and C beta-carbons of the aspartic acid coupled to the H1-antagonists are matched with those of the template in a procedure which allows the antagonist and the carboxylate group to adapt their conformation (and also their relative position) in order to optimize the overlap with the template. A six-point pharmacophoric model is derived which has stereoselective features and is furthermore able to distinguish between the so-called "cis"- and "trans"-rings mentioned in many (Q)SAR studies on H1-antagonists. Due to its stereoselectivity, the model is able to designate the absolute bioactive configuration of antihistamines such as phenindamine (S), epinastine (S), and IBF28145 (R). A further merit of this study is that a model is obtained which includes an amino acid from the receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

A series of six structural analogs of 7-methoxy-4-(aminomethyl)-coumarin (MAMC), a recently developed high-throughput substrate of P450 2D6 (CYP2D6), was synthesized to investigate the influence of N-substitution on the metabolism by cytochrome P450s, as well as on P450 selectivity. The analogs were obtained by introducing alkyl substituents at the amino group of MAMC and by replacing this moiety with a pyridine group. Competition experiments using heterologously expressed CYP2D6 demonstrated that the introduction and elongation of alkyl substituents strongly decreased the IC(50) values toward dextromethorphan O-demethylation. Metabolism studies showed that the regioselectivity of metabolism was unaffected by the varying N substituents, as only O-dealkylation of the analogs and no N-dealkylation was observed. In excellent agreement with the competition experiments, metabolism studies also showed that elongation of the alkyl chain dramatically increased the affinity of the compounds toward CYP2D6, as indicated by an up to 100-fold decrease in K(m) values. The V(max) values displayed a much less pronounced decrease with an increasing N-alkyl chain, resulting in as much as a 30-fold increase in the V(max)/K(m) value. Interestingly, due to the higher fluorescent yield of the N-alkyl metabolites compared with the metabolite of MAMC, O-dealkylation of N-methyl MAMC by CYP2D6 can be measured with a more than 3-fold higher sensitivity. Studies on P450 selectivity showed that only CYP1A2 and CYP2D6 contribute to the O-dealkylation of the N-alkyl analogs in both heterologously expressed P450s and human liver microsomes. In sharp contrast to CYP2D6, N-alkylation of MAMC did not significantly affect the K(m) values of O-dealkylation by CYP1A2, but it did result in higher V(max) values. Finally, CYP1A2 also N-dealkylated the analogs.