Ligand-guided homology modeling drives identification of novel histamine H3 receptor ligands

In this study, we report a ligand-guided homology modeling approach allowing the analysis of relevant binding site residue conformations and the identification of two novel histamine H3 receptor ligands with binding affinity in the nanomolar range. The newly developed method is based on exploiting an essential charge interaction characteristic for aminergic G-protein coupled receptors for ranking 3D receptor models appropriate for the discovery of novel compounds through virtual screening

Pharmacophore and receptor models for neurokinin receptors

Three neurokinin (NK) antagonist pharmacophore models (Models 1–3) accounting for hydrogen bonding groups in the `head' and `tail' of NK receptor ligands have been developed by use of a new procedure for treatment of hydrogen bonds during superimposition. Instead of modelling the hydrogen bond acceptor vector in the strict direction of the lone pair, an angle is allowed between the hydrogen bond acceptor direction and the ideal lone pair direction. This approach adds flexibility to hydrogen bond directions and produces more realistic RMS values. By using this approach, two novel pharmacophore models were derived (Models 2 and 3) and a hydrogen bond acceptor was added to a previously published NK2 pharmacophore model [Poulsen et al., J. Comput.-Aided Mol. Design, 16 (2002) 273] (Model 1). Model 2 as well as Model 3 are described by seven pharmacophore elements: three hydrophobic groups, three hydrogen bond acceptors and a hydrogen bond donor. Model 1 contains the same hydrophobic groups and hydrogen bond donor as Models 2 and 3, but only one hydrogen bond acceptor. The hydrogen bond acceptors and donor are represented as vectors. Two of the hydrophobic groups are always aromatic rings whereas the other hydrophobic group can be either aromatic or aliphatic. In Model 1 the antagonists bind in an extended conformation with two aromatic rings in a parallel displaced and tilted conformation. Model 2 has the same two aromatic rings in a parallel displaced conformation whereas Model 3 has the rings in an edge to face conformation. The pharmacophore models were evaluated using both a structure (NK receptor homology models) and a ligand based approach. By use of exhaustive conformational analysis (MMFFs force field and the GB/SA hydration model) and least-squares molecular superimposition studies, 21 non-peptide antagonists from several structurally diverse classes were fitted to the pharmacophore models. More antagonists could be fitted to Model 2 with a low RMS and a low conformational energy penalty than to Models 1 and 3. Pharmacophore Model 2 was also able to explain the NK1, NK2 and NK3 subtype selectivity of the compounds fitted to the model. Three NK 7TM receptor models were constructed, one for each receptor subtype. The location of the antagonist binding site in the three NK receptor models is identical. Compounds fitted to pharmacophore Model 2 could be docked into the NK1, NK2 and NK3 receptor models after adjustment of the conformation of the flexible linker connecting the head and tail. Models 1 and 3 are not compatible with the receptor models.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42969/1/10822_2004_Article_5257316.pd

A New Method for Ligand-supported Homology Modelling of Protein Binding Sites: Development and Application to the neurokinin-1 receptor

In this thesis, a novel strategy (MOBILE (Modelling Binding Sites Including Ligand Information Explicitly)) was developed that models protein binding-sites simultaneously considering information about the binding mode of bioactive ligands during the homology modelling process. As a result, protein binding-site models of higher accuracy and relevance can be generated. Starting with the (crystal) structure of one or more template proteins, in the first step several preliminary homology models of the target protein are generated using the homology modelling program MODELLER. Ligands are then placed into these preliminary models using different strategies depending on the amount of experimental information about the binding mode of the ligands. (1.) If a ligand is known to bind to the target protein and the crystal structure of the protein-ligand complex with the related template protein is available, it can be assumed that the ligand binding modes are similar in the target and template protein. Accordingly, ligands are then transferred among these structures keeping their orientation as a restraint for the subsequent modelling process. (2.) If no complex crystal structure with the template is available, the ligand(s) can be placed into the template protein structure by docking, and the resulting orientation can then be used to restrain the following protein modelling process. Alternatively, (3.) in cases where knowledge about the binding mode cannot be inferred by the template protein, ligand docking is performed into an ensemble of homology models. The ligands are placed into a crude binding-site representation via docking into averaged property fields derived from knowledge-based potentials. Once the ligands are placed, a new set of homology models is generated. However, in this step, ligand information is considered as additional restraint in terms of the knowledge-based DrugScore protein-ligand atom pair potentials. Consulting a large ensemble of produced models exhibiting di erent side-chain rotamers for the binding-site residues, a composite picture is assembled considering the individually best scored rotamers with respect to the ligand. After a local force-field optimisation, the obtained binding-site models can be used for structure-based drug design

Multifunctional opioid-derived hybrids in neuropathic pain : preclinical evidence, ideas and challenges

When the first- and second-line therapeutics used to treat neuropathic pain (NP) fail to induce effcient analgesia - which is estimated to relate to more than half of the patients-opioid drugs are prescribed. Still, the pathological changes following the nerve tissue injury, i.a. pronociceptive neuropeptide systems activation, oppose the analgesic effects of opiates, enforcing the use of relatively high therapeutic doses in order to obtain satisfying pain relief. In parallel, the repeated use of opioid agonists is associated with burdensome adverse effects due to compensatory mechanisms that arise thereafter. Rational design of hybrid drugs, in which opioid ligands are combined with other pharmacophores that block the antiopioid action of pronociceptive systems, delivers the opportunity to ameliorate the NP-oriented opioid treatment via addressing neuropathological mechanisms shared both by NP and repeated exposition to opioids. Therewith, the new dually acting drugs, tailored for the specificity of NP, can gain in efficacy under nerve injury conditions and have an improved safety profile as compared to selective opioid agonists. The current review presents the latest ideas on opioid-comprising hybrid drugs designed to treat painful neuropathy, with focus on their biological action, as well as limitations and challenges related to this therapeutic approach

In vivo antinociception of potent mu opioid agonist tetrapeptide analogues and comparison with a compact opioid agonist - neurokinin 1 receptor antagonist chimera

<p>Abstract</p> <p>Background</p> <p>An important limiting factor in the development of centrally acting pharmaceuticals is the blood-brain barrier (BBB). Transport of therapeutic peptides through this highly protective physiological barrier remains a challenge for peptide drug delivery into the central nervous system (CNS). Because the most common strategy to treat moderate to severe pain consists of the activation of opioid receptors in the brain, the development of active opioid peptide analogues as potential analgesics requires compounds with a high resistance to enzymatic degradation and an ability to cross the BBB.</p> <p>Results</p> <p>Herein we report that tetrapeptide analogues of the type H-Dmt¹-Xxx²-Yyy³-Gly⁴-NH₂are transported into the brain after intravenous and subcutaneous administration and are able to activate the μ- and δ opioid receptors more efficiently and over longer periods of time than morphine. Using the hot water tail flick test as the animal model for antinociception, a comparison in potency is presented between a side chain conformationally constrained analogue containing the benzazepine ring (BVD03, Yyy³: Aba), and a "ring opened" analogue (BVD02, Yyy³: Phe). The results show that in addition to the increased lipophilicity through amide bond N-methylation, the conformational constraint introduced at the level of the Phe³side chain causes a prolonged antinociception. Further replacement of NMe-D-Ala²by D-Arg²in the tetrapeptide sequence led to an improved potency as demonstrated by a higher and maintained antinociception for AN81 (Xxx²: D-Arg) vs. BVD03 (Xxx²: NMe-D-Ala). A daily injection of the studied opioid ligands over a time period of 5 days did however result in a substantial decrease in antinociception on the fifth day of the experiment. The compact opioid agonist - NK1 antagonist hybrid SBCHM01 could not circumvent opioid induced tolerance.</p> <p>Conclusions</p> <p>We demonstrated that the introduction of a conformational constraint has an important impact on opioid receptor activation and subsequent antinociception in vivo. Further amino acid substitution allowed to identify AN81 as an opioid ligand able to access the CNS and induce antinociception at very low doses (0.1 mg/kg) over a time period up to 7 hours. However, tolerance became apparent after repetitive i.v. administration of the investigated tetrapeptides. This side effect was also observed with the dual opioid agonist-NK1 receptor antagonist SBCHM01.</p

Modeling of Human Prokineticin Receptors: Interactions with Novel Small-Molecule Binders and Potential Off-Target Drugs

The Prokineticin receptor (PKR) 1 and 2 subtypes are novel members of family A GPCRs, which exhibit an unusually high degree of sequence similarity. Prokineticins (PKs), their cognate ligands, are small secreted proteins of ∼80 amino acids; however, non-peptidic low-molecular weight antagonists have also been identified. PKs and their receptors play important roles under various physiological conditions such as maintaining circadian rhythm and pain perception, as well as regulating angiogenesis and modulating immunity. Identifying binding sites for known antagonists and for additional potential binders will facilitate studying and regulating these novel receptors. Blocking PKRs may serve as a therapeutic tool for various diseases, including acute pain, inflammation and cancer.Ligand-based pharmacophore models were derived from known antagonists, and virtual screening performed on the DrugBank dataset identified potential human PKR (hPKR) ligands with novel scaffolds. Interestingly, these included several HIV protease inhibitors for which endothelial cell dysfunction is a documented side effect. Our results suggest that the side effects might be due to inhibition of the PKR signaling pathway. Docking of known binders to a 3D homology model of hPKR1 is in agreement with the well-established canonical TM-bundle binding site of family A GPCRs. Furthermore, the docking results highlight residues that may form specific contacts with the ligands. These contacts provide structural explanation for the importance of several chemical features that were obtained from the structure-activity analysis of known binders. With the exception of a single loop residue that might be perused in the future for obtaining subtype-specific regulation, the results suggest an identical TM-bundle binding site for hPKR1 and hPKR2. In addition, analysis of the intracellular regions highlights variable regions that may provide subtype specificity

Chemogenomics knowledgebased polypharmacology analyses of drug abuse related G-protein coupled receptors and their ligands

Drug abuse (DA) and addiction is a complex illness, broadly viewed as a neurobiological impairment with genetic and environmental factors that influence its development and manifestation. Abused substances can disrupt the activity of neurons by interacting with many proteins, particularly G-protein coupled receptors (GPCRs). A few medicines that target the central nervous system (CNS) can also modulate DA related proteins, such as GPCRs, which can act in conjunction with the controlled psychoactive substance(s) and increase side effects. To fully explore the molecular interaction networks that underlie DA and to effectively modulate the GPCRs in these networks with small molecules for DA treatment, we built a drug-abuse domain specific chemogenomics knowledgebase (DA-KB) to centralize the reported chemogenomics research information related to DA and CNS disorders in an effort to benefit researchers across a broad range of disciplines. We then focus on the analysis of GPCRs as many of them are closely related with DA. Their distribution in human tissues was also analyzed for the study of side effects caused by abused drugs. We further implement our computational algorithms/tools to explore DA targets, DA mechanisms and pathways involved in polydrug addiction and to explore polypharmacological effects of the GPCR ligands. Finally, the polypharmacology effects of GPCRs-targeted medicines for DA treatment were investigated and such effects can be exploited for the development of drugs with polypharmacophore for DA intervention. The chemogenomics database and the analysis tools will help us better understand the mechanism of drugs abuse and facilitate to design new medications for system pharmacotherapy of DA. © 2014 Xie, Wang, Liu, Ouyang, Fang and Su

Using Protein Homology Models for Structure-Based Studies: Approaches to Model Refinement

Homology modeling is a computational methodology to assign a 3-D structure to a target protein when experimental data are not available. The methodology uses another protein with a known structure that shares some sequence identity with the target as a template. The crudest approach is to thread the target protein backbone atoms over the backbone atoms of the template protein, but necessary refinement methods are needed to produce realistic models. In this mini-review anchored within the scope of drug design, we show the validity of using homology models of proteins in the discovery of binders for potential therapeutic targets. We also report several different approaches to homology model refinement, going from very simple to the most elaborate. Results show that refinement approaches are system dependent and that more elaborate methodologies do not always correlate with better performances from built homology models

Interactions of GPR54 and GPR147 receptors with RF-amide ligands

Includes bibliographical references.G protein-coupled receptors play a key role in cellular signaling by transducing extracellular signals via G proteins to elicit intracellular responses. Studies have provided evidence supporting the role of the GPCR GPR54 and its cognate peptide ligand, kisspeptin (an RFamide peptide), in the regulation of reproduction. Kisspeptin and GPR54 play a critical role in the control of the hypothalamic-pituitary-gonadal axis by regulating gonadotropin-releasing hormone secretion. Despite the physiological importance of GPR54/kisspeptin signalling, the GRP54 residues important for receptor activation and signalling have not been extensively investigated. Another hypothalamic peptide, gonadotropin inhibiting hormone (also known as RFamide-related peptide), which interacts with the GPCR GPR147, has been found to inhibit GnRH-induced gonadotropin release and is therefore also of importance in control of the HPG axis. As many of the RFamide and RFamide-related receptors and ligands can be promiscuous, there is the potential for crosstalk between the GPR54/kisspeptin and GRP147/RFRP systems (or other RFamides) which may be of importance in the regulation of reproduction. GPR54 chimeras and point mutants were constructed in order to investigate the residues important for kisspeptin binding and receptor activation. The data obtained indicate that the acidic residues within the extracellular loops of GPR54 contribute to cell surface receptor expression and play a role in receptor signalling. In order to investigate the interactions of kisspeptin/RFRP peptides at GPR147 and GPR54, binding and activation of these receptors was studied with a range of ligands and their analogs. In addition to RFRP and its analogs, kisspeptin and several kisspeptin analogs were found to act as agonists at GRP147. In contrast, of all the ligands tested, only kisspeptin was able to bind to GPR54 with high affinity and elicit a response, thus indicating that GPR54 has high specificity for kisspeptin in contrast to the more promiscuous GPR147. These data demonstrate the therapeutic potential of kisspeptin analogs, for the inhibition of gonadotropin secretion and treatment of sex steroid hormone disease. In addition, these data have identified ligand and receptor residues important for binding and activation of GRP54/GRP147 which may aid development of new analogs targeting these receptors and highlighted the importance of testing these analogs for receptor specificity

Design, Synthesis and Biological Evaluation of Two Opioid Agonist and Ca 2.2 Blocker Multitarget Ligands.

N-type voltage-dependent Ca2+ channels (CaV 2.2) are located at nerve endings in the central and peripheral nervous systems and are strongly associated with the pathological processes of cerebral ischaemia and neuropathic pain. CaV 2.2 blockers such as the omega-conotoxin MVIIA (Prialt) are analgesic and have opioid-sparing effects. With the aim to develop new multitarget analgesic compounds, we designed the first omega-conotoxin/opioid peptidomimetics based on the enkephalin-like sequence Tyr-D-Ala-Gly-Phe (for the opioid portion) and two fragments derived from the loop-2 pharmacophore of omega-conotoxin MVIIA. Antinociceptive activity evaluated in vitro and in vivo revealed differential affinity for CaV 2.2 and opioid receptors and no significant synergistic activity