R. Leurs

Histamine H3 receptor ligands break ground in a remarkable plethora of therapeutic areas

The neurotransmitter histamine exerts its action through four distinct histamine receptors. The histamine H1 and H2 receptor are well established drug targets, whereas the histamine H4 receptor is undergoing rigorous characterisation at present. The histamine H3 receptor (H3R) is a Gi/o-protein coupled receptor and is mostly expressed in the CNS. A remarkably large and different array of therapeutic areas, in which ligands for the H3R may prove useful, has been identified and a massive research undertaking is underway to substantiate the high expectations for H3R ligands. At present, several ligands for the H3R are being evaluated in clinical studies. In this review, the many potential therapeutic areas for H3R antagonists, inverse agonists and agonists is discussed. Promising medicinal chemistry and toxicological developments, as well as the advancement of several H3R ligands into the clinic, will be highlighted. This review also describes the problems that have been overcome and the questions that remain in developing H3R-related drugs. Considering the tremendous efforts by industry, it can be expected that the first H3R drugs will reach the market soon.

Fragment growing induces conformational changes in acetylcholine-binding protein: A structural and thermodynamic analysis

Optimization of fragment hits toward high-affinity lead compounds is a crucial aspect of fragment-based drug discovery (FBDD). In the current study, we have successfully optimized a fragment by growing into a ligand-inducible subpocket of the binding site of acetylcholine-binding protein (AChBP). This protein is a soluble homologue of the ligand binding domain (LBD) of Cys-loop receptors. The fragment optimization was monitored with X-ray structures of ligand complexes and systematic thermodynamic analyses using surface plasmon resonance (SPR) biosensor analysis and isothermal titration calorimetry (ITC). Using site-directed mutagenesis and AChBP from different species, we find that specific changes in thermodynamic binding profiles, are indicative of interactions with the ligand-inducible subpocket of AChBP. This study illustrates that thermodynamic analysis provides valuable information on ligand binding modes and is complementary to affinity data when guiding rational structure- and fragment-based discovery approaches.

Molecular Determinants of Ligand Binding to H4R Species Variants

The histamine H4 receptor (H4R) is the latest identified histamine receptor to emerge as a potential drug target for inflammatory diseases. Animal models are employed to validate this potential drug target. Concomitantly, various H4R orthologs have been cloned, including the human, mouse, rat, guinea pig, monkey, pig, and dog H4Rs. In this article, we expressed all these H4R orthologs in human embryonic kidney 293T cells and compared their interactions with currently used standard H4R ligands, including the H4R agonists histamine, 4-methylhistamine, guanidinylethyl isothiourea (VUF 8430), the H4R antagonists 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine (JNJ 7777120) and [(5-chloro-1H-benzimidazol-2-yl)carbonyl]-4-methylpiperazine (VUF 6002), and the inverse H4R agonist thioperamide. Most of the evaluated ligands display significantly different affinities at the different H4R orthologs. These “natural mutants” of H4R were used to study ligand-receptor interactions by using chimeric human-pig-human and pig-human-pig H4R proteins and site-directed mutagenesis. Our results are a useful reference for ligand selection for studies in animal models of diseases and offer new insights in the understanding of H4R-ligand receptor interactions.

Molecular determinants of ligand binding modes in the histamine H4 receptor: Linking ligand-based 3D-QSAR models to in silico guided receptor mutagenesis studies

The histamine H(4) receptor (H(4)R) is a G protein-coupled receptor (GPCR) that plays an important role in inflammation. Similar to the homologous histamine H(3) receptor (H(3)R), two acidic residues in the H(4)R binding pocket, D(3.32) and E(5.46), act as essential hydrogen bond acceptors of positively ionizable hydrogen bond donors in H(4)R ligands. Given the symmetric distribution of these complementary pharmacophore features in H(4)R and its ligands, different alternative ligand binding mode hypotheses have been proposed. The current study focuses on the elucidation of the molecular determinants of H(4)R-ligand binding modes by combining (3D) quantitative structure-activity relationship (QSAR), protein homology modeling, molecular dynamics simulations, and site-directed mutagenesis studies. We have designed and synthesized a series of clobenpropit (N-(4-chlorobenzyl)-S-[3-(4(5)-imidazolyl)propyl]isothiourea) derivatives to investigate H(4)R-ligand interactions and ligand binding orientations. Interestingly, our studies indicate that clobenpropit (2) itself can bind to H(4)R in two distinct binding modes, while the addition of a cyclohexyl group to the clobenpropit isothiourea moiety allows VUF5228 (5) to adopt only one specific binding mode in the H(4)R binding pocket. Our ligand-steered, experimentally supported protein modeling method gives new insights into ligand recognition by H(4)R and can be used as a general approach to elucidate the structure of protein-ligand complexes.

Nonpeptidergic allosteric antagonists differentially bind to the CXCR2 chemokine receptor.

The chemokine receptor CXCR2 is involved in different inflammatory diseases, like chronic obstructive pulmonary disease, psoriasis, rheumatoid arthritis, and ulcerative colitis; therefore, it is considered an attractive drug target. Different classes of small CXCR2 antagonists have been developed. In this study, we selected seven CXCR2 antagonists from the diarylurea, imidazolylpyrimide, and thiazolopyrimidine class and studied their mechanisms of action at human CXCR2. All compounds are able to displace 125I-CXCL8 and inhibit CXCL8-induced β-arrestin2 recruitment. Detailed studies with representatives of each class showed that these compounds displace and antagonize CXCL8, most probably via a noncompetitive, allosteric mechanism. In addition, we radiolabeled the high-affinity CXCR2 antagonist SB265610 [1-(2-bromophenyl)-3-(4-cyano-1H-benzo[d] [1,2,3]-triazol-7-yl)urea] and subjected [3H]SB265610 to a detailed analysis. The binding of this radioligand was saturable and reversible. Using [3H]SB265610, we found that compounds of the different chemical classes bind to distinct binding sites. Hence, the use of a radiolabeled low-molecular weight CXCR2 antagonist serves as a tool to investigate the different binding sites of CXCR2 antagonists in more detail.

Identification of a Novel Allosteric Binding Site in the CXCR2 Chemokine Receptor

We have shown previously that different chemical classes of small-molecule antagonists of the human chemokine CXCR2 receptor interact with distinct binding sites of the receptor. Although an intracellular binding site for diarylurea CXCR2 antagonists, such as N-(2-bromophenyl)-N′-(7-cyano-1H-benzotriazol-4-yl)urea (SB265610), and thiazolopyrimidine compounds was recently mapped by mutagenesis studies, we now report on an imidazolylpyrimidine antagonist binding pocket in the transmembrane domain of CXCR2. Using different CXCR2 orthologs, chimeric proteins, site-directed mutagenesis, and in silico modeling, we have elucidated the binding mode of this antagonist. Our in silico-guided mutagenesis studies indicate that the ligand binding cavity for imidazolylpyrimidine compounds in CXCR2 is located between transmembrane (TM) helices 3 (Phe1303.36), 5 (Ser2175.44, Phe2205.47), and 6 (Asn2686.52, Leu2716.55) and suggest that these antagonists enter CXCR2 via the TM5-TM6 interface. It is noteworthy that the same interface is postulated as the ligand entry channel in the opsin receptor and is occupied by lipid molecules in the recently solved crystal structure of the CXCR4 chemokine receptor, suggesting a general ligand entrance mechanism for nonpolar ligands to G protein-coupled receptors. The identification of a novel allosteric binding cavity in the TM domain of CXCR2, in addition to the previously identified intracellular binding site, shows the diversity in ligand recognition mechanisms by this receptor and offers new opportunities for the structure-based design of small allosteric modulators of CXCR2 in the future.

Molecular interaction fingerprint approaches for GPCR drug discovery

Protein-ligand interaction fingerprints (IFPs) are binary 1D representations of the 3D structure of protein-ligand complexes encoding the presence or absence of specific interactions between the binding pocket amino acids and the ligand. Various implementations of IFPs have been developed and successfully applied for post-processing molecular docking results for G Protein-Coupled Receptor (GPCR) ligand binding mode prediction and virtual ligand screening. Novel interaction fingerprint methods enable structural chemogenomics and polypharmacology predictions by complementing the increasing amount of GPCR structural data. Machine learning methods are increasingly used to derive relationships between bioactivity data and fingerprint descriptors of chemical and structural information of binding sites, ligands, and protein-ligand interactions. Factors that influence the application of IFPs include structure preparation, binding site definition, fingerprint similarity assessment, and data processing and these factors pose challenges as well possibilities to optimize interaction fingerprint methods for GPCR drug discovery.

Molecular pharmacology of histamine H4 receptors.

The histamine H4 receptor (H4R) is the youngest member of the histamine receptor family. Based on its predominant expression pattern in hematopoietic cells, the H4R is considered to be an interesting drug target for inflammatory disorders such as allergy and asthma. Since the identification and cloning of the H4R in 2000, drug discovery programs boosted the development of various H4R (specific) ligands. Differences between H4R orthologs in combination with available three-dimensional G protein-coupled receptor (GPCR) models have guided site-directed mutagenesis studies to gain insight in ligand binding and receptor activation. In addition, ongoing characterization of H4R-mediated signaling in transfected and native cells contributes to further unravel the (patho-) physiological functions of H4Rs.

Design and pharmacological characterization of VUF14480, a covalent partial agonist that interacts with cysteine 983.36 of the human histamine H4 receptor

The recently proposed binding mode of 2‐aminopyrimidines to the human (h) histamine H4 receptor suggests that the 2‐amino group of these ligands interacts with glutamic acid residue E1825.46 in the transmembrane (TM) helix 5 of this receptor. Interestingly, substituents at the 2‐position of this pyrimidine are also in close proximity to the cysteine residue C983.36 in TM3. We hypothesized that an ethenyl group at this position will form a covalent bond with C983.36 by functioning as a Michael acceptor. A covalent pyrimidine analogue will not only prove this proposed binding mode, but will also provide a valuable tool for H4 receptor research.

Virus-encoded G-protein-coupled receptors : constitutively active (dys)regulators of cell function and their potential as drug target

G-protein-coupled receptors encoded by herpesviruses such as EBV, HCMV and KSHV are very interesting illustrations of the (patho)physiological importance of constitutive GPCR activity. These viral proteins are expressed on the cell surface of infected cells and often constitutively activate a variety of G-proteins. For some virus-encoded GPCRs, the constitutive activity has been shown to occur in vivo, i.e., in infected cells. In this paper, we will review the occurrence of virus-encoded GPCRs and describe their known signaling properties. Moreover, we will also review the efforts, directed towards the discovery of small molecule antagonist, that so far have been mainly focused on the HCMV-encoded GPCR US28. This virus-encoded receptor might be involved in cardiovascular diseases and cancer and seems an interesting target for drug intervention.