C. de Graaf

Pharmacological modulation of chemokine receptor function

G protein‐coupled chemokine receptors and their peptidergic ligands are interesting therapeutic targets due to their involvement in various immune‐related diseases, including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, chronic obstructive pulmonary disease, HIV‐1 infection and cancer. To tackle these diseases, a lot of effort has been focused on discovery and development of small‐molecule chemokine receptor antagonists. This has been rewarded by the market approval of two novel chemokine receptor inhibitors, AMD3100 (CXCR4) and Maraviroc (CCR5) for stem cell mobilization and treatment of HIV‐1 infection respectively. The recent GPCR crystal structures together with mutagenesis and pharmacological studies have aided in understanding how small‐molecule ligands interact with chemokine receptors. Many of these ligands display behaviour deviating from simple competition and do not interact with the chemokine binding site, providing evidence for an allosteric mode of action. This review aims to give an overview of the evidence supporting modulation of this intriguing receptor family by a range of ligands, including small molecules, peptides and antibodies. Moreover, the computer‐assisted modelling of chemokine receptor–ligand interactions is discussed in view of GPCR crystal structures. Finally, the implications of concepts such as functional selectivity and chemokine receptor dimerization are considered.

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.

From heptahelical bundle to hits from the Haystack: structure-based virtual screening for GPCR ligands.

This review will focus on the construction, refinement, and validation of G-protein-coupled receptor (GPCR) structural models for the purpose of structure-based virtual screening (SBVS) and ligand design. The review will present a comparative analysis of GPCR crystal structures and their implication on GPCR (homology) modeling. The challenges associated with steps along the modeling workflow will be discussed: the use of experimental anchors to steer the modeling procedure, amino acid sequence alignment and template selection, receptor structure refinement, loop modeling, ligand-binding mode prediction, and virtual screening for novel ligands. An overview of several successful structure-based ligand discovery and design studies shows that receptor models, despite structural inaccuracies, can be efficiently used to find novel ligands for GPCRs. Moreover, the recently solved GPCR crystal structures have further increased the opportunities in structure-based ligand discovery for this pharmaceutically important protein family.

A structural chemogenomics analysis of aminergic GPCRs: lessons for histamine receptor ligand design

Chemogenomics focuses on the discovery of new connections between chemical and biological space leading to the discovery of new protein targets and biologically active molecules. G‐protein coupled receptors (GPCRs) are a particularly interesting protein family for chemogenomics studies because there is an overwhelming amount of ligand binding affinity data available. The increasing number of aminergic GPCR crystal structures now for the first time allows the integration of chemogenomics studies with high‐resolution structural analyses of GPCR‐ligand complexes.

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.

Detailed analysis of biased histamine H4 receptor signalling by JNJ 7777120 analogues

The histamine H4 receptor, originally thought to signal merely through Gαi proteins, has recently been shown to also recruit and signal via β‐arrestin2. Following the discovery that the reference antagonist indolecarboxamide JNJ 7777120 appears to be a partial agonist in β‐arrestin2 recruitment, we have identified additional biased hH4R ligands that preferentially couple to Gαi or β‐arrestin2 proteins. In this study, we explored ligand and receptor regions that are important for biased hH4R signalling.

A molecular pharmacologist's guide to G protein-coupled receptor crystallography

G protein–coupled receptor (GPCR) structural biology has progressed dramatically in the last decade. There are now over 120 GPCR crystal structures deposited in the Protein Data Bank of 32 different receptors from families scattered across the phylogenetic tree, including class B, C, and Frizzled GPCRs. These structures have been obtained in combination with a wide variety of ligands and captured in a range of conformational states. This surge in structural knowledge has enlightened research into the molecular recognition of biologically active molecules, the mechanisms of receptor activation, the dynamics of functional selectivity, and fueled structure-based drug design efforts for GPCRs. Here we summarize the innovations in both protein engineering/molecular biology and crystallography techniques that have led to these advances in GPCR structural biology and discuss how they may influence the resulting structural models. We also provide a brief molecular pharmacologist’s guide to GPCR X-ray crystallography, outlining some key aspects in the process of structure determination, with the goal to encourage noncrystallographers to interrogate structures at the molecular level. Finally, we show how chemogenomics approaches can be used to marry the wealth of existing receptor pharmacology data with the expanding repertoire of structures, providing a deeper understanding of the mechanistic details of GPCR function.

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.

Identification of Overlapping but Differential Binding Sites for the High-Affinity CXCR3 Antagonists NBI-74330 and VUF11211

CXC chemokine receptor CXCR3 and/or its main three ligands CXCL9, CXCL10, and CXCL11 are highly upregulated in a variety of diseases. As such, considerable efforts have been made to develop small-molecule receptor CXCR3 antagonists, yielding distinct chemical classes of antagonists blocking binding and/or function of CXCR3 chemokines. Although it is suggested that these compounds bind in an allosteric fashion, thus far no evidence has been provided regarding the molecular details of their interaction with CXCR3. Using site-directed mutagenesis complemented with in silico homology modeling, we report the binding modes of two high-affinity CXCR3 antagonists of distinct chemotypes: VUF11211 [(S)-5-chloro-6-(4-(1-(4-chlorobenzyl)piperidin-4-yl)-3-ethylpiperazin-1-yl)-N-ethylnicotinamide] (piperazinyl-piperidine) with a rigid elongated structure containing two basic groups and NBI-74330 [(R)-N-(1-(3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl)ethyl)-2-(4-fluoro-3-(trifluoromethyl)phenyl)-N-(pyridin-3-ylmethyl)acetamide] (8-azaquinazolinone) without any basic group. Here we show that NBI-74330 is anchored in the transmembrane minor pocket lined by helices 2 (W2.60, D2.63), 3 (F3.32), and 7 (S7.39, Y7.43), whereas VUF11211 extends from the minor pocket into the major pocket of the transmembrane domains, located between residues in helices 1 (Y1.39), 2 (W2.60), 3 (F3.32), 4 (D4.60), 6 (Y6.51), and 7 (S7.39, Y7.43). Mutation of these residues did not affect CXCL11 binding significantly, confirming the allosteric nature of the interaction of these small molecules with CXCR3. Moreover, the model derived from our in silico–guided studies fits well with the already published structure–activity relationship data on these ligands. Altogether, in this study, we show overlapping, yet different binding sites for two high-affinity CXCR3 antagonists, which offer new opportunities for the structure-based design of allosteric modulators for CXCR3.

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.