Herman D. Lim

Molecular and biochemical pharmacology of the histamine H4 receptor

The elucidation of the human genome has had a major impact on histamine receptor research. The identification of the human H4 receptor by several groups has been instrumental for a new appreciation of the role of histamine in the modulation of immune function. In this review, we summarize the historical developments and the molecular and biochemical pharmacology of the H4 receptor.

Fragment based design of new H4 receptor-ligands with anti-inflammatory properties in vivo

Using a previously reported flexible alignment model we have designed, synthesized, and evaluated a series of compounds at the human histamine H 4 receptor (H 4R) from which 2-(4-methyl-piperazin-1-yl)-quinoxaline ( 3) was identified as a new lead structure for H 4R ligands. Exploration of the structure-activity relationship (SAR) of this scaffold led to the identification of 6,7-dichloro 3-(4-methylpiperazin-1-yl)quinoxalin-2(1 H)-one (VUF 10214, 57) and 2-benzyl-3-(4-methyl-piperazin-1-yl)quinoxaline (VUF 10148, 20) as potent H 4R ligands with nanomolar affinities. In vivo studies in the rat reveal that compound 57 has significant anti-inflammatory properties in the carrageenan-induced paw-edema model.

Cloning and characterization of dominant negative splice variants of the human histamine H4 receptor

The H(4)R (histamine H(4) receptor) is the latest identified member of the histamine receptor subfamily of GPCRs (G-protein-coupled receptors) with potential functional implications in inflammatory diseases and cancer. The H(4)R is primarily expressed in eosinophils and mast cells and has the highest homology with the H(3)R. The occurrence of at least twenty different hH(3)R (human H(3)R) isoforms led us to investigate the possible existence of H(4)R splice variants. In the present paper, we report on the cloning of the first two alternatively spliced H(4)R isoforms from CD34+ cord blood-cell-derived eosinophils and mast cells. These H(4)R splice variants are localized predominantly intracellularly when expressed recombinantly in mammalian cells. We failed to detect any ligand binding, H(4)R-ligand induced signalling or constitutive activity for these H(4)R splice variants. However, when co-expressed with full-length H(4)R [H(4)R((390)) (H(4)R isoform of 390 amino acids)], the H(4)R splice variants have a dominant negative effect on the surface expression of H(4)R((390)). We detected H(4)R((390))-H(4)R splice variant hetero-oligomers by employing both biochemical (immunoprecipitation and cell-surface labelling) and biophysical [time-resolved FRET (fluorescence resonance energy transfer)] techniques. mRNAs encoding the H(4)R splice variants were detected in various cell types and expressed at similar levels to the full-length H(4)R((390)) mRNA in, for example, pre-monocytes. We conclude that the H(4)R splice variants described here have a dominant negative effect on H(4)R((390)) functionality, as they are able to retain H(4)R((390)) intracellularly and inactivate a population of H(4)R((390)), presumably via hetero-oligomerization.

Phenylalanine 169 in the Second Extracellular Loop of the Human Histamine H4 Receptor Is Responsible for the Difference in Agonist Binding between Human and Mouse H4 Receptors

Using the natural variation in histamine H4 receptor protein sequence, we tried to identify amino acids involved in the binding of H4 receptor agonists. To this end, we constructed a variety of chimeric human-mouse H4 receptor proteins to localize the domain responsible for the observed pharmacological differences between human and mouse H4 receptors in the binding of H4 receptor agonists, such as histamine, clozapine, and VUF 8430 [S-(2-guanidylethyl)-isothiourea]. After identification of a domain between the top of transmembrane domain 4 and the top of transmembrane domain 5 as being responsible for the differences in agonist affinity between human and mouse H4Rs, detailed site-directed mutagenesis studies were performed. These studies identified Phe169 in the second extracellular loop as the single amino acid responsible for the differences in agonist affinity between the human and mouse H4Rs. Phe169 is part of a Phe-Phe motif, which is also present in the recently crystallized β2-adrenergic receptor. These results point to an important role of the second extracellular loop in the agonist binding to the H4 receptor and provide a molecular explanation for the species difference between human and mouse H4 receptors.

Several down, a few to go: histamine H3 receptor ligands making the final push towards the market?

Introduction: The histamine H3 receptor (H3R) plays a pivotal role in a plethora of therapeutic areas. Blocking the H3R with antagonists/inverse agonists has been postulated to be of broad therapeutic use. Indeed, H3R antagonists/inverse agonists have been extensively evaluated in the clinic. Areas covered: Here, we address new developments, insights obtained and challenges encountered in the clinical evaluations. For recent H3R clinical candidates, the status and results of the corresponding clinical trial(s) will be discussed along with preclinical data. Main findings: In all, it becomes evident that clinical evaluation of H3R antagonists/inverse agonists is characterized by mixed results. On one hand, Pitolisant has successfully passed several Phase II trials and seems to be the most advanced compound in the clinic now, being in Phase III. On the other hand, some compounds (e.g., PF-03654647 and MK-0249) failed at Phase II clinical level for several indications. Expert opinion: A challenging feature in H3R research is the multifaceted role of the receptor at a molecular/biochemical level, which can complicate targeting by small molecules at several (pre)clinical levels. Accordingly, H3R antagonists/inverse agonists require further testing to pinpoint the determinants for clinical efficacy and to aid in the final push towards the market.

Pharmacological characterization of the new histamine H4 receptor agonist VUF 8430

Background and purpose:  We compare the pharmacological profiles of a new histamine H4 receptor agonist 2‐(2‐guanidinoethyl)isothiourea (VUF 8430) with that of a previously described H4 receptor agonist, 4‐methylhistamine.

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.

Cloning and pharmacological characterization of the dog histamine H4 receptor.

The histamine H4 receptor has been shown to have a role in chemotaxis and mediator release in various types of immune cells and has been implicated in mediating inflammation in vivo. Previous work has shown that there were differences in the histamine H4 receptor sequence of different species and these translated into changes in the pharmacology of the receptors. To help further understand the nature of these differences, we have cloned and expressed the histamine H4 receptor of dog (Canis familiaris). The dog histamine H4 receptor has a 61-71% homology with the receptors from other species, with a 71% homology to the human receptor. The affinity for histamine at the dog histamine H4 receptor is 18 nM and is 3-fold lower than the human ortholog. A number of previously described histamine H4 receptor ligands were tested for affinity at the dog histamine H4 receptor and histamine showed the highest affinity of the ligands tested. In addition, the histamine H4 receptor selective antagonist, JNJ 7777120, had a Ki value of 50 nM and acts as an antagonist at the dog receptor. In general, agonists of the human histamine H4 receptor were also agonists of the dog receptor albeit with different efficacy levels. The cloning and in vitro pharmacological characterization of the dog histamine H4 receptor provide useful information for future studies using dog models as well as in understanding the ligand-receptor interactions of the receptor.

Clobenpropit analogs as dual activity ligands for the histamine H3 and H4 receptors: synthesis, pharmacological evaluation, and cross-target QSAR studies.

Previous studies have demonstrated that clobenpropit (N-(4-chlorobenzyl)-S-[3-(4(5)-imidazolyl)propyl]isothiourea) binds to both the human histamine H(3) receptor (H(3)R) and H(4) receptor (H(4)R). In this paper, we describe the synthesis and pharmacological characterization of a series of clobenpropit analogs, which vary in the functional group adjacent to the isothiourea moiety in order to study structural requirements for H(3)R and H(4)R ligands. The compounds show moderate to high affinity for both the human H(3)R and H(4)R. Furthermore, the changes in the functional group attached to the isothiourea moiety modulate the intrinsic activity of the ligands at the H(4)R, ranging from neutral antagonism to full agonism. QSAR models have been generated in order to explain the H(3)R and H(4)R affinities.

The emerging role of the histamine H4 receptor in anti-inflammatory therapy.

Antagonists for the Histamine H(1) receptor have been on the market for decades and continue to be successfully used in the treatment of a variety of allergic conditions. The recently discovered histamine H(4) receptor subtype is emerging as a new and complementary target for treating inflammatory conditions. In this review, we describe the receptor protein, its putative role in (patho)physiology and the latest ligands that are being developed to explore the feasibility of the H(4) receptor as a drug target.