Henk Timmerman

The histamine H3 receptor: from gene cloning to H3 receptor drugs

Since the cloning of the histamine H3 receptor cDNA in 1999 by Lovenberg and co-workers, this histamine receptor has gained the interest of many pharmaceutical companies as a potential drug target for the treatment of various important disorders, including obesity, attention-deficit hyperactivity disorder, Alzheimers disease, schizophrenia, as well as for myocardial ischaemia, migraine and inflammatory diseases. Here, we discuss relevant information on this target protein and describe the development of various H3 receptor agonists and antagonists, and their effects in preclinical animal models.

Agonist-independent regulation of constitutively active G-protein-coupled receptors

G-protein-coupled receptors constitute one of the largest protein super-families in mammals. Since the cloning of the encoding genes, these important drug targets have been subjected to thorough biochemical and pharmacological studies. It has become clear that G-protein-coupled receptors not only transmit signals after stimulation by agonists but can also spontaneously couple to signal-transduction pathways. Recent findings show that constitutively active G-protein-coupled receptors can also be regulated in an agonist-independent manner, which has important implications for the interpretation of the actions of (inverse) agonists and the results of site-directed-mutagenesis studies.

Constitutive activity of the histamine H1 receptor reveals inverse agonism of histamine H1 receptor antagonists

Transient expression of the wild-type human histamine H(1) receptor in SV40-immortalised African green monkey kidney cells resulted in an agonist-independent elevation of the basal levels of the second messenger inositoltrisphospate. Several histamine H(1) receptor antagonists, including the therapeutically used anti-allergics cetirizine, loratadine and epinastine reduced this constitutive histamine H(1) receptor activity. Inverse agonism, i.e., stabilisation of an inactive conformation of the human histamine H(1) receptor, may therefore be a key component of the anti-allergic mechanism of action of clinically used antihistamines.

Selective ligands as tools to study histamine receptors

In this review the histaminergic ligands for the histamine H(1), H(2) and H(3) receptors, which are currently used as tools in pharmacological studies, are described. To study interactions with the histamine H(1) receptor, the H(1) agonist 2-aminoethylthiazole has long since been used. However, during the last decade, 2-phenylhistamine derivatives emerged with interesting binding features. So far no radiolabelled selective H(1) agonist has been commonly used. As H(1) antagonists mepyramine, triprolidine and chlorpheniramine are described together with radiolabelled H(1) antagonists [3H]mepyramine and [3H]doxepin. Special attention has been paid to the PET ligands [11C]doxepin and [11C]mepyramine and the [125I] labelled antagonists [125I]iodobolpyramine and [125I]iodoazidophenpyramine. Concerning H(2) agonists, especially dimaprit, amthamine and impromidine are discussed. There are several H(2) antagonists; amongst them cimetidine, tiotidine and ranitidine are used most frequently. Many of these antagonists behave as inverse agonists. As radiolabelled H(2) antagonists, [3H]cimetidine, [3H]tiotidine, [125I]iodoaminopotentidine and [125I]iodoazidopotentidine are included. Commonly used histamine H(3) agonists are N(alpha)-methylhistamine, (R)alpha-methylhistamine, imetit and immepip. Both methylhistamines are also available as [3H] labelled ligands. As reference compounds, often used H(3) antagonists are thioperamide, clobenpropit, iodophenpropit and impentamine. Most important radiolabelled H(3) antagonists are S-[3H]methylthioperamide, [3H]thioperamide, [125I]iodophenpropit and [125I]iodoproxyfan. The use of all these compounds as a tool in pharmacology is also discussed.

Kaposi's sarcoma-associated herpesvirus-encoded G protein-coupled receptor ORF74 constitutively activates p44/p42 MAPK and Akt via G(i) and phospholipase C-dependent signaling pathways

ABSTRACT The G protein-coupled receptor encoded by Kaposis sarcoma-associated herpesvirus, also referred to as ORF74, has been shown to stimulate oncogenic and angiogenic signaling pathways in a constitutively active manner. The biochemical routes linking ORF74 to these signaling pathways are poorly defined. In this study, we show that ORF74 constitutively activates p44/p42 mitogen-activated protein kinase (MAPK) and Akt via Gi- and phospholipase C (PLC)-mediated signaling pathways. Activation of Akt by ORF74 appears to be phosphatidylinositol 3-kinase (PI3-K) dependent but, interestingly, is also mediated by activation of protein kinase C (PKC) and p44/p42 MAPK. ORF74 may signal to Akt via p44/p42 MAPK, which can be activated by Gi, through activation of PI3-K or through PKC via the PLC pathway. Signaling of ORF74 to these proliferative and antiapoptotic signaling pathways can be further modulated positively by growth-related oncogene (GROα/CXCL1) and negatively by human gamma interferon-inducible protein 10 (IP-10/CXCL10), thus acting as an agonist and an inverse agonist, respectively. Despite the ability of the cytomegalovirus-encoded chemokine receptor US28 to constitutively activate PLC, this receptor does not increase phosphorylation of p44/p42 MAPK or Akt in COS-7 cells. Hence, ORF74 appears to signal through a larger diversity of G proteins than US28, allowing it to couple to proliferative and antiapoptotic signaling pathways. ORF74 can therefore be envisioned as an attractive target for novel treatment of Kaposis sarcoma.

New Insights into the Second Generation Antihistamines.

Second generation antihistamines are recognised as being highly effective treatments for allergy-based disease and are among the most frequently prescribed and safest drugs in the world. However, consideration of the therapeutic index or the benefit/risk ratio of the H1 receptor antagonists is of paramount importance when prescribing this class of compounds as they are used to treat non-life threatening conditions. There are many second generation antihistamines available and at first examination these appear to be comparable in terms of safety and efficacy. However, the newer antihistamines in fact represent ahererogeneous group of compounds, having markedly differing chemical structures, adverse effects, half-life, tissue distribution and metabolism, spectrum of antihistaminic properties, and varying degrees of anti-inflammatory effects. With regard to the latter, there is growing awareness that some of these compounds might represent useful adjunct medications in asthma therapy. In terms of safety issues, the current second generation grouping includes compounds with proven cardiotoxic effects and others with the potential for adverse drug interactions. Moreover, some of the second generation H1 antagonists have given cause for concern regarding their potential to cause a degree of somnolence in some individuals. It can be argued, therefore, that the present second generation grouping is too large and indistinct since this was based primarily on the concept of separating the first generation sedating compounds from nonsedating H1 antagonists. Although it is too early to talk about a third generation grouping of antihistamines, future membership of such a classification could be based on a low volume of distribution coupled with a lack of sedating effects, drug interactions and cardiotoxicity.

Identification of the First Nonpeptidergic Inverse Agonist for a Constitutively Active Viral-encoded G Protein-coupled Receptor

Human cytomegalovirus (HCMV) encodes a G protein-coupled receptor (GPCR), named US28, which shows homology to chemokine receptors and binds several chemokines with high affinity. US28 induces migration of smooth muscle cells, a feature essential for the development of atherosclerosis, and may serve as a co-receptor for human immunodeficiency virus-type 1 entry into cells. Previously, we have shown that HCMV-encoded US28 displays constitutive activity, whereas its mammalian homologs do not. In this study we have identified a small nonpeptidergic molecule (VUF2274) that inhibits US28-mediated phospholipase C activation in transiently transfected COS-7 cells and in HCMV-infected fibroblasts. Moreover, VUF2274 inhibits US28-mediated HIV entry into cells. In addition, VUF2274 fully displaces radiolabeled RANTES (regulated on activation normal T cell expressed and secreted) binding at US28, apparently with a noncompetitive behavior. Different analogues of VUF2274 have been synthesized and pharmacologically characterized, to understand which features are important for its inverse agonistic activity. Finally, by means of mutational analysis of US28, we have identified a glutamic acid in transmembrane 7 (TM 7), which is highly conserved among chemokine receptors, as a critical residue for VUF2274 binding to US28. The identification of a full inverse agonist provides an important tool to investigate the relevance of US28 constitutive activity in viral pathogenesis.

Synthesis of benzylideneacetophenones and their inhibition of lipid peroxidation

A series of benzylideneacetophenone derivatives have been synthesized and found to show potent inhibition of the lipid peroxidation (LPO) in rat liver microsomes. All 19 compounds prepared in this series are LPO inhibitors. The highest activity was found in para hydroxy derivatives with two meta tert-butyl substituents.

Constitutive Activity and Structural Instability of the Wild‐Type Human H2 Receptor

Abstract: Stable expression of the human H2 receptor in Chinese hamster ovary cells resulted in an increase in basal cyclic AMP (cAMP) production, which was inhibited by the inverse agonists cimetidine, famotidine, and ranitidine with potencies similar to those found for the rat H2 receptor. Burimamide, a neutral antagonist at the rat H2 receptor, behaved as a weak partial agonist at the human H2 receptor. Burimamide competitively antagonized both the histamine‐induced increase in cAMP and the cimetidine‐induced reduction of the basal cAMP level with apparent KB values that were similar to its H2 receptor affinity. Investigation of the modulation of receptor expression after long‐term drug treatment revealed that at low concentrations histamine induced a significant reduction in H2 receptor expression, whereas at high concentrations receptor expression was slightly increased. The partial agonist burimamide induced, like inverse agonists, an upregulation of the human H2 receptor after prolonged treatment. These findings suggest a structural instability of the constitutively active human H2 receptor in transfected Chinese hamster ovary cells. Occupation of the H2 receptor by any ligand reduces the instability, thus resulting in higher cellular expression levels.

4-Hydroxy-2,3-trans-nonenal stimulates microsomal lipid peroxidation by reducing the glutathione-dependent protection

Glutathione (GSH) protects liver microsomes against lipid peroxidation. This is probably due to the reduction of vitamin E radicals by GSH, a reaction catalyzed by a membrane-bound protein. Pretreatment of liver microsomes with 0.1 or 1mM 4-hydroxy-2,3-trans-nonenal (HNE), a major product of lipid peroxidation, reduces the GSH-dependent protection. GSH and vitamin E concentrations are not affected by this pretreatment. Pretreatment with 0.1 mM N-ethyl maleimide (NEM), a synthetic sulfhydryl reagent, resulted in a reduction similar to that with HNE of the GSH-dependent protection against lipid peroxidation. The reduction of the GSH-dependent protection by HNE and NEM is probably the result of inactivation of the membrane-bound protein by covalent binding to an essential SH group on the protein. If the GSH-dependent protection would proceed via the microsomal GSH transferase, pretreatment with NEM, which activates the microsomal GSH transferase, should enhance the GSH-dependent protection. Actually a decrease in the GSH-dependent protection is found. Apparently the GSH-dependent protection does not proceed via the microsomal GSH transferase. Also the microsomal phospholipase A2 is not involved, since addition of 0.1 mM mepacrine, an inhibitor of phospholipase A2, did not preclude the GSH-dependent protection. Once the process of lipid peroxidation, either in vivo or in vitro, has started, the protection of liver microsomes by GSH is less effective. This might be the result of formed HNE. In this way an endproduct of lipid peroxidation stimulates the process that generates this product.