Marc Gerspacher

The 7 TM G-protein-coupled receptor target family

Chemical biology approaches have a long history in the exploration of the G‐protein‐coupled receptor (GPCR) family, which represents the largest and most important group of targets for therapeutics. The analysis of the human genome revealed a significant number of new members with unknown physiological function which are today the focus of many reverse pharmacology drug‐discovery programs. As the seven hydrophobic transmembrane segments are a defining common structural feature of these receptors, and as signaling through heterotrimeric G proteins is not demonstrated in all cases, these proteins are also referred to as seven transmembrane (7 TM) or serpentine receptors. This review summarizes important historic milestones of GPCR research, from the beginning, when pharmacology was mainly descriptive, to the age of modern molecular biology, with the cloning of the first receptor and now the availability of the entire human GPCR repertoire at the sequence and protein level. It shows how GPCR‐directed drug discovery was initially based on the careful testing of a few specifically made chemical compounds and is today pursued with modern drug‐discovery approaches, including combinatorial library design, structural biology, molecular informatics, and advanced screening technologies for the identification of new compounds that activate or inhibit GPCRs specifically. Such compounds, in conjunction with other new technologies, allow us to study the role of receptors in physiology and medicine, and will hopefully result in novel therapies. We also outline how basic research on the signaling and regulatory mechanisms of GPCRs is advancing, leading to the discovery of new GPCR‐interacting proteins and thus opening new perspectives for drug development. Practical examples from GPCR expression studies, HTS (high‐throughput screening), and the design of monoamine‐related GPCR‐focused combinatorial libraries illustrate ongoing GPCR chemical biology research. Finally, we outline future progress that may relate today’s discoveries to the development of new medicines.

Potent and Selective Inhibition of Polycythemia by the Quinoxaline JAK2 Inhibitor NVP-BSK805

The recent discovery of an acquired activating point mutation in JAK2, substituting valine at amino acid position 617 for phenylalanine, has greatly improved our understanding of the molecular mechanism underlying chronic myeloproliferative neoplasms. Strikingly, the JAK2V617F mutation is found in nearly all patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia and primary myelofibrosis. Thus, JAK2 represents a promising target for the treatment of myeloproliferative neoplasms and considerable efforts are ongoing to discover and develop inhibitors of the kinase. Here, we report potent inhibition of JAK2V617F and JAK2 wild-type enzymes by a novel substituted quinoxaline, NVP-BSK805, which acts in an ATP-competitive manner. Within the JAK family, NVP-BSK805 displays more than 20-fold selectivity towards JAK2 in vitro, as well as excellent selectivity in broader kinase profiling. The compound blunts constitutive STAT5 phosphorylation in JAK2V617F-bearing cells, with concomitant suppression of cell proliferation and induction of apoptosis. In vivo, NVP-BSK805 exhibited good oral bioavailability and a long half-life. The inhibitor was efficacious in suppressing leukemic cell spreading and splenomegaly in a Ba/F3 JAK2V617F cell-driven mouse mechanistic model. Furthermore, NVP-BSK805 potently suppressed recombinant human erythropoietin-induced polycythemia and extramedullary erythropoiesis in mice and rats. Mol Cancer Ther; 9(7); 1945–55. ©2010 AACR.

Extent of salmeterol-mediated reassertion of relaxation in guinea-pig trachea pretreated with aliphatic side chain structural analogues

1 Salmeterol is a potent, selective and long acting β2‐adrenoceptor agonist. In vitro, salmeterol exerts ‘reassertion’ relaxation of airways smooth muscle. Reassertion relaxation refers to the capacity of salmeterol to cause repeated functional antagonism of induced contraction when airway smooth muscle is intermittently exposed to, then washed free from, β‐adrenoceptor antagonists such as sotalol. The mechanism(s) underlying reassertion relaxation are unknown but may relate to high affinity binding of the long aliphatic side chain of salmeterol to an accessory site, distinct from the agonist recognition site, in or near the β2‐adrenoceptor (exosite binding hypothesis). 2 In order to test the exosite hypothesis, three pure analogues of salmeterol, each exactly preserving the molecular structure of the aliphatic side chain but with zero or low efficacy at the β2‐adrenoceptor were synthesized. The effect of pre‐incubating guinea‐pig tracheal smooth muscle with these analogues on salmeterol‐induced reassertion relaxation was determined. 3 Computer Assisted Molecular Modelling of these molecules revealed that each of them exactly preserved the low energy linear conformation of the aliphatic side chain of salmeterol. Measurement of lipophilicity (octanol: water partition coefficient; log P) and direct partition into synthetic membranes (membrane partition coefficient; Kpmem) showed that all compounds had high affinity for lipids and membranes. In particular the biophysical properties of CGP 59162 (log P 1.89, Kpmem 16500) were very similar to salmeterol (log P 1.73, Kpmem 16800). 4 Two of the analogues, CGP 54103 and D 2543 (1 μm), which are structural mimics of the side chain of salmeterol, differing slightly in their length, did not prevent either the initial relaxation induced by salmeterol (0.1 μm) or the reassertion relaxation; however, it was not possible to determine whether either of these molecules occupied the β2‐adrenoceptor. 5 The third analogue, CGP 59162, which has the substituents on the active saligenin head group of salmeterol in transposed positions, itself exerted a weak β2‐adrenoceptor‐mediated relaxation antagonized by ICI 118551 (β2‐selective antagonist) but not CGP 20712 (β1‐selective antagonist) and, at higher concentrations CGP 59162 caused reassertion relaxation suggesting that it may occupy and activate the β2‐adrenoceptor in a manner analogous to salmeterol. 6 CGP 59162, at concentrations up to ten fold molar excess, did not prevent or reduce salmeterol‐induced reassertion relaxation. 7 In conclusion these data are not consistent with the existence of a distinct ‘exosite’ recognising the aliphatic side chain of salmeterol mediating reassertion.

Discovery and SAR of potent, orally available 2,8-diaryl-quinoxalines as a new class of JAK2 inhibitors.

We have designed and synthesized a novel series of 2,8-diaryl-quinoxalines as Janus kinase 2 inhibitors. Many of the inhibitors show low nanomolar activity against JAK2 and potently suppress proliferation of SET-2 cells in vitro. In addition, compounds from this series have favorable rat pharmacokinetic properties suitable for in vivo efficacy evaluation.

1-Alkyl-4-phenyl-6-alkoxy-1H-quinazolin-2-ones: a novel series of potent calcium-sensing receptor antagonists.

Parathyroid hormone (PTH) is an effective bone anabolic agent. However, only when administered by daily sc injections exposure of short duration is achieved, a prerequisite for an anabolic response. Instead of applying exogenous PTH, mobilization of endogenous stores of the hormone can be envisaged. The secretion of PTH stored in the parathyroid glands is mediated by a calcium sensing receptor (CaSR) a GPCR localized at the cell surface. Antagonists of CaSR (calcilytics) mimic a state of hypocalcaemia and stimulate PTH release to the bloodstream. Screening of the internal compound collection for inhibition of CaSR signaling function afforded 2a. In vitro potency could be improved >1000 fold by optimization of its chemical structure. The binding mode of our compounds was predicted based on molecular modeling and confirmed by testing with mutated receptors. While the compounds readily induced PTH release after iv application a special formulation was needed for oral activity. The required profile was achieved by using microemulsions. Excellent PK/PD correlation was found in rats and dogs. High levels of PTH were reached in plasma within minutes which reverted to baseline in about 1-2 h in both species.

Synthesis and SAR of a novel, potent and structurally simple LTD4 antagonist of the quinoline class.

The two geminal ethyl groups in the succinic acid moiety of CGP57698 (4-[3-(7-fluoro-2-quinolinyl-methoxy)phenyl-amino]-2,2-diethyl-4-oxo- butanoic acid) are responsible for the high in vitro and in vivo potency of this peptidoleukotriene antagonist of the quinoline type. The synthesis and structure activity relationships of CGP57698 and its analogs are described.

2-Amino-aryl-7-aryl-benzoxazoles as potent, selective and orally available JAK2 inhibitors

A series of novel benzoxazole derivatives has been designed and shown to exhibit attractive JAK2 inhibitory profiles in biochemical and cellular assays, capable of delivering compounds with favorable PK properties in rats. Synthesis and structure-activity relationship data are also provided.

Biphenyl derivatives as novel dual NK1/NK2-receptor antagonists

In a continuation of our efforts to simplify the structure of our neurokinin antagonists, a series of substituted biphenyl derivatives has been prepared. Several compounds exhibit potent affinities for both the NK(1) receptor (<10nM) and for the NK(2) receptor (<50 nM). Details on the design, synthesis, biological activities, SAR and conformational analysis of this new class of dual NK(1)/NK(2) receptor antagonists are presented.

N-[(R,R)-(E)-1-(4-chloro-benzyl)-3-(2-oxo-azepan-3-ylcarbamoyl)-allyl]-N-methyl-3,5-bis-trifluoromethyl-benzamide: an orally active neurokinin NK1/NK2 antagonist.

The stereoselective synthesis of N-[(R,R)-(E)-1-(4-chloro-benzyl)-3-(2-oxo-azepan-3-ylcarbamoyl+ ++)-allyl]-N-methyl-3,5-bis-trifluoromethyl-benzamide (4) and its NK1 and NK2 receptor binding properties are reported. In addition the potent inhibitory effects in vivo on sar9-SP- and beta-Ala-NKA-induced airway bronchoconstriction in guinea pigs are demonstrated.

Selective and combined neurokinin receptor antagonists.

Publisher Summary An exceedingly large number of structurally highly diverse potent and orally bioavailable selective neurokinins (NKs), NK1, NK2 and NK3 receptor antagonists, as well as combined neurokinin receptor antagonists are available. Based on evidence generated in numerous in vitro and animal experiments using mainly selective neurokinin antagonists great expectations have been generated for a therapeutic value of neurokinin antagonists in many central, as well as peripheral diseases. Neurokinins/tachykinins are widely distributed in the central and peripheral nervous system. Neurons are the major source of neurokinins/tachykinins, particularly in sensory, somatic, and visceral fibres with a prominent location in the peripheral endings of primary afferent capsaicin sensitive neurons.