Claus Riemer

Characterization of Ro 04‐6790 and Ro 63‐0563: potent and selective antagonists at human and rat 5‐HT6 receptors

This study describes the in vitro characterization of two potent and selective 5‐HT6 receptor antagonists at the rat and human recombinant 5‐HT6 receptor. In binding assays with [3H]‐LSD, 4‐amino‐N‐(2,6 bis‐methylamino‐pyrimidin‐4‐yl)‐benzene sulphonamide (Ro 04‐6790) and 4‐amino‐N‐(2,6 bis‐methylamino‐pyridin‐4‐yl)‐benzene sulphonamide (Ro 63‐0563) had mean pKi values ±s.e.mean at the rat 5‐HT6 receptor of 7.35±0.04 and 7.83±0.01, respectively and pKi values at the human 5‐HT6 receptor of 7.26±0.06 and 7.91±0.02, respectively. Both compounds were found to be over 100 fold selective for the 5‐HT6 receptor compared to 23 (Ro 04‐6790) and 69 (Ro 63‐0563) other receptor binding sites. In functional studies, neither compound had any significant effect on basal levels of cyclicAMP accumulation in Hela cells stably expressing the human 5‐HT6 receptor, suggesting that the compounds are neither agonists nor inverse agonists at the 5‐HT6 receptor. However, both Ro 04‐6790 and Ro 63‐0563 behaved as competitive antagonists with mean ±s.e.mean pA2 values of 6.75±0.07 and 7.10±0.09, respectively. In rats habituated to observation cages, Ro 04‐6790 produced a behavioural syndrome similar to that seen following treatment with antisense oligonucleotides designed to reduce the expression of 5‐HT6 receptors. This behavioural syndrome consisted of stretching, yawning and chewing. Ro 04‐6790 and Ro 63‐0563 represent valuable pharmacological tools for the identification of 5‐HT6 receptors in natural tissues and the study of their physiological function.

NK3 receptor antagonists: the next generation of antipsychotics?

Although current antipsychotic drugs are effective at treating the psychotic (positive) symptoms of schizophrenia, they have one or more serious side effects, including extrapyramidal symptoms, weight gain, cardiovascular liabilities and type II diabetes. However, recent data from clinical trials of selective neurokinin 3 (NK3) receptor antagonists in schizophrenia ? osanetant and talnetant ? have shown significant improvement in positive symptoms, with no major side-effects reported as yet. Here we discuss the preclinical and clinical evidence that indicates that NK3 receptor antagonists might represent a new approach to the treatment of schizophrenia and possibly other neuropsychiatric disorders.

5-HT6 receptor antagonists: lead optimisation and biological evaluation of N-aryl and N-heteroaryl 4-amino-benzene sulfonamides

RO-04-6790 (6a) has been identified in a random screen for 5-HT(6) receptor antagonists. In a medicinal chemistry optimisation program a series of analogs comprising N-heteroaryl- and N-arylbenzenesulfonamides have been synthesised and investigated for their binding affinity. Compounds with a logD profile indicative of brain penetration have been subjected to in vivo testing for reversal of a scopolamine-induced retention deficit in a passive avoidance paradigm.

Me-Talnetant and Osanetant Interact within Overlapping but Not Identical Binding Pockets in the Human Tachykinin Neurokinin 3 Receptor Transmembrane Domains

Recent clinical trials have indicated that neurokinin 3 receptor antagonists (S)-(+)-N-{{3-[1-benzoyl-3-(3,4-dichlorophenyl)-piperidin-3-yl]prop-1-yl}-4-phenylpiperidin-4-yl}-N-methylacetamine (SR142801; osanetant) and (S)-(-)-N-(α-ethylbenzyl)-3-hydroxy-2-phenylquinoline-4-carboxamide (SB223412; talnetant) may treat symptoms of schizophrenia. Using site-directed mutagenesis, rhodopsin-based modeling, [3H](S)-(-)-N-(α-ethylbenzyl)-3-methoxy-2-phenylquinoline-4-carboxamide (Me-talnetant) and [3H]osanetant binding, and functional Schild analyses, we have demonstrated the important molecular determinants of neurokinin B (NKB), Me-talnetant, and osanetant binding pockets. The residues Asn1382.57, Asn1422.61, Leu23245.49, Tyr3156.51, Phe3427.39, and Met3467.43 were found to be crucial for the NKB binding site. We observed that the M1342.53A, V1693.36M, F3427.39M, and S3417.38I/F3427.39M mutations resulted in the complete loss of [3H]Metalnetant and [3H]osanetant binding affinities and also abolished their functional potencies in an NKB-evoked accumulation of [3H]inositol phosphates assay, whereas the mutations V951.42A, N1422.61A, Y3156.51F, and M3467.43A behaved differently between the interacting modes of two antagonists. V951.42A and M3467.43A significantly decreased the affinity and potency of Me-talnetant. Y3156.51F, although not affecting Me-talnetant, led to a significant decrease in affinity and potency of osanetant. The mutation N1422.61A, which abolished the potency and affinity of osanetant, led to a significant increase in the affinity and potency of Me-talnetant. The proposed docking mode was further validated using (S)-2-(3,5-bis-trifluoromethyl-phenyl)-N-[4-(4-fluoro-2-methyl-phenyl)-6-((S)-4-methanesulfonyl-3-methyl-piperazin-1-yl)-pyridin-3-yl]-N-methyl-isobutyramide (RO49085940), from another chemical class. It is noteworthy that the mutation F3427.39A caused an 80-fold gain of RO4908594 binding affinity, but the same mutation resulted in the complete loss of the affinity of Me-talnetant and partial loss of the affinity of osanetant. These observations show that the binding pocket of Me-talnetant and osanetant are overlapping, but not identical. Taken together, our data are consistent with the proposed docking modes where Me-talnetant reaches deeply into the pocket formed by transmembrane (TM)1, -2, and -7, whereas osanetant fills the pocket TM3, -5, and -6 with its phenyl-piperidine moiety.

Discovery of Fluoromethylketone-Based Peptidomimetics as Covalent ATG4B (Autophagin-1) Inhibitors

ATG4B or autophagin-1 is a cysteine protease that cleaves ATG8 family proteins. ATG4B plays essential roles in the autophagosome formation and the autophagy pathway. Herein we disclose the design and structural modifications of a series of fluoromethylketone (FMK)-based peptidomimetics as highly potent ATG4B inhibitors. Their structure-activity relationship (SAR) and protease selectivity are also discussed.

Identification of New ATG4B Inhibitors Based on a Novel High-Throughput Screening Platform

Autophagy is an evolutionarily conserved homeostasis process through which aggregated proteins or damaged organelles are enveloped in a double-membrane structure called an autophagosome and then digested in a lysosome-dependent manner. Growing evidence suggests that malfunction of autophagy contributes to the pathogenesis of a variety of diseases, including cancer, viral infection, and neurodegeneration. However, autophagy is a complicated process, and understanding of the relevance of autophagy to disease is limited by lack of specific and potent autophagy modulators. ATG4B, a Cys-protease that cleaves ATG8 family proteins, such as LC3B, is a key protein in autophagosome formation and maturation process. A novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay measuring protease activity of ATG4B was developed, validated, and adapted into a high-throughput screening (HTS) format. HTS was then conducted with a Roche focus library of 57,000 compounds. After hit confirmation and a counterscreen to filter out fluorescence interference compounds, 267 hits were confirmed, constituting a hit rate of 0.49%. Furthermore, among 65 hits with an IC50 < 50 µM, one compound mimics the LC3 peptide substrate (-TFG-). Chemistry modification based on this particular hit gave preliminary structure activity relationship (SAR) resulting in a compound with a 10-fold increase in potency. This compound forms a stable covalent bond with Cys74 of ATG4B in a 1:1 ratio as demonstrated by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Furthermore, this compound displayed cellular ATG4B inhibition activity. Overall, the novel TR-FRET ATG4B protease assay plus counterscreen assay provides a robust platform to identify ATG4B inhibitors, which would help to elucidate the mechanism of the autophagy pathway and offer opportunities for drug discovery.

Rational design of novel pyrrolidine derivatives as orally active neurokinin-3 receptor antagonists.

The rational design of a novel series of pyrrolidine derivatives as neurokinin-3 receptor antagonists is reported starting from a selective neurokinin-1 receptor antagonist. Typical representatives in this series showed in vivo efficacy after oral administration in a NK3 mediated functional assay. This series of NK3 antagonists shows promise to deliver a novel antipsychotic.