Lactam and oxazolidinone derived potent 5-hydroxytryptamine 6 receptor antagonists.

Lactam and oxazolidinone derived potent 5-hydroxytryptamine 6 (5-HT6) receptor antagonists have been disclosed. One potent member from the lactam series, racemic compound 14 (Ki of 2.6 nM in binding assay, IC50 of 15 nM in functional cAMP antagonism assay) was separated into corresponding enantiomers that displayed the effect of chirality on binding potency (Ki of 1.6 nM and 3000 nM, respectively). The potent enantiomer displayed an IC50 of 8 nM in cAMP antagonism assay, selectivity against a number of family members as well as brain permeability in rats after 6h post oral administration.

In search of potent 5-HT6 receptor inverse agonists.

A series of non-sulfonamide/non-sulfone derived potent 5-HT6 receptor inverse agonists has been disclosed. Representative compound 9 (Ki  = 14 nm) displayed selectivity against a set of family members as well as brain permeability 6 h post-oral administration. In addition, the separated enantiomers of compound 9 displayed difference in activity indicating the influence of chirality on potency.

1-Thia-4,7-diaza-spiro[4.4]nonane-3,6-dione: a structural motif for 5-hydroxytryptamine 6 receptor antagonism.

A series of potent 5-hydroxytryptamine 6 (5-HT6) receptor antagonists based on 1-thia-4,7-diaza-spiro[4.4]nonane-3,6-dione motif has been disclosed. Enantiomers of potent racemate compound 8a (K(i) = 26 nM) displayed difference in activity (K(i) of 15 nM versus 855 nM) signaling the influence of the stereochemistry of the chiral center on potency. In addition, the potent enantiomer displayed significant selectivity in biological activities over several related family members.

Discovery of 7-arylsulfonyl-1,2,3,4, 4a,9a-hexahydro-benzo[4,5]furo[2,3-c]pyridines: identification of a potent and selective 5-HT6 receptor antagonist showing activity in rat social recognition test.

Serotoninergic neurotransmission has been implicated in modulation of learning and memory. It has been demonstrated that 5-hydroxytryptamine(6) (5-HT(6)) receptor antagonists show beneficial effect on cognition in several animal models. Based on a pharmacophore model reported in the literature, we have designed and successfully identified a 7-benzenesulfonyl-1,2,3,4-tetrahydro-benzo[4,5]furo[2,3-c]pyridine (3a) scaffold as a novel class of 5-HT(6) receptor antagonists. Despite good activity against 5-HT(6) receptor, 3a exhibited poor liver microsome stability in mouse, rat and dog. It was demonstrated that the saturation of the double bond of the tetrahydropyridine ring of 3a enhanced metabolic stability. However the resulting compound, 4a (7-phenylsulfonyl-1,2,3,4,4a,9a-hexahydro-benzo[4,5]furo[2,3-c] pyridine-HCl salt) exhibited ∼30-fold loss in potency along with introduction of two chiral centers. In our optimization process for this series, we found that substituents at the 2 or 3 positions on the distal aryl group are important for enhancing activity against 5-HT(6). Separation of enantiomers and subsequent optimization and SAR with bis substituted phenyl sulfone provided potent 5-HT(6) antagonists with improved PK profiles in rat. A potent, selective 5-HT(6)R antagonist (15k) was identified from this study which showed good oral bioavailability (F=39%) in rat with brain penetration (B/P=2.76) and in vivo activity in a rat social recognition test.

Novel brain penetrant benzofuropiperidine 5-HT6 receptor antagonists.

7-Arylsulfonyl substituted benzofuropiperidine was discovered as a novel scaffold for 5HT(6) receptor antagonists. Optimization by substitution at C-1 position led to identification of selective, orally bioavailable, brain penetrant antagonists with reduced hERG liability. An advanced analog tested in rat social recognition model showed significant activity suggesting potential utility in the enhancement of short-term memory.

Amino-terminal isoforms of the human glycine transporter GlyT1 exhibit similar pharmacology.

Alternative promoter usage and mRNA precursor splicing produce three amino-terminal isoforms of the human glycine transporter type 1 (GlyT1). To enable discovery of pharmacological tools that might distinguish them, each of these isoforms was stably expressed in CHO-K1 cells and clonal isolates were generated by limiting dilution. Glycine uptake assays were validated for two lines for each isoform, one low and one high expressor. The data show a modest trend for lower potency against higher expressing lines. IC(50) values for reference GlyT1 inhibitors ALX-5407 (Allelix), (S)-13h (Merck), and SSR504734 (Sanofi-Synthelabo) were similar across isoforms. The greatest variation was observed for ALX-5407, and its IC(50) values across isoforms were still within one log unit of each other. Antipsychotics previously shown to be weak inhibitors of GlyT1 likewise had similar potency against all three isoforms. The cell lines validated here are tools for discovering inhibitors that might distinguish among GlyT1 isoforms.

Glycine transporter (GlyT1) inhibitors with reduced residence time increase prepulse inhibition without inducing hyperlocomotion in DBA/2 mice.

Inhibition of the glycine transporter type 1 (GlyT1) leading to potentiation of the glycine site (GlyB) on the N-methyl-d-aspartate (NMDA) receptor has been proposed as a novel therapeutic approach for schizophrenia. However, sarcosine-based GlyT1 inhibitors produce undesirable side effects including compulsive walking and respiratory distress. The influence of specific biochemical properties of GlyT1 inhibitors, such as mode of inhibition and residence time, on adverse effects is unknown. Two GlyT1 inhibitors that contain a sarcosine moiety, sarcosine and ALX-5407, and two compounds that do not contain a sarcosine moiety, Roche-7 and Merck (S)-13h, were evaluated for their potency, mode of inhibition, and target residence times in vitro, and modulation of prepulse inhibition (PPI) and locomotor activity in vivo. (S)-13h and sarcosine were competitive inhibitors while ALX-5407 and Roche-7 demonstrated mixed noncompetitive inhibition. Potency of GlyT1 inhibition (ALX-5407>(S)-13h>Roche-7≫sarcosine) did not correlate with residence time on GlyT1 (sarcosine=Roche-7≪(S)-13h

Successful identification of glycine transporter inhibitors using an adaptation of a functional cell-based assay.

Glycine transporter (GlyT1) function is typically measured by radiolabeled glycine uptake using lysis methods or scintillation proximity assays (SPAs), which have limited throughput. This study shows the adaptation of the standard cell lysis method to a screening assay with improved throughput and assay characteristics. The assay takes advantage of the 384-well format, standard laboratory automation, and cryopreserved CHO-K1 cells stably overexpressing human GlyT1a transporter (CHO-K1/hGlyT1a) that were validated and banked in advance of screening. The assay was evaluated for the time course of glycine uptake, K(m), V(max), Z' factor analysis, and IC(50) value determination with reference GlyT1 inhibitors. Screening of 118,000 compounds at 10 microM identified 4556 compounds (3.9%) as inhibitors. Positive compounds (>50% inhibition) were retested in the assay at 4 inhibitor concentrations. Compounds demonstrating greater than 40% inhibition at 10 microM were considered as confirmed positives, yielding a 68% confirmation rate from the original screen. To eliminate compounds that nonspecifically inhibited glycine uptake, IC(50) values were determined in both GlyT1 and GlyT2 assays, and those compounds that inhibited GlyT2 were removed from consideration. The screening campaign identified 300 small molecules as selective GlyT1 inhibitors for lead optimization, demonstrating the utility of this cost-effective method.

Target identification and validation in drug discovery: the role of proteomics.

Proteomics, the study of cellular protein expression, is an evolving technology platform that has the potential to identify novel proteins involved in key biological processes in the cell that may serve as potential drug targets. While proteomics has considerable theoretical promise, individual cells/tissues have the potential to generate many millions of proteins while the current analytical technologies that involve the use of time-consuming two dimensional gel electrophoresis (2DIGE) and various mass spectrometry (MS) techniques are unable to handle complex biological samples without multiple high-resolution purification steps to reduce their complexity. This can significantly limit the speed of data generation and replication and requires the use of bioinformatic algorithms to reconstitute the parent proteome, a process that does not always result in a reproducible outcome. In addition, membrane bound proteins, e.g., receptors and ion channels, that are the targets of many existing drugs, are not amenable to study due, in part, to limitations in current proteomic techniques and also to these being present in low abundance and thus disproportionally represented in proteome profiles. Subproteomes with reduced complexity have been used to generate data related to specific, hypothesis-driven questions regarding target identification, protein-interaction networks and signaling pathways. However progress to date, with the exception of diagnostic proteomics in the field of cancer, has been exceedingly slow with an inability to put such studies in the context of a larger proteome, limiting the value of the information. Additionally the pathway for target validation (which can be more accurately described at the preclinical level as target confidence building) remains unclear. It is important that the ability to measure and interrogate proteomes matches expectations, avoiding a repetition of the disappointment and subsequent skepticism that accompanied what proved to be unrealistic expectations for the rapid contribution of data based on the genome maps, to biomedical research.

A homogeneous assay to assess GABA transporter activity.

This unit describes a convenient functional uptake assay for GABA transport into cell lines transiently transfected with GABA transporter-1 (GAT-1) and other GAT isoforms. This facile, homogeneous assay allows for the determination of K(m), V(max), and K(i) values. The assay utilizes commercially available microtiter plates that contain scintillant embedded in the bottom of the wells. Whereas a signal is generated as the cell accumulates the labeled neurotransmitter, label in the medium is undetected. While GABA uptake is observed in several cell lines transfected with GAT-1, K(m) values for GABA uptake may vary with the cell line. This indicates that the choice of cell line is an important consideration when conducting uptake assays.

Phosphoregulation of mixed-lineage kinase 1 activity by multiple phosphorylation in the activation loop.

Mixed-lineage kinase 1 (MLK1) is a mitogen-activated protein kinase kinase kinase capable of activating the c-Jun NH(2)-terminal kinase (JNK) pathway. Full-length MLK1 has 1104 amino acids and a domain structure identical to MLK2 and MLK3. Immunoblot and mass spectrometry show that MLK1 is threonine (and possibly serine) phosphorylated in or near the activation loop. A kinase-dead mutant is not, consistent with autophosphorylation. Mutation to alanine of any of the four serine or threonine residues in the activation loop reduces both the activity of the recombinant kinase domain and JNK pathway activation driven by full-length MLK1 expressed in mammalian cells. Furthermore, the gel mobility of the mutant MLK1s is closer to that of the kinase-dead than wild type, consistent with reduced phosphorylation. Thr312 is the key residue: MLK1[T312A] retains only basal activity (about 1-2% of wild type), and its gel mobility is indistinguishable from kinase-dead. Thr312 does not suffice, however; phosphorylation of multiple sites is necessary for full activation of MLK1. An activation mechanism consistent with these data involves phosphorylation of multiple sites in the activation loop, with phosphorylation of Thr312 required for full phosphorylation. This mechanism is broadly similar to that previously reported for MLK3 [Leung, I. W., and Lassam, N. (2001) J. Biol. Chem. 276, 1961-1967], but the key residue differs.