Beth Ann McKenna

Mixed lineage kinase activity of indolocarbazole analogues

The MLK1-3 activity for a series of analogues of the indolocarbazole K-252a is reported. Addition of 3,9-bis-alkylthiomethyl groups to K-252a results in potent and selective MLK inhibitors. The in vitro and in vivo survival promoting activity of bis-isopropylthiomethyl-K-252a (16, CEP-11004/KT-8138) is reported.

Dimebolin is a 5-HT6 antagonist with acute cognition enhancing activities.

Dimebolin (Dimebon), is a non-selective antihistamine approved in Russia for the treatment of allergy. Recently, this drug has been shown to be neuroprotective in cellular models of Alzheimers disease and Huntingtons disease, and to preserve cognitive function when chronically administered to AF64A lesioned rats. Interests in identifying the molecular targets of dimebolin have intensified with reports of efficacy in clinical trials with Alzheimers patients. Dimebolin has been found to interact with a number of molecular targets including acetylcholinesterases, N-methyl-d-aspartate receptors, and voltage-gated calcium channels, with potencies in the range of 5-50 microM. In the present study, the action of dimebolin at the serotonin 5-HT(6) receptor was investigated. Dimebolin binds with moderate affinity to both the human and rat recombinant 5-HT(6) receptor (K(i)=26.0+/-2.5 nM and 119.0+/-14.0 nM respectively) as well as the native rat 5-HT(6) receptor, and acts as an antagonist in functional cAMP assays. Furthermore, dimebolin occupies the 5-HT(6) receptor in vivo as assessed by ex vivo autoradiography, with a dose-occupancy relationship similar to that of the selective 5-HT(6) antagonist SB-399885. Finally, both SB-399885 and dimebolin produce an acute enhancement of short-term social recognition memory, although dimebolin is approximately 10-fold less potent than SB-399885. Taken together, these studies demonstrate that dimebolin antagonizes the 5-HT(6) receptor with higher affinity than other targets characterized to date, and suggest that this activity may play a role in the acute cognition enhancing effects of this compound in preclinical models and in the clinic.

Inhibition of mixed lineage kinase 3 attenuates MPP+-induced neurotoxicity in SH-SY5Y cells

The neuropathology of Parkinsons Disease has been modeled in experimental animals following MPTP treatment and in dopaminergic cells in culture treated with the MPTP neurotoxic metabolite, MPP(+). MPTP through MPP(+) activates the stress-activated c-Jun N-terminal kinase (JNK) pathway in mice and SH-SY5Y neuroblastoma cells. Recently, it was demonstrated that CEP-1347/KT7515 attenuated MPTP-induced nigrostriatal dopaminergic neuron degeneration in mice, as well as MPTP-induced JNK phosphorylation. Presumably, CEP-1347 acts through inhibition of at least one upstream kinase within the mixed lineage kinase (MLK) family since it has been shown to inhibit MLK 1, 2 and 3 in vitro. Activation of the MLK family leads to JNK activation. In this study, the potential role of MLK and the JNK pathway was examined in MPP(+)-induced cell death of differentiated SH-SY5Y cells using CEP-1347 as a pharmacological probe and dominant negative adenoviral constructs to MLKs. CEP-1347 inhibited MPP(+)-induced cell death and the morphological features of apoptosis. CEP-1347 also prevented MPP(+)-induced JNK activation in SH-SY5Y cells. Endogenous MLK 3 expression was demonstrated in SH-SY5Y cells through protein levels and RT-PCR. Adenoviral infection of SH-SY5Y cells with a dominant negative MLK 3 construct attenuated the MPP(+)-mediated increase in activated JNK levels and inhibited neuronal death following MPP(+) addition compared to cultures infected with a control construct. Adenoviral dominant negative constructs of two other MLK family members (MLK 2 and DLK) did not protect against MPP(+)-induced cell death. These studies show that inhibition of the MLK 3/JNK pathway attenuates MPP(+)-mediated SH-SY5Y cell death in culture and supports the mechanism of action of CEP-1347 as an MLK family inhibitor.

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<ALX-5407). ALX-5407 and (S)-13h induced compulsive walking, termed obstinate progression (OP), at doses that increased PPI in DBA/2 mice, demonstrating that OP was not a function of mode of inhibition or inhibitor chemotype. Sarcosine and Roche-7 increased PPI without inducing OP, suggesting that compounds with decreased GlyT1 residence time were efficacious without adverse effects. Direct activation of the GlyB site by d-serine did not produce OP. However, OP induced by (S)-13h was blocked by strychnine, a glycine receptor (GlyA) antagonist, suggesting that OP induced by GlyT1 inhibition was mediated by GlyA. Thus, GlyT1 inhibitors with short residence times demonstrated efficacy without mechanism-based adverse effects.

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 (Ki = 26 nm) displayed difference in activity (Ki 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.

Exploration of the importance of the P2-P3-NHCO-moiety in a potent di- or tripeptide inhibitor of calpain I: insights into the development of nonpeptidic inhibitors of calpain I.

Calpain I, an intracellular cysteine protease, has been implicated in the neurodegeneration following an episode of cerebral ischemia. In this paper, we report on a series of peptidomimetic ketomethylene and carbamethylene inhibitors of recombinant human calpain I (rh calpain I). Our study reveals that the -NHCO-moiety (possible hydrogen-bonding site) at the P2-P3 region of a potent tripeptide or a dipeptide inhibitor of calpain I is not a strict requirement for enzyme recognition. Compounds 7d ((R)-2-isobutyl-4-oxo-4-(9-xanthenyl)butanoic acid ((S)-1-formyl-3-methyl)butyl amide), 31 ((R)-2-isobutyl-4-(2-sulfonylnaphthyl)butyric acid ((S)1-formyl-3-methyl)butyl amide) and 34 ((R)-2-isobutyl-4-(2-sulfoxylnaphthyl)butyric acid ((S)-1-formyl-3-methyl)butyl amide) which exhibited good activity in the enzyme assay, also inhibited calpain I in a human cell line.

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.

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.

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.

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.