Compound FC-10696 Inhibits Egress of Marburg Virus.

Marburg virus (MARV) VP40 protein (mVP40) directs egress and spread of MARV, in part, by recruiting specific host WW domain-containing proteins via its conserved PPxY late (L) domain motif to facilitate efficient virus-cell separation. We reported previously that small-molecule compounds targeting the viral PPxY/host WW domain interaction inhibited VP40-mediated egress and spread. Here, we report on the antiviral potency of novel compound FC-10696, which emerged from extensive structure-activity relationship (SAR) of a previously described series of PPxY inhibitors. We show that FC-10696 inhibits egress of mVP40 virus-like particles (VLPs) and egress of authentic MARV from HeLa cells and primary human macrophages. Moreover, FC-10696 treated-mice displayed delayed onset of weight loss and clinical signs and significantly lower viral loads compared to controls, with 14% of animals surviving 21 days following a lethal MARV challenge. Thus, FC-10696 represents a first-in-class, host-oriented inhibitor effectively targeting late stages of the MARV life cycle.

Intravenously Administered Ganaxolone Blocks Diazepam-Resistant Lithium-Pilocarpine-Induced Status Epilepticus in Rats: Comparison with Allopregnanolone.

Ganaxolone (GNX) is the 3ß-methylated synthetic analog of the naturally occurring neurosteroid, allopregnanolone (ALLO). GNX is effective in a broad range of epilepsy and behavioral animal models and is currently in clinical trials designed to assess its anticonvulsant and antidepressant activities. The current studies were designed to broaden the anticonvulsant profile of GNX by evaluating its potential anticonvulsant activities following i.v. administration in treatment-resistant models of status epilepticus (SE), to establish a pharmacokinetic (PK)/pharmacodynamic (PD) relationship, and to compare its PK and anticonvulsant activities to ALLO. In PK studies, GNX had higher exposure levels, a longer half-life, slower clearance, and higher brain penetrance than ALLO. Both GNX and ALLO produced a sedating response as characterized by loss of righting reflex, but neither compound produced a full anesthetic response as animals still responded to painful stimuli. Consistent with their respective PK properties, the sedative effect of GNX was longer than that of ALLO. Unlike other nonanesthetizing anticonvulsant agents indicated for SE, both GNX and ALLO produced anticonvulsant activity in models of pharmacoresistant SE with administration delay times of up to 1 hour after seizure onset. Again, consistent with their respective PK properties, GNX produced a significantly longer anticonvulsant response. These studies show that GNX exhibited improved pharmacological characteristics versus other agents used as treatments for SE and position GNX as a uniquely acting treatment of this indication.

A Mouse Model of Furosemide-Induced Overactive Bladder.

Detailed in this unit is a mouse model of overactive bladder and urinary incontinence based on diuretic stress-induced urination. The procedure involves the use of a unique, highly sensitive, and automated urine capturing method to measure urinary latency, frequency, and void volume. Although this method was first described and validated using an anti-muscarinic drug used for treating overactive bladder, subsequent work has shown that effective non-cholinergic agents can be detected. These findings indicate good predictive value for this model regarding the possible clinical utility of test agents as treatments for overactive bladder, regardless of their site of action. © 2016 by John Wiley & Sons, Inc.

ABHD6 blockade exerts antiepileptic activity in PTZ-induced seizures and in spontaneous seizures in R6/2 mice.

The serine hydrolase α/ß-hydrolase domain 6 (ABHD6) hydrolyzes the most abundant endocannabinoid (eCB) in the brain, 2-arachidonoylglycerol (2-AG), and controls its availability at cannabinoid receptors. We show that ABHD6 inhibition decreases pentylenetetrazole (PTZ)-induced generalized tonic-clonic and myoclonic seizure incidence and severity. This effect is retained in Cnr1(-/-) or Cnr2(-/-) mice, but blocked by addition of a subconvulsive dose of picrotoxin, suggesting the involvement of GABAA receptors. ABHD6 inhibition also blocked spontaneous seizures in R6/2 mice, a genetic model of juvenile Huntington's disease known to exhibit dysregulated eCB signaling. ABHD6 blockade retained its antiepileptic activity over chronic dosing and was not associated with psychomotor or cognitive effects. While the etiology of seizures in R6/2 mice remains unsolved, involvement of the hippocampus is suggested by interictal epileptic discharges, increased expression of vGLUT1 but not vGAT, and reduced Neuropeptide Y (NPY) expression. We conclude that ABHD6 inhibition may represent a novel antiepileptic strategy.

MLR-1023 is a potent and selective allosteric activator of Lyn kinase in vitro that improves glucose tolerance in vivo.

2(1H)-pyrimidinone,5-(3-methylphenoxy) (MLR-1023) is a candidate for the treatment of type 2 diabetes. The current studies were aimed at determining the mechanism by which MLR-1023 mediates glycemic control. In these studies, we showed that MLR-1023 reduced blood glucose levels without increasing insulin secretion in vivo. We have further determined that MLR-1023 did not activate peroxisome proliferator-activated α, δ, and γ receptors or glucagon-like peptide-1 receptors or inhibit dipeptidyl peptidase-4 or α-glucosidase enzyme activity. However, in an in vitro broad kinase screen MLR-1023 activated the nonreceptor-linked Src-related tyrosine kinase Lyn. MLR-1023 increased the V(max) of Lyn with an EC(50) of 63 nM. This Lyn kinase activation was ATP binding site independent, indicating that MLR-1023 regulated the kinase through an allosteric mechanism. We have established a link between Lyn activation and blood glucose lowering with studies showing that the glucose-lowering effects of MLR-1023 were abolished in Lyn knockout mice, consistent with existing literature linking Lyn kinase and the insulin-signaling pathway. In summary, these studies describe MLR-1023 as a unique blood glucose-lowering agent and show that MLR-1023-mediated blood glucose lowering depends on Lyn kinase activity. These results, coupled with other results (J Pharmacol Exp Ther 342:23-32, 2012), suggest that MLR-1023 and Lyn kinase activation may be a new treatment modality for type 2 diabetes.

The Lyn kinase activator MLR-1023 is a novel insulin receptor potentiator that elicits a rapid-onset and durable improvement in glucose homeostasis in animal models of type 2 diabetes.

MLR-1023 [Tolimidone; CP-26154; 2(1H)-pyrimidinone, 5-(3-methylphenoxy)] is an allosteric Lyn kinase activator that reduces blood glucose levels in mice subjected to an oral glucose tolerance test (J Pharmacol Exp Ther 342:15-22, 2012). The current studies were designed to define the role of insulin in MLR-1023-mediated blood glucose lowering, to evaluate it in animal models of type 2 diabetes, and to compare it to the activities of selected existing diabetes therapeutics. Results from these studies show that in an acute oral glucose tolerance test MLR-1023 evoked a dose-dependent blood glucose-lowering response that was equivalent in magnitude to that of metformin without eliciting a hypoglycemic response. In streptozotocin-treated, insulin-depleted mice, MLR-1023 administration did not affect blood glucose levels. However, MLR-1023 potentiated the glucose-lowering activity of exogenously administered insulin, showing that MLR-1023-mediated blood glucose lowering was insulin-dependent. In a hyperinsulinemic/euglycemic clamp study, orally administered MLR-1023 increased the glucose infusion rate required to sustain blood glucose levels, demonstrating that MLR-1023 increased insulin receptor sensitivity. In chronically treated db/db mice, MLR-1023 elicited a dose-dependent and durable glucose-lowering effect, reduction in HbA1c levels and preservation of pancreatic ß-cells. The magnitude of effect was equivalent to that seen with rosiglitazone but with a faster onset of action and without causing weight gain. These studies show that MLR-1023 is an insulin receptor-potentiating agent that produces a rapid-onset and durable blood glucose-lowering activity in diabetic animals.

Synthesis and pharmacological evaluation of N-(3-(1H-indol-4-yl)-5-(2-methoxyisonicotinoyl)phenyl)methanesulfonamide (LP-261), a potent antimitotic agent.

The synthesis and optimization of a series of orally bioavailable 1-(1H-indol-4-yl)-3,5-disubstituted benzene analogues as antimitotic agents are described. A functionalized dibromobenzene intermediate was used as a key scaffold, which when modified by sequential Suzuki coupling and Buchwald-Hartwig amination provided a flexible entry to 1,3,5-trisubstituted phenyl compounds. A 1H-indol-4-yl moiety at the 1-position was determined to be a critical feature for optimal potency. The compounds have been shown to induce cell cycle arrest at the G2/M phase and demonstrate efficacy in both cell viability and cell proliferation assays. The primary site of action for these agents is revealed by their colchicine competitive inhibition of tubulin polymerization, and a computational model has been developed for the association of these compounds to tubulin. An optimized lead LP-261 significantly inhibits growth of a human non-small-cell lung tumor (NCI-H522) in a mouse xenograft model.

Mixed-lineage kinase 1 and mixed-lineage kinase 3 subtype-selective dihydronaphthyl[3,4-a]pyrrolo[3,4-c]carbazole-5-ones: optimization, mixed-lineage kinase 1 crystallography, and oral in vivo activity in 1-methyl-4-phenyltetrahydropyridine models.

The optimization of the dihydronaphthyl[3,4-a]pyrrolo[3,4-c]carbazole-5-one R(2) and R(12) positions led to the identification of the first MLK1 and MLK3 subtype-selective inhibitors within the MLK family. Compounds 14 (CEP-5104) and 16 (CEP-6331) displayed good potency for MLK1 and MLK3 inhibition with a greater than 30- to 100-fold selectivity for related family members MLK2 and DLK. Compounds 14 and 16 were orally active in vivo in a mouse MPTP biochemical efficacy model that was comparable to the first-generation pan-MLK inhibitor 1 (CEP-1347). The MLK1 structure-activity relationships were supported by the first-reported X-ray crystal structure of MLK1 bound with 16.

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

The neuropathology of Parkinson's 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.

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.

Targeting the JNK pathway for therapeutic benefit in CNS disease.

The c-Jun N-terminal Kinase (JNK) pathway leading to c-Jun phosphorylation plays a causal role in apoptosis of isolated primary embryonic neurons and of multiple neuronal cell lines following a wide variety of stimuli. Activation of this pathway may also contribute to the neuronal atrophy and death that is associated with neurodegenerative pathological conditions including Alzheimer's, Parkinson's, Huntington's Diseases and stroke. Here, the data that providelinks between the activation of the JNK pathway and its potential to play an operative role in CNS disease are reviewed. Also included is the progress on development of inhibitors targeting the JNK pathway for therapeutic benefit.

Discovery of CEP-1347/KT-7515, an inhibitor of the JNK/SAPK pathway for the treatment of neurodegenerative diseases.

Apoptosis has been proposed as a mechanism of cell death in Alzheimer's, Huntington's and Parkinson's diseases and the occurrence of apoptosis in these disorders suggests a common mechanism. Events such as oxidative stress, calcium toxicity, mitochondria defects, excitatory toxicity, and deficiency of survival factors are all postulated to play varying roles in the pathogenesis of the diseases. However, the transcription factor c-jun may play a role in the pathology and cell death processes that occur in Alzheimer's disease. Parkinson's disease (PD) is also a progressive disorder involving the specific degeneration and death of dopamine neurons in the nigrostriatal pathway. In Parkinson's disease, dopaminergic neurons in the substantia nigra are hypothesized to undergo cell death by apoptotic processes. The commonality of biochemical events and pathways leading to cell death in these diseases continues to be an area under intense investigation. The current therapy for PD and AD remains targeting replacement of lost transmitter, but the ultimate objective in neurodegenerative therapy is the functional restoration and/or cessation of progression of neuronal loss. This chapter will describe a novel approach for the treatment of neurodegenerative diseases through the development of kinase inhibitors that block the active cell death process at an early transcriptional independent step in the stress activated kinase cascade. In particular, preclinical data will be presented on the c-Jun Amino Kinase pathway inhibitor, CEP-1347/KT-7515, with respect to it's properties that make it a desirable clinical candidate for treatment of various neurodegenerative diseases.