Inhibition of calcium-calmodulin-dependent phosphodiesterase (PDE1) suppresses inflammatory responses.

A novel, potent, and highly specific inhibitor of calcium-calmodulin-dependent phosphodiesterases (PDE) of the PDE1 family, ITI-214, was used to investigate the role of PDE1 in inflammatory responses. ITI-214 dose-dependently suppressed lipopolysaccharide (LPS)-induced gene expression of pro-inflammatory cytokines in an immortalized murine microglial cell line, BV2 cells. RNA profiling (RNA-Seq) was used to analyze the impact of ITI-214 on the BV2 cell transcriptome in the absence and the presence of LPS. ITI-214 was found to regulate classes of genes that are involved in inflammation and cell migration responses to LPS exposure. The gene expression changes seen with ITI-214 treatment were distinct from those elicited by inhibitors of other PDEs with anti-inflammatory activity (e.g., a PDE4 inhibitor), indicating a distinct mechanism of action for PDE1. Functionally, ITI-214 inhibited ADP-induced migration of BV2 cells through a P2Y12-receptor-dependent pathway, possibly due to increases in the extent of cAMP and VASP phosphorylation downstream of receptor activation. Importantly, this effect was recapitulated in P2 rat microglial cells in vitro, indicating that these pathways are active in native microglial cells. These studies are the first to demonstrate that inhibition of PDE1 exerts anti-inflammatory effects through effects on microglia signaling pathways. The ability of PDE1 inhibitors to prevent or dampen excessive inflammatory responses of BV2 cells and microglia provides a basis for exploring their therapeutic utility in the treatment of neurodegenerative diseases associated with increased inflammation and microglia proliferation such as Parkinson's disease and Alzheimer's disease.

Acute Enhancement of Cardiac Function by Phosphodiesterase Type 1 Inhibition.

BACKGROUND: Phosphodiesterase type-1 (PDE1) hydrolyzes cAMP and cGMP and is constitutively expressed in the heart, although cardiac effects from its acute inhibition in vivo are largely unknown. Existing data are limited to rodents expressing mostly the cGMP-favoring PDE1A isoform. Human heart predominantly expresses PDE1C with balanced selectivity for cAMP and cGMP. Here, we determined the acute effects of PDE1 inhibition in PDE1C-expressing mammals, dogs, and rabbits, in normal and failing hearts, and explored its regulatory pathways. METHODS: Conscious dogs chronically instrumented for pressure-volume relations were studied before and after tachypacing-induced heart failure (HF). A selective PDE1 inhibitor (ITI-214) was administered orally or intravenously±dobutamine. Pressure-volume analysis in anesthetized rabbits tested the role of ß-adrenergic and adenosine receptor signaling on ITI-214 effects. Sarcomere and calcium dynamics were studied in rabbit left ventricular myocytes. RESULTS: In normal and HF dogs, ITI-214 increased load-independent contractility, improved relaxation, and reduced systemic arterial resistance, raising cardiac output without altering systolic blood pressure. Heart rate increased, but less so in HF dogs. ITI-214 effects were additive to ß-adrenergic receptor agonism (dobutamine). Dobutamine but not ITI-214 increased plasma cAMP. ITI-214 induced similar cardiovascular effects in rabbits, whereas mice displayed only mild vasodilation and no contractility effects. In rabbits, ß-adrenergic receptor blockade (esmolol) prevented ITI-214-mediated chronotropy, but inotropy and vasodilation remained unchanged. By contrast, adenosine A2B-receptor blockade (MRS-1754) suppressed ITI-214 cardiovascular effects. Adding fixed-rate atrial pacing did not alter the findings. ITI-214 alone did not affect sarcomere or whole-cell calcium dynamics, whereas ß-adrenergic receptor agonism (isoproterenol) or PDE3 inhibition (cilostamide) increased both. Unlike cilostamide, which further enhanced shortening and peak calcium when combined with isoproterenol, ITI-214 had no impact on these responses. Both PDE1 and PDE3 inhibitors increased shortening and accelerated calcium decay when combined with forskolin, yet only cilostamide increased calcium transients. CONCLUSIONS: PDE1 inhibition by ITI-214 in vivo confers acute inotropic, lusitropic, and arterial vasodilatory effects in PDE1C-expressing mammals with and without HF. The effects appear related to cAMP signaling that is different from that provided via ß-adrenergic receptors or PDE3 modulation. ITI-214, which has completed phase I trials, may provide a novel therapy for HF.

Phosphodiesterase 1: A Unique Drug Target for Degenerative Diseases and Cognitive Dysfunction.

The focus of this chapter is on the cyclic nucleotide phosphodiesterase 1 (PDE1) family. PDE1 is one member of the 11 PDE families (PDE 1-11). It is the only phosphodiesterase family that is calcium/calmodulin activated. As a result, whereas other families of PDEs 2-11 play a dominant role controlling basal levels of cyclic nucleotides, PDE1 is involved when intra-cellular calcium levels are elevated and, thus, has an "on demand" or activity-dependent involvement in the control of cyclic nucleotides in excitatory cells including neurons, cardiomyocytes and smooth muscle. As a Class 1 phosphodiesterase, PDE1 hydrolyzes the 3' bond of 3'-5'-cyclic nucleotides, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Here, we review evidence for this family of enzymes as drug targets for development of therapies aimed to address disorders of the central nervous system (CNS) and of degenerative diseases. The chapter includes sections on the potential for cognitive enhancement in mental disorders, as well as a review of PDE1 enzyme structure, enzymology, tissue distribution, genomics, inhibitors, pharmacology, clinical trials, and therapeutic indications. Information is taken from public databases. A number of excellent reviews of the phosphodiesterase family have been written as well as reviews of the PDE1 family. References cited here are not comprehensive, rather pointing to major reviews and key publications.

Preclinical profile of ITI-214, an inhibitor of phosphodiesterase 1, for enhancement of memory performance in rats.

RATIONALE: Therapeutic agents for memory enhancement in psychiatric disorders, such as schizophrenia, are urgently needed. OBJECTIVE: The aim of this study is to characterize the preclinical profile of ITI-214, a potent inhibitor of phosphodiesterase 1 (PDE1). METHODS: ITI-214 was assayed for inhibition of PDE1 versus other PDE enzyme families using recombinant human PDE enzymes and for off-target binding to 70 substrates (General SEP II diversity panel; Caliper Life Sciences). Effects of ITI-214 (0.1-10 mg/kg, po) on memory performance were assayed in rats using the novel object recognition (NOR) paradigm, with drug given at specified time points prior to or following exposure to objects in an open field. ITI-214 was evaluated for potential drug-drug interaction with risperidone in rats using conditioned avoidance response (CAR) and pharmacokinetic assessments. RESULTS: ITI-214 inhibited PDE1A (K i = 33 pmol) with >1000-fold selectivity for the nearest other PDE family (PDE4D) and displayed minimal off-target binding interactions in a 70-substrate selectivity profile. By using specific timing of oral ITI-214 administration, it was demonstrated in the NOR that ITI-214 is able to enhance acquisition, consolidation, and retrieval memory processes. All memory effects were in the absence of effects on exploratory behavior. ITI-214 did not disrupt the risperidone pharmacokinetic profile or effects in CAR. CONCLUSIONS: ITI-214 improved the memory processes of acquisition, consolidation, and retrieval across a broad dose range (0.1-10 mg/kg, po) without disrupting the antipsychotic-like activity of a clinical antipsychotic medication, specifically risperidone. Clinical development of ITI-214 is currently in progress.

Discovery of Potent and Selective Inhibitors of Phosphodiesterase 1 for the Treatment of Cognitive Impairment Associated with Neurodegenerative and Neuropsychiatric Diseases.

A diverse set of 3-aminopyrazolo[3,4-d]pyrimidinones was designed and synthesized. The structure-activity relationships of these polycyclic compounds as phosphodiesterase 1 (PDE1) inhibitors were studied along with their physicochemical and pharmacokinetic properties. Systematic optimizations of this novel scaffold culminated in the identification of a clinical candidate, (6aR,9aS)-2-(4-(6-fluoropyridin-2-yl)benzyl)-5-methyl-3-(phenylamino)-5,6a,7,8,9,9a-hexahydrocyclopenta[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4-(2H)-one phosphate (ITI-214), which exhibited picomolar inhibitory potency for PDE1, demonstrated excellent selectivity against all other PDE families and showed good efficacy in vivo. Currently, this investigational new drug is in Phase I clinical development and being considered for the treatment of several indications including cognitive deficits associated with schizophrenia and Alzheimer's disease, movement disorders, attention deficit and hyperactivity disorders, and other central nervous system (CNS) and non-CNS disorders.

Functional profile of a novel modulator of serotonin, dopamine, and glutamate neurotransmission.

RATIONALE: Schizophrenia remains among the most prevalent neuropsychiatric disorders, and current treatment options are accompanied by unwanted side effects. New treatments that better address core features of the disease with minimal side effects are needed. OBJECTIVES: As a new therapeutic approach, 1-(4-fluoro-phenyl)-4-((6bR, 10aS)-3-methyl-2,3,6b,9,10,10a-hexahydro-1H,7H-pyrido[3',4':4,5]pyrrolo[1,2,3-de]quinoxalin-8-yl)-butan-1-one (ITI-007) is currently in human clinical trials for the treatment of schizophrenia. Here, we characterize the preclinical functional activity of ITI-007. RESULTS: ITI-007 is a potent 5-HT2A receptor ligand (K i = 0.5 nM) with strong affinity for dopamine (DA) D2 receptors (K i = 32 nM) and the serotonin transporter (SERT) (K i = 62 nM) but negligible binding to receptors (e.g., H1 histaminergic, 5-HT2C, and muscarinic) associated with cognitive and metabolic side effects of antipsychotic drugs. In vivo it is a 5-HT2A antagonist, blocking (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI)-induced headtwitch in mice with an inhibitory dose 50 (ID50) = 0.09 mg/kg, per oral (p.o.), and has dual properties at D2 receptors, acting as a postsynaptic D2 receptor antagonist to block D-amphetamine hydrochloride (D-AMPH) hyperlocomotion (ID50 = 0.95 mg/kg, p.o.), yet acting as a partial agonist at presynaptic striatal D2 receptors in assays measuring striatal DA neurotransmission. Further, in microdialysis studies, this compound significantly and preferentially enhances mesocortical DA release. At doses relevant for antipsychotic activity in rodents, ITI-007 has no demonstrable cataleptogenic activity. ITI-007 indirectly modulates glutamatergic neurotransmission by increasing phosphorylation of GluN2B-type N-methyl-D-aspartate (NMDA) receptors and preferentially increases phosphorylation of glycogen synthase kinase 3ß (GSK-3ß) in mesolimbic/mesocortical dopamine systems. CONCLUSION: The combination of in vitro and in vivo activities of this compound support its development for the treatment of schizophrenia and other psychiatric and neurologic disorders.

Discovery of a tetracyclic quinoxaline derivative as a potent and orally active multifunctional drug candidate for the treatment of neuropsychiatric and neurological disorders.

We report the synthesis and structure-activity relationships of a class of tetracyclic butyrophenones that exhibit potent binding affinities to serotonin 5-HT(2A) and dopamine D2 receptors. This work has led to the discovery of 4-((6bR,10aS)-3-methyl-2,3,6b,9,10,10a-hexahydro-1H,7H-pyrido[3',4':4,5]pyrrolo[1,2,3-de]quinoxalin-8-yl)-1-(4-fluorophenyl)-butan-1-one 4-methylbenzenesulfonate (ITI-007), which is a potent 5-HT(2A) antagonist, postsynaptic D2 antagonist, and inhibitor of serotonin transporter. This multifunctional drug candidate is orally bioavailable and exhibits good antipsychotic efficacy in vivo. Currently, this investigational new drug is under clinical development for the treatment of neuropsychiatric and neurological disorders.

Muscarinic receptors acting at pre- and post-synaptic sites differentially regulate dopamine/DARPP-32 signaling in striatonigral and striatopallidal neurons.

Muscarinic receptors, activated by acetylcholine, play critical roles in the functional regulation of medium spiny neurons in the striatum. However, the muscarinic receptor signaling pathways are not fully elucidated due to their complexity. In this study, we investigated the function of muscarinic receptors in the striatum by monitoring DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of M(r) 32 kDa) phosphorylation at Thr34 (the PKA-site) using mouse striatal slices. Treatment of slices with a non-selective muscarinic receptor agonist, oxotremorine (10 µM), rapidly and transiently increased DARPP-32 phosphorylation. The increase in DARPP-32 phosphorylation was completely abolished either by a dopamine D(1) receptor antagonist (SCH23390), tetrodotoxin, genetic deletion of M5 receptors, muscarinic toxins for M1 and M4 receptors, or 6-hydroxydopamine lesioning of dopaminergic neurons, whereas it was enhanced by nicotine. Analysis in D(1)-DARPP-32-Flag/D(2)-DARPP-32-Myc transgenic mice revealed that oxotremorine increases DARPP-32 phosphorylation selectively in D(1)-type/striatonigral, but not in D(2)-type/striatopallidal, neurons. When D(1) and D(2) receptors were blocked by selective antagonists to exclude the effects of released dopamine, oxotremorine increased DARPP-32 Thr34 phosphorylation only in D(2)-type/striatopallidal neurons. This increase required activation of M1 receptors and was dependent upon adenosine A(2A) receptor activity. The results demonstrate that muscarinic receptors, especially M5 receptors, act at presynaptic dopaminergic terminals, regulate the release of dopamine in cooperation with nicotinic receptors, and activate D(1) receptor/DARPP-32 signaling in the striatonigral neurons. Muscarinic M1 receptors expressed in striatopallidal neurons interact with adenosine A(2A) receptors and activate DARPP-32 signaling.

Nerve agent exposure elicits site-specific changes in protein phosphorylation in mouse brain.

Organophosphorus (OP) compounds cause toxic symptoms, including convulsions, coma, and death, as the result of irreversible inhibition of acetylcholinesterase (AChE). The development of effective treatments to block these effects and attenuate long-term cognitive and motor disabilities that result from OP intoxication is hampered by a limited understanding of the CNS pathways responsible for these actions. We employed a candidate method (called CNSProfile) to identify changes in the phosphorylation state of key neuronal phosphoproteins evoked by the OP compound, diisopropyl fluorophosphate (DFP). Focused microwave fixation was used to preserve the phosphorylation state of phosphoproteins in brains of DFP-treated mice; hippocampus and striatum were analyzed by immunoblotting with a panel of phospho-specific antibodies. DFP exposure elicited comparable effects on phosphorylation of brain phosphoproteins in both C57BL/6 and FVB mice. DFP treatment significantly altered phosphorylation at regulatory residues on glutamate receptors, including Serine897 (S897) of the NR1 NMDA receptor. NR1 phosphorylation was bi-directionally regulated after DFP in striatum versus hippocampus. NR1 phosphorylation was reduced in striatum, but elevated in hippocampus, compared with controls. DARPP-32 phosphorylation in striatum was selectively increased at the Cdk5 kinase substrate, Threonine75 (T75). Phencynonate hydrochloride, a muscarinic cholinergic antagonist, prevented seizure-like behaviors and the observed changes in phosphorylation induced by DFP. The data reveal region-specific effects of nerve agent exposure on intracellular signaling pathways that correlate with seizure-like behavior and which are reversed by the muscarinic receptor blockade. This approach identifies specific targets for nerve agents, including substrates for Cdk5 kinase, which may be the basis for new anti-convulsant therapies.

General anesthetics selectively modulate glutamatergic and dopaminergic signaling via site-specific phosphorylation in vivo.

Isoflurane, propofol and ketamine are representative general anesthetics with distinct molecular mechanisms of action that have neuroprotective properties in models of excitotoxic ischemic damage. We characterized the effects of these agents on neuronal glutamate and dopamine signaling by profiling drug-induced changes in brain intracellular protein phosphorylation in vivo to test the hypothesis that they affect common downstream effectors. Anesthetic-treated and control mice were killed instantly by focused microwave irradiation, frontal cortex and striatum were removed, and the phosphorylation profile of specific neuronal signaling proteins was analyzed by immunoblotting with a panel of phospho-specific antibodies. At anesthetic doses that produced loss of righting reflex, isoflurane, propofol, and ketamine all reduced phosphorylation of the activating residue T183 of ERK2 (but not of ERK1); S897 of the NR1 NMDA receptor subunit; and S831 (but not S845) of the GluR1 AMPA receptor subunit in cerebral cortex. At sub-anesthetic doses, these drugs only reduced phosphorylation of ERK2. Isoflurane and ketamine also reduced phosphorylation of spinophilin at S94, but oppositely regulated phosphorylation of presynaptic (tyrosine hydroxylase) and postsynaptic (DARPP-32) markers of dopaminergic neurotransmission in striatum. These data reveal both shared and agent-specific actions of CNS depressant drugs on critical intracellular protein phosphorylation signaling pathways that integrate multiple second messenger systems. Reduced phosphorylation of ionotropic glutamate receptors by all three anesthetics indicates depression of normal glutamatergic synaptic transmission and reduced potential excitotoxicity. This novel approach indicates a role for phosphorylation-mediated down-regulation of glutamatergic synaptic transmission by general anesthetics and identifies specific in vivo targets for focused evaluation of anesthetic mechanisms.

Discovery of (S)-2-((S)-2-(3,5-difluorophenyl)-2-hydroxyacetamido)-N-((S,Z)-3-methyl-4-oxo-4,5-dihydro-3H-benzo[d][1,2]diazepin-5-yl)propanamide (BMS-433796): a gamma-secretase inhibitor with Abeta lowering activity in a transgenic mouse model of Alzheimer's disease.

We report on the design of benzodiazepinones as peptidomimetics at the carboxy terminus of hydroxyamides. Structure-activity relationships of diazepinones were investigated and orally active gamma-secretase inhibitors were synthesized. Active metabolites contributing to Abeta reduction were identified by analysis of plasma samples from Tg2576 mice. In particular, (S)-2-((S)-2-(3,5-difluorophenyl)-2-hydroxyacetamido)-N-((S,Z)-3-methyl-4-oxo-4,5-dihydro-3H-benzo[d][1,2]diazepin-5-yl)propanamide (BMS-433796) was identified with an acceptable pharmacodynamic and pharmacokinetic profile. Chronic dosing of BMS-433796 in Tg2576 mice suggested a narrow therapeutic window and Notch-mediated toxicity at higher doses.

Nicotine regulates DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of 32 kDa) phosphorylation at multiple sites in neostriatal neurons.

Nicotinic acetylcholine receptors (nAChRs) regulate dopaminergic signaling in the striatum by modulating the release of neurotransmitters. We have recently reported that nicotine stimulates the release of dopamine via alpha4beta2(*) nAChRs and/or alpha7 nAChRs, leading to the regulation of DARPP-32 at Thr34, the site involved in regulation of protein phosphatase-1 (PP-1). In this study, we investigated the regulation of DARPP-32 phosphorylation at its other sites, Thr75 [cyclin-dependent kinase-5 (Cdk5) site], Ser97 (CK2 site), and Ser130 (CK1 site), that serve to modulate Thr34 phosphorylation and dephosphorylation. In neostriatal slices, nicotine (100 microM) increased phosphorylation of DARPP-32 at Ser97 and Ser130 at an early time point (30 s) and decreased phosphorylation of DARPP-32 at Thr75 at a late time point (3 min). The increase in Ser97 and Ser130 phosphorylation was mediated through the release of dopamine via activation of alpha4beta2(*) nAChRs and alpha7 nAChRs and the subsequent activation of dopamine D1 and D2 receptors. The decrease in Thr75 phosphorylation was mediated through the release of dopamine via activation of alpha4beta2(*) nAChRs and the subsequent activation of dopamine D1 receptors. These various actions of nicotine on modulatory sites of phosphorylation would be predicted to result in a synergistic increase in the state of phosphorylation of DARPP-32 at Thr34 and thus would contribute to increased dopamine D1 receptor/DARPP-32 Thr34/PP-1 signaling.