Investigating the Effects of Exogenous and Endogenous 2-Arachidonoylglycerol on Retinal CB1 Cannabinoid Receptors and Reactive Microglia in Naive and Diseased Retina.

The endocannabinoid system (ECS) is a new target for the development of retinal disease therapeutics, whose pathophysiology involves neurodegeneration and neuroinflammation. The endocannabinoid 2-arachidonoylglycerol (2-AG) affects neurons and microglia by activating CB1/CB2 cannabinoid receptors (Rs). The aim of this study was to investigate the effects of 2-AG on the CB1R expression/downregulation and retinal neurons/reactive microglia, when administered repeatedly (4 d), in three different paradigms. These involved the 2-AG exogenous administration (a) intraperitoneally (i.p.) and (b) topically and (c) by enhancing the 2-AG endogenous levels via the inhibition (AM11920, i.p.) of its metabolic enzymes (MAGL/ABHD6). Sprague Dawley rats were treated as mentioned above in the presence or absence of CB1/CB2R antagonists and the excitatory amino acid, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Immunohistochemistry, Western blot and a 2-AG level analyses were performed. The 2-AG repeated treatment (i.p.) induced the CB1R downregulation, abolishing its neuroprotective actions. However, 2-AG attenuated the AMPA-induced activation of microglia via the CB2R, as concurred by the AM630 antagonist effect. Topically administered 2-AG was efficacious as a neuroprotectant/antiapoptotic and anti-inflammatory agent. AM11920 increased the 2-AG levels providing neuroprotection against excitotoxicity and reduced microglial activation without affecting the CB1R expression. Our findings show that 2-AG, in the three paradigms studied, displays differential pharmacological profiles in terms of the downregulation of the CB1R and neuroprotection. All treatments, however, attenuated the activation of microglia via the CB2R activation, supporting the anti-inflammatory role of 2-AG in the retina.

Design and Synthesis of Highly Potent and Specific ABHD6 Inhibitors.

Fine-tuning than complete disruption of 2-arachidonoylglycerol (2-AG) metabolism in the brain represents a promising pharmacological approach to limit potential untoward effects associated with complete blockade of monoacylglycerol lipase (MGL), the primary hydrolase of 2-AG. This could be achieved through a/b-hydrolase domain containing 6 (ABHD6) inhibition, which will provide a smaller and safer contribution to 2-AG regulation in the brain. Pharmacological studies with ABHD6 inhibitors have recently been reported, where modulation of ABHD6 activity either through CB1R-dependent or CB1R-independent processes showed promise in preclinical models of epilepsy, neuropathic pain and inflammation. Furthermore in the periphery, ABHD6 modulates 2-AG and other fatty acid monoacylglycerols (MAGs) and is implicated in Type-2 diabetes, metabolic syndrome and potentially other diseases. Herein, we report the discovery of single-digit nanomolar potent and highly specific ABHD6 inhibitors with >1000-fold selectivity against MGL and FAAH. The new ABHD6 inhibitors provide early leads to develop therapeutics for neuroprotection and the treatment of inflammation and diabetes.

Inhibition of triple negative breast cancer-associated inflammation and progression by N- acylethanolamine acid amide hydrolase (NAAA).

Triple-negative breast cancer (TNBC) is associated with high mortality due to the high expression of pro-inflammatory cytokines and lack of targeted therapies. N-acylethanolamine acid amidase (NAAA) is an N-terminal cysteine hydrolase that promotes inflammatory responses through the deactivation of Palmitoylethanolamide (PEA), an endogenous bioactive lipid mediator. Here, we examined NAAA expression in TNBC cells (MDA-MB-231 and MDA-MB-BrM2 cells) and the effects of NAAA inhibition on TNBC tumor growth, using a selective NAAA inhibitor AM11095 (IC50 = 20 nM). TNBC cells expressed elevated levels of full-length and splice mRNAs naaa variants. TNBC cells also express the N-acyl ethanol amides and elevated levels of the two fatty acid cores arachidonic (AA) and docosahexaenoic (DHA). PEA or AM11095 inhibited the secretion of IL-6 and IL-8, reduced the activation of the NF-kB pathway, decreased the expression of VEGF and Placental growth factor (PLGF) in TNBCs, and inhibited tumor cell migration in vitro. Using cellular magnetic resonance imaging (MRI), body images of mice administered with human MDA-MB-BrM2 cells treated with AM11095 showed a significant decrease in tumor numbers with a lower volume of tumors and increased mice survival. Mice untreated or treated with vehicle control showed a high number of tumors with high volumes in multiple organs. Thus, NAAA inhibition may constitute a potential therapeutic approach in the management of TNBC-associated inflammation and tumor growth.

Inhibition of triple negative breast cancer-associated inflammation, tumor growth and brain colonization by targeting monoacylglycerol lipase.

While the prevalence of breast cancer metastasis in the brain is significantly higher in triple negative breast cancers (TNBCs), there is a lack of novel and/or improved therapies for these patients. Monoacylglycerol lipase (MAGL) is a hydrolase involved in lipid metabolism that catalyzes the degradation of 2-arachidonoylglycerol (2-AG) linked to generation of pro- and anti-inflammatory molecules. Here, we targeted MAGL in TNBCs, using a potent carbamate-based inhibitor AM9928 (hMAGL IC50 = 9 nM) with prolonged pharmacodynamic effects (46 h of target residence time). AM9928 blocked TNBC cell adhesion and transmigration across human brain microvascular endothelial cells (HBMECs) in 3D co-cultures. In addition, AM9928 inhibited the secretion of IL-6, IL-8, and VEGF-A from TNBC cells. TNBC-derived exosomes activated HBMECs resulting in secretion of elevated levels of IL-8 and VEGF, which were inhibited by AM9928. Using in vivo studies of syngeneic GFP-4T1-BrM5 mammary tumor cells, AM9928 inhibited tumor growth in the mammary fat pads and attenuated blood brain barrier (BBB) permeability changes, resulting in reduced TNBC colonization in brain. Together, these results support the potential clinical application of MAGL inhibitors as novel treatments for TNBC.

N-Acylethanolamine Acid Amidase Inhibition Potentiates Morphine Analgesia and Delays the Development of Tolerance.

Opioids are essential drugs for pain management, although long-term use is accompanied by tolerance, necessitating dose escalation, and dependence. Pharmacological treatments that enhance opioid analgesic effects and/or attenuate the development of tolerance (with a desirable opioid-sparing effect in treating pain) are actively sought. Among them, N-palmitoylethanolamide (PEA), an endogenous lipid neuromodulator with anti-inflammatory and neuroprotective properties, was shown to exert anti-hyperalgesic effects and to delay the emergence of morphine tolerance. A selective augmentation in endogenous PEA levels can be achieved by inhibiting N-acylethanolamine acid amidase (NAAA), one of its primary hydrolyzing enzymes. This study aimed to test the hypothesis that NAAA inhibition, with the novel brain permeable NAAA inhibitor AM11095, modulates morphine's antinociceptive effects and attenuates the development of morphine tolerance in rats. We tested this hypothesis by measuring the pain threshold to noxious mechanical stimuli and, as a neural correlate, we conducted in vivo electrophysiological recordings from pain-sensitive locus coeruleus (LC) noradrenergic neurons in anesthetized rats. AM11095 dose-dependently (3-30 mg/kg) enhanced the antinociceptive effects of morphine and delayed the development of tolerance to chronic morphine in behaving rats. Consistently, AM11095 enhanced morphine-induced attenuation of the response of LC neurons to foot-shocks and prevented the attenuation of morphine effects following chronic treatment. Behavioral and electrophysiological effects of AM11095 on chronic morphine were paralleled by a decrease in glial activation in the spinal cord, an index of opioid-induced neuroinflammation. NAAA inhibition might represent a potential novel therapeutic approach to increase the analgesic effects of opioids and delay the development of tolerance.

Design and Structure-Activity Relationships of Isothiocyanates as Potent and Selective N-Acylethanolamine-Hydrolyzing Acid Amidase Inhibitors.

N-Acylethanolamines are signaling lipid molecules implicated in pathophysiological conditions associated with inflammation and pain. N-Acylethanolamine acid amidase (NAAA) favorably hydrolyzes lipid palmitoylethanolamide, which plays a key role in the regulation of inflammatory and pain processes. The synthesis and structure-activity relationship studies encompassing the isothiocyanate pharmacophore have produced potent low nanomolar inhibitors for hNAAA, while exhibiting high selectivity (>100-fold) against other serine hydrolases and cysteine peptidases. We have followed a target-based structure-activity relationship approach, supported by computational methods and known cocrystals of hNAAA. We have identified systemically active inhibitors with good plasma stability (t1/2 > 2 h) and microsomal stability (t1/2 ∼ 15-30 min) as pharmacological tools to investigate the role of NAAA in inflammation, pain, and drug addiction.

N-acylethanolamine-hydrolysing acid amidase: A new potential target to treat paclitaxel-induced neuropathy.

BACKGROUND: Although paclitaxel is an effective chemotherapeutic agent used to treat multiple types of cancer (e.g. breast, ovarian, neck and lung), it also elicits paclitaxel-induced peripheral neuropathy (PIPN), which represents a major dose-limiting side effect of this drug. METHODS: As the endogenously produced N-acylethanolamine, palmitoylethanolamide (PEA), reverses paclitaxel-induced mechanical hypersensitivity in mice, the main goals of this study were to examine if paclitaxel affects levels of endogenous PEA in the spinal cord of mice and whether exogenous administration of PEA provides protection from the occurrence of paclitaxel-induced mechanical hypersensitivity. We further examined whether inhibition of N-acylethanolamine-hydrolysing acid amidase (NAAA), a hydrolytic PEA enzyme, would offer protection in mouse model of PIPN. RESULTS: Paclitaxel reduced PEA levels in the spinal cord, suggesting that dysregulation of this lipid signalling system may contribute to PIPN. Consistent with this idea, repeated administration of PEA partially prevented the paclitaxel-induced mechanical hypersensitivity. We next evaluated whether the selective NAAA inhibitor, AM9053, would prevent paclitaxel-induced mechanical hypersensitivity in mice. Acute administration of AM9053 dose-dependently reversed mechanical hypersensitivity through a PPAR-α mechanism, whereas repeated administration of AM9053 fully prevented the development of PIPN, without any evidence of tolerance. Moreover, AM9053 produced a conditioned place preference in paclitaxel-treated mice, but not in control mice. This pattern of findings suggests a lack of intrinsic rewarding effects, but a reduction in the pain aversiveness induced by paclitaxel. Finally, AM9053 did not alter paclitaxel-induced cytotoxicity in lung tumour cells. CONCLUSIONS: Collectively, these studies suggest that NAAA represents a promising target to treat and prevent PIPN. SIGNIFICANCE: The present study demonstrates that the chemotherapeutic paclitaxel alters PEA levels in the spinal cord, whereas repeated exogenous PEA administration moderately alleviates PIPN in mice. Additionally, targeting NAAA, PEA's hydrolysing enzyme with a selective compound AM9053 reverses and prevents the PIPN via the PPAR-α mechanism. Overall, the data suggest that selective NAAA inhibitors denote promising future therapeutics to mitigate and prevent PIPN.

The endocannabinoid 2-arachidonoylglycerol and dual ABHD6/MAGL enzyme inhibitors display neuroprotective and anti-inflammatory actions in the in vivo retinal model of AMPA excitotoxicity.

The endocannabinoid system has been shown to be a putative therapeutic target for retinal disease. Here, we aimed to investigate the ability of the endocannabinoid 2-arachidonoylglycerol (2-AG) and novel inhibitors of its metabolic enzymes, α/ß-hydrolase domain-containing 6 (ABHD6) and monoacylglycerol lipase (MAGL), a) to protect the retina against excitotoxicity and b) the mechanisms involved in the neuroprotection. Sprague-Dawley rats, wild type and Akt2-/- C57BL/6 mice were intravitreally administered with phosphate-buffered saline or (RS)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide (AMPA). 2-AG was intravitreally co-administered with AMPA in the absence and presence of AM251 or AM630 (cannabinoid 1 and 2 receptor antagonists, respectively) or Wortmannin [Phosphoinositide 3-Kinase (PI3K)/Akt inhibitor]. Inhibitors of ABHD6 and dual ABHD6/MAGL (AM12100 and AM11920, respectively) were co-administered with AMPA intravitreally in rats. Immunohistochemistry was performed using antibodies raised against retinal neuronal markers (bNOS), microglia (Iba1) and macroglia (GFAP). TUNEL assay and real-time PCR were also employed. The CB2 receptor was expressed in rat retina (approx. 62% of CB1 expression). 2-AG attenuated the AMPA-induced increase in TUNEL+ cells. 2-AG activation of both CB1 and CB2 receptors and the PI3K/Akt downstream signaling pathway, as substantiated by the use of Akt2-/- mice, afforded neuroprotection against AMPA excitotoxicity. AM12100 and AM11920 attenuated the AMPA-induced glia activation and produced a dose-dependent partial neuroprotection, with the dual inhibitor AM11920 being more efficacious. These results show that 2-AG has the pharmacological profile of a putative therapeutic for retinal diseases characterized by neurodegeneration and neuroinflammation, when administered exogenously or by the inhibition of its metabolic enzymes.

Endocannabinoid metabolism and transport as targets to regulate intraocular pressure.

Cannabinoids are part of an endogenous signaling system found throughout the body, including the eye. Hepler and Frank showed in the early 1970s that plant cannabinoids can lower intraocular pressure (IOP), an effect since shown to occur via cannabinoid CB1 and GPR18 receptors. Endocannabinoids are synthesized and metabolized enzymatically. Enzymes implicated in endocannabinoids breakdown include monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), but also ABHD12, NAAA, and COX-2. Inhibition of MAGL activity raises levels of the endocannabinoid 2-arachidonoyl glycerol and substantially lowers IOP. Blocking other cannabinoid metabolizing enzymes or cannabinoid transporters may similarly contribute to lowering IOP and so serve as therapeutic targets for treating glaucoma. We have tested blockers for several cannabinoid-metabolizing enzymes and transporters (FABP5 and membrane reuptake) for their ability to alter ocular pressure in a murine model of IOP. Of FAAH, ABHD12, NAAA, and COX2, only FAAH was seen to play a role in regulation of IOP. Only the FAAH blocker URB597 lowered IOP, but in a temporally, diurnally, and sex-specific manner. We also tested two blockers of cannabinoid transport (SBFI-26 and WOBE437), finding that each lowered IOP in a CB1-dependent manner. Though we see a modest, limited role for FAAH, our results suggest that MAGL is the primary cannabinoid-metabolizing enzyme in regulating ocular pressure, thus pointing towards a role of 2-arachidonoyl glycerol. Interestingly, inhibition of cannabinoid transport mechanisms independent of hydrolysis may prove to be an alternative strategy to lower ocular pressure.

Design and synthesis of cyanamides as potent and selective N-acylethanolamine acid amidase inhibitors.

N-acylethanolamine acid amidase (NAAA) inhibition represents an exciting novel approach to treat inflammation and pain. NAAA is a cysteine amidase which preferentially hydrolyzes the endogenous biolipids palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). PEA is an endogenous agonist of the nuclear peroxisome proliferator-activated receptor-α (PPAR-α), which is a key regulator of inflammation and pain. Thus, blocking the degradation of PEA with NAAA inhibitors results in augmentation of the PEA/PPAR-α signaling pathway and regulation of inflammatory and pain processes. We have prepared a new series of NAAA inhibitors exploring the azetidine-nitrile (cyanamide) pharmacophore that led to the discovery of highly potent and selective compounds. Key analogs demonstrated single-digit nanomolar potency for hNAAA and showed >100-fold selectivity against serine hydrolases FAAH, MGL and ABHD6, and cysteine protease cathepsin K. Additionally, we have identified potent and selective dual NAAA-FAAH inhibitors to investigate a potential synergism between two distinct anti-inflammatory molecular pathways, the PEA/PPAR-α anti-inflammatory signaling pathway,1-4 and the cannabinoid receptors CB1 and CB2 pathways which are known for their antiinflammatory and antinociceptive properties.5-8 Our ligand design strategy followed a traditional structure-activity relationship (SAR) approach and was supported by molecular modeling studies of reported X-ray structures of hNAAA. Several inhibitors were evaluated in stability assays and demonstrated very good plasma stability (t1/2 > 2 h; human and rodents). The disclosed cyanamides represent promising new pharmacological tools to investigate the potential role of NAAA inhibitors and dual NAAA-FAAH inhibitors as therapeutic agents for the treatment of inflammation and pain.

Piperidine and piperazine inhibitors of fatty acid amide hydrolase targeting excitotoxic pathology.

FAAH inhibitors offer safety advantages by augmenting the anandamide levels "on demand" to promote neuroprotective mechanisms without the adverse psychotropic effects usually seen with direct and chronic activation of the CB1 receptor. FAAH is an enzyme implicated in the hydrolysis of the endocannabinoid N-arachidonoylethanolamine (AEA), which is a partial agonist of the CB1 receptor. Herein, we report the discovery of a new series of highly potent and selective carbamate FAAH inhibitors and their evaluation for neuroprotection. The new inhibitors showed potent nanomolar inhibitory activity against human recombinant and purified rat FAAH, were selective (>1000-fold) against serine hydrolases MGL and ABHD6 and lacked any affinity for the cannabinoid receptors CB1 and CB2. Evaluation of FAAH inhibitors 9 and 31 using the in vitro competitive activity-based protein profiling (ABPP) assay confirmed that both inhibitors were highly selective for FAAH in the brain, since none of the other FP-reactive serine hydrolases in this tissue were inhibited by these agents. Our design strategy followed a traditional SAR approach and was supported by molecular modeling studies based on known FAAH cocrystal structures. To rationally design new molecules that are irreversibly bound to FAAH, we have constructed "precovalent" FAAH-ligand complexes to identify good binding geometries of the ligands within the binding pocket of FAAH and then calculated covalent docking poses to select compounds for synthesis. FAAH inhibitors 9 and 31 were evaluated for neuroprotection in rat hippocampal slice cultures. In the brain tissue, both inhibitors displayed protection against synaptic deterioration produced by kainic acid-induced excitotoxicity. Thus, the resultant compounds produced through rational design are providing early leads for developing therapeutics against seizure-related damage associated with a variety of disorders.

The novel cannabinoid CB1 receptor agonist AM11101 increases food intake in female rats.

BACKGROUND AND PURPOSE: Δ9 -tetrahydrocannabinol (THC) acts via cannabinoid CB1 receptors to increase feeding. Here, we assessed the orexigenic effect of AM11101, a novel CB1 receptor agonist designed to have a more favourable pharmacodynamic profile than THC. EXPERIMENTAL APPROACH: The acute, orexigenic effects of AM11101 and THC were compared in female rats. Food intake and meal patterns were also examined following once daily treatment with AM11101 and THC for 7 days. KEY RESULTS: AM11101 (0.01-0.1 mg·kg-1 ) increased food intake during the first hour following both acute and chronic treatments in pre-fed and freely feeding animals. This orexigenic effect persisted for up to 4 hr, with no compensatory decrease in feeding during the subsequent 4-22 hr. THC (1 mg·kg-1 ) increased 1-hr food intake in pre-fed animals, but was less reliable than AM11101 in increasing 1-hr food intake in freely feeding animals following both acute and chronic administration. The orexigenic effect of both compounds was due to an increase in meal size, not meal number. CONCLUSIONS AND IMPLICATIONS: Our study provides the first demonstration that AM11101 increases short-term food intake via a selective increase in meal size. AM11101 promotes a more reliable orexigenic effect than THC in freely feeding animals, with no subsequent compensatory decrease in feeding. AM11101 may offer a greater efficacy than THC and its congeners in stimulating food intake in underweight clinical populations.

Inhibition of N-acylethanolamine acid amidase reduces nicotine-induced dopamine activation and reward.

Tobacco smoke is the leading preventable cause of death in the world and treatments aimed to increase success rate in smoking cessation by reducing nicotine dependence are sought. Activation of peroxisome proliferator-activated receptor-alpha (PPARα) by synthetic or endogenous agonists was shown to suppress nicotine-induced activation of mesolimbic dopamine system, one of the major neurobiological substrates of nicotine dependence, and nicotine-seeking behavior in rats and monkeys. An alternative indirect way to activate PPARα is inhibition of N-acylethanolamine acid amidase (NAAA), one of the major hydrolyzing enzyme for its endogenous agonists palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). We synthetized a novel specific brain permeable NAAA inhibitor, AM11095. We administered AM11095 to rats and carried out brain lipid analysis, a functional observational battery (FOB) to assess toxicity, in vivo electrophysiological recording from dopamine cells in the ventral tegmental area, brain microdialysis in the nucleus accumbens shell and behavioral experiments to assess its effect on nicotine -induced conditioned place preference (CPP). AM11095 (5 and 25 mg/kg, i.p.) was devoid of neurotoxic and behavioral effects and did not affect motor behavior and coordination. This NAAA inhibitor (5 mg/kg i.p.) increased OEA and PEA levels in the hippocampus and cortex, prevented nicotine-induced activation of mesolimbic dopamine neurons in the ventral tegmental area, nicotine-induced elevation of dopamine levels in the nucleus accumbens shell and decreased the expression of nicotine CPP. Our results indicate that NAAA inhibitors represent a new class of pharmacological tools to modulate brain PEA/PPARα signalling and show potential in the treatment of nicotine dependence.

Synthesis and evaluation of potent and selective MGL inhibitors as a glaucoma treatment.

Monoacylglycerol lipase (MGL) inhibition provides a potential treatment approach to glaucoma through the regulation of ocular 2-arachidonoylglycerol (2-AG) levels and the activation of CB1 receptors. Herein, we report the discovery of new series of carbamates as highly potent and selective MGL inhibitors. The new inhibitors showed potent nanomolar inhibitory activity against recombinant human and purified rat MGL, were selective (>1000-fold) against serine hydrolases FAAH and ABHD6 and lacked any affinity for the cannabinoid receptors CB1 and CB2. Protein-based 1H NMR experiments indicated that inhibitor 2 rapidly formed a covalent adduct with MGL with a residence time of about 6 h. This interconversion process "intrinsic reversibility" was exploited by modifications of the ligand's size (length and bulkiness) to generate analogs with "tunable' adduct residence time (τ). Inhibitor 2 was evaluated in a normotensive murine model for assessing intraocular pressure (IOP), which could lead to glaucoma, a major cause of blindness. Inhibitor 2 was found to decrease ocular pressure by ∼4.5 mmHg in a sustained manner for at least 12 h after a single ocular application, underscoring the potential for topically-administered MGL inhibitors as a novel therapeutic target for the treatment of glaucoma.

Oximes short-acting CB1 receptor agonists.

New oximes short-acting CB1 agonists were explored by the introduction of an internal oxime and polar groups at the C3 alkyl tail of Δ8-THC. The scope of the research was to drastically alter two important physicochemical properties hydrophobicity (log P) and topological surface area (tPSA) of the compound, which play a critical role in tissue distribution and sequestration (depot effect). Key synthesized analogs demonstrated sub-nanomolar affinity for CB1, marked reduction in hydrophobicity (ClogP∼2.5-3.5 vs 9.09 of Δ8-THC-DMH), and found to function as either agonists (trans-oximes) or neutral antagonists (cis-oximes) in a cAMP functional assay. All oxime analogs showed comparable affinity at the CB2 receptor, but surprisingly they were found to function as inverse agonists for CB2. In behavioral studies (i.e. analgesia, hypothermia) trans-oxime 8a exhibited a predictable fast onset (∼20 min) and short duration of pharmacological action (∼180 min), in contrast to the very prolonged duration of Δ8-THC-DMH (>24 h), thus limiting the potential for severe psychotropic side-effects associated with persistent activation of the CB1 receptor. We have conducted 100 ns molecular dynamic (MD) simulations of CB1 complexes with AM11542 (CB1 agonist) and both trans-8a and cis-8b isomeric oximes. These studies revealed that the C3 alkyl tail of cis-8b orientated within the CB1 binding pocket in a manner that triggered a conformational change that stabilized the CB1 receptor at its inactive-state (antagonistic functional effect). In contrast, the trans-8a isomer's conformation was coincided with that of the AM11542 CB1 agonist-bound structure, stabilizing the CB1 receptor at the active-state (agonistic functional effect). We have selected oxime trans-8a based on its potency for CB1, and favorable pharmacodynamic profile, such as fast onset and predictable duration of pharmacological action, for evaluation in pre-clinical models of anorexia nervosa.

Novel triazines as potent and selective phosphodiesterase 10A inhibitors.

The identification of highly potent and orally active triazines for the inhibition of PDE10A is reported. The new analogs exhibit low-nanomolar potency for PDE10A, demonstrate high selectivity against all other members of the PDE family, and show desired drug-like properties. Employing structure-based drug design approaches, we investigated the selectivity of PDE10A inhibitors against other known PDE isoforms, by methodically exploring the various sub-regions of the PDE10A ligand binding pocket. A systematic assessment of the ADME and pharmacokinetic properties of the newly synthesized compounds has led to the design of drug-like candidates with good brain permeability and desirable drug kinetics (t(1/2), bioavailability, clearance). Compound 66 was highly potent for PDE10A (IC(50)=1.4 nM), demonstrated high selectivity (>200×) for the other PDEs, and was efficacious in animal models of psychoses; reversal of MK-801 induced hyperactivity (MED=0.1mg/kg) and conditioned avoidance responding (CAR; ID(50)=0.2 mg/kg).

Highly potent, selective, and orally active phosphodiesterase 10A inhibitors.

The identification of highly potent and orally active phenylpyrazines for the inhibition of PDE10A is reported. The new analogues exhibit subnanomolar potency for PDE10A, demonstrate high selectivity against all other members of the PDE family, and show desired druglike properties. Employing structure-based drug design approaches, we methodically explored two key regions of the binding pocket of the PDE10A enzyme to alter the planarity of the parent compound 1 and optimize its affinity for PDE10A. Bulky substituents at the C9 position led to elimination of the mutagenicity of 1, while a crucial hydrogen bond interaction with Glu716 markedly enhanced its potency and selectivity. A systematic assessment of the ADME and PK properties of the new analogues led to druglike development candidates. One of the more potent compounds, 96, displayed an IC(50) for PDE10A of 0.7 nM and was active in predictive antipsychotic animal models.

New pyrazolyl and thienyl aminohydantoins as potent BACE1 inhibitors: exploring the S2' region.

The proteolytic enzyme ß-secretase (BACE1) plays a central role in the synthesis of the pathogenic ß-amyloid in Alzheimer's disease. SAR studies of the S2' region of the BACE1 ligand binding pocket with pyrazolyl and thienyl P2' side chains are reported. These analogs exhibit low nanomolar potency for BACE1, and demonstrate >50- to 100-fold selectivity for the structurally related aspartyl proteases BACE2 and cathepsin D. Small groups attached at the nitrogen of the P2' pyrazolyl moiety, together with the P3 pyrimidine nucleus projecting into the S3 region of the binding pocket, are critical components to ligand's potency and selectivity. P2' thiophene side chain analogs are highly potent BACE1 inhibitors with excellent selectivity against cathepsin D, but only modest selectivity against BACE2. The cell-based activity of these new analogs tracked well with their increased molecular binding with EC(50) values of 0.07-0.2 µM in the ELISA assay for the most potent analogs.

Design and synthesis of aminohydantoins as potent and selective human ß-secretase (BACE1) inhibitors with enhanced brain permeability.

The identification of small molecule aminohydantoins as potent and selective human ß-secretase inhibitors is reported. These analogs exhibit good brain permeability (40-70%), low nanomolar potency for BACE1, and demonstrate >100-fold selectivity for the structurally related aspartyl proteases cathepsin D, renin and pepsin. Alkyl and alkoxy groups at the meta-position of the P1 phenyl, which extend toward the S3 region of the enzyme, have contributed to the ligand's reduced affinity for the efflux transporter protein P-gp, and decreased topological polar surface area, thus resulting in enhanced brain permeability. A fluorine substitution at the para-position of the P1 phenyl has contributed to 100-fold decrease of CYP3A4 inhibition and enhancement of compound metabolic stability. The plasma and brain protein binding properties of these new analogs are affected by substitutions at the P1 phenyl moiety. Higher compound protein binding was observed in the brain than in the plasma. Two structurally diverse potent BACE1 inhibitors (84 and 89) reduced 30% plasma Aß40 in the Tg2576 mice in vivo model at 30 mg/kg p.o..

Discovery of imidazo[1,5-a]pyrido[3,2-e]pyrazines as a new class of phosphodiesterase 10A inhibitiors.

Novel imidazo[1,5-a]pyrido[3,2-e]pyrazines have been synthesized and characterized as both potent and selective phosphodiesterase 10A (PDE10A) inhibitors. For in vitro characterization, inhibition of PDE10A mediated cAMP hydrolysis was used and a QSAR model was established to analyze substitution effects. The outcome of this analysis was complemented by the crystal structure of PDE10A in complex with compound 49. Qualitatively new interactions between inhibitor and binding site were found, contrasting with previously published crystal structures of papaverine-like inhibitors. In accordance with the known antipsychotic potential of PDE10A inhibitors, MK-801 induced stereotypy and hyperactivity in rats were reversed by selected compounds. Thus, a promising compound class has been identified for the treatment of schizophrenia that could circumvent side effects connected with current therapies.