Inhibitor Report for: JJKK-048

Triple negative breast cancer (TNBC) is among the most aggressive and deadly cancer subtypes. Intra-tumoral hypoxia is associated with aggressiveness and drug resistance in TNBC. One of the underlying mechanisms of hypoxia-induced drug resistance is the elevated expression of efflux transporters such as breast cancer resistant protein (ABCG2). In the present study, we investigated the possibility of ameliorating ABCG2-mediated drug resistance in hypoxic TNBC cells by monoacylglycerol lipase (MAGL) inhibition and the consequent downregulation of ABCG2 expression. The effect of MAGL inhibition on ABCG2 expression, function, and efficacy of regorafenib, an ABCG2 substrate was investigated in cobalt dichloride (CoCl(2)) induced pseudohypoxic TNBC (MDA-MB-231) cells, using quantitative targeted absolute proteomics, qRT-PCR, anti-cancer drug accumulation in the cells, cell invasiveness and resazurin-based cell viability assays. Our results showed that hypoxia-induced ABCG2 expression led to low regorafenib intracellular concentrations, reduced the anti-invasiveness efficacy, and elevated half maximal inhibitory concentration (IC(50)) of regorafenib in vitro MDA-MB-231 cells. MAGL inhibitor, JJKK048, reduced ABCG2 expression, increased regorafenib cell accumulation, which led to higher regorafenib efficacy. In conclusion, hypoxia-induced regorafenib resistance due to ABCG2 over-expression in TNBC cells can be ameliorated by MAGL inhibition.

Monoacylglycerol lipase (MAGL) is a serine hydrolase that acts as a principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). In addition to terminating the signaling function of 2-AG, MAGL liberates arachidonic acid to be used as a primary source for neuroinflammatory prostaglandin synthesis in the brain. MAGL activity also contributes to cancer pathogenicity by producing precursors for tumor-promoting bioactive lipids. Pharmacological inhibitors of MAGL provide valuable tools for characterization of MAGL and 2-AG signaling pathways. They also hold great therapeutic potential to treat several pathophysiological conditions, such as pain, neurodegenerative disorders, and cancer. We have previously reported piperidine triazole urea, {4-[bis-(benzo[d][1,3]dioxol-5-yl)methyl]-piperidin-1-yl}(1H-1,2,4-triazol-1-yl)m ethanone (JJKK-048), to be an ultrapotent and highly selective inhibitor of MAGL in vitro. Here, we characterize in vivo effects of JJKK-048. Acute in vivo administration of JJKK-048 induced a massive increase in mouse brain 2-AG levels without affecting brain anandamide levels. JJKK-048 appeared to be extremely potent in vivo. Activity-based protein profiling revealed that JJKK-048 maintains good selectivity toward MAGL over other serine hydrolases. Our results are also the first to show that JJKK-048 promoted significant analgesia in a writhing test with a low dose that did not cause cannabimimetic side effects. At a high dose, JJKK-048 induced analgesia both in the writhing test and in the tail-immersion test, as well as hypomotility and hyperthermia, but not catalepsy.

Monoacylglycerol lipase (MAGL) terminates the signaling function of the endocannabinoid, 2-arachidonoylglycerol (2-AG). During 2-AG hydrolysis, MAGL liberates arachidonic acid, feeding the principal substrate for the neuroinflammatory prostaglandins. In cancer cells, MAGL redirects lipid stores toward protumorigenic signaling lipids. Thus MAGL inhibitors may have great therapeutic potential. Although potent and increasingly selective MAGL inhibitors have been described, their number is still limited. Here, we have characterized piperazine and piperidine triazole ureas that combine the high potency attributable to the triazole leaving group together with the bulky aromatic benzodioxolyl moiety required for selectivity, culminating in compound JJKK-048 that potently (IC50 < 0.4 nM) inhibited human and rodent MAGL. JJKK-048 displayed low cross-reactivity with other endocannabinoid targets. Activity-based protein profiling of mouse brain and human melanoma cell proteomes suggested high specificity also among the metabolic serine hydrolases.

Triple negative breast cancer (TNBC) is among the most aggressive and deadly cancer subtypes. Intra-tumoral hypoxia is associated with aggressiveness and drug resistance in TNBC. One of the underlying mechanisms of hypoxia-induced drug resistance is the elevated expression of efflux transporters such as breast cancer resistant protein (ABCG2). In the present study, we investigated the possibility of ameliorating ABCG2-mediated drug resistance in hypoxic TNBC cells by monoacylglycerol lipase (MAGL) inhibition and the consequent downregulation of ABCG2 expression. The effect of MAGL inhibition on ABCG2 expression, function, and efficacy of regorafenib, an ABCG2 substrate was investigated in cobalt dichloride (CoCl(2)) induced pseudohypoxic TNBC (MDA-MB-231) cells, using quantitative targeted absolute proteomics, qRT-PCR, anti-cancer drug accumulation in the cells, cell invasiveness and resazurin-based cell viability assays. Our results showed that hypoxia-induced ABCG2 expression led to low regorafenib intracellular concentrations, reduced the anti-invasiveness efficacy, and elevated half maximal inhibitory concentration (IC(50)) of regorafenib in vitro MDA-MB-231 cells. MAGL inhibitor, JJKK048, reduced ABCG2 expression, increased regorafenib cell accumulation, which led to higher regorafenib efficacy. In conclusion, hypoxia-induced regorafenib resistance due to ABCG2 over-expression in TNBC cells can be ameliorated by MAGL inhibition.

2-Arachidonoylglycerol (2-AG) and anandamide are two major endocannabinoids produced, released and eliminated by metabolic pathways. Anticonvulsive effect of 2-AG and CB1 receptor is well-established. Herein, we designed to investigate the anticonvulsive influence of key components of the 2-AG and anandamide metabolism. Tonic-clonic seizures were induced by an injection of Pentylenetetrazol (80 mg/kg, i.p.) in adult male Wistar rats. Delay and duration for the seizure stages were considered for analysis. Monoacylglycerol lipase blocker (JJKK048; 1 mg/kg) or alpha/beta hydroxylase domain 6 blocker (WWL70; 5 mg/kg) were administrated alone or with 2-AG to evaluate the anticonvulsive potential of these enzymes. To determine the CB1 receptor involvement, its blocker (MJ15; 3 mg/kg) was administrated associated with JJKK048 or WWL70. To assess anandamide anticonvulsive effect, anandamide membrane transporter blocker (LY21813240; 2.5 mg/kg) was used alone or associated with MJ15. Also, fatty acid amide hydrolase blocker (URB597; 1 mg/kg; to prevent intracellular anandamide hydrolysis) were used alone or with AMG21629 (transient receptor potential vanilloid; TRPV1 antagonist; 3 mg/kg). All compounds were dissolved in DMSO and injected i.p., before the Pentylenetetrazol. Both JJKK048 and WWL70 revealed anticonvulsive effect. Anticonvulsive effect of JJKK048 but not WWL70 was CB1 receptor dependent. LY2183240 showed CB1 receptor dependent anticonvulsive effect. However, URB597 revealed a TRPV1 dependent proconvulsive effect. It seems extracellular accumulation of 2-AG or anandamide has anticonvulsive effect through the CB1 receptor, while intracellular anandamide accumulation is proconvulsive through TRPV1.

Monoacylglycerol lipase (MAGL) is a serine hydrolase that acts as a principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). In addition to terminating the signaling function of 2-AG, MAGL liberates arachidonic acid to be used as a primary source for neuroinflammatory prostaglandin synthesis in the brain. MAGL activity also contributes to cancer pathogenicity by producing precursors for tumor-promoting bioactive lipids. Pharmacological inhibitors of MAGL provide valuable tools for characterization of MAGL and 2-AG signaling pathways. They also hold great therapeutic potential to treat several pathophysiological conditions, such as pain, neurodegenerative disorders, and cancer. We have previously reported piperidine triazole urea, {4-[bis-(benzo[d][1,3]dioxol-5-yl)methyl]-piperidin-1-yl}(1H-1,2,4-triazol-1-yl)m ethanone (JJKK-048), to be an ultrapotent and highly selective inhibitor of MAGL in vitro. Here, we characterize in vivo effects of JJKK-048. Acute in vivo administration of JJKK-048 induced a massive increase in mouse brain 2-AG levels without affecting brain anandamide levels. JJKK-048 appeared to be extremely potent in vivo. Activity-based protein profiling revealed that JJKK-048 maintains good selectivity toward MAGL over other serine hydrolases. Our results are also the first to show that JJKK-048 promoted significant analgesia in a writhing test with a low dose that did not cause cannabimimetic side effects. At a high dose, JJKK-048 induced analgesia both in the writhing test and in the tail-immersion test, as well as hypomotility and hyperthermia, but not catalepsy.

Monoacylglycerol lipase (MAGL) terminates the signaling function of the endocannabinoid, 2-arachidonoylglycerol (2-AG). During 2-AG hydrolysis, MAGL liberates arachidonic acid, feeding the principal substrate for the neuroinflammatory prostaglandins. In cancer cells, MAGL redirects lipid stores toward protumorigenic signaling lipids. Thus MAGL inhibitors may have great therapeutic potential. Although potent and increasingly selective MAGL inhibitors have been described, their number is still limited. Here, we have characterized piperazine and piperidine triazole ureas that combine the high potency attributable to the triazole leaving group together with the bulky aromatic benzodioxolyl moiety required for selectivity, culminating in compound JJKK-048 that potently (IC50 < 0.4 nM) inhibited human and rodent MAGL. JJKK-048 displayed low cross-reactivity with other endocannabinoid targets. Activity-based protein profiling of mouse brain and human melanoma cell proteomes suggested high specificity also among the metabolic serine hydrolases.