Targeting glucosylceramide synthase induces antiproliferative and proapoptotic effects in osimertinib-resistant NSCLC cell models.

The EGFR tyrosine kinase inhibitor osimertinib has been approved for the first-line treatment of EGFR-mutated Non-Small Cell Lung Cancer (NSCLC) patients. Despite its efficacy, patients develop resistance. Mechanisms of resistance are heterogeneous and not fully understood, and their characterization is essential to find new strategies to overcome resistance. Ceramides are well-known regulators of apoptosis and are converted into glucosylceramides (GlcCer) by glucosylceramide synthase (GCS). A higher content of GlcCers was observed in lung pleural effusions from NSCLC patients and their role in osimertinib-resistance has not been documented. The aim of this study was to determine the therapeutic potential of inhibiting GCS in NSCLC EGFR-mutant models resistant to osimertinib in vitro and in vivo. Lipidomic analysis showed a significant increase in the intracellular levels of glycosylceramides, including GlcCers in osimertinib resistant clones compared to sensitive cells. In resistant cells, the GCS inhibitor PDMP caused cell cycle arrest, inhibition of 2D and 3D cell proliferation, colony formation and migration capability, and apoptosis induction. The intratumoral injection of PDMP completely suppressed the growth of OR xenograft models. This study demonstrated that dysregulation of ceramide metabolism is involved in osimertinib-resistance and targeting GCS may be a promising therapeutic strategy for patients progressed to osimertinib.

Social stress-induced depressive-like symptoms and changes in gut microbial and lipidomic profiles are prevented by pharmacological inhibition of FAAH activity in male rats.

Pharmacological inhibition of fatty acid amide hydrolase (FAAH) activity has antidepressant-like effects in preclinical models of stress. In this study, we investigated whether the antidepressant-like effects of FAAH inhibition are associated with corresponding changes in gut microbial and lipidomic profiles, which are emerging as critical components in the pathophysiology of depression. Adult male Wistar rats experienced five weeks of repeated social defeat or control procedure and were treated with the FAAH inhibitor URB694 (0.3 mg/kg/day, i.p.) or vehicle starting from the third week. Repeated social defeat induced the emergence of depressive-like behavioral (sucrose preference reduction and passive coping behaviors in the forced swim test) and neuroendocrine (increased corticosterone levels) changes, which were prevented by URB694 treatment. Repeated social defeat also provoked a significant variation in gut microbiota (changes in the relative abundance of 14 bacterial taxa) and lipidic (e.g., glycerophospholipids) composition. These stress-induced changes were prevented by URB694 treatment. These findings indicate that inhibition of FAAH activity with URB694 blocks the co-occurrence of depressive-like behavioral and neuroendocrine changes and alterations in gut microbial and lipid composition in rats exposed to repeated social defeat. In conclusion, these results suggest that the gut microbiota-lipid crosstalk may represent a novel biological target for FAAH inhibitors to enhance stress resilience.

Enhancement of peripheral fatty acyl ethanolamide signaling prevents stress-induced social avoidance and anxiety-like behaviors in male rats.

RATIONALE: Exposure to traumatic events can lead to alterations in social and anxiety-related behaviors. Emerging evidence suggests that peripheral host-defense processes are implicated in the expression of stress-induced behavioral responses and may be targeted to mitigate the negative sequalae of stress exposure. OBJECTIVES: In this study, we used the peripherally restricted FAAH inhibitor URB937 to investigate the effects of the fatty acyl ethanolamide (FAE) family of lipid mediators - which include the endocannabinoid anandamide and the endogenous PPAR-α agonists, oleoylethanolamide and palmitoylethanolamide - on behavioral and peripheral biochemical responses to two ethologically distinct rat models of stress. METHODS: Male adult rats were exposed to acute social defeat, a model of psychological stress (Experiment 1), or to the predator odor 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), a test of innate predator-evoked fear (Experiment 2), and subsequently treated with URB937 (1 or 3 mg/kg, intraperitoneal) or vehicle. Behavioral analyses were conducted 24 h (Experiment 1) or 7 days (Experiment 2) after exposure. RESULTS: URB937 administration prevented the emergence of both social avoidance behavior after social defeat stress and anxiety-related behaviors after TMT exposure. Further, URB937 administration blocked social defeat-induced transient increase in plasma concentrations of pro-inflammatory cytokines and the elevation in plasma corticosterone levels observed 24 h after social defeat CONCLUSIONS: Enhancement of peripheral FAAH-regulated lipid signaling prevents the emergence of stress-induced social avoidance and anxiety-like behaviors in male rats through mechanisms that may involve an attenuation of peripheral cytokine release induced by stress exposure.

Combined targeting of fatty acid amide hydrolase and melatonin receptors promotes neuroprotection and stimulates inflammation resolution in rats.

BACKGROUND AND PURPOSE: Devising novel strategies to therapeutically favour inflammation resolution and provide neuroprotection is an unmet clinical need. Enhancing endocannabinoid tone by inhibiting the catabolic enzyme fatty acid amide hydrolase (FAAH), or stimulating melatonin receptors has therapeutic potential to treat neuropathological states in which neuroinflammation plays a central role. EXPERIMENTAL APPROACH: A rodent hippocampal explant model of inflammatory injury was used to assess the effects of UCM1341, a dual-acting compound with FAAH inhibitory action and agonist activity at melatonin receptors, against neuroinflammatory damage. FAAH activity was measured by a radiometric assay, and N-acylethanolamine levels were assessed by HPLC-MS/MS methods. FAAH distribution, evolution of inflammation and the contribution of UCM1341 to the expression of proteins controlling macrophage behaviour were investigated by biochemical and confocal analyses. KEY RESULTS: UCM1341 exhibited greater neuroprotection against neuroinflammatory degeneration, compared with the reference compounds URB597 (FAAH inhibitor) and melatonin. During neuroinflammation, UCM1341 augmented the levels of anandamide and N-oleoylethanolamine, but not N-palmitoylethanolamine, up-regulated PPAR-α levels, attenuated demyelination and prevented the release of TNF-α. UCM1341 modulated inflammatory responses by contributing to microglia/macrophage polarization, stimulating formation of lipid-laden macrophages and regulating expression of proteins controlling cholesterol metabolism and efflux. The neuroprotective effects of UCM1341 were prevented by PPARα, TRPV1 and melatonin receptor antagonists. CONCLUSION AND IMPLICATIONS: UCM1341, by enhancing endocannabinoid and melatoninergic signalling, benefits neuroprotection and stimulates inflammation resolution pathways. Our findings provide an encouraging prospect of therapeutically targeting endocannabinoid and melatoninergic systems in inflammatory demyelinating states in the CNS.

Expanding the knowledge around antitubercular 5-(2-aminothiazol-4-yl)isoxazole-3-carboxamides: Hit-to-lead optimization and release of a novel antitubercular chemotype via scaffold derivatization.

Tuberculosis is one of the deadliest infectious diseases in the world, and the increased number of multidrug-resistant and extensively drug-resistant strains is a reason for concern. We have previously reported a series of substituted 5-(2-aminothiazol-4-yl)isoxazole-3-carboxamides with growth inhibitory activity against Mycobacterium tuberculosis strains and low propensity to be substrate of efflux pumps. Encouraged by these preliminary results, we have undertaken a medicinal chemistry campaign to determine the metabolic fate of these compounds and to delineate a reliable body of Structure-Activity Relationships. Keeping intact the (thiazol-4-yl)isoxazole-3-carboxamide core, as it is deemed to be the pharmacophore of the molecule, we have extensively explored the structural modifications able to confer good activity and avoid rapid clearance. Also, a small set of analogues based on isostere manipulation of the 2-aminothiazole were prepared and tested, with the aim to disclose novel antitubercular chemotypes. These studies, combined, were instrumental in designing improved compounds such as 42g and 42l, escaping metabolic degradation by human liver microsomes and, at the same time, maintaining good antitubercular activity against both drug-susceptible and drug-resistant strains.

A Sensitive Ultrahigh-Performance Liquid Chromatography/Tandem Mass Spectrometry Method for the Simultaneous Analysis of Phytocannabinoids and Endocannabinoids in Plasma and Brain.

Introduction: Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are major chemical constituents of cannabis, which may interact either directly or indirectly with the endocannabinoid and endocannabinoid-like ("paracannabinoid") systems, two lipid-based signaling complexes that play important roles in physiology. Legislative changes emphasize the need to understand how THC and CBD might impact endocannabinoid and paracannabinoid signaling, and to develop analytical approaches to study such impact. In this study, we describe a sensitive and accurate method for the simultaneous quantification of THC, its main oxidative metabolites [11-hydroxy-Δ9-THC (11-OH-THC) and 11-nor-9-carboxy-Δ9-THC (11-COOH-THC)], CBD, and a representative set of endocannabinoid [anandamide and 2-arachidonoyl-sn-glycerol (2-AG)] and paracannabinoid [palmitoylethanolamide (PEA) and oleoylethanolamide (OEA)] compounds. Analyte separation relies on the temperature-dependent shape selectivity properties of polymerically bonded C18 stationary phases. Materials and Methods: Analytes are extracted from tissues using acetonitrile precipitation followed by phospholipid removal. The ultrahigh-performance liquid chromatography/tandem mass spectrometry protocol utilizes a commercially available C18 polymeric-bonded phase column and a simple gradient elution system. Results: Ten-point calibration curves show excellent linearity (R2>0.99) over a wide range of analyte concentrations (0.02-500 ng/mL). Lowest limits of quantification are 0.05 ng/mL for anandamide, 0.1 ng/mL for 11-OH-THC and OEA, 0.2 ng/mL for THC and CBD, 0.5 ng/mL for 11-COOH-THC, 1.0 ng/mL for 2-AG, and 2.0 ng/mL for PEA. The lowest limits of detection are 0.02 ng/mL for anandamide, 0.05 ng/mL for 11-OH-THC and OEA, 0.1 ng/mL for THC and CBD, 0.2 ng/mL for 11-COOH-THC, 0.5 ng/mL for 2-AG, and 1.0 ng/mL for PEA. Conclusions: An application of the method is presented, which showed that phytocannabinoid administration elevates endocannabinoid levels in plasma and brain of adolescent male and female mice.

Identification of Human Alanine-Glyoxylate Aminotransferase Ligands as Pharmacological Chaperones for Variants Associated with Primary Hyperoxaluria Type 1.

Primary hyperoxaluria type I (PH1) is a rare kidney disease due to the deficit of alanine:glyoxylate aminotransferase (AGT), a pyridoxal-5'-phosphate-dependent enzyme responsible for liver glyoxylate detoxification, which in turn prevents oxalate formation and precipitation as kidney stones. Many PH1-associated missense mutations cause AGT misfolding. Therefore, the use of pharmacological chaperones (PCs), small molecules that promote correct folding, represents a useful therapeutic option. To identify ligands acting as PCs for AGT, we first performed a small screening of commercially available compounds. We tested each molecule by a dual approach aimed at defining the inhibition potency on purified proteins and the chaperone activity in cells expressing a misfolded variant associated with PH1. We then performed a chemical optimization campaign and tested the resulting synthetic molecules using the same approach. Overall, the results allowed us to identify a promising hit compound for AGT and draw conclusions about the requirements for optimal PC activity.

Protein-Protein Interaction Inhibitors Targeting the Eph-Ephrin System with a Focus on Amino Acid Conjugates of Bile Acids.

The role of the Eph-ephrin system in the etiology of pathological conditions has been consolidated throughout the years. In this context, approaches directed against this signaling system, intended to modulate its activity, can be strategic therapeutic opportunities. Currently, the most promising class of compounds able to interfere with the Eph receptor-ephrin protein interaction is composed of synthetic derivatives of bile acids. In the present review, we summarize the progresses achieved, in terms of chemical expansions and structure-activity relationships, both in the steroidal core and the terminal carboxylic acid group, along with the pharmacological characterization for the most promising Eph-ephrin antagonists in in vivo settings.

A sulfonyl fluoride derivative inhibits EGFRL858R/T790M/C797S by covalent modification of the catalytic lysine.

The emergence of the C797S mutation in EGFR is a frequent mechanism of resistance to osimertinib in the treatment of non-small cell lung cancer (NSCLC). In the present work, we report the design, synthesis and biochemical characterization of UPR1444 (compound 11), a new sulfonyl fluoride derivative which potently and irreversibly inhibits EGFRL858R/T790M/C797S through the formation of a sulfonamide bond with the catalytic residue Lys745. Enzymatic assays show that compound 11 displayed an inhibitory activity on EGFRWT comparable to that of osimertinib, and it resulted more selective than the sulfonyl fluoride probe XO44, recently reported to inhibit a significant part of the kinome. Neither compound 11 nor XO44 inhibited EGFRdel19/T790M/C797S triple mutant. When tested in Ba/F3 cells expressing EGFRL858R/T790M/C797S, compound 11 resulted significantly more potent than osimertinib at inhibiting both EGFR autophosphorylation and proliferation, even if the inhibition of EGFR autophosphorylation by compound 11 in Ba/F3 cells was not long lasting.

N-(Anilinoethyl)amide Melatonergic Ligands with Improved Water Solubility and Metabolic Stability.

The MT2 -selective melatonin receptor ligand UCM765 (N-(2-((3-methoxyphenyl)(phenyl)amino)ethyl)acetamide), showed interesting sleep inducing, analgesic and anxiolytic properties in rodents, but suffers from low water solubility and modest metabolic stability. To overcome these limitations, different strategies were investigated, including modification of metabolically liable sites, introduction of hydrophilic substituents and design of more basic derivatives. Thermodynamic solubility, microsomal stability and lipophilicity of new compounds were experimentally evaluated, together with their MT1 and MT2 binding affinities. Introduction of a m-hydroxymethyl substituent on the phenyl ring of UCM765 and replacement of the replacement of the N,N-diphenyl-amino scaffold with a N-methyl-N-phenyl-amino one led to highly soluble compounds with good microsomal stability and receptor binding affinity. Docking studies into the receptor crystal structure provided a rationale for their binding affinity. Pharmacokinetic characterization in rats highlighted higher plasma concentrations for the N-methyl-N-phenyl-amino derivative, consistent with its improved microsomal stability and makes this compound worthy of consideration for further pharmacological investigation.

Chemical modification of NSC12 leads to a specific FGF-trap with antitumor activity in multiple myeloma.

Inhibition of FGF/FGFR signaling is a promising strategy for the treatment of malignances dependent from FGF stimulation, including multiple myeloma (MM). The steroidal derivative NSC12 (compound 1) is a pan-FGF trap endowed with antitumor activity in vivo. Chemical modifications of compound 1 were explored to investigate structure-activity relationships, focusing on the role of the bis(trifluoromethyl)1,3-propanediol chain, the stereochemistry at C20 and functionalization of C3 position. Our studies unveiled compound 25b, the pregnane 3-keto 20R derivative of compound 1 as an effective agent, blocking the proliferation of MM cells in vitro by inhibiting FGF-dependent receptor activation and slowing MM growth in vivo. Importantly, the absence of the hydroxyl group at C3 prevents binding to estrogen receptors, which might concur to the antitumor activity observed for compound 1, leading to a specific FGF/FGFR system inhibitor, and further supporting the role of FGFR in anticancer therapy in MM.

Palladium Catalyst Recycling for Heck-Cassar-Sonogashira Cross-Coupling Reactions in Green Solvent/Base Blend.

The identification of a green, versatile, user-friendly, and efficient methodology is necessary to facilitate the use of Heck-Cassar-Sonogashira (HCS) cross-coupling reaction in drug discovery and industrial production in the pharmaceutical segment. The Heck-Cassar and Sonogashira protocols, using N-hydroxyethylpyrrolidone (HEP)/water/N,N,N',N'-tetramethyl guanidine (TMG) as green solvent/base mixture and sulfonated phosphine ligands, allowed to recycle the catalyst, always guaranteeing high yields and fast conversion under mild conditions, with aryl iodides, bromides, and triflates. No catalyst leakage or metal contamination of the final product were observed during the HCS recycling. To our knowledge, a turnover number (TON) up to 2375, a turnover frequency (TOF) of 158 h-1 , and a process mass intensity (PMI) around 7 that decreased around 3 after solvent, base, and palladium recovery, represent one of the best results to date using a sustainable protocol. The Heck-Cassar protocol using sSPhos was successfully applied to the telescoped synthesis of Erlotinib (TON: 1380; TOF: 46 h-1 ).

Metabolic Soft Spot and Pharmacokinetics: Functionalization of C-3 Position of an Eph-Ephrin Antagonist Featuring a Bile Acid Core as an Effective Strategy to Obtain Oral Bioavailability in Mice.

UniPR129, an L-ß-homotryptophan conjugate of the secondary bile acid lithocholic acid (LCA), acts as an effective protein-protein interaction (PPI) inhibitor of the Eph-ephrin system but suffers from a poor oral bioavailability in mice. To improve UniPR129 bioavailability, a metabolic soft spot, i.e., the 3α-hydroxyl group on the LCA steroidal ring, was functionalized to 3-hydroxyimine. In vitro metabolism of UniPR129 and 3-hydroxyimine derivative UniPR500 was compared in mouse liver subcellular fractions, and main metabolites were profiled by high resolution (HR-MS) and tandem (MS/MS) mass spectrometry. In mouse liver microsomes (MLM), UniPR129 was converted into several metabolites: M1 derived from the oxidation of the 3-hydroxy group to 3-oxo, M2-M7, mono-hydroxylated metabolites, M8-M10, di-hydroxylated metabolites, and M11, a mono-hydroxylated metabolite of M1. Phase II reactions were only minor routes of in vitro biotransformation. UniPR500 shared several metabolic pathways with parent UniPR129, but it showed higher stability in MLM, with a half-life (t1/2) of 60.4 min, if compared to a t1/2 = 16.8 min for UniPR129. When orally administered to mice at the same dose, UniPR500 showed an increased systemic exposure, maintaining an in vitro valuable pharmacological profile as an EphA2 receptor antagonist and an overall improvement in its physico-chemical profile (solubility, lipophilicity), if compared to UniPR129. The present work highlights an effective strategy for the pharmacokinetic optimization of aminoacid conjugates of bile acids as small molecule Eph-ephrin antagonists.

The GABAB receptor positive allosteric modulator COR659: In vitro metabolism, in vivo pharmacokinetics in rats, synthesis and pharmacological characterization of metabolically protected derivatives.

We report an in vitro phase I metabolism study on COR659 (1), a 2-acylaminothiophene derivative able to suppress alcohol and chocolate self-administration in rats, likely via positive allosteric modulation of the GABAB receptor and antagonism/inverse agonism at the cannabinoid CB1 receptor. Given the identification of the methyl ester group at C-3 of the thiophene ring as a metabolic soft spot, we also report the chemical optimization project aimed to balance metabolic stability with in vitro and in vivo potency on a set of 3-substituted COR659 analogues. High performance liquid chromatography coupled to tandem and high resolution mass spectrometry was employed for the characterization of in vitro metabolism and in vivo pharmacokinetics of COR659 in rats. In vitro [35S]GTPγS binding assays on stimulated GABAB and CB1 receptors, in combination with alcohol and chocolate self-administration experiments in rats, were employed to assess the pharmacological profile of this novel set of analogues, using COR659 as reference compound. Eight metabolites of COR659 were discovered in liver microsomal incubates; two of them (M1, M2) were identified by comparison with synthetic reference standards. M2, oxidation product of methyl group at C-5 of the thiophene ring, was a major metabolite in vitro, but showed a low systemic exposure in vivo. M1, cleavage product of the methyl ester group at C-3, revealed in vitro an unusual mechanism of metabolism by a NADPH-dependent route and, in vivo, it maintained high and persistent levels in plasma, which could represent a potential pharmacokinetic and toxicological issue. In the novel set of COR659 analogues, those bearing branched alkyl substituents on the ester group, showed an improved in vitro metabolic stability (2-4), had an in vitro GABAB PAM (2-4) and/or CB1 partial agonist/antagonist profile (2-3) and maintained the ability to reduce alcohol (2-4) and/or chocolate (4) self-administration in rats. Both PK and PD data ruled out any involvement of metabolite M1 in the in vivo potency of COR659 and 4. The present results, therefore, highlight the importance to design and synthesize novel compounds endowed with the dual activity profile and devoid of metabolic liabilities.

Design, Synthesis, and Physicochemical and Pharmacological Profiling of 7-Hydroxy-5-oxopyrazolo[4,3-b]pyridine-6-carboxamide Derivatives with Antiosteoarthritic Activity In Vivo.

The hallmark of joint diseases, such as osteoarthritis (OA), is pain, originating from both inflammatory and neuropathic components, and compounds able to modulate the signal transduction pathways of the cannabinoid type-2 receptor (CB2R) can represent a helpful option in the treatment of OA. In this perspective, a set of 18 cannabinoid type-2 receptor (CB2R) ligands was developed based on an unprecedented structure. With the aim of improving the physicochemical properties of previously reported 4-hydroxy-2-quinolone-3-carboxamides, a structural optimization program led to the discovery of isosteric 7-hydroxy-5-oxopyrazolo[4,3-b]pyridine-6-carboxamide derivatives. These new compounds are endowed with high affinity for the CB2R and moderate to good selectivity over the cannabinoid type-1 receptor (CB1R), associated with good physicochemical characteristics. As to the functional activity at the CB2R, compounds able to act either as agonists or as inverse agonists/antagonists were discovered. Among them, compound 51 emerged as a potent CB2R agonist able to reduce pain in rats carrying OA induced by injection of monoiodoacetic acid (MIA).

Optimization of EphA2 antagonists based on a lithocholic acid core led to the identification of UniPR505, a new 3α-carbamoyloxy derivative with antiangiogenetic properties.

The EphA2 receptor has been validated in animal models as new target for treating tumors depending on angiogenesis and vasculogenic mimicry. In the present work, we extended our current knowledge on structure-activity relationship (SAR) data of two related classes of antagonists of the EphA2 receptor, namely 5ß-cholan-24-oic acids and 5ß-cholan-24-oyl l-ß-homotryptophan conjugates, with the aim to develop new antiangiogenic compounds able to efficiently prevent the formation of blood vessels. As a result of our exploration, we identified UniPR505, N-[3α-(Ethylcarbamoyl)oxy-5ß-cholan-24-oyl]-l-ß-homo-tryptophan (compound 14), as a submicromolar antagonist of the EphA2 receptor capable to block EphA2 phosphorylation and to inhibit neovascularization in a chorioallantoic membrane (CAM) assay.

Antidepressant-like effects of pharmacological inhibition of FAAH activity in socially isolated female rats.

Pharmacological inhibition of the enzyme fatty acid amide hydrolase (FAAH), which terminates signaling of the endocannabinoid N-arachidonoylethanolamine (or anandamide, AEA), exerts favourable effects in rodent models of stress-related depression. Yet although depression seems to be more common among women than men and in spite of some evidence of sex differences in treatment efficacy, preclinical development of FAAH inhibitors for the pharmacotherapy of stress-related depression has been predominantly conducted in male animals. Here, adult female rats were exposed to six weeks of social isolation and, starting from the second week, treated with the FAAH inhibitor URB694 (0.3 mg/kg/day, i.p.) or vehicle. Compared to pair-housed females, socially isolated female rats treated with vehicle developed behavioral (mild anhedonia, passive stress coping) and physiological (reduced body weight gain, elevated plasma corticosterone levels) alterations. Moreover, prolonged social isolation provoked a reduction in brain-derived neurotrophic factor (BDNF) and AEA levels within the hippocampus. Together, these changes are indicative of an increased risk of developing a depressive-like state. Conversely, pharmacological inhibition of FAAH activity with URB694 restored both AEA and BDNF levels within the hippocampus of socially isolated rats and prevented the development of behavioral and physiological alterations. These results suggest a potential interplay between AEA-mediated signaling and hippocampal BDNF in the pathogenesis of depression-relevant behaviors and physiological alterations and antidepressant action of FAAH inhibition in socially isolated female rats.

Drug-gut microbiota metabolic interactions: the case of UniPR1331, selective antagonist of the Eph-ephrin system, in mice.

The interest on the role of gut microbiota in the biotransformation of drugs and xenobiotics has grown over the last decades and a deeper understanding of the mutual interactions is expected to help future improvements in the fields of drug development, toxicological risk assessment and precision medicine. In this paper, a microbiome drug metabolism case is presented, involving a lipophilic small molecule, N-(3ß-hydroxy-Δ5-cholen-24-oyl)-l-tryptophan, UniPR1331, active as antagonist of the Eph-ephrin system and effective in vivo in a murine orthotopic model of glioblastoma multiforme (GBM). Following the administration of a single 30 mg/kg dose (p.o.) to mice, maximal plasma levels were reached 30 min after dosing and rapidly declined thereafter. To explain the observed in vivo behaviour, in vitro phase I and II metabolism assays were conducted employing mouse and human liver subcellular fractions and profiling main metabolites by means of tandem (HPLC-ESI-MS/MS) and high resolution mass spectrometry (HPLC-ESI-HR-MS). In the presence of in vitro mouse liver fractions, UniPR1331 showed a low phase I metabolic clearance, despite the identification of a 3-oxo and several hydroxylated metabolites. Conversely, after oral administration of UniPR1331 to mice, a novel isobaric metabolite was detected that (i) was subjected, as parent UniPR1331, to enterohepatic circulation (ii) had not been previously identified in vitro in mouse liver microsomes and (iii) was not observed forming after intraperitoneal (i.p.) administration of UniPR1331. An in vitro faecal fermentation assay produced the same chemical entity supporting a major role of gut microbiota in the in vivo clearance of UniPR1331.

Benzisothiazolinone Derivatives as Potent Allosteric Monoacylglycerol Lipase Inhibitors That Functionally Mimic Sulfenylation of Regulatory Cysteines.

We describe a set of benzisothiazolinone (BTZ) derivatives that are potent inhibitors of monoacylglycerol lipase (MGL), the primary degrading enzyme for the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Structure-activity relationship studies evaluated various substitutions on the nitrogen atom and the benzene ring of the BTZ nucleus. Optimized derivatives with nanomolar potency allowed us to investigate the mechanism of MGL inhibition. Site-directed mutagenesis and mass spectrometry experiments showed that BTZs interact in a covalent reversible manner with regulatory cysteines, Cys201 and Cys208, causing a reversible sulfenylation known to modulate MGL activity. Metadynamics simulations revealed that BTZ adducts favor a closed conformation of MGL that occludes substrate recruitment. The BTZ derivative 13 protected neuronal cells from oxidative stimuli and increased 2-AG levels in the mouse brain. The results identify Cys201 and Cys208 as key regulators of MGL function and point to the BTZ scaffold as a useful starting point for the discovery of allosteric MGL inhibitors.