Identification of a Dual Autophagy and REV-ERB Inhibitor with in Vivo Anticancer Efficacy.

The autophagy process appears as a promising target for anticancer interventions. Chloroquine (CQ) and its derivative hydroxychloroquine (HCQ) are the only FDA-approved autophagy flux inhibitors. Although diverse anticancer clinical trials are providing encouraging results, several limitations associated with the need of high dosage and long-term administration of these autophagy inhibitors are also emerging. We showed that the inhibition of REV-ERB, a nuclear receptor regulating circadian rhythm and metabolism, enhances CQ-mediated cancer cell death and identified a class of dual inhibitors of autophagy and REV-ERB displaying an in vitro anticancer activity against diverse tumor cells greatly higher than CQ. Herein, we describe our lead optimization strategy that led to the identification of compound 24 as a dual autophagy and REV-ERB inhibitor, showing improved potency in blocking autophagy, enhanced toxicity against cancer cells, optimal drug-like properties, and efficacy in a mouse xenograft model of melanoma as a single anticancer agent.

Potentiation of endocannabinoids and other lipid amides prevents hyperalgesia and inflammation in a pre-clinical model of migraine.

Targeting fatty acid amide hydrolase (FAAH) is a promising therapeutic strategy to combat certain forms of pain, including migraine headache. FAAH inhibitors, such as the O-biphenyl-3-yl carbamate URB597, have been shown to produce anti-hyperalgesic effects in animal models of migraine. The objective of this study was to investigate the behavioral and biochemical effects of compounds ARN14633 and ARN14280, two URB597 analogs with improved solubility and bioavailability, in a migraine-specific rat model in which trigeminal hyperalgesia is induced by nitroglycerin (NTG) administration. ARN14633 (1 mg/kg, i.p.) and ARN14280 (3 mg/kg, i.p.) were administered to adult male Sprague-Dawley rats 3 hours after NTG injection. One hour after the administration of either compound, rats were subjected to the orofacial formalin test. ARN14633 and ARN14280 attenuated NTG-induced nocifensive behavior and reduced transcription of genes encoding neuronal nitric oxide synthase, pain mediators peptides (calcitonin gene-related peptide, substance P) and pro-inflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta and 6) in the trigeminal ganglion, cervical spinal cord and medulla. Finally, both compounds strongly elevated levels of endocannabinoids and/or other FAAH substrates in cervical spinal cord and medulla, and, to a lesser extent, in the trigeminal ganglia. The results indicate that the novel global FAAH inhibitors ARN14633 and ARN14280 elicit significant anti-hyperalgesic effects in a migraine-specific animal model and inhibit the associated peptidergic-inflammatory response. Although the precise mechanism underlying these effects remains to be elucidated, our results support further investigational studies of FAAH blockade as a potential therapeutic strategy to treat migraine conditions.

Design, Synthesis, and Biological Evaluation of a Series of Oxazolone Carboxamides as a Novel Class of Acid Ceramidase Inhibitors.

Acid ceramidase (AC) is a cysteine hydrolase that plays a crucial role in the metabolism of lysosomal ceramides, important members of the sphingolipid family, a diversified class of bioactive molecules that mediate many biological processes ranging from cell structural integrity, signaling, and cell proliferation to cell death. In the effort to expand the structural diversity of the existing collection of AC inhibitors, a novel class of substituted oxazol-2-one-3-carboxamides were designed and synthesized. Herein, we present the chemical optimization of our initial hits, 2-oxo-4-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 8a and 2-oxo-5-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 12a, which resulted in the identification of 5-[4-fluoro-2-(1-methyl-4-piperidyl)phenyl]-2-oxo-N-pentyl-oxazole-3-carboxamide 32b as a potent AC inhibitor with optimal physicochemical and metabolic properties, showing target engagement in human neuroblastoma SH-SY5Y cells and a desirable pharmacokinetic profile in mice, following intravenous and oral administration. 32b enriches the arsenal of promising lead compounds that may therefore act as useful pharmacological tools for investigating the potential therapeutic effects of AC inhibition in relevant sphingolipid-mediated disorders.

Lead Optimization of Benzoxazolone Carboxamides as Orally Bioavailable and CNS Penetrant Acid Ceramidase Inhibitors.

Sphingolipids (SphLs) are a diverse class of molecules that are regulated by a complex network of enzymatic pathways. A disturbance in these pathways leads to lipid accumulation and initiation of several SphL-related disorders. Acid ceramidase is one of the key enzymes that regulate the metabolism of ceramides and glycosphingolipids, which are important members of the SphL family. Herein, we describe the lead optimization studies of benzoxazolone carboxamides resulting in piperidine 22m, where we demonstrated target engagement in two animal models of neuropathic lysosomal storage diseases (LSDs), Gaucher's and Krabbe's diseases. After daily intraperitoneal administration at 90 mg kg-1, 22m significantly reduced the brain levels of the toxic lipids glucosylsphingosine (GluSph) in 4L;C* mice and galactosylsphingosine (GalSph) in Twitcher mice. We believe that 22m is a lead molecule that can be further developed for the correction of severe neurological LSDs where GluSph or GalSph play a significant role in disease pathogenesis.

Kernel-Based, Partial Least Squares Quantitative Structure-Retention Relationship Model for UPLC Retention Time Prediction: A Useful Tool for Metabolite Identification.

We propose a new QSRR model based on a Kernel-based partial least-squares method for predicting UPLC retention times in reversed phase mode. The model was built using a combination of classical (physicochemical and topological) and nonclassical (fingerprints) molecular descriptors of 1383 compounds, encompassing different chemical classes and structures and their accurately measured retention time values. Following a random splitting of the data set into a training and a test set, we tested the ability of the model to predict the retention time of all the compounds. The best predicted/experimental R2 value was higher than 0.86, while the best Q2 value we observed was close to 0.84. A comparison of our model with traditional and simpler MLR and PLS regression models shows that KPLS better performs in term of correlation (R2), prediction (Q2), and support to MetID peak assignment. The KPLS model succeeded in two real-life MetID tasks by correctly predicting elution order of Phase I metabolites, including isomeric monohydroxylated compounds. We also show in this paper that the model's predictive power can be extended to different gradient profiles, by simple mathematical extrapolation using a known equation, thus offering very broad flexibility. Moreover, the current study includes a deep investigation of different types of chemical descriptors used to build the structure-retention relationship.

Second-Generation Non-Covalent NAAA Inhibitors are Protective in a Model of Multiple Sclerosis.

Palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are endogenous lipid mediators that suppress inflammation. Their actions are terminated by the intracellular cysteine amidase, N-acylethanolamine acid amidase (NAAA). Even though NAAA may offer a new target for anti-inflammatory therapy, the lipid-like structures and reactive warheads of current NAAA inhibitors limit the use of these agents as oral drugs. A series of novel benzothiazole-piperazine derivatives that inhibit NAAA in a potent and selective manner by a non-covalent mechanism are described. A prototype member of this class (8) displays high oral bioavailability, access to the central nervous system (CNS), and strong activity in a mouse model of multiple sclerosis (MS). This compound exemplifies a second generation of non-covalent NAAA inhibitors that may be useful in the treatment of MS and other chronic CNS disorders.

Potent multitarget FAAH-COX inhibitors: Design and structure-activity relationship studies.

Non-steroidal anti-inflammatory drugs (NSAIDs) exert their pharmacological effects by inhibiting cyclooxygenase (COX)-1 and COX-2. Though widely prescribed for pain and inflammation, these agents have limited utility in chronic diseases due to serious mechanism-based adverse events such as gastrointestinal damage. Concomitant blockade of fatty acid amide hydrolase (FAAH) enhances the therapeutic effects of the NSAIDs while attenuating their propensity to cause gastrointestinal injury. This favorable interaction is attributed to the accumulation of protective FAAH substrates, such as the endocannabinoid anandamide, and suggests that agents simultaneously targeting COX and FAAH might provide an innovative strategy to combat pain and inflammation with reduced side effects. Here, we describe the rational design and structure-active relationship (SAR) properties of the first class of potent multitarget FAAH-COX inhibitors. A focused SAR exploration around the prototype 10r (ARN2508) led to the identification of achiral (18b) as well as racemic (29a-c and 29e) analogs. Absolute configurational assignment and pharmacological evaluation of single enantiomers of 10r are also presented. (S)-(+)-10r is the first highly potent and selective chiral inhibitor of FAAH-COX with marked in vivo activity, and represents a promising lead to discover novel analgesics and anti-inflammatory drugs.

Fluorine nuclear magnetic resonance-based assay in living mammalian cells.

Nuclear magnetic resonance (NMR)-based screening has been recognized as a powerful approach for the identification and characterization of molecules interacting with pharmaceutical targets. Indeed, several NMR methods have been developed and successfully applied to many drug discovery projects. Whereas most of these approaches have targeted isolated biomolecular receptors, very few cases are reported with the screening performed in intact cells and cell extracts. Here we report the first successful application of the fluorine NMR-based assay n-FABS (n-fluorine atoms for biochemical screening) in living mammalian cells expressing the membrane protein fatty acid amide hydrolase (FAAH). This method allows the identification of both weak and potent inhibitors and the measurement of their potency in a physiological environment.

A Double Whammy: Targeting Both Fatty Acid Amide Hydrolase (FAAH) and Cyclooxygenase (COX) To Treat Pain and Inflammation.

Pain states that arise from non-resolving inflammation, such as inflammatory bowel disease or arthritis, pose an unusually difficult challenge for therapy because of the complexity and heterogeneity of their underlying mechanisms. It has been suggested that key nodes linking interactive pathogenic pathways of non-resolving inflammation might offer novel targets for the treatment of inflammatory pain. Nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit the cyclooxygenase (COX)-mediated production of pain- and inflammation-inducing prostanoids, are a common first-line treatment for this condition, but their use is limited by mechanism-based side effects. The endogenous levels of anandamide, an endocannabinoid mediator with analgesic and tissue-protective functions, are regulated by fatty acid amide hydrolase (FAAH). This review outlines the pharmacological and chemical rationale for the simultaneous inhibition of COX and FAAH activities with designed multitarget agents. Preclinical studies indicate that such agents may combine superior anti-inflammatory efficacy with reduced toxicity.

Benzoxazolone Carboxamides as Potent Acid Ceramidase Inhibitors: Synthesis and Structure-Activity Relationship (SAR) Studies.

Ceramides are lipid-derived intracellular messengers involved in the control of senescence, inflammation, and apoptosis. The cysteine amidase, acid ceramidase (AC), hydrolyzes these substances into sphingosine and fatty acid and, by doing so, regulates their signaling activity. AC inhibitors may be useful in the treatment of pathological conditions, such as cancer, in which ceramide levels are abnormally reduced. Here, we present a systematic SAR investigation of the benzoxazolone carboxamides, a recently described class of AC inhibitors that display high potency and systemic activity in mice. We examined a diverse series of substitutions on both benzoxazolone ring and carboxamide side chain. Several modifications enhanced potency and stability, and one key compound with a balanced activity-stability profile (14) was found to inhibit AC activity in mouse lungs and cerebral cortex after systemic administration. The results expand our arsenal of AC inhibitors, thereby facilitating the use of these compounds as pharmacological tools and their potential development as drug leads.

Synthesis and in Vitro Anticancer Activity of the First Class of Dual Inhibitors of REV-ERBß and Autophagy.

Autophagy inhibition is emerging as a promising anticancer strategy. We recently reported that the circadian nuclear receptor REV-ERBß plays an unexpected role in sustaining cancer cell survival when the autophagy flux is compromised. We also identified 4-[[[1-(2-fluorophenyl)cyclopentyl]amino]methyl]-2-[(4-methylpiperazin-1-yl)methyl]phenol, 1 (ARN5187), as a novel dual inhibitor of REV-ERBß and autophagy. 1 had improved cytotoxicity against BT-474 breast cancer cells compared to chloroquine, a clinically relevant autophagy inhibitor. Here, we present the results of structure-activity studies, based around 1, that disclose the first class of dual inhibitors of REV-ERBß and autophagy. This study led to identification of 18 and 28, which were more effective REV-ERBß antagonists than 1 and were more cytotoxic to BT-474. The combination of optimal chemical and structural moieties of these analogs generated 30, which elicited 15-fold greater REV-ERBß inhibitory and cytotoxic activities compared to 1. Furthermore, 30 induced death in a panel of tumor cell lines at doses 5-50 times lower than an equitoxic amount of chloroquine but did not affect the viability of normal mammary epithelial cells.

3,4-Dihydro-1,3,5-triazin-2(1H)-ones as the First Dual BACE-1/GSK-3ß Fragment Hits against Alzheimer's Disease.

One of the main obstacles toward the discovery of effective anti-Alzheimer drugs is the multifactorial nature of its etiopathology. Therefore, the use of multitarget-directed ligands has emerged as particularly suitable. Such ligands, able to modulate different neurodegenerative pathways, for example, amyloid and tau cascades, as well as cognitive and neurogenic functions, are fostered to come. In this respect, we report herein on the first class of BACE-1/GSK-3ß dual inhibitors based on a 3,4-dihydro-1,3,5-triazin-2(1H)-one skeleton, whose hit compound 1 showed interesting properties in a preliminary investigation. Notably, compound 2, endowed with well-balanced potencies against the two isolated enzymes (IC50 of 16 and 7 µM against BACE-1 and GSK-3ß, respectively), displayed effective neuroprotective and neurogenic activities and no neurotoxicity in cell-based assays. It also showed good brain permeability in a pharmacokinetic assessment in mice. Overall, triazinone derivatives, thanks to the simultaneous modulation of multiple points of the diseased network, might emerge as suitable candidates to be tested in in vivo Alzheimer's disease models.

Multitarget fatty acid amide hydrolase/cyclooxygenase blockade suppresses intestinal inflammation and protects against nonsteroidal anti-inflammatory drug-dependent gastrointestinal damage.

The ability of nonsteroidal anti-inflammatory drugs (NSAIDs) to inhibit cyclooxygenase (Cox)-1 and Cox-2 underlies the therapeutic efficacy of these drugs, as well as their propensity to damage the gastrointestinal (GI) epithelium. This toxic action greatly limits the use of NSAIDs in inflammatory bowel disease (IBD) and other chronic pathologies. Fatty acid amide hydrolase (FAAH) degrades the endocannabinoid anandamide, which attenuates inflammation and promotes GI healing. Here, we describe the first class of systemically active agents that simultaneously inhibit FAAH, Cox-1, and Cox-2 with high potency and selectivity. The class prototype 4: (ARN2508) is potent at inhibiting FAAH, Cox-1, and Cox-2 (median inhibitory concentration: FAAH, 0.031 ± 0.002 µM; Cox-1, 0.012 ± 0.002 µM; and Cox-2, 0.43 ± 0.025 µM) but does not significantly interact with a panel of >100 off targets. After oral administration in mice, ARN2508 engages its intended targets and exerts profound therapeutic effects in models of intestinal inflammation. Unlike NSAIDs, ARN2508 causes no gastric damage and indeed protects the GI from NSAID-induced damage through a mechanism that requires FAAH inhibition. Multitarget FAAH/Cox blockade may provide a transformative approach to IBD and other pathologies in which FAAH and Cox are overactive.

O-(triazolyl)methyl carbamates as a novel and potent class of fatty acid amide hydrolase (FAAH) inhibitors.

Inhibition of fatty acid amide hydrolase (FAAH) activity is under investigation as a valuable strategy for the treatment of several disorders, including pain and drug addiction. A number of potent FAAH inhibitors belonging to different chemical classes have been disclosed to date; O-aryl carbamates are one of the most representative families. In the search for novel FAAH inhibitors, a series of O-(1,2,3-triazol-4-yl)methyl carbamate derivatives were designed and synthesized exploiting a copper- catalyzed [3+2] cycloaddition reaction between azides and alkynes (click chemistry). Exploration of the structure-activity relationships within this new class of compounds identified potent inhibitors of both rat and human FAAH with IC50 values in the single-digit nanomolar range. In addition, these derivatives showed improved stability in rat plasma and kinetic solubility in buffer with respect to the lead compound. Based on the results of the study, the novel analogues identified can be considered to be promising starting point for the development of new FAAH inhibitors with improved drug-like properties.

Multitarget drug discovery for Alzheimer's disease: triazinones as BACE-1 and GSK-3ß inhibitors.

Cumulative evidence strongly supports that the amyloid and tau hypotheses are not mutually exclusive, but concomitantly contribute to neurodegeneration in Alzheimer's disease (AD). Thus, the development of multitarget drugs which are involved in both pathways might represent a promising therapeutic strategy. Accordingly, reported here in is the discovery of 6-amino-4-phenyl-3,4-dihydro-1,3,5-triazin-2(1H)-ones as the first class of molecules able to simultaneously modulate BACE-1 and GSK-3ß. Notably, one triazinone showed well-balanced in vitro potencies against the two enzymes (IC50 of (18.03±0.01) µM and (14.67±0.78) µM for BACE-1 and GSK-3ß, respectively). In cell-based assays, it displayed effective neuroprotective and neurogenic activities and no neurotoxicity. It also showed good brain permeability in a preliminary pharmacokinetic assessment in mice. Overall, triazinones might represent a promising starting point towards high quality lead compounds with an AD-modifying potential.

Structural determinants of peripheral O-arylcarbamate FAAH inhibitors render them dual substrates for Abcb1 and Abcg2 and restrict their access to the brain.

The blood-brain barrier (BBB) is the main entry route for chemicals into the mammalian central nervous system (CNS). Two transmembrane transporters of the ATP-binding cassette (ABC) family - breast cancer resistance protein (ABCG2 in humans, Abcg2 in rodents) and P-glycoprotein (ABCB1 in humans, Abcb1 in rodents) - play a key role in mediating this process. Pharmacological and genetic evidence suggests that Abcg2 prevents CNS access to a group of highly potent and selective O-arylcarbamate fatty-acid amidohydrolase (FAAH) inhibitors, which include the compound URB937 (cyclohexylcarbamic acid 3'-carbamoyl-6-hydroxybiphenyl-3-yl ester). To define structure-activity relationships of the interaction of these molecules with Abcg2, in the present study we tested various peripherally restricted and non-restricted O-arylcarbamate FAAH inhibitors for their ability to serve as transport substrates in monolayer cultures of Madin-Darby Canine Kidney-II (MDCKII) cells over-expressing Abcg2. Surprisingly, we found that the majority of compounds tested - even those able to enter the CNS in vivo - were substrates for Abcg2 in vitro. Additional experiments in MDCKII cells overexpressing ABCB1 revealed that only those compounds that were dual substrates for ABCB1 and Abcg2 in vitro were also peripherally restricted in vivo. The extent of such restriction seems to depend upon other physicochemical features of the compounds, in particular the polar surface area. Consistent with these in vitro results, we found that URB937 readily enters the brain in dual knockout mice lacking both Abcg2 and Abcb1, whereas it is either partially or completely excluded from the brain of mice lacking either transporter alone. The results suggest that Abcg2 and Abcb1 act together to restrict the access of URB937 to the CNS.

Fluorine NMR-based screening on cell membrane extracts.

The possibility of measuring the action of inhibitors of specific enzymatic reactions in intact cells, cell lysates or membrane preparations represents a major advance in the lead discovery process. Despite the relevance of assaying in physiological conditions, only a small number of biophysical techniques, often requiring complex set-up, are applicable to these sample types. Here, we demonstrate the first application of n-fluorine atoms for biochemical screening (n-FABS), a homogeneous and versatile assay based on (19) F NMR spectroscopy, to the detection of high- and low-affinity inhibitors of a membrane enzyme in cell extracts and determination of their IC50 values. Our approach can allow the discovery of novel binding fragments against targets known to be difficult to purify or where membrane-association is required for activity. These results pave the way for future applications of the methodology to these relevant and complex biological systems.

Development of fragment-based n-FABS NMR screening applied to the membrane enzyme FAAH.

Despite the recognized importance of membrane proteins as pharmaceutical targets, the reliable identification of fragment hits that are able to bind these proteins is still a major challenge. Among different ¹9F NMR spectroscopic methods, n-fluorine atoms for biochemical screening (n-FABS) is a highly sensitive technique that has been used efficiently for fragment screening, but its application for membrane enzymes has not been reported yet. Herein, we present the first successful application of n-FABS to the discovery of novel fragment hits, targeting the membrane-bound enzyme fatty acid amide hydrolase (FAAH), using a library of fluorinated fragments generated based on the different local environment of fluorine concept. The use of the recombinant fusion protein MBP-FAAH and the design of compound 11 as a suitable novel fluorinated substrate analogue allowed n-FABS screening to be efficiently performed using a very small amount of enzyme. Notably, we have identified 19 novel fragment hits that inhibit FAAH with a median effective concentration (IC50) in the low mM-µM range. To the best of our knowledge, these results represent the first application of a ¹9F NMR fragment-based functional assay to a membrane protein.

Synthesis and structure-activity relationship studies of O-biphenyl-3-yl carbamates as peripherally restricted fatty acid amide hydrolase inhibitors.

The peripherally restricted fatty acid amide hydrolase (FAAH) inhibitor URB937 (3, cyclohexylcarbamic acid 3'-carbamoyl-6-hydroxybiphenyl-3-yl ester) is extruded from the brain and spinal cord by the Abcg2 efflux transporter. Despite its inability to enter the central nervous system (CNS), 3 exerts profound antinociceptive effects in mice and rats, which result from the inhibition of FAAH in peripheral tissues and the consequent enhancement of anandamide signaling at CB1 cannabinoid receptors localized on sensory nerve endings. In the present study, we examined the structure-activity relationships (SAR) for the biphenyl region of compound 3, focusing on the carbamoyl and hydroxyl groups in the distal and proximal phenyl rings. Our SAR studies generated a new series of peripherally restricted FAAH inhibitors and identified compound 35 (cyclohexylcarbamic acid 3'-carbamoyl-5-hydroxybiphenyl-3-yl ester) as the most potent brain-impermeant FAAH inhibitor disclosed to date.

Discovery of a new class of highly potent inhibitors of acid ceramidase: synthesis and structure-activity relationship (SAR).

Acid ceramidase (AC) is an intracellular cysteine amidase that catalyzes the hydrolysis of the lipid messenger ceramide. By regulating ceramide levels in cells, AC may contribute to the regulation of cancer cell proliferation and senescence and to the response to cancer therapy. We recently identified the antitumoral agent carmofur (4a) as the first nanomolar inhibitor of intracellular AC activity (rat AC, IC50 = 0.029 µM). In the present work, we expanded our initial structure-activity relationship (SAR) studies around 4a by synthesizing and testing a series of 2,4-dioxopyrimidine-1-carboxamides. Our investigations provided a first elucidation of the structural features of uracil derivatives that are critical for AC inhibition and led us to identify the first single-digit nanomolar inhibitors of this enzyme. The present results confirm that substituted 2,4-dioxopyrimidine-1-carboxamides are a novel class of potent inhibitors of AC. Selected compounds of this class may represent useful probes to further characterize the functional roles of AC.