RESUMO
The cannabinoid receptor type 2 (CB2R) is a G protein-coupled receptor with therapeutic potential for the treatment of inflammatory disorders. Fluorescent probes are desirable to study its receptor localization, expression and occupancy. Previously, we have reported a photoaffinity probe LEI-121 that stabilized the inactive conformation of the CB2R. Here, we report the structure-based design of a novel bifunctional probe that captures the active conformation of the CB2R upon irradiation with light. An alkyne handle was incorporated to visualize the receptor using click-chemistry with fluorophore-azides. These probes may hold promise to study different receptor conformations in relation to their cellular localization and function.
Assuntos
Canabinoides , Corantes Fluorescentes , Receptores de Canabinoides , Corantes Fluorescentes/química , Conformação Molecular , Receptores Acoplados a Proteínas GRESUMO
Phenotypic screening is a powerful approach to identify novel antibiotics, but elucidation of the targets responsible for the antimicrobial activity is often challenging in the case of compounds with a polypharmacological mode of action. Here, we show that activity-based protein profiling maps the target interaction landscape of a series of 1,3,4-oxadiazole-3-ones identified in a phenotypic screen to have high antibacterial potency against multidrug-resistant Staphylococcus aureus. In situ competitive and comparative chemical proteomics with a tailor-made activity-based probe, in combination with transposon and resistance studies, revealed several cysteine and serine hydrolases as relevant targets. Our data showcase oxadiazolones as a novel antibacterial chemotype with a polypharmacological mode of action, in which FabH, FphC, and AdhE play a central role.
Assuntos
Antibacterianos , Staphylococcus aureus Resistente à Meticilina , Antibacterianos/farmacologia , Antibacterianos/química , Proteômica , Testes de Sensibilidade Microbiana , Staphylococcus aureusRESUMO
ß-glucosidases [GBA1 (glucocerebrosidase) and GBA2] are ubiquitous essential enzymes. Lysosomal GBA1 and cytosol-facing GBA2 degrade glucosylceramide (GlcCer); GBA1 deficiency causes Gaucher disease, a lysosomal storage disorder characterized by lysosomal accumulation of GlcCer, which is partly converted to glucosylsphingosine (GlcSph). GBA1 and GBA2 also may transfer glucose from GlcCer to cholesterol, yielding glucosylated cholesterol (GlcChol). Here, we aimed to clarify the role of zebrafish Gba2 in glycosphingolipid metabolism during Gba1 deficiency in zebrafish (Danio rerio), which are able to survive total Gba1 deficiency. We developed Gba1 (gba1-/-), Gba2 (gba2-/-), and double (gba1-/-:gba2-/-) zebrafish knockouts using CRISPR/Cas9 and explored the effects of both genetic and pharmacological interventions on GlcCer metabolism in individual larvae. Activity-based probes and quantification of relevant glycolipid metabolites confirmed enzyme deficiency. GlcSph increased in gba1-/- larvae (0.09 pmol/fish) but did not increase more in gba1-/-:gba2-/- larvae. GlcCer was comparable in gba1-/- and WT larvae but increased in gba2-/- and gba1-/-:gba2-/- larvae. Independent of Gba1 status, GlcChol was low in all gba2-/- larvae (0.05 vs. 0.18 pmol/fish in WT). Pharmacologic inactivation of zebrafish Gba1 comparably increased GlcSph. Inhibition of GlcCer synthase (GCS) in Gba1-deficient larvae reduced GlcCer and GlcSph, and concomitant inhibition of GCS and Gba2 with iminosugars also reduced excessive GlcChol. Finally, overexpression of human GBA1 and injection of recombinant GBA1 both decreased GlcSph. We determined that zebrafish larvae offer an attractive model to study glucosidase actions in glycosphingolipid metabolism in vivo, and we identified distinguishing characteristics of zebrafish Gba2 deficiency.
Assuntos
Glucosilceramidase/deficiência , Glicoesfingolipídeos/metabolismo , Modelos Biológicos , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/metabolismo , beta-Glucosidase/metabolismo , Animais , Células Cultivadas , Glucosilceramidase/metabolismo , Peixe-Zebra , beta-Glucosidase/deficiênciaRESUMO
Diacylglycerol lipases (DAGL) produce the endocannabinoid 2-arachidonoylglycerol, a key modulator of neurotransmitter release. Chemical tools that visualize endogenous DAGL activity are desired. Here, we report the design, synthesis and application of a triazole urea probe for DAGL equipped with a norbornene as a biorthogonal handle. The activity and selectivity of the probe was assessed with activity-based protein profiling. This probe was potent against endogenous DAGLα (IC50 = 5 nM) and it was successfully applied as a two-step activity-based probe for labeling of DAGLα using an inverse electron-demand Diels-Alder ligation in living cells.
Assuntos
Lipase Lipoproteica/química , Lipase Lipoproteica/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular Tumoral , Reação de Cicloadição , Teoria da Densidade Funcional , Endocanabinoides/química , Humanos , Lipase Lipoproteica/antagonistas & inibidores , Camundongos , Sondas Moleculares/química , Sondas Moleculares/toxicidade , Norbornanos/química , Proteoma , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Triazóis/química , Ureia/químicaRESUMO
Human nonlysosomal glucosylceramidase (GBA2) is one of several enzymes that controls levels of glycolipids and whose activity is linked to several human disease states. There is a major need to design or discover selective GBA2 inhibitors both as chemical tools and as potential therapeutic agents. Here, we describe the development of a fluorescence polarization activity-based protein profiling (FluoPol-ABPP) assay for the rapid identification, from a 350+ library of iminosugars, of GBA2 inhibitors. A focused library is generated based on leads from the FluoPol-ABPP screen and assessed on GBA2 selectivity offset against the other glucosylceramide metabolizing enzymes, glucosylceramide synthase (GCS), lysosomal glucosylceramidase (GBA), and the cytosolic retaining ß-glucosidase, GBA3. Our work, yielding potent and selective GBA2 inhibitors, also provides a roadmap for the development of high-throughput assays for identifying retaining glycosidase inhibitors by FluoPol-ABPP on cell extracts containing recombinant, overexpressed glycosidase as the easily accessible enzyme source.
Assuntos
Ensaios Enzimáticos/métodos , Inibidores Enzimáticos/farmacologia , Polarização de Fluorescência/métodos , Imino Açúcares/farmacologia , beta-Glucosidase/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/química , Glucosilceramidase , Humanos , Imino Açúcares/química , beta-Glucosidase/metabolismoRESUMO
In this paper, a new synthetic route toward 6-hydroxysphingosine and α-hydroxy ceramide is described. The synthesis employs a cross-metathesis to unite a sphingosine head allylic alcohol with a long-chain fatty acid alkene that also bears an allylic alcohol group. To allow for a productive CM coupling, the sphingosine head allylic alcohol was protected with a cyclic carbonate moiety and a reactive CM catalyst system, consisting of Grubbs II catalyst and CuI, was employed.
Assuntos
Ceramidas/química , Propanóis/química , Esfingosina/análogos & derivados , Alcenos/síntese química , Catálise , Estrutura Molecular , Esfingosina/síntese química , Esfingosina/química , EstereoisomerismoRESUMO
Monoacylglycerol lipase (MAGL) is the key enzyme for the hydrolysis of endocannabinoid 2-arachidonoylglycerol (2-AG). The central role of MAGL in the metabolism of 2-AG makes it an attractive therapeutic target for a variety of disorders, including inflammation-induced tissue injury, pain, multiple sclerosis, and cancer. Previously, we reported LEI-515, an aryl sulfoxide, as a peripherally restricted, covalent reversible MAGL inhibitor that reduced neuropathic pain and inflammation in preclinical models. Here, we describe the structure-activity relationship (SAR) of aryl sulfoxides as MAGL inhibitors that led to the identification of LEI-515. Optimization of the potency of high-throughput screening (HTS) hit 1 yielded compound ±43. However, ±43 was not metabolically stable due to its ester moiety. Replacing the ester group with α-CF2 ketone led to the identification of compound ±73 (LEI-515) as a metabolically stable MAGL inhibitor with subnanomolar potency. LEI-515 is a promising compound to harness the therapeutic potential of MAGL inhibition.
Assuntos
Inibidores Enzimáticos , Monoacilglicerol Lipases , Sulfóxidos , Monoacilglicerol Lipases/antagonistas & inibidores , Monoacilglicerol Lipases/metabolismo , Relação Estrutura-Atividade , Humanos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese química , Sulfóxidos/química , Sulfóxidos/farmacologia , Sulfóxidos/síntese química , Animais , Microssomos Hepáticos/metabolismo , Ensaios de Triagem em Larga EscalaRESUMO
Cannabinoid CB2 receptor (CB2R) agonists are investigated as therapeutic agents in the clinic. However, their molecular mode-of-action is not fully understood. Here, we report the discovery of LEI-102, a CB2R agonist, used in conjunction with three other CBR ligands (APD371, HU308, and CP55,940) to investigate the selective CB2R activation by binding kinetics, site-directed mutagenesis, and cryo-EM studies. We identify key residues for CB2R activation. Highly lipophilic HU308 and the endocannabinoids, but not the more polar LEI-102, APD371, and CP55,940, reach the binding pocket through a membrane channel in TM1-TM7. Favorable physico-chemical properties of LEI-102 enable oral efficacy in a chemotherapy-induced nephropathy model. This study delineates the molecular mechanism of CB2R activation by selective agonists and highlights the role of lipophilicity in CB2R engagement. This may have implications for GPCR drug design and sheds light on their activation by endogenous ligands.
Assuntos
Agonistas de Receptores de Canabinoides , Canabinoides , Agonistas de Receptores de Canabinoides/farmacologia , Receptores de Canabinoides , Canabinoides/farmacologia , Receptor CB1 de Canabinoide/genética , Receptor CB2 de Canabinoide/genéticaRESUMO
Monoacylglycerol lipase (MAGL) regulates endocannabinoid 2-arachidonoylglycerol (2-AG) and eicosanoid signalling. MAGL inhibition provides therapeutic opportunities but clinical potential is limited by central nervous system (CNS)-mediated side effects. Here, we report the discovery of LEI-515, a peripherally restricted, reversible MAGL inhibitor, using high throughput screening and a medicinal chemistry programme. LEI-515 increased 2-AG levels in peripheral organs, but not mouse brain. LEI-515 attenuated liver necrosis, oxidative stress and inflammation in a CCl4-induced acute liver injury model. LEI-515 suppressed chemotherapy-induced neuropathic nociception in mice without inducing cardinal signs of CB1 activation. Antinociceptive efficacy of LEI-515 was blocked by CB2, but not CB1, antagonists. The CB1 antagonist rimonabant precipitated signs of physical dependence in mice treated chronically with a global MAGL inhibitor (JZL184), and an orthosteric cannabinoid agonist (WIN55,212-2), but not with LEI-515. Our data support targeting peripheral MAGL as a promising therapeutic strategy for developing safe and effective anti-inflammatory and analgesic agents.
Assuntos
Monoacilglicerol Lipases , Monoglicerídeos , Animais , Camundongos , Rimonabanto , Endocanabinoides , Analgésicos/farmacologia , Receptor CB1 de Canabinoide , Camundongos Endogâmicos C57BLRESUMO
Three different photoprobes were synthesized to label beta-glucosidases; one probe was based on glucose, two probes on the iminosugar deoxynojirimycin. The affinity of the probes for three different beta-glucosidases was determined. Furthermore, their labeling efficiencies, binding specificities through competition with deoxynojirimycin, and binding specificities in the presence of cell lysate, were evaluated. Especially one showed very high affinity towards non-lysosomal glucoceramidase (IC(50)=20 nM).
Assuntos
Glucosilceramidase/análise , Glucosilceramidase/metabolismo , Imino Açúcares/química , Lisossomos/enzimologia , Bactérias/química , Glucose/química , Glucose/metabolismo , Glucosilceramidase/química , Células HeLa , Humanos , Imino Açúcares/metabolismo , Fotoquímica , Ligação ProteicaRESUMO
In the recent past sugar-derived cyclopropylamines were proposed as structurally new glycosidase inhibitors. In this Letter we report our efforts in the synthesis of a set of alpha-glucose configured oxabicyclo[4.1.0] heptanes, based on this hypothesis, bearing an amine substituent on the propyl ring and reveal that their inhibitory potential towards a range of mammalian glucosidases is modest.
Assuntos
Aminas/síntese química , Aminas/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/síntese química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Glucosidases/antagonistas & inibidores , Aminas/química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Conformação Molecular , Relação Estrutura-AtividadeRESUMO
The discovery of the glycosphingolipids is generally attributed to Johan L. W. Thudichum, who in 1884 published on the chemical composition of the brain. In his studies he isolated several compounds from ethanolic brain extracts which he coined cerebrosides. He subjected one of these, phrenosin (now known as galactosylceramide), to acid hydrolysis, and this produced three distinct components. One he identified as a fatty acid and another proved to be an isomer of D-glucose, which is now known as D-galactose. The third component, with an "alkaloidal nature", presented "many enigmas" to Thudichum, and therefore he named it sphingosine, after the mythological riddle of the Sphinx. Today, sphingolipids and their glycosidated derivatives are the subjects of intense study aimed at elucidating their role in the structural integrity of the cell membrane, their participation in recognition and signaling events, and in particular their involvement in pathological processes that are at the basis of human disease (for example, sphingolipidoses and diabetes type 2). This Review details some of the recent findings on the biosynthesis, function, and degradation of glycosphingolipids in man, with a focus on the glycosphingolipid glucosylceramide. Special attention is paid to the clinical relevance of compounds directed at interfering with the factors responsible for glycosphingolipid metabolism.
Assuntos
Glicoesfingolipídeos/metabolismo , Membrana Celular , Galactosilceramidas/metabolismo , Doença de Gaucher/terapia , Glucosilceramidase/antagonistas & inibidores , Glucosilceramidase/metabolismo , Glucosiltransferases/antagonistas & inibidores , Glucosiltransferases/metabolismo , Glicoesfingolipídeos/biossíntese , Glicoesfingolipídeos/farmacologia , Humanos , Esfingolipídeos/biossíntese , Esfingolipídeos/metabolismoRESUMO
The fact that GPCRs might function in a dimeric fashion is currently well accepted. For GnRHR, a GPCR that regulates gonadotropin release, there is evidence that the receptor also functions as a dimer. We here describe the design and synthesis of a set of dimeric GnRHR antagonists in order to understand the interaction of dimeric ligands to the receptor and to address the question whether GnRHR dimerization is a prerequisite for signalling. Biological evaluation of the compounds shows no discrimination between monomeric and dimeric-ligands in respect to binding affinities, however, the dimeric ligands appear to have different functional properties.
Assuntos
Benzeno/síntese química , Benzeno/farmacologia , Pargilina/química , Receptores LHRH/antagonistas & inibidores , Receptores LHRH/metabolismo , Aminoácidos/química , Animais , Benzeno/química , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Bases de Dados de Proteínas , Dimerização , Humanos , Iodetos/química , Ligantes , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Introduction: Δ9-Tetrahydrocannabinol (THC), the principle psychoactive ingredient in Cannabis, is widely used for its therapeutic effects in a large variety of diseases, but it also has numerous neurological side effects. The cannabinoid receptors (CBRs) are responsible to a large extent for these, but not all biological responses are mediated via the CBRs. Objectives: The identification of additional target proteins of THC to enable a better understanding of the (adverse) physiological effects of THC. Methods: In this study, a chemical proteomics approach using a two-step photoaffinity probe is applied to identify potential proteins that may interact with THC. Results: Photoaffinity probe 1, containing a diazirine as a photocrosslinker, and a terminal alkyne as a ligation handle, was synthesized in 14 steps. It demonstrated high affinity for both CBRs. Subsequently, two-step photoaffinity labeling in neuroblastoma cells led to identification of four potential novel protein targets of THC. The identification of these putative protein hits is a first step towards a better understanding of the protein interaction profile of THC, which could ultimately lead to the development of novel therapeutics based on THC.
RESUMO
Iminosugars are carbohydrate mimics that are useful as molecular probes to dissect metabolism in plants. To analyse the effects of iminosugar derivatives on germination and seedling growth, we screened a library of 390 N-substituted iminosugar analogues against Arabidopsis and the small cereal Eragrostis tef (Tef). The most potent compound identified in both systems, N-5-(adamantane-1-yl-ethoxy)pentyl- L-ido-deoxynojirimycin (L-ido-AEP-DNJ), inhibited root growth in agar plate assays by 92% and 96% in Arabidopsis and Tef respectively, at 10 µM concentration. Phenocopying the effect of L-ido-AEP-DNJ with the commercial inhibitor (PDMP) implicated glucosylceramide synthase as the target responsible for root growth inhibition. L-ido-AEP-DNJ was twenty-fold more potent than PDMP. Liquid chromatography-mass spectrometry (LC-MS) analysis of ceramide:glucosylceramide ratios in inhibitor-treated Arabidopsis seedlings showed a decrease in the relative quantity of the latter, confirming that glucosylceramide synthesis is perturbed in inhibitor-treated plants. Bioinformatic analysis of glucosylceramide synthase indicates gene conservation across higher plants. Previous T-DNA insertional inactivation of glucosylceramide synthase in Arabidopsis caused seedling lethality, indicating a role in growth and development. The compounds identified herein represent chemical alternatives that can overcome issues caused by genetic intervention. These inhibitors offer the potential to dissect the roles of glucosylceramides in polyploid crop species.
Assuntos
Arabidopsis/efeitos dos fármacos , Grão Comestível/efeitos dos fármacos , Eragrostis/efeitos dos fármacos , Glucosiltransferases/antagonistas & inibidores , Raízes de Plantas/crescimento & desenvolvimento , Açúcares/química , Açúcares/farmacologia , Animais , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Avaliação Pré-Clínica de Medicamentos , Grão Comestível/genética , Grão Comestível/crescimento & desenvolvimento , Grão Comestível/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Eragrostis/genética , Eragrostis/crescimento & desenvolvimento , Eragrostis/metabolismo , Glucosilceramidas/metabolismo , Raízes de Plantas/efeitos dos fármacosRESUMO
Inhibitors of diacylglycerol lipases and α,ß-hydrolase domain containing protein 6 (ABHD6) are potential leads for the development of therapeutic agents for metabolic and neurodegenerative disorders. Here, we report the enantioselective synthesis and structure activity relationships of triazole ureas featuring chiral, hydroxylated 2-benzylpiperidines as dual inhibitors of DAGLα and ABHD6. The chirality of the carbon bearing the C2 substituent, as well as the position of the hydroxyl (tolerated at C5, but not at C3) has profound influence on the inhibitory activity of both DAGLα and ABHD6, as established using biochemical assays and competitive activity-based protein profiling on mouse brain extracts.
RESUMO
Triazole ureas constitute a versatile class of irreversible inhibitors that target serine hydrolases in both cells and animal models. We have previously reported that triazole ureas can act as selective and CNS-active inhibitors for diacylglycerol lipases (DAGLs), enzymes responsible for the biosynthesis of 2-arachidonoylglycerol (2-AG) that activates cannabinoid CB1 receptor. Here, we report the enantio- and diastereoselective synthesis and structure-activity relationship studies. We found that 2,4-substituted triazole ureas with a biphenylmethanol group provided the most optimal scaffold. Introduction of a chiral ether substituent on the 5-position of the piperidine ring provided ultrapotent inhibitor 38 (DH376) with picomolar activity. Compound 38 temporarily reduces fasting-induced refeeding of mice, thereby emulating the effect of cannabinoid CB1-receptor inverse agonists. This was mirrored by 39 (DO34) but also by the negative control compound 40 (DO53) (which does not inhibit DAGL), which indicates the triazole ureas may affect the energy balance in mice through multiple molecular targets.
Assuntos
Ingestão de Alimentos , Inibidores Enzimáticos/farmacologia , Jejum , Lipase Lipoproteica/antagonistas & inibidores , Triazóis/química , Ureia/química , Animais , Células HEK293 , Humanos , Camundongos , Relação Estrutura-AtividadeRESUMO
The chemo- and regioselective TEMPO/BAIB-mediated oxidation of 2,6- and 3,6-dihydroxy 1-thio glycopyranosides to the corresponding 1-thio uronic acid lactones is described. These locked 1-thio glycuronides can directly be used as donors in glycosidation reactions using the Ph(2)SO/Tf(2)O reagent system. Alternatively, selective opening of the lactone bridge liberates a hydroxyl function for ensuing glycosylations.
Assuntos
Lactonas/síntese química , Oligossacarídeos/síntese química , Ácidos Urônicos/síntese química , Estrutura Molecular , Oxirredução , EstereoisomerismoRESUMO
Glucosylceramide metabolism and the enzymes involved have attracted significant interest in medicinal chemistry, because aberrations in the levels of glycolipids that are derived from glucosylceramide are causative in a range of human diseases including lysosomal storage disorders, typeâ 2 diabetes, and neurodegenerative diseases. Selective modulation of one of the glycoprocessing enzymes involved in glucosylceramide metabolism-glucosylceramide synthase (GCS), acid glucosylceramidase (GBA1), or neutral glucosylceramidase (GBA2)-is therefore an attractive research objective. In this study we took two established GCS inhibitors, one based on deoxynojirimycin and the other a ceramide analogue, and merged characteristic features to obtain hybrid compounds. The resulting 39-compound library does not contain new GCS inhibitors; however, a potent (200â nm) GBA1 inhibitor was identified that has little activity toward GBA2 and might therefore serve as a lead for further biomedical development as a selective GBA1 modulator.
Assuntos
Inibidores Enzimáticos/síntese química , Glucosiltransferases/antagonistas & inibidores , 1-Desoxinojirimicina/síntese química , 1-Desoxinojirimicina/química , 1-Desoxinojirimicina/metabolismo , Ceramidas/síntese química , Ceramidas/química , Ceramidas/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Glucosamina/análogos & derivados , Glucosamina/síntese química , Glucosamina/química , Glucosamina/metabolismo , Glucosiltransferases/metabolismo , Humanos , Concentração Inibidora 50 , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
This work details the evaluation of a number of N-alkylated deoxynojirimycin derivatives on their merits as dual glucosylceramide synthase/neutral glucosylceramidase inhibitors. Building on our previous work, we synthesized a series of D-gluco and L-ido-configured iminosugars N-modified with a variety of hydrophobic functional groups. We found that iminosugars featuring N-pentyloxymethylaryl substituents are considerably more potent inhibitors of glucosylceramide synthase than their aliphatic counterparts. In a next optimization round, we explored a series of biphenyl-substituted iminosugars of both configurations (D-gluco and L-ido) with the aim to introduce structural features known to confer metabolic stability to drug-like molecules. From these series, two sets of molecules emerge as lead series for further profiling. Biphenyl-substituted L-ido-configured deoxynojirimycin derivatives are selective for glucosylceramidase and the nonlysosomal glucosylceramidase, and we consider these as leads for the treatment of neuropathological lysosomal storage disorders. Their D-gluco-counterparts are also potent inhibitors of intestinal glycosidases, and because of this characteristic, we regard these as the prime candidates for type 2 diabetes therapeutics.