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1.
Nat Chem Biol ; 16(6): 667-675, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32393901

RESUMO

N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus-pituitary-adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior.


Assuntos
Comportamento Animal/efeitos dos fármacos , Inibidores Enzimáticos/química , Metabolismo dos Lipídeos/efeitos dos fármacos , Fosfatidiletanolaminas/metabolismo , Fosfolipase D/antagonistas & inibidores , Amidoidrolases/metabolismo , Animais , Proteínas Sanguíneas/metabolismo , Encéfalo/metabolismo , Antagonistas de Receptores de Canabinoides/metabolismo , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Medo/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Receptores de Canabinoides/metabolismo , Transdução de Sinais
2.
Sci Rep ; 14(1): 6229, 2024 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-38486006

RESUMO

Distinct platelet activation patterns are elicited by the tyrosine kinase-linked collagen receptor glycoprotein VI (GPVI) and the G-protein coupled protease-activated receptors (PAR1/4) for thrombin. This is reflected in the different platelet Ca2+ responses induced by the GPVI agonist collagen-related peptide (CRP) and the PAR1/4 agonist thrombin. Using a 96 well-plate assay with human Calcium-6-loaded platelets and a panel of 22 pharmacological inhibitors, we assessed the cytosolic Ca2+ signaling domains of these receptors and developed an automated Ca2+ curve algorithm. The algorithm was used to evaluate an ultra-high throughput (UHT) based screening of 16,635 chemically diverse small molecules with orally active physicochemical properties for effects on platelets stimulated with CRP or thrombin. Stringent agonist-specific selection criteria resulted in the identification of 151 drug-like molecules, of which three hit compounds were further characterized. The dibenzyl formamide derivative ANO61 selectively modulated thrombin-induced Ca2+ responses, whereas the aromatic sulfonyl imidazole AF299 and the phenothiazine ethopropazine affected CRP-induced responses. Platelet functional assays confirmed selectivity of these hits. Ethopropazine retained its inhibitory potential in the presence of plasma, and suppressed collagen-dependent thrombus buildup at arterial shear rate. In conclusion, targeting of platelet Ca2+ signaling dynamics in a screening campaign has the potential of identifying novel platelet-inhibiting molecules.


Assuntos
Cálcio , Fenotiazinas , Inibidores da Agregação Plaquetária , Humanos , Inibidores da Agregação Plaquetária/farmacologia , Cálcio/metabolismo , Trombina/metabolismo , Sinalização do Cálcio , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptor PAR-1/metabolismo , Plaquetas/metabolismo , Ativação Plaquetária , Cálcio da Dieta/farmacologia , Agregação Plaquetária
3.
Chem Sci ; 15(21): 8227-8241, 2024 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-38817593

RESUMO

The three human SNM1 metallo-ß-lactamase fold nucleases (SNM1A-C) play key roles in DNA damage repair and in maintaining telomere integrity. Genetic studies indicate that they are attractive targets for cancer treatment and to potentiate chemo- and radiation-therapy. A high-throughput screen for SNM1A inhibitors identified diverse pharmacophores, some of which were shown by crystallography to coordinate to the di-metal ion centre at the SNM1A active site. Structure and turnover assay-guided optimization enabled the identification of potent quinazoline-hydroxamic acid containing inhibitors, which bind in a manner where the hydroxamic acid displaces the hydrolytic water and the quinazoline ring occupies a substrate nucleobase binding site. Cellular assays reveal that SNM1A inhibitors cause sensitisation to, and defects in the resolution of, cisplatin-induced DNA damage, validating the tractability of MBL fold nucleases as cancer drug targets.

4.
Nat Commun ; 14(1): 8039, 2023 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-38052772

RESUMO

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 C57BL
5.
iScience ; 25(1): 103718, 2022 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-35072010

RESUMO

Antiplatelet drugs targeting G-protein-coupled receptors (GPCRs), used for the secondary prevention of arterial thrombosis, coincide with an increased bleeding risk. Targeting ITAM-linked receptors, such as the collagen receptor glycoprotein VI (GPVI), is expected to provide a better antithrombotic-hemostatic profile. Here, we developed and characterized an ultra-high-throughput (UHT) method based on intracellular [Ca2+]i increases to differentiate GPVI and GPCR effects on platelets. In 96-, 384-, or 1,536-well formats, Calcium-6-loaded human platelets displayed a slow-prolonged or fast-transient [Ca2+]i increase when stimulated with the GPVI agonist collagen-related peptide or with thrombin and other GPCR agonists, respectively. Semi-automated curve fitting revealed five parameters describing the Ca2+ responses. Verification of the UHT assay was done with a robustness compound library and clinically relevant platelet inhibitors. Taken together, these results present proof of principle of distinct receptor-type-dependent Ca2+ signaling curves in platelets, which allow identification of new inhibitors in a UHT way.

6.
SLAS Discov ; 26(2): 192-204, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32734803

RESUMO

The European Lead Factory (ELF) consortium provides European academics and small and medium enterprises access to ~0.5 million unique compounds, a state-of-the-art ultra-high-throughput screening (u-HTS) platform, and industrial early drug discovery (DD) expertise with the aim of delivering innovative DD starting points. From 2013 to 2018, 154 proposals for eight target classes in seven therapeutic areas were submitted to the ELF consortium, 88 of which were accepted by the selection committee. During this period, 76 primary assays based on seven different readout technologies were optimized and mainly miniaturized to 1536-well plates. In total, 72 u-HTS campaigns were carried out, and follow-up work including hit triage through orthogonal, deselection, selectivity, and biophysical assays were finalized. This ambitious project showed that besides the quality of the compound library and the primary assay, the success of centralized u-HTS of large compound libraries across many target classes, various assay types, and different readout technologies is also largely dependent on the capacity and flexibility of the automation on one hand and the hit-triaging phase on the other, particularly because of undesired compound-assay interference. Thus far, the delivered hit lists from the ELF consortium have resulted in spinoffs, patents, in vivo proof of concepts, preclinical development programs, peer-reviewed publications, PhD theses, and much more, demonstrating early success indications.


Assuntos
Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Pesquisa , Automação , Biotecnologia/métodos , Desenho de Fármacos , Descoberta de Drogas/normas , Europa (Continente) , Ensaios de Triagem em Larga Escala/normas , Humanos , Revisão da Pesquisa por Pares , Parcerias Público-Privadas , Bibliotecas de Moléculas Pequenas
7.
SLAS Discov ; 25(9): 1018-1025, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32418491

RESUMO

Arginase-1, which converts the amino acid L-arginine into L-ornithine and urea, is a promising new drug target for cancer immunotherapy, as it has a role in the regulation of T-cell immunity in the tumor microenvironment. To enable the discovery of small-molecule Arginase-1 inhibitors by high-throughput screening, we developed a novel homogeneous (mix-and-measure) fluorescence-based activity assay. The assay measures the conversion of L-arginine into L-ornithine by a decrease in fluorescent signal due to quenching of a fluorescent probe, Arginase Gold. This way, inhibition of Arginase-1 results in a gain of signal when compared with the uninhibited enzyme. Side-by-side profiling of reference inhibitors in the fluorescence-based assay and a colorimetric urea formation assay revealed similar potencies and the same potency rank order among the two assay formats. The fluorescence-based assay was successfully automated for high-throughput screening of a small-molecule library in 384-well format with a good Z'-factor and hit confirmation rate. Finally, we show that the assay can be used to study the binding kinetics of inhibitors.


Assuntos
Arginase/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Neoplasias/terapia , Arginase/antagonistas & inibidores , Arginase/imunologia , Arginina/genética , Arginina/metabolismo , Fluorescência , Humanos , Neoplasias/imunologia , Ornitina/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
8.
ChemMedChem ; 15(1): 79-95, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31675166

RESUMO

Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease that belongs to the family of tissue kallikreins. Aberrant expression of KLK6 has been found in different cancers and neurodegenerative diseases, and KLK6 is currently studied as a potential target in these pathologies. We report a novel series of KLK6 inhibitors discovered in a high-throughput screen within the European Lead Factory program. Structure-guided design based on docking studies enabled rapid progression of a hit cluster to inhibitors with improved potency, selectivity and pharmacokinetic properties. In particular, inhibitors 32 ((5R)-3-(4-carbamimidoylphenyl)-N-((S)-1-(naphthalen-1-yl)propyl)-2-oxooxazolidine-5-carboxamide) and 34 ((5R)-3-(6-carbamimidoylpyridin-3-yl)-N-((1S)-1-(naphthalen-1-yl)propyl)-2-oxooxazolidine-5-carboxamide) have single-digit nanomolar potency against KLK6, with over 25-fold and 100-fold selectivities against the closely related enzyme trypsin, respectively. The most potent compound, 32, effectively reduces KLK6-dependent invasion of HCT116 cells. The high potency in combination with good solubility and low clearance of 32 make it a good chemical probe for KLK6 target validation in vitro and potentially in vivo.


Assuntos
Calicreínas/antagonistas & inibidores , Fármacos Neuroprotetores/síntese química , Oxazolidinonas/química , Sítios de Ligação , Movimento Celular/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Células HCT116 , Meia-Vida , Humanos , Concentração Inibidora 50 , Calicreínas/metabolismo , Simulação de Acoplamento Molecular , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Oxazolidinonas/metabolismo , Oxazolidinonas/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade
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