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1.
Bioorg Chem ; 146: 107295, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38513326

RESUMO

A concise asymmetric synthesis of clickable enantiomeric pyrrolidines was achieved using Crabbé-Ma allenation. The synthesized iminosugars were grafted by copper-free strain-promoted alkyne-azide cycloaddition onto phosphorus dendrimers. The hexavalent and dodecavalent pyrrolidines were evaluated as ß-glucocerebrosidase inhibitors. The level of inhibition suggests that monofluorocyclooctatriazole group may contribute to the affinity for the protein leading to potent multivalent inhibitors. Docking studies were carried out to rationalize these results. Then, the iminosugars clusters were evaluated as pharmacological chaperones in Gaucher patients' fibroblasts. An increase in ß-glucocerebrosidase activity was observed with hexavalent and dodecavalent pyrrolidines at concentrations as low as 1 µM and 0.1 µM, respectively. These iminosugar clusters constitute the first example of multivalent pyrrolidines acting as pharmacological chaperones against Gaucher disease.


Assuntos
Doença de Gaucher , Imino Açúcares , Humanos , Doença de Gaucher/tratamento farmacológico , Glucosilceramidase , Pirrolidinas/farmacologia , Inibidores Enzimáticos/farmacologia
2.
J Med Chem ; 66(20): 13918-13945, 2023 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-37816126

RESUMO

A series of 25 chiral anti-cancer lipidic alkynylcarbinols (LACs) were devised by introducing an (hetero)aromatic ring between the aliphatic chain and the dialkynylcarbinol warhead. The resulting phenyl-dialkynylcarbinols (PACs) exhibit enhanced stability, while retaining cytotoxicity against HCT116 and U2OS cell lines with IC50 down to 40 nM for resolved eutomers. A clickable probe was used to confirm the PAC prodrug behavior: upon enantiospecific bio-oxidation of the carbinol by the HSD17B11 short-chain dehydrogenase/reductase (SDR), the resulting ynones covalently modify cellular proteins, leading to endoplasmic reticulum stress, ubiquitin-proteasome system inhibition, and apoptosis. Insights into the design of LAC prodrugs specifically bioactivated by HSD17B11 vs its paralogue HSD17B13 were obtained. The HSD17B11/HSD17B13-dependent cytotoxicity of PACs was exploited to develop a cellular assay to identify specific inhibitors of these enzymes. A docking study was performed with the HSD17B11 AlphaFold model, providing a molecular basis of the SDR substrates mimicry by PACs. The safety profile of a representative PAC was established in mice.


Assuntos
Alcinos , Antineoplásicos , Camundongos , Animais , Alcinos/farmacologia , Alcinos/química , Antineoplásicos/farmacologia , Antineoplásicos/química , Acetileno , Estrutura Molecular , Lipídeos/química , Linhagem Celular Tumoral
3.
Chemistry ; 29(53): e202301210, 2023 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-37313991

RESUMO

The first phosphorus dendrimers built on a cyclotriphosphazene core and decorated with six or twelve monofluorocyclooctyne units were prepared. A simple stirring allowed the grafting of N-hexyl deoxynojirimycin inhitopes onto their surface by copper-free strain promoted alkyne-azide cycloaddition click reaction. The synthesized iminosugars clusters were tested as multivalent inhibitors of the biologically relevant enzymes ß-glucocerebrosidase and acid α-glucosidase, involved in Gaucher and Pompe lysosomal storage diseases, respectively. For both enzymes, all the multivalent compounds were more potent than the reference N-hexyl deoxynojirimycin. Remarkably, the final dodecavalent compound proved to be one of the best ß-glucocerebrosidase inhibitors described to date. These cyclotriphosphazene-based deoxynojirimycin dendrimers were then evaluated as pharmacological chaperones against Gaucher disease. Not only did these multivalent constructs cross the cell membranes but they were also able to increase ß-glucocerebrosidase activity in Gaucher cells. Notably, dodecavalent compound allowed a 1.4-fold enzyme activity enhancement at a concentration as low as 100 nM. These new monofluorocyclooctyne-presenting dendrimers may further find numerous applications in the synthesis of multivalent objects for biological and pharmacological purposes.


Assuntos
Dendrímeros , Doença de Gaucher , Humanos , Doença de Gaucher/tratamento farmacológico , Glucosilceramidase/metabolismo , Glucosilceramidase/uso terapêutico , Inibidores Enzimáticos/metabolismo
4.
Front Physiol ; 13: 1055023, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36518111

RESUMO

Purpose: In severe chronic obstructive pulmonary disease (COPD) patients, the application of an inspiratory pressure support (IPS) during exercise increases exercise tolerance and the benefit of exercise training during pulmonary rehabilitation (PR). Moreover, it improves quadriceps endurance after a session of cycling exercise suggesting a reduced muscle fatigue. We looked for the persistence of this effect after PR and sought an association between the improved quadriceps endurance with IPS and the training load during PR. Patients and methods: We studied 20 patients with severe COPD (6 in stage 3and 14 in stage 4 of GOLD) before and after PR. As part of a PR program, patients completed 16 cycling sessions over 6 weeks with the addition of IPS during exercise. As a surrogate of muscular fatigue, quadriceps endurance was measured at 70% of maximal strength in a control condition, after a constant work rate exercise test (CWR) with IPS (TlimQ IPS) or with a sham ventilation (TlimQsham), in a random order. These tests were repeated similarly at the end of PR. Results: PR was associated with a significant increase in maximal power output, cycling endurance, quadriceps strength and endurance. Session training load (power output x duration of the session) increased by 142% during the course of the program. Before PR, CWR duration increases with IPS compared to sham ventilation (Δtime = +244s, p = 0.001). Compared to control condition, post-exercise TlimQ reduction was lower with IPS at isotime than at the end of CWR or than with sham ventilation (-9 ± 21%, -18 ± 16% and -23 ± 18%, respectively, p = 0.09, p < 0.0001 and p < 0.0001). After PR, the post-exercise decrease of TlimQ was reduced after IPS compared to sham (-9 ± 18% vs. -21 ± 17%, respectively, p = 0.004). No relationship was found between the prevention of quadriceps fatigue and the training load. Conclusion: In severe COPD patients, the beneficial effect of a ventilator support on quadriceps endurance persisted after PR with IPS. However, it was not related to the increase in training load, and could not predict the training response to non-invasive ventilation during exercise.

5.
Elife ; 112022 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-35535493

RESUMO

Hundreds of cytotoxic natural or synthetic lipidic compounds contain chiral alkynylcarbinol motifs, but the mechanism of action of those potential therapeutic agents remains unknown. Using a genetic screen in haploid human cells, we discovered that the enantiospecific cytotoxicity of numerous terminal alkynylcarbinols, including the highly cytotoxic dialkynylcarbinols, involves a bioactivation by HSD17B11, a short-chain dehydrogenase/reductase (SDR) known to oxidize the C-17 carbinol center of androstan-3-alpha,17-beta-diol to the corresponding ketone. A similar oxidation of dialkynylcarbinols generates dialkynylketones, that we characterize as highly protein-reactive electrophiles. We established that, once bioactivated in cells, the dialkynylcarbinols covalently modify several proteins involved in protein-quality control mechanisms, resulting in their lipoxidation on cysteines and lysines through Michael addition. For some proteins, this triggers their association to cellular membranes and results in endoplasmic reticulum stress, unfolded protein response activation, ubiquitin-proteasome system inhibition and cell death by apoptosis. Finally, as a proof-of-concept, we show that generic lipidic alkynylcarbinols can be devised to be bioactivated by other SDRs, including human RDH11 and HPGD/15-PGDH. Given that the SDR superfamily is one of the largest and most ubiquitous, this unique cytotoxic mechanism-of-action could be widely exploited to treat diseases, in particular cancer, through the design of tailored prodrugs.


Assuntos
Antineoplásicos , Redutases-Desidrogenases de Cadeia Curta , Antineoplásicos/farmacologia , Estresse do Retículo Endoplasmático , Humanos , Lipídeos , Resposta a Proteínas não Dobradas
6.
Molecules ; 26(23)2021 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-34885805

RESUMO

Pompe disease (PD), a lysosomal storage disease, is caused by mutations of the GAA gene, inducing deficiency in the acid alpha-glucosidase (GAA). This enzymatic impairment causes glycogen burden in lysosomes and triggers cell malfunctions, especially in cardiac, smooth and skeletal muscle cells and motor neurons. To date, the only approved treatment available for PD is enzyme replacement therapy (ERT) consisting of intravenous administration of rhGAA. The limitations of ERT have motivated the investigation of new therapies. Pharmacological chaperone (PC) therapy aims at restoring enzymatic activity through protein stabilization by ligand binding. PCs are divided into two classes: active site-specific chaperones (ASSCs) and the non-inhibitory PCs. In this review, we summarize the different pharmacological chaperones reported against PD by specifying their PC class and activity. An emphasis is placed on the recent use of these chaperones in combination with ERT.


Assuntos
Doença de Depósito de Glicogênio Tipo II/tratamento farmacológico , Animais , Terapia de Reposição de Enzimas , Doença de Depósito de Glicogênio Tipo II/enzimologia , Humanos , Preparações Farmacêuticas/química , alfa-Glucosidases/metabolismo
7.
Pharmaceuticals (Basel) ; 14(12)2021 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-34959681

RESUMO

The mycolic acid biosynthetic pathway represents a promising source of pharmacological targets in the fight against tuberculosis. In Mycobacterium tuberculosis, mycolic acids are subject to specific chemical modifications introduced by a set of eight S-adenosylmethionine dependent methyltransferases. Among these, Hma (MmaA4) is responsible for the introduction of oxygenated modifications. Crystallographic screening of a library of fragments allowed the identification of seven ligands of Hma. Two mutually exclusive binding modes were identified, depending on the conformation of residues 147-154. These residues are disordered in apo-Hma but fold upon binding of the S-adenosylmethionine (SAM) cofactor as well as of analogues, resulting in the formation of the short η1-helix. One of the observed conformations would be incompatible with the presence of the cofactor, suggesting that allosteric inhibitors could be designed against Hma. Chimeric compounds were designed by fusing some of the bound fragments, and the relative binding affinities of initial fragments and evolved compounds were investigated using molecular dynamics simulation and generalised Born and Poisson-Boltzmann calculations coupled to the surface area continuum solvation method. Molecular dynamics simulations were also performed on apo-Hma to assess the structural plasticity of the unliganded protein. Our results indicate a significant improvement in the binding properties of the designed compounds, suggesting that they could be further optimised to inhibit Hma activity.

8.
Sci Rep ; 11(1): 18042, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34508141

RESUMO

Owing to their role in activating enzymes essential for bacterial viability and pathogenicity, phosphopantetheinyl transferases represent novel and attractive drug targets. In this work, we examined the inhibitory effect of the aminido-urea 8918 compound against the phosphopantetheinyl transferases PptAb from Mycobacterium abscessus and PcpS from Pseudomonas aeruginosa, two pathogenic bacteria associated with cystic fibrosis and bronchiectasis, respectively. Compound 8918 exhibits inhibitory activity against PptAb but displays no activity against PcpS in vitro, while no antimicrobial activity against Mycobacterium abscessus or Pseudomonas aeruginosa could be detected. X-ray crystallographic analysis of 8918 bound to PptAb-CoA alone and in complex with an acyl carrier protein domain in addition to the crystal structure of PcpS in complex with CoA revealed the structural basis for the inhibition mechanism of PptAb by 8918 and its ineffectiveness against PcpS. Finally, in crystallo screening of potent inhibitors from the National Cancer Institute library identified a hydroxypyrimidinethione derivative that binds PptAb. Both compounds could serve as scaffolds for the future development of phosphopantetheinyl transferases inhibitors.


Assuntos
Proteínas de Bactérias/química , Inibidores Enzimáticos/química , Pirimidinonas/química , Transferases (Outros Grupos de Fosfato Substituídos)/química , Ureia/química , Proteínas de Bactérias/antagonistas & inibidores , Sítios de Ligação , Inibidores Enzimáticos/farmacologia , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mycobacterium abscessus/enzimologia , Ligação Proteica , Pseudomonas aeruginosa/enzimologia , Proteínas Recombinantes , Relação Estrutura-Atividade , Especificidade por Substrato , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Ureia/análogos & derivados , Ureia/farmacologia
9.
Org Biomol Chem ; 18(39): 7852-7861, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-32975266

RESUMO

A concise and asymmetric synthesis of the enantiomeric pyrrolidines 2 and ent-2 are herein reported. Both enantiomers were assessed as ß-GCase inhibitors. While compound ent-2 acted as a poor competitive inhibitor, its enantiomer 2 proved to be a potent non-competitive inhibitor. Docking studies were carried out to substantiate their respective protein binding mode. Both pyrrolidines were also able to enhance lysosomal ß-GCase residual activity in N370S homozygous Gaucher fibroblasts. Notably, the non-competitive inhibitor 2 displayed an enzyme activity enhancement comparable to that of reference compounds IFG and NN-DNJ. This work highlights the impact of inhibitors chirality on their protein binding mode and shows that, beyond competitive inhibitors, the study of non-competitive ones can lead to the identification of new relevant parmacological chaperones.


Assuntos
Doença de Gaucher
10.
Molecules ; 25(14)2020 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-32660097

RESUMO

Protein misfolding induced by missense mutations is the source of hundreds of conformational diseases. The cell quality control may eliminate nascent misfolded proteins, such as enzymes, and a pathological loss-of-function may result from their early degradation. Since the proof of concept in the 2000s, the bioinspired pharmacological chaperone therapy became a relevant low-molecular-weight compound strategy against conformational diseases. The first-generation pharmacological chaperones were competitive inhibitors of mutant enzymes. Counterintuitively, in binding to the active site, these inhibitors stabilize the proper folding of the mutated protein and partially rescue its cellular function. The main limitation of the first-generation pharmacological chaperones lies in the balance between enzyme activity enhancement and inhibition. Recent research efforts were directed towards the development of promising second-generation pharmacological chaperones. These non-inhibitory ligands, targeting previously unknown binding pockets, limit the risk of adverse enzymatic inhibition. Their pharmacophore identification is however challenging and likely requires a massive screening-based approach. This review focuses on second-generation chaperones designed to restore the cellular activity of misfolded enzymes. It intends to highlight, for a selected set of rare inherited metabolic disorders, the strategies implemented to identify and develop these pharmacologically relevant small organic molecules as potential drug candidates.


Assuntos
Ativadores de Enzimas/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Chaperonas Moleculares/uso terapêutico , Ativadores de Enzimas/química , Inibidores Enzimáticos/química , Humanos , Chaperonas Moleculares/química , Mutação , Dobramento de Proteína
11.
Bioorg Med Chem Lett ; 30(2): 126796, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31757669

RESUMO

Dysregulation of the ceramide transport protein CERT is associated to diseases such as cancer. In search for new CERT START domain ligands, N-dodecyl-deoxynojirimycin (N-dodecyl-DNJ) iminosugar was found to display, as a ceramide mimic, significant protein recognition. To reinforce the lipophilic interactions and strengthen this protein binding, a docking study was carried out in order to select the optimal position on which to introduce an additional O-alkyl chain on N-dodecyl-DNJ. Analysis of the calculated poses for three different regioisomers indicated an optimal calculated interaction pattern for N,O3-didodecyl-DNJ. The two most promising regioisomers were prepared by a divergent route and their binding to the CERT START domain was evaluated with fluorescence intensity (FLINT) binding assay. N,O3-didodecyl-DNJ was confirmed to be a new binder prototype with level of protein recognition in the FLINT assay comparable to the best known ligands from the alkylated HPA-12 series. This work opens promising perspectives for the development of new inhibitors of CERT-mediated ceramide trafficking.


Assuntos
Glucosamina/análogos & derivados , Proteínas Serina-Treonina Quinases/química , 1-Desoxinojirimicina/química , 1-Desoxinojirimicina/metabolismo , Sítios de Ligação , Ceramidas/metabolismo , Glucosamina/química , Glucosamina/metabolismo , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/metabolismo , Humanos , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios Proteicos , Proteínas Serina-Treonina Quinases/metabolismo , Estereoisomerismo , Termodinâmica
12.
Chembiochem ; 19(23): 2438-2442, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30303294

RESUMO

The first biologically relevant clickable probe related to the antitumor marine lipid jaspine B is reported. The concise synthetic route to both enantiomers relied on the supercritical fluid chromatography (SFC) enantiomeric resolution of racemic materials. The eutomeric dextrogyre derivative represents the first jaspine B analogue with enhanced cytotoxicity with IC50 down to 30 nm. These enantiomeric probes revealed a chiralitydependent cytoplasmic imaging of U2OS cancer cells by in situ click labeling.


Assuntos
Alcinos/química , Antineoplásicos/química , Corantes Fluorescentes/química , Sondas Moleculares/química , Esfingosina/análogos & derivados , Alcinos/síntese química , Alcinos/toxicidade , Antineoplásicos/síntese química , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Química Click , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/toxicidade , Humanos , Sondas Moleculares/síntese química , Sondas Moleculares/toxicidade , Esfingosina/síntese química , Esfingosina/toxicidade , Estereoisomerismo
13.
Bioorg Med Chem ; 25(6): 1984-1989, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28237558

RESUMO

The enigmatical dichotomy between the two CERT/GPBP protein isoforms, their vast panel of biological implications and the scarcity of known antagonist series call for new ligand chemotypes identification. We report the design of iminosugar-based ceramide mimics for the development of new START domain ligands potentially targeting either protein isoforms. Strategic choice of (i) an iminoxylitol core structure and (ii) the positioning of two dodecyl residues led to an extent of protein binding comparable to that of the natural cargo lipid ceramide or the archetypical inhibitor HPA-12. Molecular docking study evidenced a possible mode of protein binding fully coherent with the one observed in crystalline co-structures of known ligands. The present study thus paves the way for cellular CERT inhibition studies en route to the development of pharmacological tools aiming at deciphering the respective function and therapeutic potential of the two CERT/GPBP protein isoforms.


Assuntos
Imino Açúcares/química , Mimetismo Molecular , Isoformas de Proteínas/metabolismo , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Desenho de Fármacos , Imino Açúcares/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Espectroscopia de Prótons por Ressonância Magnética , Espectrometria de Massas por Ionização por Electrospray
14.
Chemistry ; 22(49): 17514-17525, 2016 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-27628428

RESUMO

In 2001, two years before the disclosure of the CERT-associated Cer transfer machinery, N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)alkanamides (HPAs) were described as the first, and to date unique, family of intracellular Cer trafficking inhibitors. The dodecanamide derivative, HPA-12, turned out to be a benchmark as a cellular inhibitor of CERT-mediated de novo sphingomyelin biosynthesis. In only 15 years after its first disclosure, this compound has prompted a growing number of biological and chemical studies. Its initial chemical development closely paralleled the study of the CERT protein. It was only after its structural revision in 2011 that HPA-12 received broad attention from the synthetic chemistry community, leading to novel analogues with enhanced protein binding. This Minireview aims at presenting an exhaustive report of the syntheses of HPA-12 and analogues. Biological activities of this CERT inhibitor and structure-activity relationships are also presented to afford a comprehensive overview of the chemistry and biology of the HPA series.


Assuntos
Amidas/química , Ceramidas/química , Movimento Celular , Relação Estrutura-Atividade
15.
Chemistry ; 22(19): 6676-86, 2016 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-27031925

RESUMO

A series of 12 analogues of the Cer transfer protein (CERT) antagonist HPA-12 with long aliphatic chains were prepared as their (1R,3S)-syn and (1R,3R)-anti stereoisomers from pivotal chiral oxoamino acids. The enantioselective access to these intermediates as well as their ensuing transformation relied on a practical crystallization-induced asymmetric transformation (CIAT) process. Sonogashira coupling followed by triple bond reduction and thiophene ring hydrodesulfurization (HDS) into the corresponding alkane moieties was then implemented to complete the synthetic routes delivering the targeted HPA-12 analogues in concise 4- to 6-step reaction sequences. Ten compounds were evaluated regarding their ability to bind to the CERT START domain by using the recently developed time-resolved FRET-based homogeneous (HTR-FRET) binding assay. The introduction of a lipophilic appendage on the phenyl moiety led to an overall 10- to 1000-fold enhancement of the protein binding, with the highest effect being observed for a n-hexyl residue in the meta position. The importance of the phenyl ring for the activity was indicated by the reduced potency of the 3-deoxyphytoceramide aliphatic analogues. The 1,3-syn stereoisomers were systematically more potent than their 1,3-anti analogues. In silico studies were used to rationalized these trends, leading to a model of protein recognition coherent with the stronger binding of (1R,3S)-syn HPAs.


Assuntos
Amidas/química , Ceramidas/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Tiofenos/química , Amidas/metabolismo , Transporte Biológico , Ceramidas/metabolismo , Ligantes , Modelos Moleculares , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade
16.
Chem Biodivers ; 12(7): 1115-25, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26172331

RESUMO

A new sphingolipid hybrid molecule was designed to assemble, within a tail-to-tail double-chain structure, the ceramide hydrophilic moiety and the tetrahydrofuran pharmacophore of jaspine B, a natural product known to interfere with sphingolipid metabolism. This compound was prepared through acylation of sphingosine with a jaspine B derivative bearing a COOH group in the terminal position of the aliphatic backbone. This new hybrid molecule was evaluated for its capacities to affect melanoma cell viability and sphingolipid metabolism. While retaining the cytotoxicity of ceramide itself, this compound was shown to lower the sphingomyelin cellular levels and significantly enhance the production of sphingosine-1-phosphate, thus representing a novel sphingolipid metabolism modulator.


Assuntos
Produtos Biológicos/farmacologia , Ceramidas/farmacologia , Esfingolipídeos/metabolismo , Esfingosina/análogos & derivados , Animais , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ceramidas/química , Ceramidas/metabolismo , Relação Dose-Resposta a Droga , Humanos , Camundongos , Conformação Molecular , Esfingolipídeos/química , Esfingosina/química , Esfingosina/metabolismo , Esfingosina/farmacologia , Relação Estrutura-Atividade
17.
Bioorg Med Chem ; 23(9): 2004-9, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25818765

RESUMO

The first unified synthetic route to the four enantiopure HPA-12 stereoisomers in multi-gram scale is reported based on Crystallization-Induced Asymmetric Transformation (CIAT) technology. This preparative stereoselective synthesis allowed the unprecedented comparative evaluation of HPA-12 stereoisomers regarding their interaction with the CERT START domain. In vitro binding assay coupled to in silico docking approach indicate a possible interaction for the four derivatives. The first TR-FRET homogeneous-phase assay was developed to quantify their binding to the START domain, allowing complete determination of HPA-12 EC50. Results indicate that not only the (1R,3S) lead to the strongest binding, but that both 1R and 3S stereocenters similarly contribute to extent of recognition This automated homogenous assay further opens up promising prospect for the identification of novel potential CERT antagonist by means of high throughput screening.


Assuntos
Amidas/síntese química , Amidas/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Amidas/química , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Conformação Molecular , Estereoisomerismo , Relação Estrutura-Atividade
18.
J Biomol Screen ; 20(6): 779-87, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25716975

RESUMO

Sphingomyelin (SM) metabolism deregulation was recently associated with cell metastasis and chemoresistance, and several pharmacological strategies targeting SM metabolism have emerged. The ceramide (Cer) generated in the endoplasmic reticulum (ER) is transferred to the Golgi apparatus to be transformed into SM. CERamide Transfer (CERT) protein is responsible for the nonvesicular trafficking of Cer to Golgi. Blocking the CERT-mediated ER-to-Golgi Cer transfer is an interesting antioncogenic therapeutic approach. Here, we developed a protein-lipid interaction assay for the identification of new CERT-Cer interaction inhibitors. Frequently used for protein-protein interaction by enzymatic and analyte dosage assays, homogeneous time-resolved fluorescence technology was adapted for the first time to a lipid-protein binding assay. This test was developed for high-throughput screening, and a library of 672 molecules was screened. Seven hits were identified, and their inhibitory effect quantified by EC50 measurements showed binding inhibition three orders of magnitude more potent than that of HPA12, the unique known CERT antagonist to date. Each compound was tested on an independent test, confirming its high affinity and pharmacological potential.


Assuntos
Proteínas de Transporte/metabolismo , Ceramidas/metabolismo , Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proteínas de Transporte/química , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Humanos , Cinética , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Transporte Proteico/efeitos dos fármacos , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas
19.
Chembiochem ; 15(17): 2522-8, 2014 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-25256104

RESUMO

A highly compartmentalized enzymatic network regulates the pro-apoptotic and proliferative effects of sphingolipids. Over-conversion of ceramide (Cer) correlates with insensitivity to apoptosis signaling (in response to chemotherapy) and to drug resistance of cancer cells. De novo sphingomyelin biosynthesis relies on non-vesicular ceramide trafficking by the CERT (CERamide Transfer) protein. Therefore, blocking CERT transfer, thus leading to increased intracellular ceramide availability, represents a potential anticancer strategy. Our study is based on the implementation of an in vitro binding assay, supported by in silico molecular docking. It constitutes the first attempt to explore at the molecular level for the identification of novel CERT ligands. This approach is the first step toward in silico design and optimization of CERT inhibitor candidates, potentially relevant as innovative ceramide-transfer-targeting therapeutic agents.


Assuntos
Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Ceramidas/metabolismo , Transporte Biológico/efeitos dos fármacos , Ligantes , Modelos Moleculares , Conformação Molecular
20.
Lung ; 192(5): 775-9, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25064631

RESUMO

INTRODUCTION: The prevalence of pulmonary restriction increases in the elderly and detection could be challenging due to the difficulty in measuring lung volumes in older patients. The recently published Global Lung Function Initiative (GLI) equations were found to predict better restriction in middle-aged patients compared to ERS'93 norms. However, the ability of the GLI equations to detect pulmonary restriction in older patients has not been investigated. PATIENTS AND METHODS: We extracted spirometric data in patients older than 85 years from the database of our pulmonary function testing laboratory. The population with pulmonary restriction was defined as those having a total lung capacity value (TLC) below the lower limit of normal (LLN) using ERS'93 equations. We then compared the ability of the ERS'93 and GLI equations to detect this when the forced vital capacity (FVC) was below the LLN. RESULTS: We analyzed data from 285 patients. A true restrictive defect was found in 66 patients (23%). Sensitivity to detect a reduced TLC was higher when calculated from the GLI than the ERS'93 equations, (70 vs 45%). By contrast, specificity was lower (74 vs 89%, respectively); there was no difference in the negative predictive value (89 and 84%). Using receiver operating curves, both sets of equations performed similarly to detect spirometric restriction. CONCLUSIONS: In conclusion, both sets of equations similarly predicted a pulmonary restriction in older subjects. The high negative predictive value of the GLI equations thus allows for static lung volume measurement to be avoided in older patients when the FCV exceeds the LLN whatever the predicted equation used.


Assuntos
Pneumopatias/fisiopatologia , Pulmão/fisiopatologia , Modelos Biológicos , Capacidade Vital , Fatores Etários , Idoso de 80 Anos ou mais , Área Sob a Curva , Bases de Dados Factuais , Feminino , França , Humanos , Pneumopatias/diagnóstico , Medidas de Volume Pulmonar , Masculino , Valor Preditivo dos Testes , Curva ROC , Espirometria
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