RESUMO
MALT1 paracaspase is central for lymphocyte antigen-dependent responses including NF-κB activation. We discovered nanomolar, selective allosteric inhibitors of MALT1 that bind by displacing the side chain of Trp580, locking the protease in an inactive conformation. Interestingly, we had previously identified a patient homozygous for a MALT1 Trp580-to-serine mutation who suffered from combined immunodeficiency. We show that the loss of tryptophan weakened interactions between the paracaspase and C-terminal immunoglobulin MALT1 domains resulting in protein instability, reduced protein levels and functions. Upon binding of allosteric inhibitors of increasing potency, we found proportionate increased stabilization of MALT1-W580S to reach that of wild-type MALT1. With restored levels of stable MALT1 protein, the most potent of the allosteric inhibitors rescued NF-κB and JNK signaling in patient lymphocytes. Following compound washout, MALT1 substrate cleavage was partly recovered. Thus, a molecular corrector rescues an enzyme deficiency by substituting for the mutated residue, inspiring new potential precision therapies to increase mutant enzyme activity in other deficiencies.
Assuntos
Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/antagonistas & inibidores , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Regulação da Expressão Gênica , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/terapia , Linfócitos/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/ultraestrutura , NF-kappa B/metabolismo , Proteínas de Neoplasias , Transdução de SinaisRESUMO
Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is essential for immune responses triggered by antigen receptors but the contribution of its paracaspase activity is not fully understood. Here, we studied how MALT1 proteolytic function regulates T-cell activation and fate after engagement of the T-cell receptor pathway. We show that MLT-827, a potent and selective MALT1 paracaspase inhibitor, does not prevent the initial phase of T-cell activation, in contrast to the pan-protein kinase C inhibitor AEB071. However, MLT-827 strongly impacted cell expansion after activation. We demonstrate this is the consequence of profound inhibition of IL-2 production as well as reduced expression of the IL-2 receptor alpha subunit (CD25), resulting from defective canonical NF-κB activation and accelerated mRNA turnover mechanisms. Accordingly, MLT-827 revealed a unique transcriptional fingerprint of MALT1 protease activity, providing evidence for broad control of T-cell signaling pathways. Altogether, this first report with a potent and selective inhibitor elucidates how MALT1 paracaspase activity integrates several T-cell activation pathways and indirectly controls gamma-chain receptor dependent survival, to impact on T-cell expansion.
Assuntos
Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , NF-kappa B/metabolismo , Linfócitos T/imunologia , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Imunomodulação , Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Ativação Linfocitária , Proteólise , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de SinaisRESUMO
Starting from a weak screening hit, potent and selective inhibitors of the MALT1 protease function were elaborated. Advanced compounds displayed high potency in biochemical and cellular assays. Compounds showed activity in a mechanistic Jurkat T cell activation assay as well as in the B-cell lymphoma line OCI-Ly3, which suggests potential use of MALT1 inhibitors in the treatment of autoimmune diseases as well as B-cell lymphomas with a dysregulated NF-κB pathway. Initially, rat pharmacokinetic properties of this compound series were dominated by very high clearance which could be linked to amide cleavage. Using a rat hepatocyte assay a good in vitro-in vivo correlation could be established which led to the identification of compounds with improved PK properties.
Assuntos
Antineoplásicos/farmacologia , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/antagonistas & inibidores , Piperidinas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Hepatócitos/efeitos dos fármacos , Humanos , Células Jurkat , Microssomos/efeitos dos fármacos , Estrutura Molecular , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Piperidinas/síntese química , Piperidinas/química , Proteólise/efeitos dos fármacos , Ratos , Relação Estrutura-AtividadeRESUMO
CSN5 is the zinc metalloprotease subunit of the COP9 signalosome (CSN), which is an important regulator of cullin-RING E3 ubiquitin ligases (CRLs). CSN5 is responsible for the cleavage of NEDD8 from CRLs, and blocking deconjugation of NEDD8 traps the CRLs in a hyperactive state, thereby leading to auto-ubiquitination and ultimately degradation of the substrate recognition subunits. Herein, we describe the discovery of azaindoles as a new class of CSN5 inhibitors, which interact with the active-site zinc ion of CSN5 through an unprecedented binding mode. The best compounds inhibited CSN5 with nanomolar potency, led to degradation of the substrate recognition subunit Skp2 in cells, and reduced the viability of HCT116 cells.
Assuntos
Complexo do Signalossomo COP9/antagonistas & inibidores , Indóis/metabolismo , Zinco/metabolismo , Sítios de Ligação , Complexo do Signalossomo COP9/genética , Complexo do Signalossomo COP9/metabolismo , Domínio Catalítico , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Células HCT116 , Humanos , Indóis/química , Indóis/farmacologia , Simulação de Acoplamento Molecular , Proteína NEDD8/química , Proteína NEDD8/metabolismo , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Quinases Associadas a Fase S/química , Proteínas Quinases Associadas a Fase S/metabolismo , Zinco/químicaRESUMO
Hit generation is a crucial step in drug discovery that will determine the speed and chance of success of identifying drug candidates. Many strategies are now available to identify chemical starting points, or hits, and each biological target warrants a tailored approach. In this set of best practices, we detail the essential approaches for target centric hit generation and the opportunities and challenges they come with. We then provide guidance on how to validate hits to ensure medicinal chemistry is only performed on compounds and scaffolds that engage the target of interest and have the desired mode of action. Finally, we discuss the design of integrated hit generation strategies that combine several approaches to maximize the chance of identifying high quality starting points to ensure a successful drug discovery campaign.
Assuntos
Química Farmacêutica , Descoberta de Drogas , BiologiaRESUMO
Phenotypic drug discovery has a long track record of delivering innovative drugs and has received renewed attention in the last few years. The promise of this approach, however, comes with several challenges that should be addressed to avoid wasting time and resources on drugs with undesired modes of action or, worse, false-positive hits. In this set of best practices, we go over the essential steps of phenotypic drug discovery and provide guidance on how to increase the chance of success in identifying validated and relevant chemical starting points for optimization: selecting the right assay, selecting the right compound screening library and developing appropriate hit validation assays. Then, we highlight the importance of initiating studies to determine the mode of action of the identified hits early and present the current state of the art.
Assuntos
Química Farmacêutica , Descoberta de Drogas , Europa (Continente) , Humanos , Fenótipo , EstudantesRESUMO
As part of an initiative aimed to share best practices in Medicinal Chemistry, the European Federation for Medicinal Chemistry (EFMC) is preparing a series of webinars and slide sets focused on the early phase of drug discovery. This educational material is freely accessible through the EFMC. The main target audiences are students or early career scientists and we also believe it will be valuable for experienced practitioners. The first of the series is focused on the generation and validation of high-quality chemical probes, which are critical for drug discovery and more broadly to further our understanding of human biology and disease.
Assuntos
Química Farmacêutica/educação , Descoberta de Drogas/educação , Indicadores e Reagentes/normas , Agências Internacionais , Sociedades Científicas , Webcasts como Assunto , Europa (Continente) , Humanos , Indicadores e Reagentes/químicaRESUMO
The paracaspase MALT1 has gained increasing interest as a target for the treatment of subsets of lymphomas as well as autoimmune diseases, and there is a need for suitable compounds to explore the therapeutic potential of this target. Here, we report the optimization of the in vivo potency of pyrazolopyrimidines, a class of highly selective allosteric MALT1 inhibitors. High doses of the initial lead compound led to tumor stasis in an activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) xenograft model, but this compound suffered from a short in vivo half-life and suboptimal potency in whole blood. Guided by metabolism studies, we identified compounds with reduced metabolic clearance and increased in vivo half-life. In the second optimization step, masking one of the hydrogen-bond donors of the central urea moiety through an intramolecular interaction led to improved potency in whole blood. This was associated with improved in vivo potency in a mechanistic model of B cell activation. The optimized compound led to tumor regression in a CARD11 mutant ABC-DLBCL lymphoma xenograft model.
Assuntos
Sangue/metabolismo , Inibidores de Caspase/uso terapêutico , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/antagonistas & inibidores , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Ureia/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Inibidores de Caspase/síntese química , Inibidores de Caspase/metabolismo , Inibidores de Caspase/farmacocinética , Linhagem Celular Tumoral , Feminino , Meia-Vida , Humanos , Camundongos Endogâmicos BALB C , Camundongos SCID , Microssomos Hepáticos/metabolismo , Neoplasias/tratamento farmacológico , Pirazóis/síntese química , Pirazóis/metabolismo , Pirazóis/farmacocinética , Pirimidinas/síntese química , Pirimidinas/metabolismo , Pirimidinas/farmacocinética , Ratos Sprague-Dawley , Ovinos , Ureia/síntese química , Ureia/metabolismo , Ureia/farmacocinética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
MALT1 plays a central role in immune cell activation by transducing NF-κB signaling, and its proteolytic activity represents a key node for therapeutic intervention. Two cycles of scaffold morphing of a high-throughput biochemical screening hit resulted in the discovery of MLT-231, which enabled the successful pharmacological validation of MALT1 allosteric inhibition in preclinical models of humoral immune responses and B-cell lymphomas. Herein, we report the structural activity relationships (SARs) and analysis of the physicochemical properties of a pyrazolopyrimidine-derived compound series. In human T-cells and B-cell lymphoma lines, MLT-231 potently and selectively inhibits the proteolytic activity of MALT1 in NF-κB-dependent assays. Both in vitro and in vivo profiling of MLT-231 support further optimization of this in vivo tool compound toward preclinical characterization.
Assuntos
Inibidores de Caspase/uso terapêutico , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Ureia/análogos & derivados , Ureia/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Inibidores de Caspase/síntese química , Inibidores de Caspase/farmacologia , Descoberta de Drogas , Feminino , Humanos , Imunidade Humoral/efeitos dos fármacos , Masculino , Camundongos Endogâmicos BALB C , Estrutura Molecular , Pirazóis/síntese química , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/síntese química , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Ureia/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Genetic disruption or short-term pharmacological inhibition of MALT1 protease is effective in several preclinical models of autoimmunity and B cell malignancies. Despite these protective effects, the severe reduction in regulatory T cells (Tregs) and the associated IPEX-like pathology occurring upon congenital disruption of the MALT1 protease in mice has raised concerns about the long-term safety of MALT1 inhibition. Here we describe the results of a series of toxicology studies in rat and dog species using MLT-943, a novel potent and selective MALT1 protease inhibitor. While MLT-943 effectively prevented T cell-dependent B cell immune responses and reduced joint inflammation in the collagen-induced arthritis rat pharmacology model, in both preclinical species, pharmacological inhibition of MALT1 was associated with a rapid and dose-dependent reduction in Tregs and resulted in the progressive appearance of immune abnormalities and clinical signs of an IPEX-like pathology. At the 13-week time point, rats displayed severe intestinal inflammation associated with mast cell activation, high serum IgE levels, systemic T cell activation and mononuclear cell infiltration in multiple tissues. Importantly, using thymectomized rats we demonstrated that MALT1 protease inhibition affects peripheral Treg frequency independently of effects on thymic Treg output and development. Our data confirm the therapeutic potential of MALT1 protease inhibitors but highlight the safety risks and challenges to consider before potential application of such inhibitors into the clinic.
Assuntos
Diabetes Mellitus Tipo 1/congênito , Diarreia/etiologia , Doenças Genéticas Ligadas ao Cromossomo X/etiologia , Doenças do Sistema Imunitário/congênito , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/antagonistas & inibidores , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Diabetes Mellitus Tipo 1/etiologia , Cães , Feminino , Humanos , Doenças do Sistema Imunitário/etiologia , Inflamação/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Linfócitos T Reguladores/imunologiaRESUMO
OBJECTIVE: Fcγ receptors (FcγR) play important roles in both protective and pathogenic immune responses. The assembly of the CBM signalosome encompassing caspase recruitment domain-containing protein 9, B cell CLL/lymphoma 10, and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT-1) is required for optimal FcγR-induced canonical NF-κB activation and proinflammatory cytokine release. This study was undertaken to clarify the relevance of MALT-1 protease activity in FcγR-driven events and evaluate the therapeutic potential of selective MALT-1 protease inhibitors in FcγR-mediated diseases. METHODS: Using genetic and pharmacologic disruption of MALT-1 scaffolding and enzymatic activity, we assessed the relevance of MALT-1 function in murine and human primary myeloid cells upon stimulation with immune complexes (ICs) and in murine models of autoantibody-driven arthritis and immune thrombocytopenic purpura (ITP). RESULTS: MALT-1 protease function is essential for optimal FcγR-induced production of proinflammatory cytokines by various murine and human myeloid cells stimulated with ICs. In contrast, MALT-1 protease inhibition did not affect the Syk-dependent, FcγR-mediated production of reactive oxygen species or leukotriene B4 . Notably, pharmacologic MALT-1 protease inhibition in vivo reduced joint inflammation in the murine K/BxN serum-induced arthritis model (mean area under the curve for paw swelling of 45.42% versus 100% in control mice; P = 0.0007) but did not affect platelet depletion in a passive model of ITP. CONCLUSION: Our findings indicate a specific contribution of MALT-1 protease activity to FcγR-mediated events and suggest that MALT-1 protease inhibitors have therapeutic potential in a subset of FcγR-driven inflammatory disorders.
Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/imunologia , Receptores de IgG/imunologia , Animais , Complexo Antígeno-Anticorpo/metabolismo , Plaquetas/metabolismo , Citocinas/imunologia , Modelos Animais de Doenças , Humanos , Camundongos , Células Mieloides/metabolismoRESUMO
We examined the effect of simultaneously incorporating proline or proline-amino acid chimeras in positions 9, 10, and/or 11 of substance P, on the affinity for the two NK-1 binding sites and on second-messenger activation. Because these 3-substituted prolines constrain not only the (phi,psi) values of the peptide backbone, but also the chi space of the amino acid side chain, we were able to gather data on the structural requirements for high-affinity binding to the NK-1 receptor. We were able to confirm that this C-terminal component is crucial and that it should adopt an extended conformation close to a polyproline II structure when bound to the receptor. The partial additivity of these constraints, more specifically, for the NK-1M site, suggests that the peptide backbone flexibility around the hinge-point residue Gly9 is essential to subtly position crucial side chains.
Assuntos
Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Receptores da Neurocinina-1/metabolismo , Substância P/química , Substância P/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Modelos Lineares , Modelos Moleculares , Prolina/química , Ligação Proteica , Conformação ProteicaRESUMO
The COP9 signalosome (CSN) is a central component of the activation and remodelling cycle of cullin-RING E3 ubiquitin ligases (CRLs), the largest enzyme family of the ubiquitin-proteasome system in humans. CRLs are implicated in the regulation of numerous cellular processes, including cell cycle progression and apoptosis, and aberrant CRL activity is frequently associated with cancer. Remodelling of CRLs is initiated by CSN-catalysed cleavage of the ubiquitin-like activator NEDD8 from CRLs. Here we describe CSN5i-3, a potent, selective and orally available inhibitor of CSN5, the proteolytic subunit of CSN. The compound traps CRLs in the neddylated state, which leads to inactivation of a subset of CRLs by inducing degradation of their substrate recognition module. CSN5i-3 differentially affects the viability of tumour cell lines and suppresses growth of a human xenograft in mice. Our results provide insights into how CSN regulates CRLs and suggest that CSN5 inhibition has potential for anti-tumour therapy.
Assuntos
Antineoplásicos/farmacologia , Azepinas/farmacologia , Complexo do Signalossomo COP9/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Linfoma Anaplásico de Células Grandes/tratamento farmacológico , Pirazóis/farmacologia , Ubiquitina-Proteína Ligases/genética , Animais , Antineoplásicos/síntese química , Azepinas/síntese química , Complexo do Signalossomo COP9/genética , Complexo do Signalossomo COP9/metabolismo , Feminino , Células HCT116 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/metabolismo , Linfoma Anaplásico de Células Grandes/patologia , Camundongos , Camundongos SCID , Terapia de Alvo Molecular , Proteína NEDD8/genética , Proteína NEDD8/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise/efeitos dos fármacos , Pirazóis/síntese química , Células THP-1 , Carga Tumoral/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
RATIONAL: Homeostasis of vascular barriers depends upon sphingosine 1-phosphate (S1P) signaling via the S1P1 receptor. Accordingly, S1P1 competitive antagonism is known to reduce vascular barrier integrity with still unclear pathophysiological consequences. This was explored in the present study using NIBR-0213, a potent and selective S1P1 competitive antagonist. RESULTS: NIBR-0213 was tolerated at the efficacious oral dose of 30 mg/kg BID in the rat adjuvant-induced arthritis (AiA) model, with no sign of labored breathing. However, it induced dose-dependent acute vascular pulmonary leakage and pleural effusion that fully resolved within 3-4 days, as evidenced by MRI monitoring. At the supra-maximal oral dose of 300 mg/kg QD, NIBR-0213 impaired lung function (with increased breathing rate and reduced tidal volume) within the first 24 hrs. Two weeks of NIBR-0213 oral dosing at 30, 100 and 300 mg/kg QD induced moderate pulmonary changes, characterized by alveolar wall thickening, macrophage accumulation, fibrosis, micro-hemorrhage, edema and necrosis. In addition to this picture of chronic inflammation, perivascular edema and myofiber degeneration observed in the heart were also indicative of vascular leakage and its consequences. CONCLUSIONS: Overall, these observations suggest that, in the rat, the lung is the main target organ for the S1P1 competitive antagonism-induced acute vascular leakage, which appears first as transient and asymptomatic but could lead, upon chronic dosing, to lung remodeling with functional impairments. Hence, this not only raises the question of organ specificity in the homeostasis of vascular barriers, but also provides insight into the pre-clinical evaluation of a potential safety window for S1P1 competitive antagonists as drug candidates.
Assuntos
Compostos de Anilina/farmacologia , Artrite Experimental/fisiopatologia , Permeabilidade Capilar/efeitos dos fármacos , Dipeptídeos/farmacologia , Inflamação/fisiopatologia , Lisofosfolipídeos/metabolismo , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Esfingosina/análogos & derivados , Adjuvantes Imunológicos/toxicidade , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/tratamento farmacológico , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Homeostase/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Esfingosina/metabolismoRESUMO
3-Hydroxyanthranilic acid 3,4-dioxygenase (EC 1.13.11.6; HADO) was purified to homogeneity from beef liver with the use of two dye columns (Cibacron Blue and Reactive Green 19) and hydroxyapatite. Two active peaks of enzyme were isolated from the hydroxyapatite column or by nondenaturing chromatofocusing of the enzyme prior to hydroxyapatite. The two active forms moved with different electrophoretic mobilities when they were subjected to nondenaturing polyacrylamide gel electrophoresis, regardless of the method of isolation. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), however, these species had apparently identical mobilities and have, therefore, close molecular mass. Analysis by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry gave them a molecular mass of 32566 and 32515 Da, respectively, for the species with apparent pI values of 5.60 and 4.98, respectively, suggesting that they differ only in the presence or absence of the iron cofactor. The N-terminal group appears to be blocked as no amino-terminal sequence was possible from direct Edman degradation. A new inactivator of the enzyme, 6-chloro-3-hydroxyanthranilic acid, was synthesized and was shown to exhibit time-dependent inactivation. A possible mechanism for inactivation is proposed.
Assuntos
Dioxigenases , Fígado/enzimologia , Oxigenases/isolamento & purificação , 3-Hidroxiantranilato 3,4-Dioxigenase , Animais , Bovinos , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Cinética , Oxigenases/antagonistas & inibidores , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Botulinum neurotoxins are the most potent toxins known to date. They are zinc-metalloproteases able to cleave selectively an essential component of neurotransmitter exocytosis, causing the syndrome of botulism characterized by a flaccid paralysis. There is a great interest in designing antagonists of the action of these toxins. One way is to inhibit their catalytic activity. In this study, we report the design of such inhibitors directed toward BoNT/B. A study of the S(1) subsite specificity, using several beta-amino thiols, has shown that this subsite prefers a p-carboxybenzyl moiety. The specificity of the S(1)' and S(2)' subsites was studied using two libraries of pseudotripeptides containing the S(1) synthon derived from the best beta-amino thiol tested. Finally, a selection of various non natural amino acids for the recognition of the "prime" domain led to the most potent inhibitor of BoNT/B described to date with a K(i) value of 20 nM.
Assuntos
Benzoatos/síntese química , Toxinas Botulínicas/antagonistas & inibidores , Metaloendopeptidases/antagonistas & inibidores , Oligopeptídeos/síntese química , Inibidores de Proteases/síntese química , Benzoatos/química , Toxinas Botulínicas/química , Toxinas Botulínicas Tipo A , Metaloendopeptidases/química , Modelos Moleculares , Oligopeptídeos/química , Inibidores de Proteases/química , Relação Estrutura-Atividade , Compostos de EnxofreRESUMO
A prodrug approach to optimize the oral exposure of a series of sphingosine 1-phosphate receptor 1 (S1P(1)) antagonists for chronic efficacy studies led to the discovery of (S)-2-{[3'-(4-chloro-2,5-dimethylphenylsulfonylamino)-3,5-dimethylbiphenyl-4-carbonyl]methylamino}-4-dimethylaminobutyric acid methyl ester 14. Methyl ester prodrug 14 is hydrolyzed in vivo to the corresponding carboxylic acid 15, a potent and selective S1P(1) antagonist. Oral administration of the prodrug 14 induces sustained peripheral blood lymphocyte reduction in rats. In a rat cardiac transplantation model coadministration of a nonefficacious dose of prodrug 14 with a nonefficacious dose of sotrastaurin (19), a protein kinase C inhibitor, or everolimus (20), an mTOR inhibitor, effectively prolonged the survival time of rat cardiac allografts. This demonstrates that clinically useful immunomodulation mediated by the S1P(1) receptor can be achieved with an S1P(1) antagonist generated in vivo after oral administration of its prodrug.
Assuntos
Aminobutiratos/síntese química , Transplante de Coração , Linfócitos/efeitos dos fármacos , Pró-Fármacos/síntese química , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Sulfonamidas/síntese química , Administração Oral , Aminobutiratos/administração & dosagem , Aminobutiratos/farmacologia , Animais , Linfócitos/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Estrutura Molecular , Pró-Fármacos/administração & dosagem , Pró-Fármacos/farmacologia , Ratos , Ratos Endogâmicos Lew , Sulfonamidas/administração & dosagem , Sulfonamidas/farmacologiaRESUMO
Lymphocyte trafficking is critically regulated by the Sphingosine 1-phosphate receptor-1 (S1P(1)), a G protein-coupled receptor that has been highlighted as a promising therapeutic target in autoimmunity. Fingolimod (FTY720, Gilenya) is a S1P(1) receptor agonist that has recently been approved for the treatment of multiple sclerosis (MS). Here, we report the discovery of NIBR-0213, a potent and selective S1P(1) antagonist that induces long-lasting reduction of peripheral blood lymphocyte counts after oral dosing. NIBR-0213 showed comparable therapeutic efficacy to fingolimod in experimental autoimmune encephalomyelitis (EAE), a model of human MS. These data provide convincing evidence that S1P(1) antagonists are effective in EAE. In addition, the profile of NIBR-0213 makes it an attractive candidate to further study the consequences of S1P(1) receptor antagonism and to differentiate the effects from those of S1P(1) agonists.
Assuntos
Compostos de Anilina/farmacologia , Compostos de Anilina/uso terapêutico , Dipeptídeos/farmacologia , Dipeptídeos/uso terapêutico , Encefalomielite Autoimune Experimental/tratamento farmacológico , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Administração Oral , Compostos de Anilina/administração & dosagem , Compostos de Anilina/química , Animais , Células CHO , Cricetinae , Cricetulus , Dipeptídeos/administração & dosagem , Dipeptídeos/química , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Estrutura Molecular , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Receptores de Esfingosina-1-Fosfato , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
We present here a comprehensive analysis of proteases in the peptide substrate space and demonstrate its applicability for lead discovery. Aligned octapeptide substrates of 498 proteases taken from the MEROPS peptidase database were used for the in silico analysis. A multiple-category naïve Bayes model, trained on the two-dimensional chemical features of the substrates, was able to classify the substrates of 365 (73%) proteases and elucidate statistically significant chemical features for each of their specific substrate positions. The positional awareness of the method allows us to identify the most similar substrate positions between proteases. Our analysis reveals that proteases from different families, based on the traditional classification (aspartic, cysteine, serine, and metallo), could have substrates that differ at the cleavage site (P1-P1') but are similar away from it. Caspase-3 (cysteine protease) and granzyme B (serine protease) are previously known examples of cross-family neighbors identified by this method. To assess whether peptide substrate similarity between unrelated proteases could reliably translate into the discovery of low molecular weight synthetic inhibitors, a lead discovery strategy was tested on two other cross-family neighbors--namely cathepsin L2 and matrix metallo proteinase 9, and calpain 1 and pepsin A. For both these pairs, a naïve Bayes classifier model trained on inhibitors of one protease could successfully enrich those of its neighbor from a different family and vice versa, indicating that this approach could be prospectively applied to lead discovery for a novel protease target with no known synthetic inhibitors.