RESUMO
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
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
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
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
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
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.
Assuntos
Biologia Computacional/métodos , Peptídeo Hidrolases/química , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Teorema de Bayes , Simulação por Computador , Humanos , Oligopeptídeos/química , Peptídeo Hidrolases/metabolismo , Estrutura Terciária de Proteína , Ratos , Reprodutibilidade dos Testes , Proteínas Virais/química , Proteínas Virais/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.