RESUMO
Intravenously administered cyclic dinucleotides and other STING agonists are hampered by low cellular uptake and poor circulatory half-life. Here we report the covalent conjugation of cyclic dinucleotides to poly(ß-amino ester) nanoparticles through a cathepsin-sensitive linker. This is shown to increase stability and loading, thereby expanding the therapeutic window in multiple syngeneic tumour models, enabling the study of how the long-term fate of the nanoparticles affects the immune response. In a melanoma mouse model, primary tumour clearance depends on the STING signalling by host cells-rather than cancer cells-and immune memory depends on the spleen. The cancer cells act as a depot for the nanoparticles, releasing them over time to activate nearby immune cells to control tumour growth. Collectively, this work highlights the importance of nanoparticle structure and nano-biointeractions in controlling immunotherapy efficacy.
Assuntos
Melanoma , Nanopartículas , Neoplasias , Animais , Camundongos , Polímeros/farmacologia , Neoplasias/tratamento farmacológico , Transdução de Sinais , Nanopartículas/uso terapêutico , Nanopartículas/químicaRESUMO
Aminoacyl transfer RNA (tRNA) synthetases (aaRSs) are attractive drug targets, and we present class I and II aaRSs as previously unrecognized targets for adenosine 5'-monophosphate-mimicking nucleoside sulfamates. The target enzyme catalyzes the formation of an inhibitory amino acid-sulfamate conjugate through a reaction-hijacking mechanism. We identified adenosine 5'-sulfamate as a broad-specificity compound that hijacks a range of aaRSs and ML901 as a specific reagent a specific reagent that hijacks a single aaRS in the malaria parasite Plasmodium falciparum, namely tyrosine RS (PfYRS). ML901 exerts whole-life-cycle-killing activity with low nanomolar potency and single-dose efficacy in a mouse model of malaria. X-ray crystallographic studies of plasmodium and human YRSs reveal differential flexibility of a loop over the catalytic site that underpins differential susceptibility to reaction hijacking by ML901.
Assuntos
Antimaláricos , Malária Falciparum , Terapia de Alvo Molecular , Plasmodium falciparum , Biossíntese de Proteínas , Proteínas de Protozoários , Tirosina-tRNA Ligase , Adenosina/análogos & derivados , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Cristalografia por Raios X , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Camundongos , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Biossíntese de Proteínas/efeitos dos fármacos , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Ácidos Sulfônicos/química , Tirosina-tRNA Ligase/química , Tirosina-tRNA Ligase/metabolismoRESUMO
Stimulator of Interferon Genes (STING) plays an important role in innate immunity by inducing type I interferon production upon infection with intracellular pathogens. STING activation can promote increased T-cell activation and inflammation in the tumor microenvironment, resulting in antitumor immunity. Natural and synthetic cyclic dinucleotides (CDNs) are known to activate STING, and several synthetic CDN molecules are being investigated in the clinic using an intratumoral administration route. Here, we describe the identification of STING agonist 15a, a cyclic dinucleotide structurally diversified from natural ligands with optimized properties for systemic intravenous (iv) administration. Our studies have shown that STING activation by 15a leads to an acute innate immune response as measured by cytokine secretion and adaptive immune response via activation of CD8+ cytotoxic T-cells, which ultimately provides robust antitumor efficacy.
Assuntos
Proteínas de Membrana/agonistas , Nucleotídeos Cíclicos/química , Pirimidinas/química , Administração Intravenosa , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Meia-Vida , Humanos , Imunoterapia , Proteínas de Membrana/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Neoplasias/patologia , Neoplasias/terapia , Nucleotídeos Cíclicos/metabolismo , Nucleotídeos Cíclicos/uso terapêutico , Fosfatos/química , Ratos , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
SUMOylation is a reversible post-translational modification that regulates protein function through covalent attachment of small ubiquitin-like modifier (SUMO) proteins. The process of SUMOylating proteins involves an enzymatic cascade, the first step of which entails the activation of a SUMO protein through an ATP-dependent process catalyzed by SUMO-activating enzyme (SAE). Here, we describe the identification of TAK-981, a mechanism-based inhibitor of SAE which forms a SUMO-TAK-981 adduct as the inhibitory species within the enzyme catalytic site. Optimization of selectivity against related enzymes as well as enhancement of mean residence time of the adduct were critical to the identification of compounds with potent cellular pathway inhibition and ultimately a prolonged pharmacodynamic effect and efficacy in preclinical tumor models, culminating in the identification of the clinical molecule TAK-981.
Assuntos
Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Neoplasias/tratamento farmacológico , Ácidos Sulfônicos/uso terapêutico , Sumoilação/efeitos dos fármacos , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Humanos , Camundongos , Estrutura Molecular , Ligação Proteica , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Relação Estrutura-Atividade , Ácidos Sulfônicos/síntese química , Ácidos Sulfônicos/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Investigations of a biaryl ether scaffold identified tetrahydronaphthalene Raf inhibitors with good in vivo activity; however these compounds had affinity toward the hERG potassium channel. Herein we describe our work to eliminate this hERG activity via alteration of the substituents on the benzoic amide functionality. The resulting compounds have improved selectivity against the hERG channel, good pharmacokinetic properties and potently inhibit the Raf pathway in vivo.
Assuntos
Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Tetra-Hidronaftalenos/química , Animais , Linhagem Celular Tumoral , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Mutagênese , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Ligação Proteica , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Tetra-Hidronaftalenos/farmacocinética , Tetra-Hidronaftalenos/uso terapêutico , Transplante HeterólogoRESUMO
Inhibition of mutant B-Raf signaling, through either direct inhibition of the enzyme or inhibition of MEK, the direct substrate of Raf, has been demonstrated preclinically to inhibit tumor growth. Very recently, treatment of B-Raf mutant melanoma patients with a selective B-Raf inhibitor has resulted in promising preliminary evidence of antitumor activity. This article describes the design and optimization of tetrahydronaphthalene-derived compounds as potent inhibitors of the Raf pathway in vitro and in vivo. These compounds possess good pharmacokinetic properties in rodents and inhibit B-Raf mutant tumor growth in mouse xenograft models.
Assuntos
Antineoplásicos/síntese química , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Tetra-Hidronaftalenos/síntese química , Administração Oral , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Disponibilidade Biológica , Cristalografia por Raios X , Desenho de Fármacos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/enzimologia , Melanoma Experimental/patologia , Camundongos , Camundongos Nus , Modelos Moleculares , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Estereoisomerismo , Relação Estrutura-Atividade , Tetra-Hidronaftalenos/química , Tetra-Hidronaftalenos/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
MLN4924 is a potent and selective small molecule NEDD8-activating enzyme (NAE) inhibitor. In most cancer cells tested, inhibition of NAE leads to induction of DNA rereplication, resulting in DNA damage and cell death. However, in preclinical models of activated B cell-like (ABC) diffuse large B-cell lymphoma (DLBCL), we show that MLN4924 induces an alternative mechanism of action. Treatment of ABC DLBCL cells with MLN4924 resulted in rapid accumulation of pIkappaBalpha, decrease in nuclear p65 content, reduction of nuclear factor-kappaB (NF-kappaB) transcriptional activity, and G(1) arrest, ultimately resulting in apoptosis induction, events consistent with potent NF-kappaB pathway inhibition. Treatment of germinal-center B cell-like (GCB) DLBCL cells resulted in an increase in cellular Cdt-1 and accumulation of cells in S-phase, consistent with cells undergoing DNA rereplication. In vivo administration of MLN4924 to mice bearing human xenograft tumors of ABC- and GCB-DLBCL blocked NAE pathway biomarkers and resulted in complete tumor growth inhibition. In primary human tumor models of ABC-DLBCL, MLN4924 treatment resulted in NF-kappaB pathway inhibition accompanied by tumor regressions. This work describes a novel mechanism of targeted NF-kappaB pathway modulation in DLBCL and provides strong rationale for clinical development of MLN4924 against NF-kappaB-dependent lymphomas.
Assuntos
Ciclopentanos/farmacologia , Centro Germinativo/efeitos dos fármacos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/patologia , NF-kappa B/metabolismo , Pirimidinas/farmacologia , Ubiquitinas/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Western Blotting , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Feminino , Citometria de Fluxo , Centro Germinativo/metabolismo , Centro Germinativo/patologia , Humanos , Linfoma Difuso de Grandes Células B/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteína NEDD8 , NF-kappa B/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitinas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The NEDD8-activating enzyme (NAE) initiates a protein homeostatic pathway essential for cancer cell growth and survival. MLN4924 is a selective inhibitor of NAE currently in clinical trials for the treatment of cancer. Here, we show that MLN4924 is a mechanism-based inhibitor of NAE and creates a covalent NEDD8-MLN4924 adduct catalyzed by the enzyme. The NEDD8-MLN4924 adduct resembles NEDD8 adenylate, the first intermediate in the NAE reaction cycle, but cannot be further utilized in subsequent intraenzyme reactions. The stability of the NEDD8-MLN4924 adduct within the NAE active site blocks enzyme activity, thereby accounting for the potent inhibition of the NEDD8 pathway by MLN4924. Importantly, we have determined that compounds resembling MLN4924 demonstrate the ability to form analogous adducts with other ubiquitin-like proteins (UBLs) catalyzed by their cognate-activating enzymes. These findings reveal insights into the mechanism of E1s and suggest a general strategy for selective inhibition of UBL conjugation pathways.
Assuntos
Monofosfato de Adenosina/metabolismo , Ciclopentanos/metabolismo , Inibidores Enzimáticos/metabolismo , Pirimidinas/metabolismo , Ubiquitinas/metabolismo , Monofosfato de Adenosina/química , Sítios de Ligação , Ligação Competitiva , Linhagem Celular Tumoral , Cristalografia por Raios X , Ciclopentanos/química , Ciclopentanos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Proteína NEDD8 , Estrutura Terciária de Proteína , Pirimidinas/química , Pirimidinas/farmacologia , Ubiquitinas/químicaRESUMO
The clinical development of an inhibitor of cellular proteasome function suggests that compounds targeting other components of the ubiquitin-proteasome system might prove useful for the treatment of human malignancies. NEDD8-activating enzyme (NAE) is an essential component of the NEDD8 conjugation pathway that controls the activity of the cullin-RING subtype of ubiquitin ligases, thereby regulating the turnover of a subset of proteins upstream of the proteasome. Substrates of cullin-RING ligases have important roles in cellular processes associated with cancer cell growth and survival pathways. Here we describe MLN4924, a potent and selective inhibitor of NAE. MLN4924 disrupts cullin-RING ligase-mediated protein turnover leading to apoptotic death in human tumour cells by a new mechanism of action, the deregulation of S-phase DNA synthesis. MLN4924 suppressed the growth of human tumour xenografts in mice at compound exposures that were well tolerated. Our data suggest that NAE inhibitors may hold promise for the treatment of cancer.