RESUMO
Systemic administration of small molecule toll-like receptor (TLR)-7 agonists leads to potent activation of innate immunity and to the generation of anti-tumor immune responses. However, activation of TLRs with small molecule agonists may lead to the induction of TLR tolerance, defined as a state of hyporesponsiveness to subsequent agonism, which may limit immune activation, the generation of anti-tumor responses and clinical response. Our data reveal that dose scheduling impacts on the efficacy of systemic therapy with the selective TLR7 agonist, 6-amino-2-(butylamino)-9-((6-(2-(dimethylamino)ethoxy)pyridin-3-yl)methyl)-7,9-dihydro-8H-purin-8-one (DSR-6434). In a preclinical model of renal cell cancer, systemic administration of DSR-6434 dosed once weekly resulted in a significant anti-tumor response. However, twice weekly dosing of DSR-6434 led to the induction of TLR tolerance, and no anti-tumor response was observed. We show that TLR7 tolerance was independent of type I interferon (IFN) negative feedback because induction of TLR7 tolerance was also observed in IFN-α/ß receptor knockout mice treated with DSR-6434. Moreover, our data demonstrate that treatment of bone marrow-derived plasmacytoid dendritic cells (BM-pDC) with DSR-6434 led to downregulation of TLR7 expression. From our data, dose scheduling of systemically administered TLR7 agonists can impact on anti-tumor activity through the induction of TLR tolerance. Furthermore, TLR7 expression on pDC may be a useful biomarker of TLR7 tolerance and aid in the optimization of dosing schedules involving systemically administered TLR7 agonists.
Assuntos
Adenina/análogos & derivados , Carcinoma de Células Renais/imunologia , Glicoproteínas de Membrana/metabolismo , Receptor 7 Toll-Like/metabolismo , Adenina/administração & dosagem , Adenina/farmacologia , Animais , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Protocolos Clínicos , Citotoxicidade Imunológica , Humanos , Tolerância Imunológica , Imunidade Inata , Interferon Tipo I/metabolismo , Glicoproteínas de Membrana/agonistas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Experimentais , Transdução de Sinais , Receptor 7 Toll-Like/agonistasRESUMO
Although topical TLR7 therapies such as imiquimod have proved successful in the treatment of dermatological malignancy, systemic delivery may be required for optimal immunotherapy of nondermatological tumors. We report that intravenous delivery of the novel small molecule TLR7 agonist, DSR-6434, leads to the induction of type 1 interferon and activation of T and B lymphocytes, NK and NKT cells. Our data demonstrate that systemic administration of DSR-6434 enhances the efficacy of ionizing radiation (IR) and leads to improved survival in mice bearing either CT26 or KHT tumors. Of the CT26 tumor-bearing mice that received combined therapy, 55% experienced complete tumor resolution. Our data reveal that these long-term surviving mice have a significantly greater frequency of tumor antigen specific CD8(+) T cells when compared to age-matched tumor-naïve cells. To evaluate therapeutic effects on spontaneous metastases, we showed that combination of DSR-6434 with local IR of the primary tumor significantly reduced metastatic burden in the lung, when compared to time-matched cohorts treated with IR alone. The data demonstrate that systemic administration of the novel TLR7 agonist DSR-6434 in combination with IR primes an antitumor CD8(+) T-cell response leading to improved survival in syngeneic models of colorectal carcinoma and fibrosarcoma. Importantly, efficacy extends to sites outside of the field of irradiation, reducing metastatic load. Clinical evaluation of systemic TLR7 therapy in combination with IR for the treatment of solid malignancy is warranted.
Assuntos
Adenina/análogos & derivados , Imunoterapia/métodos , Glicoproteínas de Membrana/agonistas , Neoplasias/radioterapia , Receptor 7 Toll-Like/agonistas , Adenina/administração & dosagem , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/efeitos da radiação , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Interferon gama/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/efeitos da radiação , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Knockout , Metástase Neoplásica , Transplante de Neoplasias , Radiação Ionizante , Baço/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/efeitos da radiaçãoRESUMO
For patients with non-small-cell lung cancer (NSCLC) tumors without currently targetable molecular alterations, standard-of-care treatment is immunotherapy with anti-PD-(L)1 checkpoint inhibitors, alone or with platinum-doublet therapy. However, not all patients derive durable benefit and resistance to immune checkpoint blockade is common. Understanding mechanisms of resistance-which can include defects in DNA damage response and repair pathways, alterations or functional mutations in STK11/LKB1, alterations in antigen-presentation pathways, and immunosuppressive cellular subsets within the tumor microenvironment-and developing effective therapies to overcome them, remains an unmet need. Here the phase 2 umbrella HUDSON study evaluated rational combination regimens for advanced NSCLC following failure of anti-PD-(L)1-containing immunotherapy and platinum-doublet therapy. A total of 268 patients received durvalumab (anti-PD-L1 monoclonal antibody)-ceralasertib (ATR kinase inhibitor), durvalumab-olaparib (PARP inhibitor), durvalumab-danvatirsen (STAT3 antisense oligonucleotide) or durvalumab-oleclumab (anti-CD73 monoclonal antibody). Greatest clinical benefit was observed with durvalumab-ceralasertib; objective response rate (primary outcome) was 13.9% (11/79) versus 2.6% (5/189) with other regimens, pooled, median progression-free survival (secondary outcome) was 5.8 (80% confidence interval 4.6-7.4) versus 2.7 (1.8-2.8) months, and median overall survival (secondary outcome) was 17.4 (14.1-20.3) versus 9.4 (7.5-10.6) months. Benefit with durvalumab-ceralasertib was consistent across known immunotherapy-refractory subgroups. In ATM-altered patients hypothesized to harbor vulnerability to ATR inhibition, objective response rate was 26.1% (6/23) and median progression-free survival/median overall survival were 8.4/22.8 months. Durvalumab-ceralasertib safety/tolerability profile was manageable. Biomarker analyses suggested that anti-PD-L1/ATR inhibition induced immune changes that reinvigorated antitumor immunity. Durvalumab-ceralasertib is under further investigation in immunotherapy-refractory NSCLC.ClinicalTrials.gov identifier: NCT03334617.
Assuntos
Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Indóis , Neoplasias Pulmonares , Morfolinas , Pirimidinas , Sulfonamidas , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Platina/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Anticorpos Monoclonais , Antineoplásicos/uso terapêutico , Biomarcadores , Antígeno B7-H1 , Microambiente TumoralRESUMO
The kinase ataxia telangiectasia mutated and rad3 related (ATR) is a key regulator of the DNA-damage response and the apical kinase which orchestrates the cellular processes that repair stalled replication forks (replication stress) and associated DNA double-strand breaks. Inhibition of repair pathways mediated by ATR in a context where alternative pathways are less active is expected to aid clinical response by increasing replication stress. Here we describe the development of the clinical candidate 2 (AZD6738), a potent and selective sulfoximine morpholinopyrimidine ATR inhibitor with excellent preclinical physicochemical and pharmacokinetic (PK) characteristics. Compound 2 was developed improving aqueous solubility and eliminating CYP3A4 time-dependent inhibition starting from the earlier described inhibitor 1 (AZ20). The clinical candidate 2 has favorable human PK suitable for once or twice daily dosing and achieves biologically effective exposure at moderate doses. Compound 2 is currently being tested in multiple phase I/II trials as an anticancer agent.
Assuntos
Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Sulfóxidos/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Fenômenos Químicos , Ensaios Clínicos como Assunto , Feminino , Humanos , Indóis , Camundongos , Modelos Moleculares , Conformação Molecular , Morfolinas , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Pirimidinas/química , Pirimidinas/farmacocinética , Sulfonamidas , Sulfóxidos/química , Sulfóxidos/farmacocinética , Distribuição TecidualRESUMO
Strategies to augment anti-cancer immune responses have recently demonstrated therapeutic utility. To date clinical success has been achieved through targeting co-inhibitory checkpoints such as CTLA-4, PD-1, and PD-L1. However, approaches that target co-activatory pathways are also being actively being developed. Here we report that the novel TLR7-selective agonist DSR-29133 is well tolerated in mice and leads to acute immune activation. Administration of DSR-29133 leads to the induction of IFNα/γ, IP-10, TNFα, IL-1Ra and IL-12p70, and to a reduction in tumor burden in syngeneic models of renal cancer (Renca), metastatic osteosarcoma (LM8) and colorectal cancer (CT26). Moreover, we show that the efficacy of DSR-29133 was significantly improved when administered in combination with low-dose fractionated radiotherapy (RT). Effective combination therapy required weekly administration of DSR-29133 commencing on day 1 of a fractionated RT treatment cycle, whereas no enhancement of radiation response was observed when DSR-29133 was administered at the end of the fractionated RT cycle. Combined therapy resulted in curative responses in a high proportion of mice bearing established CT26 tumors which was dependent on the activity of CD8+ T-cells but independent of CD4+ T-cells and NK/NKT cells. Moreover, long-term surviving mice originally treated with DSR-29133 and RT were protected by a tumor-specific memory immune response which could prevent tumor growth upon rechallenge. These results demonstrate that DSR-29133 is a potent selective TLR7 agonist that when administered intravenously can induce anti-tumor immune responses that can be further enhanced through combination with low-dose fractionated RT.
Assuntos
Adenina/análogos & derivados , Antineoplásicos/farmacologia , Quimiorradioterapia/métodos , Neoplasias Experimentais/tratamento farmacológico , Receptor 7 Toll-Like/agonistas , Adenina/farmacologia , Administração Intravenosa , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Fracionamento da Dose de Radiação , Humanos , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/radioterapiaRESUMO
Efforts to apply nanotechnology in cancer have focused almost exclusively on the delivery of cytotoxic drugs to improve therapeutic index. There has been little consideration of molecularly targeted agents, in particular kinase inhibitors, which can also present considerable therapeutic index limitations. We describe the development of Accurin polymeric nanoparticles that encapsulate the clinical candidate AZD2811, an Aurora B kinase inhibitor, using an ion pairing approach. Accurins increase biodistribution to tumor sites and provide extended release of encapsulated drug payloads. AZD2811 nanoparticles containing pharmaceutically acceptable organic acids as ion pairing agents displayed continuous drug release for more than 1 week in vitro and a corresponding extended pharmacodynamic reduction of tumor phosphorylated histone H3 levels in vivo for up to 96 hours after a single administration. A specific AZD2811 nanoparticle formulation profile showed accumulation and retention in tumors with minimal impact on bone marrow pathology, and resulted in lower toxicity and increased efficacy in multiple tumor models at half the dose intensity of AZD1152, a water-soluble prodrug of AZD2811. These studies demonstrate that AZD2811 can be formulated in nanoparticles using ion pairing agents to give improved efficacy and tolerability in preclinical models with less frequent dosing. Accurins specifically, and nanotechnology in general, can increase the therapeutic index of molecularly targeted agents, including kinase inhibitors targeting cell cycle and oncogenic signal transduction pathways, which have to date proved toxic in humans.
Assuntos
Aurora Quinases/antagonistas & inibidores , Nanopartículas/química , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Aurora Quinases/metabolismo , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Linhagem Celular Tumoral , Liberação Controlada de Fármacos , Feminino , Humanos , Masculino , Espectrometria de Massas , Camundongos , Camundongos SCID , Organofosfatos/química , Organofosfatos/farmacocinética , Organofosfatos/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Quinazolinas/química , Quinazolinas/farmacocinética , Quinazolinas/farmacologia , Ratos Nus , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Evaluation of the effects of purine C-8 substitution within a series of CDK1/2-selective O(6)-cyclohexylmethylguanine derivatives revealed that potency decreases initially with increasing size of the alkyl substituent. Structural analysis showed that C-8 substitution is poorly tolerated, and to avoid unacceptable steric interactions, these compounds adopt novel binding modes. Thus, 2-amino-6-cyclohexylmethoxy-8-isopropyl-9H-purine adopts a "reverse" binding mode where the purine backbone has flipped 180°. This provided a novel lead chemotype from which we have designed more potent CDK2 inhibitors using, in the first instance, quantum mechanical energy calculations. Introduction of an ortho-tolyl or ortho-chlorophenyl group at the purine C-8 position restored the potency of these "reverse" binding mode inhibitors to that of the parent 2-amino-6-cyclohexylmethoxy-9H-purine. By contrast, the corresponding 8-(2-methyl-3-sulfamoylphenyl)-purine derivative exhibited submicromolar CDK2-inhibitory activity by virtue of engineered additional interactions with Asp86 and Lys89 in the reversed binding mode, as confirmed by X-ray crystallography.
Assuntos
Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Inibidores de Proteínas Quinases/síntese química , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
ATR is an attractive new anticancer drug target whose inhibitors have potential as chemo- or radiation sensitizers or as monotherapy in tumors addicted to particular DNA-repair pathways. We describe the discovery and synthesis of a series of sulfonylmorpholinopyrimidines that show potent and selective ATR inhibition. Optimization from a high quality screening hit within tight SAR space led to compound 6 (AZ20) which inhibits ATR immunoprecipitated from HeLa nuclear extracts with an IC50 of 5 nM and ATR mediated phosphorylation of Chk1 in HT29 colorectal adenocarcinoma tumor cells with an IC50 of 50 nM. Compound 6 potently inhibits the growth of LoVo colorectal adenocarcinoma tumor cells in vitro and has high free exposure in mouse following moderate oral doses. At well tolerated doses 6 leads to significant growth inhibition of LoVo xenografts grown in nude mice. Compound 6 is a useful compound to explore ATR pharmacology in vivo.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Morfolinas/síntese química , Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/síntese química , Animais , Antineoplásicos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia , Cristalografia por Raios X , Descoberta de Drogas , Feminino , Células HeLa , Humanos , Camundongos , Modelos Moleculares , Morfolinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The docking of flexible small molecule ligands to large flexible protein targets is addressed in this article using a two-stage simulation-based method. The methodology presented is a hybrid approach where the first component is a dock of the ligand to the protein binding site, based on deriving sets of simultaneously satisfied intermolecular hydrogen bonds using graph theory and a recursive distance geometry algorithm. The output structures are reduced in number by cluster analysis based on distance similarities. These structures are submitted to a modified Monte Carlo algorithm using the AMBER-AA molecular mechanics force field with the Generalized Born/Surface Area (GB/SA) continuum model. This solvent model is not only less expensive than an explicit representation, but also yields increased sampling. Sampling is also increased using a rotamer library to direct some of the protein side-chain movements along with large dihedral moves. Finally, a softening function for the nonbonded force field terms is used, enabling the potential energy function to be slowly turned on throughout the course of the simulation. The docking procedure is optimized, and the results are presented for a single complex of the arabinose binding protein. It was found that for a rigid receptor model, the X-ray binding geometry was reproduced and uniquely identified based on the associated potential energy. However, when side-chain flexibility was included, although the X-ray structure was identified, it was one of three possible binding geometries that were energetically indistinguishable. These results suggest that on relaxing the constraint on receptor flexibility, the docking energy hypersurface changes from being funnel-like to rugged. A further 14 complexes were then examined using the optimized protocol. For each complex the docking methodology was tested for a fully flexible ligand, both with and without protein side-chain flexibility. For the rigid protein docking, 13 out of the 15 test cases were able to find the experimental binding mode; this number was reduced to 11 for the flexible protein docking. However, of these 11, in the majority of cases the experimental binding mode was not uniquely identified, but was present in a cluster of low energy structures that were energetically indistinguishable. These results not only support the presence of a rugged docking energy hypersurface, but also suggest that it may be necessary to consider the possibility of more than one binding conformation during ligand optimization.
Assuntos
Algoritmos , Simulação por Computador , Modelos Moleculares , Proteínas/química , Aminoácidos/química , Proteínas de Transporte/química , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Ligação de Hidrogênio , Ligantes , Estrutura Molecular , Método de Monte Carlo , Conformação Proteica , TermodinâmicaRESUMO
SAR studies led to the identification of 4-(3-benzoylamino-6-methyl-anilino)quinazolines as potent and selective inhibitors of p38 MAP kinase. Further optimisation led to the identification of a series of 4-(3-benzoylamino-6-methyl-anilino)pyrimidines as potent inhibitors of the p38 MAP kinase signalling pathway in vitro and in vivo.
Assuntos
Compostos de Anilina/síntese química , Pirimidinas/síntese química , Quinazolinas/síntese química , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Trifosfato de Adenosina/química , Administração Oral , Compostos de Anilina/farmacocinética , Compostos de Anilina/farmacologia , Animais , Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/farmacocinética , Anti-Inflamatórios não Esteroides/farmacologia , Antirreumáticos/síntese química , Antirreumáticos/farmacocinética , Antirreumáticos/farmacologia , Sítios de Ligação , Disponibilidade Biológica , Cristalografia por Raios X , Humanos , Técnicas In Vitro , Modelos Moleculares , Ligação Proteica , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Quinazolinas/farmacocinética , Quinazolinas/farmacologia , Ratos , Transdução de Sinais , Relação Estrutura-Atividade , Fatores de Tempo , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
Using a high-throughput screening campaign, we identified the 4,6-bis anilino pyrimidines as inhibitors of the cyclin-dependent kinase, CDK4. Herein we describe the further chemical modification and use of X-ray crystallography to develop potent and selective in vitro inhibitors of CDK4.
Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Proteínas Proto-Oncogênicas , Pirimidinas/farmacologia , Animais , Antineoplásicos/síntese química , Sítios de Ligação , Cristalografia por Raios X , Quinase 4 Dependente de Ciclina , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Pirimidinas/síntese química , Relação Estrutura-AtividadeRESUMO
Through chemical modification and X-ray crystallography we identified the 2,4-bis anilino pyrimidines as potent inhibitors of CDK4. Herein, we describe the optimisation of this series.
Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Proteínas Proto-Oncogênicas , Pirimidinas/farmacologia , Trifosfato de Adenosina/química , Animais , Antineoplásicos/síntese química , Sítios de Ligação , Cristalografia por Raios X , Quinase 4 Dependente de Ciclina , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Pirimidinas/síntese química , Relação Estrutura-AtividadeRESUMO
High-throughput screening identified the imidazo[1,2-a]pyridine and bisanilinopyrimidine series as inhibitors of the cyclin-dependent kinase CDK4. Comparison of their experimentally-determined binding modes and emerging structure-activity trends led to the development of potent and selective imidazo[1,2-a]pyridine inhibitors for CDK4 and in particular CDK2.