RESUMO
Glucocorticoids (GCs) are excellent anti-inflammatory drugs but are dose-limited by on-target toxicity. We sought to solve this problem by delivering GCs to immune cells with antibody-drug conjugates (ADCs) using antibodies containing site-specific incorporation of a non-natural amino acid, novel linker chemistry for in vitro and in vivo stability, and existing and novel glucocorticoid receptor (GR) agonists as payloads. We directed fluticasone propionate to human antigen-presenting immune cells to afford GR activation that was dependent on the targeted antigen. However, mechanism of action studies pointed to accumulation of free payload in the tissue culture supernatant as the dominant driver of activity and indeed administration of the ADC to human CD74 transgenic mice failed to activate GR target genes in splenic B cells. Suspecting dissipation of released payload, we designed an ADC bearing a novel GR agonist payload with reduced permeability which afforded cell-intrinsic activity in human B cells. Our work shows that antibody-targeting offers significant potential for rescuing existing and new dose-limited drugs outside the field of oncology.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos de Diferenciação de Linfócitos B/imunologia , Linfócitos B/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Glucocorticoides/administração & dosagem , Antígenos de Histocompatibilidade Classe II/imunologia , Imunoconjugados/uso terapêutico , Animais , Anti-Inflamatórios/uso terapêutico , Linfócitos B/efeitos dos fármacos , Desenvolvimento de Medicamentos , Estabilidade de Medicamentos , Fluticasona/administração & dosagem , Humanos , Camundongos , Camundongos Transgênicos , Receptores de Glucocorticoides/agonistasRESUMO
Synthesis and SAR studies of novel triazolobenzazepinones as gamma secretase modulators (GSMs) are presented in this communication. Starting from our azepinone leads, optimization studies toward improving central lowering of Aß42 led to the discovery of novel benzo-fused azepinones. Several benzazepinones were profiled in vivo and found to lower brain Aß42 levels in Sprague Dawley rats and transgenic APP-YAC mice in a dose-dependent manner after a single oral dose. Compound 34 was further progressed into a pilot study in our cisterna-magna-ported rhesus monkey model, where we observed robust lowering of CSF Aß42 levels.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Descoberta de Drogas , Macaca mulatta , Camundongos , Camundongos Transgênicos , Ratos , Ratos Sprague-DawleyRESUMO
The triazolyl amide γ-secretase modulators are potent alternatives to the cinnamyl amides that have entered the clinic for the treatment of Alzheimer's disease. Herein we build on the lead benzoazepinones described in our prior communication with imidazomethoxyarene moiety alternatives that offer opportunities to fine tune physical properties as well as address hERG binding and PK. Both half-life and bioavailability were significantly improved, especially in dog, with robust brain Aß42 lowering maintained in both transgenic mouse and rat.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/farmacocinética , Animais , Disponibilidade Biológica , Camundongos , Camundongos Transgênicos , RatosRESUMO
BACKGROUND: The impact of anti-TNF, corticosteroid and analgesic therapy on inflammation and pain was evaluated in a novel mono-arthritic multi-flare rat Streptococcal Cell Wall (SCW) model using Etanercept, Dexamethasone and Buprenorphine. METHODS: Multiple flares of arthritis were induced with an intra-articular injection of SCW in the hind ankle on day 1, followed by intravenous challenges on days 21 and 42. Inflammation and pain were monitored in the hind paws. Cytokine profiling, cell phenotyping, bioluminescence imaging and histopathological evaluation were also performed. RESULTS: Local injection of SCW caused a rapid onset of inflammation and pain in the injected ankle which resolved within 4 days (Flare 1). Intravenous injection 20 days after sensitization resulted in an increase in ankle diameter and pain, which partially resolved in 8 days (Flare 2). The subsequent intra-venous injection in the same animals 14 days after resulted in a more chronic disease with inflammation and pain persisting over a period of 10 days (Flare 3). In Flare 2, therapeutic administration of Dexamethasone inhibited paw swelling (95%; P<0.001) and pain (55%; P<0.05). Therapeutic administration of Buprenorphine inhibited pain (80%; P<0.001) without affecting paw swelling (0%). Prophylactic administration of Etanercept in Flare 2 inhibited paw swelling (≥60%; P<0.001) and pain by ≥30%. Expression of IL-1ß, IL-6, MCP-1 and CINC was reduced by >50% (P<0.001). Treatment with Etanercept in Flare 3 inhibited paw swelling by 60% (P<0.001) and pain by 25%. Prior treatment with Etanercept in Flare 2 followed by re-administration in Flare 3 led to a complete loss in the efficacy of Etanercept. Systemic exposure of Etanercept corroborated with lack of efficacy. Dexamethasone inhibited inflammation and pain in both Flares 2 and 3 (P<0.001). CONCLUSIONS: We established a novel multi-flare SCW arthritis model enabling drug intervention in different stages of disease. We show for the first time the evaluation of inflammation and pain simultaneously in this model. Etanercept and Dexamethasone inhibited inflammation, pain and proinflammatory cytokines in this model. Taken together, this model facilitates the assessment of anti-rheumatic agents targeting inflammation and pain in the multiple flare paradigm and offers a powerful tool for drug discovery.
Assuntos
Antirreumáticos/uso terapêutico , Artrite Experimental/tratamento farmacológico , Parede Celular , Imunoglobulina G/uso terapêutico , Dor/tratamento farmacológico , Receptores do Fator de Necrose Tumoral/uso terapêutico , Streptococcus , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/patologia , Etanercepte , Feminino , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/patologia , Dor/induzido quimicamente , Dor/patologia , Ratos , Ratos Endogâmicos LewRESUMO
Cancer immunotherapy has revolutionized the treatment of lung adenocarcinoma (LUAD); however, a significant proportion of patients do not respond. Recent transcriptomic studies to understand determinants of immunotherapy response have pinpointed stromal-mediated resistance mechanisms. To gain a better understanding of stromal biology at the cellular and molecular level in LUAD, we performed single-cell RNA sequencing of 256,379 cells, including 13,857 mesenchymal cells, from 9 treatment-naïve patients. Among the mesenchymal cell subsets, FAP+PDPN+ cancer-associated fibroblasts (CAF) and ACTA2+MCAM+ pericytes were enriched in tumors and differentiated from lung-resident fibroblasts. Imaging mass cytometry revealed that both subsets were topographically adjacent to the perivascular niche and had close spatial interactions with endothelial cells (EC). Modeling of ligand and receptor interactomes between mesenchymal and ECs identified that NOTCH signaling drives these cell-to-cell interactions in tumors, with pericytes and CAFs as the signal receivers and arterial and PLVAPhigh immature neovascular ECs as the signal senders. Either pharmacologically blocking NOTCH signaling or genetically depleting NOTCH3 levels in mesenchymal cells significantly reduced collagen production and suppressed cell invasion. Bulk RNA sequencing data demonstrated that NOTCH3 expression correlated with poor survival in stroma-rich patients and that a T cell-inflamed gene signature only predicted survival in patients with low NOTCH3. Collectively, this study provides valuable insights into the role of NOTCH3 in regulating tumor stroma biology, warranting further studies to elucidate the clinical implications of targeting NOTCH3 signaling. SIGNIFICANCE: NOTCH3 signaling activates tumor-associated mesenchymal cells, increases collagen production, and augments cell invasion in lung adenocarcinoma, suggesting its critical role in remodeling tumor stroma.
Assuntos
Adenocarcinoma de Pulmão , Fibroblastos Associados a Câncer , Neoplasias Pulmonares , Invasividade Neoplásica , Receptor Notch3 , Análise de Célula Única , Células Estromais , Microambiente Tumoral , Humanos , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/genética , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Comunicação Celular , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Receptor Notch3/metabolismo , Receptor Notch3/genética , Transdução de Sinais , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
Spleen tyrosine kinase (SYK) is a key activator of signaling pathways downstream of multiple surface receptors implicated in asthma. SYK function has been extensively studied in mast cells downstream of the high-affinity IgE receptor, FcεR1. Preclinical studies have demonstrated a role for SYK in models of allergic inflammation, but a role in airway constriction has not been demonstrated. Here, we have used a potent and selective pharmacological inhibitor of SYK to determine the role of SYK in allergen-mediated inflammation and airway constriction in preclinical models. Attenuation of allergic airway responses was evaluated in a rat passive anaphylaxis model and rat and sheep inhaled allergen challenge models, as well as an ex vivo model of allergen-mediated airway constriction in rats and cynomolgus monkeys. Pharmacological inhibition of SYK dose-dependently blocked IgE-mediated tracheal plasma extravasation in rats. In a rat ovalbumin-sensitized airway challenge model, oral dosing with an SYK inhibitor led to a dose-dependent reduction in lung inflammatory cells. Ex vivo analysis of allergen-induced airway constriction in ovalbumin-sensitized brown Norway rats showed a complete attenuation with treatment of a SYK inhibitor, as well as a complete block of allergen-induced serotonin release. Similarly, allergen-mediated airway constriction was attenuated in ex vivo studies from nonhuman primate lungs. Intravenous administration of an SYK inhibitor attenuated both early- and late-phase allergen-induced increases in airway resistance in an Ascaris-sensitive sheep allergen challenge model. These data support a key role for SYK signaling in mediating allergic airway responses.
Assuntos
Alérgenos/administração & dosagem , Asma/prevenção & controle , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Ascaris suum/imunologia , Asma/etiologia , Asma/fisiopatologia , Broncoconstrição/efeitos dos fármacos , Broncoconstrição/imunologia , Broncoconstrição/fisiologia , Degranulação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Macaca fascicularis , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Ovalbumina/imunologia , Proteínas Tirosina Quinases/fisiologia , Ratos , Ratos Endogâmicos BN , Ratos Sprague-Dawley , Ovinos , Transdução de Sinais/efeitos dos fármacos , Quinase SykRESUMO
Genetic mutation of the leucine-rich repeat kinase 2 (LRRK2) protein has been associated with Parkinson's disease (PD), a disabling and progressive neurodegenerative disorder that is devoid of efficacious disease-modifying therapies. Herein, we describe the invention of an amidoisoquinoline (IQ)-derived LRRK2 inhibitor lead chemical series. Knowledge-, structure-, and property-based drug design in concert with rigorous application of in silico calculations and presynthesis predictions enabled the prioritization of molecules with favorable CNS "drug-like" physicochemical properties. This resulted in the discovery of compound 8, which was profiled extensively before human ether-a-go-go (hERG) ion channel inhibition halted its progression. Strategic reduction of lipophilicity and basicity resulted in attenuation of hERG ion channel inhibition while maintaining a favorable CNS efflux transporter profile. Further structure- and property-based optimizations resulted in the discovery of preclinical candidate MK-1468. This exquisitely selective LRRK2 inhibitor has a projected human dose of 48 mg BID and a preclinical safety profile that supported advancement toward GLP toxicology studies.
Assuntos
Doença de Parkinson , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/química , Encéfalo/metabolismo , Mutação , Canais Iônicos/metabolismoRESUMO
Cytotoxic CD8+ T cells are the primary effector cells mediating anti-tumor responses. In vivo monitoring of CD8+ T cells has broad implications for the development of novel cancer therapies. Here we describe the development of a genetically engineered mouse model (GEMM) in which CD8+ T cells are labeled with an optical reporter, enabling in vivo, longitudinal monitoring using bioluminescence imaging (BLI). Firefly luciferase (Luc2), human diphtheria toxin receptor (DTR), and enhanced green fluorescence protein (eGFP) cDNAs are engineered under the CD8α promoter to generate a transgenic mouse line. Luciferase mRNA and CD8α mRNA were generally correlated in various tissues from these mice. Sorted splenic CD8+ T cells, CD4+ T cells and CD3- non-T cells verified that the luciferase signal is specific to CD8+ T cells. In vivo imaging showed that luciferase signal was detected in various immune organs, such as lymph nodes, thymus, and spleen, and the detection was confirmed by ex vivo examination. Administration of diphtheria toxin markedly reduced luciferase signal systemically, confirming the function of the DTR. In the MC38 mouse syngeneic model, we observed significant increases in CD8+ T cells with mDX400 treatment, an anti PD-1 mouse monoclonal antibody that correlated with tumor growth inhibition. This novel reporter GEMM is a valuable drug discovery tool for profiling compounds and understanding mechanisms of action in immunotherapy of cancer.
Assuntos
Linfócitos T CD8-Positivos , Luciferases , Animais , Anticorpos Monoclonais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Genes Reporter/genética , Luciferases/genética , Luciferases/metabolismo , Medições Luminescentes , Camundongos , Camundongos Transgênicos , RNA Mensageiro/metabolismoRESUMO
Janus kinase (JAK) inhibitors, such as tofacitinib (Xeljanz) and filgotinib (Jyseleca), have been approved for treatment of ulcerative colitis with several other JAK inhibitors in late-stage clinical trials for inflammatory bowel disease (IBD). Despite their impressive efficacy, the risk of adverse effects accompanying the use of JAK inhibitors has brought the entire class under scrutiny, leading to them receiving an FDA black box warning. In this study we investigated whether ileocolonic-targeted delivery of a pan-JAK inhibitor, tofacitinib, can lead to increased tissue exposure and reduced systemic exposure compared to untargeted formulations. The stability of tofacitinib in the presence of rat colonic microbiota was first confirmed. Next, in vivo computed tomography imaging was performed in rats to determine the transit time and disintegration site of ileocolonic-targeted capsules compared to gastric release capsules. Pharmacokinetic studies demonstrated that systemic drug exposure was significantly decreased, and colonic tissue exposure increased at 10 mg/kg tofacitinib dosed in ileocolonic-targeted capsules compared to gastric release capsules and an oral solution. Finally, in a rat model of LPS-induced colonic inflammation, targeted tofacitinib capsules significantly reduced concentrations of proinflammatory interleukin 6 in colonic tissue compared to a vehicle-treated control (p = 0.0408), unlike gastric release tofacitinib capsules and orally administered dexamethasone. Overall, these results support further development of ileocolonic-targeted tofacitinib, and potentially other specific JAK inhibitors in pre-clinical and clinical development, for the treatment of IBD.
RESUMO
Targeting the programmed death 1/programmed death ligand 1 (PD-1/PD-L1) pathway with immunotherapy has revolutionized the treatment of many cancers. Somatic tumor mutational burden (TMB) and T-cell-inflamed gene expression profile (GEP) are clinically validated pan-tumor genomic biomarkers that can predict responsiveness to anti-PD-1/PD-L1 monotherapy in many tumor types. We analyzed the association between these biomarkers and the efficacy of PD-1 inhibitor in 11 commonly used preclinical syngeneic tumor mouse models using murinized rat anti-mouse PD-1 DX400 antibody muDX400, a surrogate for pembrolizumab. Response to muDX400 treatment was broadly classified into three categories: highly responsive, partially responsive, and intrinsically resistant to therapy. Molecular and cellular profiling validated differences in immune cell infiltration and activation in the tumor microenvironment of muDX400-responsive tumors. Baseline and on-treatment genomic analysis showed an association between TMB, murine T-cell-inflamed gene expression profile (murine-GEP), and response to muDX400 treatment. We extended our analysis to investigate a canonical set of cancer and immune biology-related gene signatures, including signatures of angiogenesis, myeloid-derived suppressor cells, and stromal/epithelial-to-mesenchymal transition/TGFß biology previously shown to be inversely associated with the clinical efficacy of immune checkpoint blockade. Finally, we evaluated the association between murine-GEP and preclinical efficacy with standard-of-care chemotherapy or antiangiogenic agents that previously demonstrated promising clinical activity, in combination with muDX400. Our profiling studies begin to elucidate the underlying biological mechanisms of response and resistance to PD-1/PD-L1 blockade represented by these models, thereby providing insight into which models are most appropriate for the evaluation of orthogonal combination strategies.
Assuntos
Antígeno B7-H1 , Imunoterapia , Neoplasias , Receptor de Morte Celular Programada 1 , Animais , Antígeno B7-H1/antagonistas & inibidores , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Inibidores de Checkpoint Imunológico , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Microambiente TumoralRESUMO
Inhibition of leucine-rich repeat kinase 2 (LRRK2) kinase activity represents a genetically supported, chemically tractable, and potentially disease-modifying mechanism to treat Parkinson's disease. Herein, we describe the optimization of a novel series of potent, selective, central nervous system (CNS)-penetrant 1-heteroaryl-1H-indazole type I (ATP competitive) LRRK2 inhibitors. Type I ATP-competitive kinase physicochemical properties were integrated with CNS drug-like properties through a combination of structure-based drug design and parallel medicinal chemistry enabled by sp3-sp2 cross-coupling technologies. This resulted in the discovery of a unique sp3-rich spirocarbonitrile motif that imparted extraordinary potency, pharmacokinetics, and favorable CNS drug-like properties. The lead compound, 25, demonstrated exceptional on-target potency in human peripheral blood mononuclear cells, excellent off-target kinase selectivity, and good brain exposure in rat, culminating in a low projected human dose and a pre-clinical safety profile that warranted advancement toward pre-clinical candidate enabling studies.
Assuntos
Doença de Parkinson , Ratos , Humanos , Animais , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Doença de Parkinson/tratamento farmacológico , Indazóis/farmacologia , Indazóis/uso terapêutico , Leucócitos Mononucleares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/química , Encéfalo/metabolismo , Trifosfato de AdenosinaRESUMO
Centrosome- and microtubule-associated proteins have been shown to be important for maintaining the neural progenitor pool during neocortical development by regulating the mitotic spindle. It remains unclear whether these proteins may control neurogenesis by regulating other microtubule-dependent processes such as nuclear migration. Here, we identify Cep120, a centrosomal protein preferentially expressed in neural progenitors during neocortical development. We demonstrate that silencing Cep120 in the developing neocortex impairs both interkinetic nuclear migration (INM), a characteristic pattern of nuclear movement in neural progenitors, and neural progenitor self-renewal. Furthermore, we show that Cep120 interacts with transforming acidic coiled-coil proteins (TACCs) and that silencing TACCs also causes defects in INM and neural progenitor self-renewal. Our data suggest a critical role for Cep120 and TACCs in both INM and neurogenesis. We propose that sustaining INM may be a mechanism by which microtubule-regulating proteins maintain the neural progenitor pool during neocortical development.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Movimento Celular/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Neurônios/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Animais , Proteínas de Ciclo Celular/genética , Núcleo Celular/metabolismo , Eletroporação/métodos , Embrião de Mamíferos , Feminino , Técnicas In Vitro , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Neocórtex/citologia , Neocórtex/embriologia , Neocórtex/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Gravidez , Transporte Proteico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Tempo , Transfecção/métodosRESUMO
Motor learning and neuro-adaptations to drugs of abuse rely upon neuronal signaling in the striatum. Cyclin-dependent kinase 5 (Cdk5) regulates striatal dopamine neurotransmission and behavioral responses to cocaine. Although the role for Cdk5 in neurodegeneration in the cortex and hippocampus and in hippocampal-dependent learning has been demonstrated, its dysregulation in the striatum has not been examined. Here we show that strong activation of striatal NMDA receptors produced p25, the truncated form of the Cdk5 co-activator p35. Furthermore, inducible overexpression of p25 in the striatum prevented locomotor sensitization to cocaine and attenuated motor coordination and learning. This corresponded with reduced dendritic spine density, increased neuro-inflammation, altered dopamine signaling, and shifted Cdk5 specificity with regard to physiological and aberrant substrates, but no apparent loss of striatal neurons. Thus, dysregulation of Cdk5 dramatically affects striatal-dependent brain function and may be relevant to non-neurodegenerative disorders involving dopamine neurotransmission.
Assuntos
Cocaína/farmacologia , Corpo Estriado/enzimologia , Quinase 5 Dependente de Ciclina/fisiologia , Dendritos/efeitos dos fármacos , Aprendizagem , Locomoção , Animais , Comportamento Animal , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Dendritos/fisiologia , Camundongos , Camundongos Transgênicos , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
The development of a novel series of purines as gamma-secretase modulators for potential use in the treatment of Alzheimer's disease is disclosed herein. Optimization of a previously disclosed pyrimidine series afforded a series of potent purine-based gamma-secretase modulators with 300- to 2000-fold in vitro selectivity over inhibition of Notch cleavage and that selectively reduces Alphabeta42 in an APP-YAC transgenic mouse model.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/antagonistas & inibidores , Fragmentos de Peptídeos/antagonistas & inibidores , Purinas/química , Purinas/uso terapêutico , Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Humanos , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/metabolismo , Purinas/farmacologia , Receptores Notch/metabolismo , Relação Estrutura-AtividadeRESUMO
Cancer-associated fibroblasts (CAF) represent a functionally heterogeneous population of activated fibroblasts that constitutes a major component of tumor stroma. Although CAFs have been shown to promote tumor growth and mediate resistance to chemotherapy, the mechanisms by which they may contribute to immune suppression within the tumor microenvironment (TME) in lung squamous cell carcinoma (LSCC) remain largely unexplored. Here, we identified a positive correlation between CAF and monocytic myeloid cell abundances in 501 primary LSCCs by mining The Cancer Genome Atlas data sets. We further validated this finding in an independent cohort using imaging mass cytometry and found a significant spatial interaction between CAFs and monocytic myeloid cells in the TME. To delineate the interplay between CAFs and monocytic myeloid cells, we used chemotaxis assays to show that LSCC patient-derived CAFs promoted recruitment of CCR2+ monocytes via CCL2, which could be reversed by CCR2 inhibition. Using a three-dimensional culture system, we found that CAFs polarized monocytes to adopt a myeloid-derived suppressor cell (MDSC) phenotype, characterized by robust suppression of autologous CD8+ T-cell proliferation and IFNγ production. We further demonstrated that inhibiting IDO1 and NADPH oxidases, NOX2 and NOX4, restored CD8+ T-cell proliferation by reducing reactive oxygen species (ROS) generation in CAF-induced MDSCs. Taken together, our study highlights a pivotal role of CAFs in regulating monocyte recruitment and differentiation and demonstrated that CCR2 inhibition and ROS scavenging abrogate the CAF-MDSC axis, illuminating a potential therapeutic path to reversing the CAF-mediated immunosuppressive microenvironment.
Assuntos
Fibroblastos Associados a Câncer/imunologia , Carcinoma de Células Escamosas/imunologia , Neoplasias Pulmonares/imunologia , Monócitos/imunologia , Células Supressoras Mieloides/imunologia , Espécies Reativas de Oxigênio/metabolismo , Idoso , Idoso de 80 Anos ou mais , Linfócitos T CD8-Positivos/imunologia , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proliferação de Células , Células Cultivadas , Feminino , Humanos , Terapia de Imunossupressão , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , NADPH Oxidase 2/imunologia , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/imunologia , NADPH Oxidase 4/metabolismo , Receptores CCR2/imunologia , Receptores CCR2/metabolismo , Transdução de Sinais , Microambiente TumoralRESUMO
The clinical success of anti-IL-17 monoclonal antibodies (i.e., Cosentyx and Taltz) has validated Th17 pathway modulation for the treatment of autoimmune diseases. The nuclear hormone receptor RORγt is a master regulator of Th17 cells and affects the production of a host of cytokines, including IL-17A, IL-17F, IL-22, IL-26, and GM-CSF. Substantial interest has been spurred across both academia and industry to seek small molecules suitable for RORγt inhibition. A variety of RORγt inhibitors have been reported in the past few years, the majority of which are orthosteric binders. Here we disclose the discovery and optimization of a class of inhibitors, which bind differently to an allosteric binding pocket. Starting from a weakly active hit 1, a tool compound 14 was quickly identified that demonstrated superior potency, selectivity, and off-target profile. Further optimization focused on improving metabolic stability. Replacing the benzoic acid moiety with piperidinyl carboxylate, modifying the 4-aza-indazole core in 14 to 4-F-indazole, and incorporating a key hydroxyl group led to the discovery of 25, which possesses exquisite potency and selectivity, as well as an improved pharmacokinetic profile suitable for oral dosing.
RESUMO
Aberrant processing of the amyloid precursor protein (APP) and the subsequent accumulation of amyloid beta (Abeta) peptide has been widely established as a central event in Alzheimer's disease (AD) pathogenesis. The sequential cleavage steps required for the generation of Abeta are well outlined; however, there is a relative dearth of knowledge pertaining to signaling pathways and molecular mechanisms that can modulate this process. Here, we demonstrate a novel role for p25/cyclin-dependent kinase 5 (Cdk5) in regulating APP processing, Abeta peptide generation, and intraneuronal Abeta accumulation in inducible p25 transgenic and compound PD-APP transgenic mouse models that demonstrate deregulated Cdk5 activity and a neurodegenerative phenotype. Induction of p25 resulted in enhanced forebrain Abeta levels before any evidence of neuropathology in these mice. Intracellular Abeta accumulated in perinuclear regions and distended axons within the forebrains of these mice. Evidence for modulations in axonal transport or beta-site APP cleaving enzyme 1 protein levels and activity are presented as mechanisms that may account for the Abeta accumulation caused by p25/Cdk5 deregulation. Collectively, these findings delineate a novel pathological mechanism involving aberrant APP processing by p25/Cdk5 and have important implications in AD pathogenesis.
Assuntos
Peptídeos beta-Amiloides/biossíntese , Quinase 5 Dependente de Ciclina/fisiologia , Neurônios/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Neurônios/química , Neurônios/patologia , Fosfotransferases/fisiologiaRESUMO
Interleukin-1 receptor associated kinase 4 (IRAK4) is an essential signal transducer downstream of the IL-1R and TLR superfamily, and selective inhibition of the kinase activity of the protein represents an attractive target for the treatment of inflammatory diseases. A series of 5-amino-N-(1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamides was developed via sequential modifications to the 5-position of the pyrazolopyrimidine ring and the 3-position of the pyrazole ring. Replacement of substituents responsible for poor permeability and improvement of physical properties guided by cLogD led to the identification of IRAK4 inhibitors with excellent potency, kinase selectivity, and pharmacokinetic properties suitable for oral dosing.
RESUMO
Rheumatoid arthritis (RA) is a chronic autoimmune disease resulting in joint inflammation, pain, and eventual bone loss. Bone loss and remodeling caused by symmetric polyarthritis, the hallmark of RA, is readily detectable by bone mineral density (BMD) measurement using micro-CT. Abnormalities in these measurements over time reflect the underlying pathophysiology of the bone. To evaluate the efficacy of anti-rheumatic agents in animal models of arthritis, we developed a high throughput knee and ankle joint imaging assay to measure BMD as a translational biomarker. A bone sample holder was custom designed for micro-CT scanning, which significantly increased assay throughput. Batch processing 3-dimensional image reconstruction, followed by automated image cropping, significantly reduced image processing time. In addition, we developed a novel, automated image analysis method to measure BMD and bone volume of knee and ankle joints. These improvements significantly increased the throughput of ex vivo bone sample analysis, reducing data turnaround from 5 days to 24 hours for a study with 200 rat hind limbs. Taken together, our data demonstrate that BMD, as quantified by micro-CT, is a robust efficacy biomarker with a high degree of sensitivity. Our innovative approach toward evaluation of BMD using optimized image acquisition and novel image processing techniques in preclinical models of RA enables high throughput assessment of anti-rheumatic agents offering a powerful tool for drug discovery.
Assuntos
Artrite Reumatoide/patologia , Densidade Óssea , Colágeno/administração & dosagem , Microtomografia por Raio-X/métodos , Animais , Artrite Reumatoide/diagnóstico por imagem , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/prevenção & controle , Modelos Animais de Doenças , Feminino , Ratos , Ratos Endogâmicos LewRESUMO
Development of a series of highly kinome-selective spleen tyrosine kinase (Syk) inhibitors with favorable druglike properties is described. Early leads were discovered through X-ray crystallographic analysis, and a systematic survey of cores within a selected chemical space focused on ligand binding efficiency. Attenuation of hERG ion channel activity inherent within the initial chemotype was guided through modulation of physicochemical properties including log D, PSA, and pKa. PSA proved most effective for prospective compound design. Further profiling of an advanced compound revealed bacterial mutagenicity in the Ames test using TA97a Salmonella strain, and subsequent study demonstrated that this mutagenicity was pervasive throughout the series. Identification of intercalation as a likely mechanism for the mutagenicity-enabled modification of the core scaffold. Implementation of a DNA binding assay as a prescreen and models in DNA allowed resolution of the mutagenicity risk, affording molecules with favorable potency, selectivity, pharmacokinetic, and off-target profiles.