RESUMO
Chimeric antigen receptor (CAR) T cells with a long-lived memory phenotype are correlated with durable, complete remissions in patients with leukemia. However, not all CAR T cell products form robust memory populations, and those that do can induce chronic B cell aplasia in patients. To address these challenges, we previously developed a switchable CAR (sCAR) T cell system that allows fully tunable, on/off control over engineered cellular activity. To further evaluate the platform, we generated and assessed different murine sCAR constructs to determine the factors that afford efficacy, persistence, and expansion of sCAR T cells in a competent immune system. We find that sCAR T cells undergo significant in vivo expansion, which is correlated with potent antitumor efficacy. Most importantly, we show that the switch dosing regimen not only allows control over B cell populations through iterative depletion and repopulation, but that the "rest" period between dosing cycles is the key for induction of memory and expansion of sCAR T cells. These findings introduce rest as a paradigm in enhancing memory and improving the efficacy and persistence of engineered T cell products.
Assuntos
Bioengenharia/métodos , Imunoterapia Adotiva/métodos , Animais , Antígenos CD19/imunologia , Linfócitos B/imunologia , Citocinas/metabolismo , Citotoxicidade Imunológica/imunologia , Feminino , Região de Troca de Imunoglobulinas/genética , Região de Troca de Imunoglobulinas/imunologia , Ativação Linfocitária/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Modelos Biológicos , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologiaRESUMO
OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is a disease of unmet medical need. While immunotherapy with chimeric antigen receptor T (CAR-T) cells has shown much promise in haematological malignancies, their efficacy for solid tumours is challenged by the lack of tumour-specific antigens required to avoid on-target, off-tumour effects. Switchable CAR-T cells whereby activity of the CAR-T cell is controlled by dosage of a tumour antigen-specific recombinant Fab-based 'switch' to afford a fully tunable response may overcome this translational barrier. DESIGN: In this present study, we have used conventional and switchable CAR-T cells to target the antigen HER2, which is upregulated on tumour cells, but also present at low levels on normal human tissue. We used patient-derived xenograft models derived from patients with stage IV PDAC that mimic the most aggressive features of PDAC, including severe liver and lung metastases. RESULTS: Switchable CAR-T cells followed by administration of the switch directed against human epidermal growth factor receptor 2 (HER2)-induced complete remission in difficult-to-treat, patient-derived advanced pancreatic tumour models. Switchable HER2 CAR-T cells were as effective as conventional HER2 CAR-T cells in vivo testing a range of different CAR-T cell doses. CONCLUSION: These results suggest that a switchable CAR-T system is efficacious against aggressive and disseminated tumours derived from patients with advanced PDAC while affording the potential safety of a control switch.
Assuntos
Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/terapia , Imunoterapia Adotiva/métodos , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Animais , Antígenos de Neoplasias/genética , Biópsia por Agulha , Carcinoma Ductal Pancreático/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Imunoterapia/métodos , Invasividade Neoplásica/patologia , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias Pancreáticas/imunologia , Receptor ErbB-2/genética , Estatísticas não Paramétricas , Resultado do Tratamento , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Chimeric antigen receptor T (CAR-T) cell therapy has produced impressive results in clinical trials for B-cell malignancies. However, safety concerns related to the inability to control CAR-T cells once infused into the patient remain a significant challenge. Here we report the engineering of recombinant antibody-based bifunctional switches that consist of a tumor antigen-specific Fab molecule engrafted with a peptide neo-epitope, which is bound exclusively by a peptide-specific switchable CAR-T cell (sCAR-T). The switch redirects the activity of the bio-orthogonal sCAR-T cells through the selective formation of immunological synapses, in which the sCAR-T cell, switch, and target cell interact in a structurally defined and temporally controlled manner. Optimized switches specific for CD19 controlled the activity, tissue-homing, cytokine release, and phenotype of sCAR-T cells in a dose-titratable manner in a Nalm-6 xenograft rodent model of B-cell leukemia. The sCAR-T-cell dosing regimen could be tuned to provide efficacy comparable to the corresponding conventional CART-19, but with lower cytokine levels, thereby offering a method of mitigating cytokine release syndrome in clinical translation. Furthermore, we demonstrate that this methodology is readily adaptable to targeting CD20 on cancer cells using the same sCAR-T cell, suggesting that this approach may be broadly applicable to heterogeneous and resistant tumor populations, as well as other liquid and solid tumor antigens.
Assuntos
Antígenos CD19/imunologia , Antígenos de Neoplasias/imunologia , Imunoterapia Adotiva/métodos , Leucemia de Células B/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T , Subpopulações de Linfócitos T/imunologia , Animais , Azidas , Linfócitos B/imunologia , Linfócitos B/patologia , Fatores de Transcrição de Zíper de Leucina Básica/imunologia , Linhagem Celular Tumoral , Citocinas/metabolismo , Citotoxicidade Imunológica , Relação Dose-Resposta Imunológica , Feminino , Genes Reporter , Vetores Genéticos , Humanos , Imunoterapia Adotiva/efeitos adversos , Ativação Linfocitária , Linfopenia/etiologia , Linfopenia/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Fenilalanina/análogos & derivados , Engenharia de Proteínas/métodos , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas de Saccharomyces cerevisiae/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Relação Estrutura-Atividade , Subpopulações de Linfócitos T/transplante , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Phosphodiesterase 4 (PDE4) inhibitors are approved for the treatment of some moderate to severe inflammatory conditions. However, dose-limiting side effects in the central nervous system and gastrointestinal tract, including nausea, emesis, headache, and diarrhea, have impeded the broader therapeutic application of PDE4 inhibitors. We sought to exploit the wealth of validation surrounding PDE4 inhibition by improving the therapeutic index through generation of an antibody-drug conjugate (ADC) that selectively targets immune cells through the CD11a antigen. The resulting ADC consisted of a human αCD11a antibody (based on efalizumab clone hu1124) conjugated to an analog of the highly potent PDE4 inhibitor GSK256066. Both the human αCD11a ADC and a mouse surrogate αCD11a ADC (based on the M17 clone) rapidly internalized into immune cells and suppressed lipololysaccharide (LPS)-induced TNFα secretion in primary human monocytes and mouse peritoneal cells, respectively. In a carrageenan-induced air pouch inflammation mouse model, treatment with the ADC significantly reduced inflammatory cytokine production in the air pouch exudate. Overall, these results provide compelling evidence for the feasibility of delivering drugs with anti-inflammatory activity selectively to the immune compartment via CD11a and the development of tissue-targeted PDE4 inhibitors as a promising therapeutic modality for treating inflammatory diseases.
Assuntos
Aminoquinolinas/metabolismo , Antígenos CD11/metabolismo , Imunoconjugados/administração & dosagem , Inflamação/imunologia , Inibidores da Fosfodiesterase 4/metabolismo , Sulfonas/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoconjugados/farmacologia , Lipopolissacarídeos/efeitos adversos , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Peritônio/efeitos dos fármacos , Peritônio/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The treatment of patients with acute myeloid leukemia (AML) with targeted immunotherapy is challenged by the heterogeneity of the disease and a lack of tumor-exclusive antigens. Conventional immunotherapy targets for AML such as CD33 and CD123 have been proposed as targets for chimeric antigen receptor (CAR)-engineered T-cells (CAR-T-cells), a therapy that has been highly successful in the treatment of B-cell leukemia and lymphoma. However, CD33 and CD123 are present on hematopoietic stem cells, and targeting with CAR-T-cells has the potential to elicit long-term myelosuppression. C-type lectin-like molecule-1 (CLL1 or CLEC12A) is a myeloid lineage antigen that is expressed by malignant cells in more than 90% of AML patients. CLL1 is not expressed by healthy Hematopoietic Stem Cells (HSCs), and is therefore a promising target for CAR-T-cell therapy. Here, we describe the development and optimization of an anti-CLL1 CAR-T-cell with potent activity on both AML cell lines and primary patient-derived AML blasts in vitro while sparing healthy HSCs. Furthermore, in a disseminated mouse xenograft model using the CLL1-positive HL60 cell line, these CAR-T-cells completely eradicated tumor, thus supporting CLL1 as a promising target for CAR-T-cells to treat AML while limiting myelosuppressive toxicity.
Assuntos
Lectinas Tipo C/antagonistas & inibidores , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores Mitogênicos/antagonistas & inibidores , Proteínas Recombinantes de Fusão , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Modelos Animais de Doenças , Feminino , Humanos , Imunoterapia Adotiva/métodos , Lectinas Tipo C/imunologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Camundongos , Receptores de Antígenos de Linfócitos T/genética , Receptores Mitogênicos/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Chimeric antigen receptor T (CAR-T) cells have demonstrated promising results against hematological malignancies, but have encountered significant challenges in translation to solid tumors. To overcome these hurdles, we have developed a switchable CAR-T cell platform in which the activity of the engineered cell is controlled by dosage of an antibody-based switch. Herein, we apply this approach to Her2-expressing breast cancers by engineering switch molecules through site-specific incorporation of FITC or grafting of a peptide neo-epitope (PNE) into the anti-Her2 antibody trastuzumab (clone 4D5). We demonstrate that both switch formats can be readily optimized to redirect CAR-T cells (specific for the corresponding FITC or PNE) to Her2-expressing tumor cells, and afford dose-titratable activation of CAR-T cells exâ vivo and complete clearance of the tumor in rodent xenograft models. This strategy may facilitate the application of immunotherapy to solid tumors by affording comparable efficacy with improved safety owing to switch-based control of the CAR-T response.
Assuntos
Neoplasias da Mama/terapia , Genes de Troca , Imunoterapia , Receptores de Antígenos de Linfócitos T , Animais , Relação Dose-Resposta a Droga , Feminino , Genes de Troca/genética , Xenoenxertos , Humanos , Camundongos , Receptor ErbB-2/efeitos dos fármacos , Receptor ErbB-2/metabolismoRESUMO
The liver has a central role in glucose homeostasis, as it has the distinctive ability to produce and consume glucose. On feeding, glucose influx triggers gene expression changes in hepatocytes to suppress endogenous glucose production and convert excess glucose into glycogen or fatty acids to be stored in adipose tissue. This process is controlled by insulin, although debate exists as to whether insulin acts directly or indirectly on the liver. In addition to stimulating pancreatic insulin release, glucose also regulates the activity of ChREBP, a transcription factor that modulates lipogenesis. Here we describe another mechanism whereby glucose determines its own fate: we show that glucose binds and stimulates the transcriptional activity of the liver X receptor (LXR), a nuclear receptor that coordinates hepatic lipid metabolism. d-Glucose and d-glucose-6-phosphate are direct agonists of both LXR-alpha and LXR-beta. Glucose activates LXR at physiological concentrations expected in the liver and induces expression of LXR target genes with efficacy similar to that of oxysterols, the known LXR ligands. Cholesterol homeostasis genes that require LXR for expression are upregulated in liver and intestine of fasted mice re-fed with a glucose diet, indicating that glucose is an endogenous LXR ligand. Our results identify LXR as a transcriptional switch that integrates hepatic glucose metabolism and fatty acid synthesis.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Glucose/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Benzoatos/farmacologia , Benzilaminas/farmacologia , Linhagem Celular Tumoral , Colesterol/metabolismo , Proteínas de Ligação a DNA/agonistas , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Jejum , Ácidos Graxos/biossíntese , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Glucose/farmacologia , Glucose-6-Fosfato/metabolismo , Glucose-6-Fosfato/farmacologia , Homeostase/genética , Humanos , Ligantes , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Receptores X do Fígado , Camundongos , Receptores Nucleares Órfãos , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Elementos de Resposta/genética , Receptores X de Retinoides/química , Receptores X de Retinoides/genética , Receptores X de Retinoides/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genéticaRESUMO
T cells are critical mediators of antitumor immunity and a major target for cancer immunotherapy. Antibody blockade of inhibitory receptors such as PD-1 can partially restore the activity of tumor-infiltrating lymphocytes (TILs). However, the activation signals required to promote TIL responses are less well characterized. Here we show that the antitumor activity of CD8 and γδ TIL is supported by interactions between junctional adhesion molecule-like protein (JAML) on T cells and its ligand coxsackie and adenovirus receptor (CXADR) within tumor tissue. Loss of JAML through knockout in mice resulted in accelerated tumor growth that was associated with an impaired γδ TIL response and increased CD8 TIL dysfunction. In mouse tumor models, therapeutic treatment with an agonistic anti-JAML antibody inhibited tumor growth, improved γδ TIL activation, decreased markers of CD8 TIL dysfunction, and significantly improved response to anti-PD-1 checkpoint blockade. Thus, JAML represents a novel therapeutic target to enhance both CD8 and γδ TIL immunity.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Imunoterapia/métodos , Melanoma Experimental/patologia , Animais , Linfócitos T CD8-Positivos/patologia , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/genética , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Melanoma/genética , Melanoma/mortalidade , Melanoma/patologia , Melanoma Experimental/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/genética , Neoplasias/mortalidade , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologiaRESUMO
Transcriptional coregulators, which mediate chromatin-dependent transcriptional signaling, represent tractable targets to modulate tumorigenic gene expression programs with small molecules. Genetic loss-of-function studies have recently implicated the transcriptional coactivator, ENL, as a selective requirement for the survival of acute leukemia and highlighted an essential role for its chromatin reader YEATS domain. Motivated by these discoveries, we executed a screen of nearly 300,000 small molecules and identified an amido-imidazopyridine inhibitor of the ENL YEATS domain (IC50 = 7 µM). Improvements to the initial screening hit were enabled by adopting and expanding upon a SuFEx-based approach to high-throughput medicinal chemistry, ultimately demonstrating that it is compatible with cell-based drug discovery. Through these efforts, we discovered SR-0813, a potent and selective ENL/AF9 YEATS domain inhibitor (IC50 = 25 nM). Armed with this tool and a first-in-class ENL PROTAC, SR-1114, we detailed the biological response of AML cells to pharmacological ENL disruption for the first time. Most notably, we discovered that ENL YEATS inhibition is sufficient to selectively suppress ENL target genes, including HOXA9/10, MYB, MYC, and a number of other leukemia proto-oncogenes. Cumulatively, our study establishes YEATS domain inhibition as a viable approach to disrupt the pathogenic function of ENL in acute leukemia and provides the first thoroughly characterized chemical probe for the ENL YEATS domain.
RESUMO
Despite the development of next-generation antiandrogens, metastatic castration-resistant prostate cancer (mCRPC) remains incurable. Here, we describe a unique semisynthetic bispecific antibody that uses site-specific unnatural amino acid conjugation to combine the potency of a T cell-recruiting anti-CD3 antibody with the specificity of an imaging ligand (DUPA) for prostate-specific membrane antigen. This format enabled optimization of structure and function to produce a candidate (CCW702) with specific, potent in vitro cytotoxicity and improved stability compared with a bispecific single-chain variable fragment format. In vivo, CCW702 eliminated C4-2 xenografts with as few as three weekly subcutaneous doses and prevented growth of PCSD1 patient-derived xenograft tumors in mice. In cynomolgus monkeys, CCW702 was well tolerated up to 34.1 mg/kg per dose, with near-complete subcutaneous bioavailability and a PK profile supporting testing of a weekly dosing regimen in patients. CCW702 is being evaluated in a first in-human clinical trial for men with mCRPC who had progressed on prior therapies (NCT04077021).
Assuntos
Anticorpos Biespecíficos , Neoplasias de Próstata Resistentes à Castração , Animais , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Complexo CD3/uso terapêutico , Linhagem Celular Tumoral , Ensaios Clínicos como Assunto , Humanos , Ligantes , Masculino , Camundongos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Linfócitos TRESUMO
ENL is a transcriptional coactivator that recruits elongation machinery to active cis-regulatory elements upon binding of its YEATS domain-a chromatin reader module-to acylated lysine side chains. Discovery chemistry for the ENL YEATS domain is highly motivated by its significance in acute leukemia pathophysiology, but cell-based assays able to support large-scale screening or hit validation efforts do not presently exist. Here, we report on the discovery of a target engagement assay that allows for high-throughput ligand discovery in living cells. This assay is based on the cellular thermal shift assay (CETSA) but does not require exposing cells to elevated temperatures, as small-molecule ligands are able to stabilize the ENL YEATS domain at 37 °C. By eliminating temperature shifts, we developed a simplified target engagement assay that requires just two steps: drug treatment and luminescence detection. To demonstrate its value for higher throughput applications, we miniaturized the assay to a 1536-well format and screened 37â¯120 small molecules, ultimately identifying an acyl-lysine-competitive ENL/AF9 YEATS domain inhibitor.
Assuntos
Bioensaio/métodos , Ensaios de Triagem em Larga Escala/métodos , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Linhagem Celular Tumoral , Descoberta de Drogas , Células HEK293 , Humanos , Ligantes , Ligação Proteica , Domínios Proteicos/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Elongação da Transcrição/antagonistas & inibidoresRESUMO
Stimulator of interferon genes (STING) links innate immunity to biological processes ranging from antitumor immunity to microbiome homeostasis. Mechanistic understanding of the anticancer potential for STING receptor activation is currently limited by metabolic instability of the natural cyclic dinucleotide (CDN) ligands. From a pathway-targeted cell-based screen, we identified a non-nucleotide, small-molecule STING agonist, termed SR-717, that demonstrates broad interspecies and interallelic specificity. A 1.8-angstrom cocrystal structure revealed that SR-717 functions as a direct cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) mimetic that induces the same "closed" conformation of STING. SR-717 displayed antitumor activity; promoted the activation of CD8+ T, natural killer, and dendritic cells in relevant tissues; and facilitated antigen cross-priming. SR-717 also induced the expression of clinically relevant targets, including programmed cell death 1 ligand 1 (PD-L1), in a STING-dependent manner.
Assuntos
Antineoplásicos/farmacologia , Materiais Biomiméticos/farmacologia , Proteínas de Membrana/metabolismo , Nucleotídeos Cíclicos/farmacologia , Animais , Antígeno B7-H1/metabolismo , Materiais Biomiméticos/química , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Cristalografia por Raios X , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Camundongos , Nucleotídeos Cíclicos/química , Conformação Proteica/efeitos dos fármacosRESUMO
The gene for the Thermotoga maritima Trk potassium transporter component TrkA was originally thought to be a frameshift mutation and not to encode a functional protein. However, expression from this gene yielded a complex consisting of two distinct proteins designated TM1088A and -B. Genetic complementation of Escherichia coli mutants unable to transport potassium suggests that TM1088A/B is part of a functional Trk potassium transporter complex with the membrane protein TM1089. The protein structure for TM1088A shows a characteristic Rossmann fold indicating an NAD+ binding site and has structural similarity to potassium channel-related proteins. Ligand binding studies indicated that ATP, ADP, and AMP stabilized TM1088A to a much greater degree than NADH and NAD, consistent with the crystal structure of TM1088A, which contains a bound AMP natural ligand at the characteristic GXGXXG nucleotide binding site. Mutation of single and all glycines at this nucleotide binding site eliminated in vitro protein stabilization by the ligand, yet these mutated proteins could still functionally complement the E. coli potassium uptake mutants. We predict that this new two-subunit class of TrkA proteins is present in a number of organisms. A further subclass of the predicted two-subunit TrkA proteins lack an identifiable membrane-spanning subunit of the Trk K+ transporter. This class, as exemplified by Mycobacterium tuberculosis, did not complement E. coli potassium transport with the native E. coli TrkH; thus, it may require a novel TrkH-like protein for activity or provide an alternate function in vivo.
Assuntos
Proteínas de Bactérias/metabolismo , Mutação da Fase de Leitura , Canais de Potássio/metabolismo , Potássio/metabolismo , Thermotoga maritima/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Bactérias/classificação , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Escherichia coli/genética , Escherichia coli/metabolismo , Teste de Complementação Genética , Dados de Sequência Molecular , Filogenia , Canais de Potássio/química , Canais de Potássio/genética , Ligação Proteica , Alinhamento de Sequência , Thermotoga maritima/química , Thermotoga maritima/genéticaRESUMO
Retinol-binding protein-4 (RBP4) is an emerging candidate drug target for type 2 diabetes and lipofuscin-mediated macular degeneration. The retinoic acid derivative fenretinide (N-(4-hydroxyphenyl) retinamide; HPR) exerts therapeutic effects in mouse models of obesity, diabetes, and Stargardt's disease by targeting RBP4. Fenretinide competes with retinoids for RBP4 binding, disrupts RBP4-transthyretin (TTR) complexes, and results in urinary secretion of RBP4 and systemic depletion of retinol. To enable the search for nonretinoid molecules with fenretinide-like activities we developed a HTS-compatible homogeneous TR-FRET assay monitoring the displacement of retinoic acid derivatives from RBP4 in high-density 384-well and 1536-well microtiter plate formats. The retinoid displacement assay proved to be highly sensitive and robust after miniaturization with IC(50)s for fenretinide and retinol ranging around 50 and 100 nM, respectively, and Z'-factors around 0.7. In addition, a surface plasmon resonance (SPR)-based secondary assay was developed to interrogate small molecule RBP4 binders for their ability to modulate the RBP4-TTR interaction. Finally, a 1.6 x 10(6) compound library was screened against the retinoid displacement assay. Several potent retinoid competitors were identified that also appeared to disrupt RBP4-TTR complexes. Some of these compounds could potentially serve as valuable tools to further probe RBP4 biology in the future.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Pré-Albumina/análise , Retinoides/análise , Proteínas Plasmáticas de Ligação ao Retinol/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Avaliação Pré-Clínica de Medicamentos , Humanos , Estrutura Molecular , Pré-Albumina/química , Pré-Albumina/metabolismo , Ligação Proteica , Retinoides/química , Retinoides/metabolismo , Fatores de TempoRESUMO
PCSK9 (proprotein convertase subtilisin/kexin 9) is a secreted serine protease that regulates cholesterol homoeostasis by inducing post-translational degradation of hepatic LDL-R [LDL (low-density lipoprotein) receptor]. Intramolecular autocatalytic processing of the PCSK9 zymogen in the endoplasmic reticulum results in a tightly associated complex between the prodomain and the catalytic domain. Although the autocatalytic processing event is required for proper secretion of PCSK9, the requirement of proteolytic activity in the regulation of LDL-R is currently unknown. Co-expression of the prodomain and the catalytic domain in trans allowed for production of a catalytically inactive secreted form of PCSK9. This catalytically inactive PCSK9 was characterized and shown to be functionally equivalent to the wild-type protein in lowering cellular LDL uptake and LDL-R levels. These findings suggest that, apart from autocatalytic processing, the protease activity of PCSK9 is not necessary for LDL-R regulation.
Assuntos
Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Linhagem Celular , Humanos , Mutação/genética , Serina/genética , Serina/metabolismo , Serina Endopeptidases/genéticaRESUMO
BtDyP from Bacteroides thetaiotaomicron (strain VPI-5482) and TyrA from Shewanella oneidensis are dye-decolorizing peroxidases (DyPs), members of a new family of heme-dependent peroxidases recently identified in fungi and bacteria. Here, we report the crystal structures of BtDyP and TyrA at 1.6 and 2.7 A, respectively. BtDyP assembles into a hexamer, while TyrA assembles into a dimer; the dimerization interface is conserved between the two proteins. Each monomer exhibits a two-domain, alpha+beta ferredoxin-like fold. A site for heme binding was identified computationally, and modeling of a heme into the proposed active site allowed for identification of residues likely to be functionally important. Structural and sequence comparisons with other DyPs demonstrate a conservation of putative heme-binding residues, including an absolutely conserved histidine. Isothermal titration calorimetry experiments confirm heme binding, but with a stoichiometry of 0.3:1 (heme:protein).
Assuntos
Proteínas de Bactérias/química , Corantes/metabolismo , Sequência Conservada , Heme/metabolismo , Complexos Multienzimáticos/química , Peroxidases/química , Dobramento de Proteína , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/fisiologia , Bacteroides/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Dados de Sequência Molecular , Complexos Multienzimáticos/fisiologia , Peroxidases/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Shewanella/enzimologiaRESUMO
Glutathione S-transferases (GSTs) comprise a diverse superfamily of enzymes found in organisms from all kingdoms of life. GSTs are involved in diverse processes, notably small-molecule biosynthesis or detoxification, and are frequently also used in protein engineering studies or as biotechnology tools. Here, we report the high-resolution X-ray structure of Atu5508 from the pathogenic soil bacterium Agrobacterium tumefaciens (atGST1). Through use of comparative sequence and structural analysis of the GST superfamily, we identified local sequence and structural signatures, which allowed us to distinguish between different GST classes. This approach enables GST classification based on structure, without requiring additional biochemical or immunological data. Consequently, analysis of the atGST1 crystal structure suggests a new GST class, distinct from previously characterized GSTs, which would make it an attractive target for further biochemical studies.