RESUMO
Although immune checkpoint inhibitors targeting T-cell immunoregulatory proteins have revolutionized cancer treatment, they are effective only in a limited number of patients, and new strategies are needed to enhance tumor responses to immunotherapies. Deletion of protein tyrosine phosphatase non-receptor type 2 (Ptpn2), a regulator of growth factor and cytokine signaling pathways, has been shown to sensitize murine B16F10 melanoma cells to IFNγ and anti-PD-1 immunotherapy. Here, we investigated the potential therapeutic utility of small-molecule PTPN2 inhibitors. Ten inhibitors were synthesized on the basis of in silico modeling and structure-based design and functionally tested in vitro and in vivo. We show that the inhibitors had little effect on B16F10 cells alone, but effectively sensitized the tumor cells to IFNγ treatment in vitro and to anti-PD-1 therapy in vivo. Under both conditions, Ptpn2 inhibitor cotreatment suppressed B16F10 cell growth and enhanced Stat1 phosphorylation and expression of IFNγ response genes. In vivo, PTPN2 inhibitor cotreatment significantly reduced melanoma and colorectal tumor growth and enhanced mouse survival compared with anti-PD-1 treatment alone, and this was accompanied by increased tumor infiltration by granzyme B+ CD8+ T cells. Similar results were obtained with representative murine and human colon cancer and lung cancer cell lines. Collectively, these results demonstrate that small-molecule inhibitors of PTPN2 may have clinical utility as sensitizing agents for immunotherapy-resistant cancers. Significance: To enhance the effectiveness of immunotherapies in resistant or nonresponsive cancers, it is important to develop inhibitors of enzymes that negatively influence the outcome of treatments. We have designed and evaluated small-molecule inhibitors of PTPN2 demonstrating that these compounds may have clinical utility as sensitizing agents for immunotherapy-resistant cancers.
Assuntos
Linfócitos T CD8-Positivos , Melanoma , Humanos , Camundongos , Animais , Linfócitos T CD8-Positivos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Melanoma/tratamento farmacológico , Interferon gama , Imunoterapia/métodosRESUMO
Aberrant regulation of N6-methyladenosine (m6A) RNA modification has been implicated in the progression of multiple diseases, including cancer. Previously, we identified a small molecule inhibitor of the m6A demethylase fat mass- and obesity-associated protein (FTO), which removes both m6A and N6,2'-O-dimethyladenosine (m6Am) RNA modifications. In this work, we describe the rational design and optimization of a new class of FTO inhibitors derived from our previous lead FTO-04 with nanomolar potency and high selectivity against the homologous m6A RNA demethylase ALKBH5. The oxetanyl class of compounds comprise competitive inhibitors of FTO with potent antiproliferative effects in glioblastoma, acute myeloid leukemia, and gastric cancer models where lead FTO-43 demonstrated potency comparable to clinical chemotherapeutic 5-fluorouracil. Furthermore, FTO-43 increased m6A and m6Am levels in a manner comparable to FTO knockdown in gastric cancer cells and regulated Wnt/PI3K-Akt signaling pathways. The oxetanyl class contains significantly improved anticancer agents with a variety of applications beyond glioblastoma.
Assuntos
Antineoplásicos , Glioblastoma , Neoplasias Gástricas , Adenosina/metabolismo , Adenosina/farmacologia , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Antineoplásicos/farmacologia , Glioblastoma/tratamento farmacológico , Humanos , Fosfatidilinositol 3-Quinases , RNA , RNA Mensageiro/metabolismo , Neoplasias Gástricas/tratamento farmacológicoRESUMO
Ribonucleotide reductase (RR) is an essential multi-subunit enzyme found in all living organisms; it catalyzes the rate-limiting step in dNTP synthesis, namely, the conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates. As expression levels of human RR (hRR) are high during cell replication, hRR has long been considered an attractive drug target for a range of proliferative diseases, including cancer. While there are many excellent reviews regarding the structure, function, and clinical importance of hRR, recent years have seen an increase in novel approaches to inhibiting hRR that merit an updated discussion of the existing inhibitors and strategies to target this enzyme. In this review, we discuss the mechanisms and clinical applications of classic nucleoside analog inhibitors of hRRM1 (large catalytic subunit), including gemcitabine and clofarabine, as well as inhibitors of the hRRM2 (free radical housing small subunit), including triapine and hydroxyurea. Additionally, we discuss novel approaches to targeting RR and the discovery of new classes of hRR inhibitors.
Assuntos
Neoplasias , Ribonucleotídeo Redutases , Domínio Catalítico , Difosfatos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Neoplasias/tratamento farmacológico , Ribonucleotídeo Redutases/metabolismoRESUMO
The emergence of a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), presents an urgent public health crisis. Without available targeted therapies, treatment options remain limited for COVID-19 patients. Using medicinal chemistry and rational drug design strategies, we identify a 2-phenyl-1,2-benzoselenazol-3-one class of compounds targeting the SARS-CoV-2 main protease (Mpro). FRET-based screening against recombinant SARS-CoV-2 Mpro identified six compounds that inhibit proteolysis with nanomolar IC50 values. Preincubation dilution experiments and molecular docking determined that the inhibition of SARS-CoV-2 Mpro can occur by either covalent or noncovalent mechanisms, and lead E04 was determined to inhibit Mpro competitively. Lead E24 inhibited viral replication with a nanomolar EC50 value (844 nM) in SARS-CoV-2-infected Vero E6 cells and was further confirmed to impair SARS-CoV-2 replication in human lung epithelial cells and human-induced pluripotent stem cell-derived 3D lung organoids. Altogether, these studies provide a structural framework and mechanism of Mpro inhibition that should facilitate the design of future COVID-19 treatments.
Assuntos
Antivirais/farmacologia , Benzotiazóis/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Descoberta de Drogas , SARS-CoV-2/efeitos dos fármacos , Animais , Antivirais/síntese química , Antivirais/química , Benzotiazóis/química , COVID-19/metabolismo , Chlorocebus aethiops , Proteases 3C de Coronavírus/isolamento & purificação , Proteases 3C de Coronavírus/metabolismo , Cristalografia por Raios X , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/química , Relação Dose-Resposta a Droga , Transferência Ressonante de Energia de Fluorescência , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , SARS-CoV-2/enzimologia , Células Vero , Replicação Viral/efeitos dos fármacos , Tratamento Farmacológico da COVID-19RESUMO
N6-methyladenosine (m6A) has emerged as the most abundant mRNA modification that regulates gene expression in many physiological processes. m6A modification in RNA controls cellular proliferation and pluripotency and has been implicated in the progression of multiple disease states, including cancer. RNA m6A methylation is controlled by a multiprotein "writer" complex including the enzymatic factor methyltransferase-like protein 3 (METTL3) that regulates methylation and two "eraser" proteins, RNA demethylase ALKBH5 (ALKBH5) and fat mass- and obesity-associated protein (FTO), that demethylate m6A in transcripts. FTO can also demethylate N6,2'-O-dimethyladenosine (m6Am), which is found adjacent to the m7G cap structure in mRNA. FTO has recently gained interest as a potential cancer target, and small molecule FTO inhibitors such as meclofenamic acid have been shown to prevent tumor progression in both acute myeloid leukemia and glioblastoma in vivo models. However, current FTO inhibitors are unsuitable for clinical applications due to either poor target selectivity or poor pharmacokinetics. In this work, we describe the structure-based design, synthesis, and biochemical evaluation of a new class of FTO inhibitors. Rational design of 20 small molecules with low micromolar IC50's and specificity toward FTO over ALKBH5 identified two competitive inhibitors FTO-02 and FTO-04. Importantly, FTO-04 prevented neurosphere formation in patient-derived glioblastoma stem cells (GSCs) without inhibiting the growth of healthy neural stem cell-derived neurospheres. Finally, FTO-04 increased m6A and m6Am levels in GSCs consistent with FTO inhibition. These results support FTO-04 as a potential new lead for treatment of glioblastoma.
Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato/antagonistas & inibidores , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Pirimidinas/farmacologia , Adenosina/análogos & derivados , Adenosina/química , Dioxigenase FTO Dependente de alfa-Cetoglutarato/química , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Sítios de Ligação , Desmetilação/efeitos dos fármacos , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Humanos , Simulação de Acoplamento Molecular , Células-Tronco Neoplásicas , Ligação Proteica , Pirimidinas/síntese química , Pirimidinas/metabolismo , RNA/química , RNA/metabolismoRESUMO
Although immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment, many patients do not respond or develop resistance to ICB. N6 -methylation of adenosine (m6A) in RNA regulates many pathophysiological processes. Here, we show that deletion of the m6A demethylase Alkbh5 sensitized tumors to cancer immunotherapy. Alkbh5 has effects on m6A density and splicing events in tumors during ICB. Alkbh5 modulates Mct4/Slc16a3 expression and lactate content of the tumor microenvironment and the composition of tumor-infiltrating Treg and myeloid-derived suppressor cells. Importantly, a small-molecule Alkbh5 inhibitor enhanced the efficacy of cancer immunotherapy. Notably, the ALKBH5 gene mutation and expression status of melanoma patients correlate with their response to immunotherapy. Our results suggest that m6A demethylases in tumor cells contribute to the efficacy of immunotherapy and identify ALKBH5 as a potential therapeutic target to enhance immunotherapy outcome in melanoma, colorectal, and potentially other cancers.
Assuntos
Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Vacinas Anticâncer/imunologia , Lactatos/metabolismo , Melanoma/metabolismo , Receptor de Morte Celular Programada 1/imunologia , Linfócitos T Reguladores/fisiologia , Homólogo AlkB 5 da RNA Desmetilase/genética , Anticorpos , Citocinas/genética , Citocinas/metabolismo , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/terapia , Metiltransferases/genética , Metiltransferases/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Células Supressoras Mieloides/fisiologia , Sítios de Splice de RNA , Splicing de RNA , Simportadores/genética , Simportadores/metabolismo , Transcriptoma , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVE: Across a correlational cross-sectional and experimental study, we investigate how contextual variables, such as majority group perceptions, interact with individual variables, such as the relationship between ethnic and national identity, to ultimately influence intergroup relations between bicultural individuals and White Americans. METHOD: Across 2 studies (Study 1, n = 187; Study 2, n = 176), bicultural participants completed a battery of surveys measuring bicultural identity integration, impressions of majority group attitudes, and behavioral intentions targeted at majority group members. Additionally, majority group attitudes were experimentally manipulated in Study 2. RESULTS: Individuals with integrated bicultural identities were more likely to approach and less likely to avoid majority members. Although perceptions of negative majority group attitudes are typically associated with negative intergroup relations, individuals with greater harmony (Study 1) and blendedness (Study 2) between their ethnic and national identities were buffered from these adverse effects. CONCLUSIONS: These findings highlight the importance of both individual and contextual factors in intergroup relations. The distinct effects of blendedness and harmony across self-report and experimental studies have theoretical and practical implications for our understanding of bicultural identity integration. Finally, implications for encouraging more positive intercultural relationships between majority and minority group members will be discussed. (PsycInfo Database Record (c) 2020 APA, all rights reserved).
Assuntos
Grupos Minoritários/psicologia , Identificação Social , Estudantes/psicologia , População Branca/psicologia , Adulto , Estudos Transversais , Etnicidade , Feminino , Humanos , Masculino , Grupos Minoritários/estatística & dados numéricos , Estudantes/estatística & dados numéricos , Inquéritos e Questionários , População Branca/estatística & dados numéricos , Adulto JovemRESUMO
The HIV-1 accessory protein Vif, which counteracts the antiviral action of the DNA-editing cytidine deaminase APOBEC3G (A3G), is an attractive and yet unexploited therapeutic target. Vif reduces the virion incorporation of A3G by targeting the restriction factor for proteasomal degradation in the virus-producing cell. Compounds that inhibit Vif-mediated degradation of A3G in cells targeted by HIV-1 would represent a novel antiviral therapeutic. We previously described small molecules with activity consistent with Vif antagonism. In this study, we derived inhibitor escape HIV-1 variants to characterize the mechanism by which these novel agents inhibit virus replication. Here we show that resistance to these agents is dependent on an amino acid substitution in Vif (V142I) and on a point mutation that likely upregulates transcription by modifying the lymphocyte enhancing factor 1 (LEF-1) binding site. Molecular modeling demonstrated a docking site in the Vif-Elongin C complex that is disrupted by these inhibitors. This docking site is lost when Vif acquires the V142I mutation that leads to inhibitor resistance. Competitive fitness experiments indicated that the V142I Vif and LEF-1 binding site mutations created a virus that is better adapted to growing in the presence of A3G than the wild-type virus.IMPORTANCE Although antiretroviral therapy can suppress HIV-1 replication effectively, virus reservoirs persist in infected individuals and virus replication rapidly rebounds if therapy is interrupted. Currently, there is a need for therapeutic approaches that eliminate, reduce, or control persistent viral reservoirs if a cure is to be realized. This work focuses on the preclinical development of novel, small-molecule inhibitors of the HIV-1 Vif protein. Vif inhibitors represent a new class of antiretroviral drugs that may expand treatment options to more effectively suppress virus replication or to drive HIV-1 reservoirs to a nonfunctional state by harnessing the activity of the DNA-editing cytidine deaminase A3G, a potent, intrinsic restriction factor expressed in macrophage and CD4+ T cells. In this study, we derived inhibitor escape variants to characterize the mechanism by which these novel agents inhibit virus replication and to provide evidence for target validation.
Assuntos
Antivirais/farmacologia , Farmacorresistência Viral , HIV-1/efeitos dos fármacos , Mutação de Sentido Incorreto , Replicação Viral/efeitos dos fármacos , Produtos do Gene vif do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Desaminase APOBEC-3G/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Linhagem Celular , HIV-1/genética , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Simulação de Acoplamento Molecular , Mutação Puntual , Produtos do Gene vif do Vírus da Imunodeficiência Humana/química , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genéticaRESUMO
Ribonucleotide reductase (RR), an established cancer target, is usually inhibited by antimetabolites, which display multiple cross-reactive effects. Recently, we discovered a naphthyl salicyl acyl hydrazone-based inhibitor (NSAH or E-3a) of human RR (hRR) binding at the catalytic site (C-site) and inhibiting hRR reversibly. We herein report the synthesis and biochemical characterization of 25 distinct analogs. We designed each analog through docking to the C-site of hRR based on our 2.7 Å X-ray crystal structure (PDB ID: 5TUS). Broad tolerance to minor structural variations preserving inhibitory potency is observed. E-3f (82% yield) displayed an in vitro IC50 of 5.3 ± 1.8 µM against hRR, making it the most potent in this series. Kinetic assays reveal that E-3a, E-3c, E-3t, and E-3w bind and inhibit hRR through a reversible and competitive mode. Target selectivity toward the R1 subunit of hRR is established, providing a novel way of inhibition of this crucial enzyme.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Hidrazonas/síntese química , Hidrazonas/farmacologia , Ribonucleotídeo Redutases/antagonistas & inibidores , Técnicas de Química Sintética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Hidrazonas/química , Simulação de Acoplamento Molecular , Conformação Proteica , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Relação Estrutura-AtividadeRESUMO
Human ribonucleotide reductase (hRR) is crucial for DNA replication and maintenance of a balanced dNTP pool, and is an established cancer target. Nucleoside analogs such as gemcitabine diphosphate and clofarabine nucleotides target the large subunit (hRRM1) of hRR. These drugs have a poor therapeutic index due to toxicity caused by additional effects, including DNA chain termination. The discovery of nonnucleoside, reversible, small-molecule inhibitors with greater specificity against hRRM1 is a key step in the development of more effective treatments for cancer. Here, we report the identification and characterization of a unique nonnucleoside small-molecule hRR inhibitor, naphthyl salicylic acyl hydrazone (NSAH), using virtual screening, binding affinity, inhibition, and cell toxicity assays. NSAH binds to hRRM1 with an apparent dissociation constant of 37 µM, and steady-state kinetics reveal a competitive mode of inhibition. A 2.66-Å resolution crystal structure of NSAH in complex with hRRM1 demonstrates that NSAH functions by binding at the catalytic site (C-site) where it makes both common and unique contacts with the enzyme compared with NDP substrates. Importantly, the IC50 for NSAH is within twofold of gemcitabine for growth inhibition of multiple cancer cell lines, while demonstrating little cytotoxicity against normal mobilized peripheral blood progenitor cells. NSAH depresses dGTP and dATP levels in the dNTP pool causing S-phase arrest, providing evidence for RR inhibition in cells. This report of a nonnucleoside reversible inhibitor binding at the catalytic site of hRRM1 provides a starting point for the design of a unique class of hRR inhibitors.
Assuntos
Hidrazonas/farmacologia , Naftalenos/farmacologia , Ribonucleotídeo Redutases/antagonistas & inibidores , Salicilatos/farmacologia , Domínio Catalítico , Ciclo Celular/efeitos dos fármacos , Cristalografia por Raios X , Nucleotídeos de Desoxiadenina/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Hidrazonas/química , Naftalenos/química , Ribonucleosídeo Difosfato Redutase , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Salicilatos/química , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismoRESUMO
Ribonucleotide reductase (RR) catalyzes the rate-limiting step of dNTP synthesis and is an established cancer target. Drugs targeting RR are mainly nucleoside in nature. In this study, we sought to identify non-nucleoside small-molecule inhibitors of RR. Using virtual screening, binding affinity, inhibition, and cell toxicity, we have discovered a class of small molecules that alter the equilibrium of inactive hexamers of RR, leading to its inhibition. Several unique chemical categories, including a phthalimide derivative, show micromolar IC50s and KDs while demonstrating cytotoxicity. A crystal structure of an active phthalimide binding at the targeted interface supports the noncompetitive mode of inhibition determined by kinetic studies. Furthermore, the phthalimide shifts the equilibrium from dimer to hexamer. Together, these data identify several novel non-nucleoside inhibitors of human RR which act by stabilizing the inactive form of the enzyme.
Assuntos
Antineoplásicos/química , Ribonucleotídeo Redutases/antagonistas & inibidores , Proteínas Supressoras de Tumor/antagonistas & inibidores , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Simulação por Computador , Cristalografia por Raios X , Bases de Dados de Compostos Químicos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Simulação de Acoplamento Molecular , Ftalimidas/química , Ftalimidas/farmacologia , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Ribonucleosídeo Difosfato Redutase , Ribonucleotídeo Redutases/química , Relação Estrutura-Atividade , Proteínas Supressoras de Tumor/químicaRESUMO
Prior research suggests that cultural groups vary on an overarching dimension of independent versus interdependent social orientation, with European Americans being more independent, or less interdependent, than Asians. Drawing on recent evidence suggesting that the dopamine D4 receptor gene (DRD4) plays a role in modulating cultural learning, we predicted that carriers of DRD4 polymorphisms linked to increased dopamine signaling (7- or 2-repeat alleles) would show higher levels of culturally dominant social orientations, compared with noncarriers. European Americans and Asian-born Asians (total N = 398) reported their social orientation on multiple scales. They were also genotyped for DRD4. As in earlier work, European Americans were more independent, and Asian-born Asians more interdependent. This cultural difference was significantly more pronounced for carriers of the 7- or 2-repeat alleles than for noncarriers. Indeed, no cultural difference was apparent among the noncarriers. Implications for potential coevolution of genes and culture are discussed.
Assuntos
Povo Asiático/genética , Povo Asiático/psicologia , Cognição/fisiologia , Receptores de Dopamina D4/genética , Comportamento Social , População Branca/genética , População Branca/psicologia , Alelos , Evolução Cultural , Feminino , Genótipo , Humanos , Masculino , Repetições Minissatélites , Polimorfismo Genético , Inquéritos e Questionários , Adulto JovemRESUMO
The anatomic pathology discipline is slowly moving toward a digital workflow, where pathologists will evaluate whole-slide images on a computer monitor rather than glass slides through a microscope. One of the driving factors in this workflow is computer-assisted scoring, which depends on appropriate selection of regions of interest. With advances in tissue pattern recognition techniques, a more precise region of the tissue can be evaluated, no longer bound by the pathologist's patience in manually outlining target tissue areas. Pathologists use entire tissues from which to determine a score in a region of interest when making manual immunohistochemistry assessments. Tissue pattern recognition theoretically offers this same advantage; however, error rates exist in any tissue pattern recognition program, and these error rates contribute to errors in the overall score. To provide a real-world example of tissue pattern recognition, 11 HER2-stained upper gastrointestinal malignancies with high heterogeneity were evaluated. HER2 scoring of gastric cancer was chosen due to its increasing importance in gastrointestinal disease. A method is introduced for quantifying the error rates of tissue pattern recognition. The trade-off between fully sampling tumor with a given tissue pattern recognition error rate versus randomly sampling a limited number of fields of view with higher target accuracy was modeled with a Monte-Carlo simulation. Under most scenarios, stereological methods of sampling-limited fields of view outperformed whole-slide tissue pattern recognition approaches for accurate immunohistochemistry analysis. The importance of educating pathologists in the use of statistical sampling is discussed, along with the emerging role of hybrid whole-tissue imaging and stereological approaches.