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1.
Artigo em Inglês | MEDLINE | ID: mdl-34816323

RESUMO

Natural killer (NK) cells are cytotoxic lymphocytes that play a major role in the innate immune system. NK cells exhibit potent cytotoxic activity against cancer cells and virally infected cells without antigen priming. These unique cytotoxic properties make NK cells a promising therapeutic against cancer. Limitations of NK cell therapy include deficiencies in high clinical efficacy often due to a need for a high NK cell to target cell ratio to achieve effective killing. In order to address the suboptimal efficacy of current adoptive NK cell therapy, a high throughput screen (HTS) was designed and performed to identify drug-like compounds that increase NK cytotoxic activity against tumor cells without affecting the normal cells. This screen was performed in a 384-well plate format utilizing an expanded primary NK cell product and ovarian cancer cells as a target cell (TC) line. Of the 8000 diverse small molecules screened, 16 hits were identified (0.2% hit rate) based on both a robust Z (RZ) score < -3 and a greater than 10% increase in NK cell killing. A validation screen had a confirmation rate of 70%. Select compounds were further validated and characterized by additional cytotoxicity assays including activity against multiple blood cancer and solid tumor cell lines, with no effect on primary human T cells. This work demonstrates that high-throughput screening can be reliably used to identify compounds that increase NK tumoricidal activity in vitro that can be further investigated and translated for potential clinical application. Précis: Our work led to the identification of promising compound that potently increases NK cell-mediated killing of a variety of different cancer cells, but no impact on the killing of normal cells. This compound demonstrates the utility of this assay.

2.
Molecules ; 26(19)2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34641449

RESUMO

We discovered SW033291 in a high throughput chemical screen aimed at identifying 15-prostaglandin dehydrogenase (15-PGDH) modulators. The compound exhibited inhibitory activity in in vitro biochemical and cell-based assays of 15-PGDH activity. We subsequently demonstrated that this compound, and several analogs thereof, are effective in in vivo mouse models of bone marrow transplant, colitis, and liver regeneration, where increased levels of PGE2 positively potentiate tissue regeneration. To better understand the binding of SW033291, we carried out docking studies for both the substrate, PGE2, and an inhibitor, SW033291, to 15-PGDH. Our models suggest similarities in the ways that PGE2 and SW033291 interact with key residues in the 15-PGDH-NAD+ complex. We carried out molecular dynamics simulations (MD) of SW033291 bound to this complex, in order to understand the dynamics of the binding interactions for this compound. The butyl side chain (including the sulfoxide) of SW033291 participates in crucial binding interactions that are similar to those observed for the C15-OH and the C16-C20 alkyl chain of PGE2. In addition, interactions with residues Ser138, Tyr151, and Gln148 play key roles in orienting and stabilizing SW033291 in the binding site and lead to enantioselectivity for the R-enantiomer. Finally, we compare the binding mode of (R)-S(O)-SW033291 with the binding interactions of published 15-PGDH inhibitors.


Assuntos
Hidroxiprostaglandina Desidrogenases/antagonistas & inibidores , Piridinas/química , Piridinas/farmacologia , Tiofenos/química , Tiofenos/farmacologia , Sítios de Ligação , Humanos , Simulação de Dinâmica Molecular
3.
Chem Sci ; 12(30): 10388-10394, 2021 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-34377425

RESUMO

The spiroindimicins are a unique class of chlorinated indole alkaloids characterized by three heteroaromatic rings structured around a congested spirocyclic stereocenter. Here, we report the first total synthesis of (+)-spiroindimicin A, which bears a challenging C-3'/C-5''-linked spiroindolenine. We detail our initial efforts to effect a biomimetic oxidative spirocyclization from its proposed natural precursor, lynamicin D, and describe how these studies shaped our final abiotic 9-step solution to this complex alkaloid built around a key Pd-catalyzed asymmetric spirocyclization. Scalable access to spiroindimicins A, H, and their congeners has enabled discovery of their activity against several parasites relevant to human health, providing potential starting points for new therapeutics for the neglected tropical diseases leishmaniasis and African sleeping sickness.

4.
J Med Chem ; 64(15): 10951-10966, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34260245

RESUMO

Influenza viruses cause approximately half a million deaths every year worldwide. Vaccines are available but partially effective, and the number of antiviral medications is limited. Thus, it is crucial to develop therapeutic strategies to counteract this major pathogen. Influenza viruses enter the host cell via their hemagglutinin (HA) proteins. The HA subtypes of influenza A virus are phylogenetically classified into groups 1 and 2. Here, we identified an inhibitor of the HA protein, a tertiary aryl sulfonamide, that prevents influenza virus entry and replication. This compound shows potent antiviral activity against diverse H1N1, H5N1, and H3N2 influenza viruses encoding HA proteins from both groups 1 and 2. Synthesis of derivatives of this aryl sulfonamide identified moieties important for antiviral activity. This compound may be considered as a lead for drug development with the intent to be used alone or in combination with other influenza A virus antivirals to enhance pan-subtype efficacy.


Assuntos
Antivirais/farmacologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/efeitos dos fármacos , Sulfonamidas/farmacologia , Antivirais/síntese química , Antivirais/química , Relação Dose-Resposta a Droga , Estrutura Molecular , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
5.
J Med Chem ; 64(5): 2739-2761, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33620219

RESUMO

Malaria control programs continue to be threatened by drug resistance. To identify new antimalarials, we conducted a phenotypic screen and identified a novel tetrazole-based series that shows fast-kill kinetics and a relatively low propensity to develop high-level resistance. Preliminary structure-activity relationships were established including identification of a subseries of related amides with antiplasmodial activity. Assaying parasites with resistance to antimalarials led us to test whether the series had a similar mechanism of action to chloroquine (CQ). Treatment of synchronized Plasmodium falciparum parasites with active analogues revealed a pattern of intracellular inhibition of hemozoin (Hz) formation reminiscent of CQ's action. Drug selections yielded only modest resistance that was associated with amplification of the multidrug resistance gene 1 (pfmdr1). Thus, we have identified a novel chemical series that targets the historically druggable heme polymerization pathway and that can form the basis of future optimization efforts to develop a new malaria treatment.


Assuntos
Amidas/farmacologia , Antimaláricos/farmacologia , Hemoglobinas/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Tetrazóis/farmacologia , Amidas/síntese química , Amidas/farmacocinética , Antimaláricos/síntese química , Antimaláricos/farmacocinética , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Hemeproteínas/antagonistas & inibidores , Células Hep G2 , Humanos , Estrutura Molecular , Testes de Sensibilidade Parasitária , Plasmodium falciparum/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/farmacocinética , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Tetrazóis/síntese química , Tetrazóis/farmacocinética
6.
J Med Chem ; 63(22): 14054-14066, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33180487

RESUMO

A phenotypic high-throughput screen identified a benzamide small molecule with activity against small cell lung cancer cells. A "clickable" benzamide probe was designed that irreversibly bound a single 50 kDa cellular protein, identified by mass spectrometry as ß-tubulin. Moreover, the anti-cancer potency of a series of benzamide analogs strongly correlated with probe competition, indicating that ß-tubulin was the functional target. Additional evidence suggested that benzamides covalently modified Cys239 within the colchicine binding site. Consistent with this mechanism, benzamides impaired growth of microtubules formed with ß-tubulin harboring Cys239, but not ß3 tubulin encoding Ser239. We therefore designed an aldehyde-containing analog capable of trapping Ser239 in ß3 tubulin, presumably as a hemiacetal. Using a forward genetics strategy, we identified benzamide-resistant cell lines harboring a Thr238Ala mutation in ß-tubulin sufficient to induce compound resistance. The disclosed chemical probes are useful to identify other colchicine site binders, a frequent target of structurally diverse small molecules.


Assuntos
Antineoplásicos/farmacologia , Benzamidas/química , Colchicina/metabolismo , Microtúbulos/efeitos dos fármacos , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/química , Antineoplásicos/química , Sítios de Ligação , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Microtúbulos/metabolismo , Conformação Proteica , Carcinoma de Pequenas Células do Pulmão/patologia , Relação Estrutura-Atividade , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química
7.
Sci Rep ; 10(1): 20662, 2020 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-33244070

RESUMO

PDA is a major cause of US cancer-related deaths. Oncogenic Kras presents in 90% of human PDAs. Kras mutations occur early in pre-neoplastic lesions but are insufficient to cause PDA. Other contributing factors early in disease progression include chronic pancreatitis, alterations in epigenetic regulators, and tumor suppressor gene mutation. GPCRs activate heterotrimeric G-proteins that stimulate intracellular calcium and oncogenic Kras signaling, thereby promoting pancreatitis and progression to PDA. By contrast, Rgs proteins inhibit Gi/q-coupled GPCRs to negatively regulate PDA progression. Rgs16::GFP is expressed in response to caerulein-induced acinar cell dedifferentiation, early neoplasia, and throughout PDA progression. In genetically engineered mouse models of PDA, Rgs16::GFP is useful for pre-clinical rapid in vivo validation of novel chemotherapeutics targeting early lesions in patients following successful resection or at high risk for progressing to PDA. Cultured primary PDA cells express Rgs16::GFP in response to cytotoxic drugs. A histone deacetylase inhibitor, TSA, stimulated Rgs16::GFP expression in PDA primary cells, potentiated gemcitabine and JQ1 cytotoxicity in cell culture, and Gem + TSA + JQ1 inhibited tumor initiation and progression in vivo. Here we establish the use of Rgs16::GFP expression for testing drug combinations in cell culture and validation of best candidates in our rapid in vivo screen.


Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Células Acinares/efeitos dos fármacos , Células Acinares/metabolismo , Células Acinares/patologia , Adenocarcinoma/metabolismo , Animais , Cálcio/metabolismo , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma Ductal Pancreático/metabolismo , Desdiferenciação Celular/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Ceruletídeo/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Progressão da Doença , Proteínas de Ligação ao GTP/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Camundongos , Ductos Pancreáticos/efeitos dos fármacos , Ductos Pancreáticos/metabolismo , Neoplasias Pancreáticas/metabolismo , Pancreatite/tratamento farmacológico , Pancreatite/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas RGS/metabolismo , Transdução de Sinais/efeitos dos fármacos
8.
Biochem J ; 477(17): 3253-3269, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32776146

RESUMO

The carbohydrate response element binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in glucose-mediated induction of genes involved in hepatic glycolysis and lipogenesis. In response to fluctuating blood glucose levels ChREBP activity is regulated mainly by nucleocytoplasmic shuttling of ChREBP. Under high glucose ChREBP binds to importin α and importin ß and translocates into the nucleus to initiate transcription. We have previously shown that the nuclear localization signal site (NLS) for ChREBP is bipartite with the NLS extending from Arg158 to Lys190. Here, we report the 2.5 Šcrystal structure of the ChREBP-NLS peptide bound to importin α. The structure revealed that the NLS binding is monopartite, with the amino acid residues K171RRI174 from the ChREBP-NLS interacting with ARM2-ARM5 on importin α. We discovered that importin α also binds to the primary binding site of the 14-3-3 proteins with high affinity, which suggests that both importin α and 14-3-3 are each competing with the other for this broad-binding region (residues 117-196) on ChREBP. We screened a small compound library and identified two novel compounds that inhibit the ChREBP-NLS/importin α interaction, nuclear localization, and transcription activities of ChREBP. These candidate molecules support developing inhibitors of ChREBP that may be useful in treatment of obesity and the associated diseases.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Sinais de Localização Nuclear/química , alfa Carioferinas/química , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Cristalografia por Raios X , Células Hep G2 , Humanos , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , alfa Carioferinas/genética , alfa Carioferinas/metabolismo
9.
J Med Chem ; 63(17): 9773-9786, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32787093

RESUMO

A series of N-acyl benzothiazoles shows selective and potent cytotoxicity against cancer cell lines expressing cytochrome P450 4F11. A prodrug form is metabolized by cancer cells into an active inhibitor of stearoyl-CoA desaturase (SCD). Substantial variation on the acyl portion of the inhibitors allowed the identification of (R)-27, which balanced potency, solubility, and lipophilicity to allow proof-of-concept studies in mice. The prodrugs were activated inside the tumor, where they can arrest tumor growth. Together, these observations offer promise that a tumor-activated prodrug strategy might exploit the essentiality of SCD for tumor growth, while avoiding toxicity associated with systemic SCD inhibition.


Assuntos
Benzotiazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Estearoil-CoA Dessaturase/antagonistas & inibidores , Animais , Benzotiazóis/farmacocinética , Linhagem Celular Tumoral , Família 4 do Citocromo P450/metabolismo , Feminino , Humanos , Camundongos , Pró-Fármacos/metabolismo , Distribuição Tecidual
10.
Biofabrication ; 12(3): 035019, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32408287

RESUMO

High-throughput screening (HTS) is a well-established approach for tumor-specific drug development because of its high efficiency and customizable selection of antineoplastic drugs. However, there is still a lack of an appropriate cell-based HTS specific for migratory cancer cells. In the study presented here, we created a novel assay (mHTS): a single-cell-level screening method targeting migratory cancer cells and can be applied in a high-throughput manner. This mHTS platform is based on microchannel devices (providing physical confinement during cell migration and limit migrating cells' proliferation rate) assembled 96-well plate (fitting to HTS manner). To determine the feasibility of this assay, we quantified the anti-migratory and anti-viability effects of several molecules (Cytochalasin D, Doxorubicin and AZD-6244) on migrating (creeping inside microchannel) glioblastoma multiforme (GBM) cells. After analyzing migration screening data that was collected on a single-cell-level, we were able to compare those drug's effects on cancer cells' migration velocity and uncovered the migration inhibiting potential of AZD (500 nM and 1000 nM). Viability data based on single-cell-level screening also allowed us to further understand the same drug's different lethality toward migrating and normal 2D cultured cancer cells. The Pre-classification of subpopulations enables us to study the heterogeneity of cancer and ensures our method's feasibility for a high-throughput manner. All these results proved our mHTS platform is suitable for single-cell-level anti-migration drug screening and has potential feasibility in promoting the development of anti-migratory-cancer-drug in a high-throughput manner.


Assuntos
Movimento Celular , Ensaios de Triagem em Larga Escala , Neoplasias/patologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Estudos de Viabilidade , Humanos , Camundongos
11.
PLoS Pathog ; 16(4): e1008407, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32240278

RESUMO

Influenza A viruses are human pathogens with limited therapeutic options. Therefore, it is crucial to devise strategies for the identification of new classes of antiviral medications. The influenza A virus genome is constituted of 8 RNA segments. Two of these viral RNAs are transcribed into mRNAs that are alternatively spliced. The M1 mRNA encodes the M1 protein but is also alternatively spliced to yield the M2 mRNA during infection. M1 to M2 mRNA splicing occurs at nuclear speckles, and M1 and M2 mRNAs are exported to the cytoplasm for translation. M1 and M2 proteins are critical for viral trafficking, assembly, and budding. Here we show that gene knockout of the cellular protein NS1-BP, a constituent of the M mRNA speckle-export pathway and a binding partner of the virulence factor NS1 protein, inhibits M mRNA nuclear export without altering bulk cellular mRNA export, providing an avenue to preferentially target influenza virus. We performed a high-content, image-based chemical screen using single-molecule RNA-FISH to label viral M mRNAs followed by multistep quantitative approaches to assess cellular mRNA and cell toxicity. We identified inhibitors of viral mRNA biogenesis and nuclear export that exhibited no significant activity towards bulk cellular mRNA at non-cytotoxic concentrations. Among the hits is a small molecule that preferentially inhibits nuclear export of a subset of viral and cellular mRNAs without altering bulk cellular mRNA export. These findings underscore specific nuclear export requirements for viral mRNAs and phenocopy down-regulation of the mRNA export factor UAP56. This RNA export inhibitor impaired replication of diverse influenza A virus strains at non-toxic concentrations. Thus, this screening strategy yielded compounds that alone or in combination may serve as leads to new ways of treating influenza virus infection and are novel tools for studying viral RNA trafficking in the nucleus.


Assuntos
Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Antivirais/farmacologia , Núcleo Celular/virologia , Vírus da Influenza A/metabolismo , Influenza Humana/virologia , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Vírus da Influenza A/genética , RNA Mensageiro/genética , RNA Viral/genética , Replicação Viral/efeitos dos fármacos
12.
Cell Chem Biol ; 27(5): 622-634.e6, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32330442

RESUMO

Destabilizing domains (DDs), such as a mutated form of Escherichia coli dihydrofolate reductase (ecDHFR), confer instability and promote protein degradation. However, when combined with small-molecule stabilizers (e.g., the antibiotic trimethoprim), DDs allow positive regulation of fusion protein abundance. Using a combinatorial screening approach, we identified and validated 17 unique 2,4-diaminopyrimidine/triazine-based ecDHFR DD stabilizers, at least 15 of which were ineffective antibiotics against E. coli and S. aureus. Identified stabilizers functioned in vivo to control an ecDHFR DD-firefly luciferase in the mouse eye and/or the liver. Next, stabilizers were leveraged to perform synergistic dual functions in vitro (HeLa cell death sensitization) and in vivo (repression of ocular inflammation) by stabilizing a user-defined ecDHFR DD while also controlling endogenous signaling pathways. Thus, these newly identified pharmacological chaperones allow for simultaneous control of compound-specific endogenous and user-defined genetic pathways, the combination of which may provide synergistic effects in complex biological scenarios.


Assuntos
Antibacterianos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Antagonistas do Ácido Fólico/farmacologia , Pirimidinas/farmacologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Animais , Antibacterianos/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Feminino , Antagonistas do Ácido Fólico/química , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pirimidinas/química , Tetra-Hidrofolato Desidrogenase/química , Triazinas/química , Triazinas/farmacologia , Trimetoprima/análogos & derivados , Trimetoprima/farmacologia
13.
JCI Insight ; 5(1)2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31941841

RESUMO

Autosis is a distinct form of cell death that requires both autophagy genes and the Na+,K+-ATPase pump. However, the relationship between the autophagy machinery and Na+,K+-ATPase is unknown. We explored the hypothesis that Na+,K+-ATPase interacts with the autophagy protein Beclin 1 during stress and autosis-inducing conditions. Starvation increased the Beclin 1/Na+,K+-ATPase interaction in cultured cells, and this was blocked by cardiac glycosides, inhibitors of Na+,K+-ATPase. Increases in Beclin 1/Na+,K+-ATPase interaction were also observed in tissues from starved mice, livers of patients with anorexia nervosa, brains of neonatal rats subjected to cerebral hypoxia-ischemia (HI), and kidneys of mice subjected to renal ischemia/reperfusion injury (IRI). Cardiac glycosides blocked the increased Beclin 1/Na+,K+-ATPase interaction during cerebral HI injury and renal IRI. In the mouse renal IRI model, cardiac glycosides reduced numbers of autotic cells in the kidney and improved clinical outcome. Moreover, blockade of endogenous cardiac glycosides increased Beclin 1/Na+,K+-ATPase interaction and autotic cell death in mouse hearts during exercise. Thus, Beclin 1/Na+,K+-ATPase interaction is increased in stress conditions, and cardiac glycosides decrease this interaction and autosis in both pathophysiological and physiological settings. This crosstalk between cellular machinery that generates and consumes energy during stress may represent a fundamental homeostatic mechanism.


Assuntos
Autofagia/fisiologia , Proteína Beclina-1/metabolismo , Isquemia/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Inanição/metabolismo , Animais , Morte Celular/fisiologia , Células Cultivadas , Glicosídeos , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão
14.
J Steroid Biochem Mol Biol ; 185: 17-26, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30071248

RESUMO

AIM: Vitamin D deficiency in rodents negatively affects glucose-stimulated insulin secretion (GSIS) and human epidemiological studies connect poor vitamin D status with type 2 diabetes. Previous studies performed primarily in rat islets have shown that vitamin D can enhance GSIS. However the molecular pathways linking vitamin D and insulin secretion are currently unknown. Therefore, experiments were undertaken to elucidate the transcriptional role(s) of the vitamin D receptor (VDR) in islet function. METHODS: Human and mouse islets were cultured with vehicle or 1,25-dihydroxyvitamin-D3 (1,25D3) and then subjected to GSIS assays. Insulin expression, insulin content, glucose uptake and glucose-stimulated calcium influx were tested. Microarray analysis was performed. In silico analysis was used to identify VDR response elements (VDRE) within target genes and their activity was tested using reporter assays. RESULTS: Vdr mRNA is abundant in islets and Vdr expression is glucose-responsive. Preincubation of mouse and human islets with 1,25D3 enhances GSIS and increases glucose-stimulated calcium influx. Microarray analysis identified the R-type voltage-gated calcium channel (VGCC) gene, Cacna1e, which is highly upregulated by 1,25D3 in human and mouse islets and contains a conserved VDRE in intron 7. Results from GSIS assays suggest that 1,25D3 might upregulate a variant of R-type VGCC that is resistant to chemical inhibition. CONCLUSION: These results suggest that the role of 1,25D3 in regulating calcium influx acts through the R-Type VGCC during GSIS, thereby modulating the capacity of beta cells to secrete insulin.


Assuntos
Calcitriol/metabolismo , Canais de Cálcio Tipo R/metabolismo , Cálcio/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Receptores de Calcitriol/metabolismo , Animais , Linhagem Celular Tumoral , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Deficiência de Vitamina D/patologia
15.
Cancer Res ; 78(21): 6196-6208, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30185546

RESUMO

Cancer testis antigens (CTA) are expressed in testis and placenta and anomalously activated in a variety of tumors. The mechanistic contribution of CTAs to neoplastic phenotypes remains largely unknown. Using a chemigenomics approach, we find that the CTA HORMAD1 correlates with resistance to the mitochondrial complex I inhibitor piericidin A in non-small cell lung cancer (NSCLC). Resistance was due to a reductive intracellular environment that attenuated the accumulation of free radicals. In human lung adenocarcinoma (LUAD) tumors, patients expressing high HORMAD1 exhibited elevated mutational burden and reduced survival. HORMAD1 tumors were enriched for genes essential for homologous recombination (HR), and HORMAD1 promoted RAD51-filament formation, but not DNA resection, during HR. Accordingly, HORMAD1 loss enhanced sensitivity to γ-irradiation and PARP inhibition, and HORMAD1 depletion significantly reduced tumor growth in vivo These results suggest that HORMAD1 expression specifies a novel subtype of LUAD, which has adapted to mitigate DNA damage. In this setting, HORMAD1 could represent a direct target for intervention to enhance sensitivity to DNA-damaging agents or as an immunotherapeutic target in patients.Significance: This study uses a chemigenomics approach to demonstrate that anomalous expression of the CTA HORMAD1 specifies resistance to oxidative stress and promotes HR to support tumor cell survival in NSCLC. Cancer Res; 78(21); 6196-208. ©2018 AACR.


Assuntos
Adenocarcinoma de Pulmão/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Proteínas de Ciclo Celular/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/diagnóstico , Células A549 , Adenocarcinoma de Pulmão/metabolismo , Animais , Antígenos de Neoplasias/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Dano ao DNA , Reparo do DNA , Feminino , Radicais Livres , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Mutagênicos , Transplante de Neoplasias , Estresse Oxidativo , Prognóstico , Recombinação Genética
16.
J Med Chem ; 61(12): 5199-5221, 2018 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-29869888

RESUMO

Stearoyl-CoA desaturase (SCD) catalyzes the first step in the conversion of saturated fatty acids to unsaturated fatty acids. Unsaturated fatty acids are required for membrane integrity and for cell proliferation. For these reasons, inhibitors of SCD represent potential treatments for cancer. However, systemically active SCD inhibitors result in skin toxicity, which presents an obstacle to their development. We recently described a series of oxalic acid diamides that are converted into active SCD inhibitors within a subset of cancers by CYP4F11-mediated metabolism. Herein, we describe the optimization of the oxalic acid diamides and related N-acyl ureas and an analysis of the structure-activity relationships related to metabolic activation and SCD inhibition.


Assuntos
Família 4 do Citocromo P450/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Estearoil-CoA Dessaturase/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos , Ácido Oxálico/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Estearoil-CoA Dessaturase/metabolismo , Relação Estrutura-Atividade
17.
ACS Chem Biol ; 13(8): 2247-2260, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-29878747

RESUMO

Autophagy, a lysosomal degradation pathway, plays a crucial role in cellular homeostasis, development, immunity, tumor suppression, metabolism, prevention of neurodegeneration, and lifespan extension. Thus, pharmacological stimulation of autophagy may be an effective approach for preventing or treating certain human diseases and/or aging. We sought to establish a method for developing new chemical compounds that specifically induce autophagy. To do this, we developed two assays to identify compounds that target a key regulatory node of autophagy induction-specifically, the binding of Bcl-2 (a negative regulator of autophagy) to Beclin 1 (an allosteric modulator of the Beclin 1/VPS34 lipid kinase complex that functions in autophagy initiation). These assays use either a split-luciferase assay to measure Beclin 1/Bcl-2 binding in cells or an AlphaLISA assay to directly measure direct Beclin 1/Bcl-2 binding in vitro. We screened two different chemical compound libraries, comprising ∼300 K compounds, to identify small molecules that disrupt Beclin 1/Bcl-2 binding and induce autophagy. Three novel compounds were identified that directly inhibit Beclin 1/Bcl-2 interaction with an IC50 in the micromolar range and increase autophagic flux. These compounds do not demonstrate significant cytotoxicity, and they exert selectivity for disruption of Bcl-2 binding to the BH3 domain of Beclin 1 compared with the BH3 domain of the pro-apoptotic Bcl-2 family members, Bax and Bim. Thus, we have identified candidate molecules that serve as lead templates for developing potent and selective Beclin 1/Bcl-2 inhibitors that may be clinically useful as autophagy-inducing agents.


Assuntos
Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células HeLa , Humanos , Mapas de Interação de Proteínas/efeitos dos fármacos
18.
Nat Commun ; 9(1): 2050, 2018 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-29784984

RESUMO

The originally published version of this Article contained an error in the spelling of the author Nathaniel W. Oswald, which was incorrectly given as Nathaniel W. Olswald. This has now been corrected in both the PDF and HTML versions of the Article.

19.
Cell ; 173(4): 864-878.e29, 2018 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-29681454

RESUMO

Diversity in the genetic lesions that cause cancer is extreme. In consequence, a pressing challenge is the development of drugs that target patient-specific disease mechanisms. To address this challenge, we employed a chemistry-first discovery paradigm for de novo identification of druggable targets linked to robust patient selection hypotheses. In particular, a 200,000 compound diversity-oriented chemical library was profiled across a heavily annotated test-bed of >100 cellular models representative of the diverse and characteristic somatic lesions for lung cancer. This approach led to the delineation of 171 chemical-genetic associations, shedding light on the targetability of mechanistic vulnerabilities corresponding to a range of oncogenotypes present in patient populations lacking effective therapy. Chemically addressable addictions to ciliogenesis in TTC21B mutants and GLUT8-dependent serine biosynthesis in KRAS/KEAP1 double mutants are prominent examples. These observations indicate a wealth of actionable opportunities within the complex molecular etiology of cancer.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Pulmonares/patologia , Bibliotecas de Moléculas Pequenas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Família 4 do Citocromo P450/deficiência , Família 4 do Citocromo P450/genética , Descoberta de Drogas , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Glucocorticoides/farmacologia , Proteínas Facilitadoras de Transporte de Glucose/antagonistas & inibidores , Proteínas Facilitadoras de Transporte de Glucose/genética , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor Notch2/genética , Receptor Notch2/metabolismo , Receptores de Glucocorticoides/antagonistas & inibidores , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo
20.
Antiviral Res ; 150: 193-201, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29294299

RESUMO

Ebola virus (EBOV) is an enveloped negative-sense, single-stranded RNA virus of the filovirus family that causes severe disease in humans. Approved therapies for EBOV disease are lacking. EBOV RNA synthesis is carried out by a virus-encoded complex with RNA-dependent RNA polymerase activity that is required for viral propagation. This complex and its activities are therefore potential antiviral targets. To identify potential lead inhibitors of EBOV RNA synthesis, a library of small molecule compounds was screened against a previously established assay of EBOV RNA synthesis, the EBOV minigenome assay (MGA), in 384 well microplate format. The screen identified 56 hits that inhibited EBOV MGA activity by more than 70% while exhibiting less than 20% cell cytotoxicity. Inhibitory chemical scaffolds included angelicin derivatives, derivatives of the antiviral compound GSK983 and benzoquinolines. Structure-activity relationship (SAR) studies of the benzoquinoline scaffold produced ∼50 analogs and led to identification of an optimized compound, SW456, with a submicromolar IC50 in the EBOV MGA and antiviral activity against infectious EBOV in cell culture. The compound was also active against a MGA for another deadly filovirus, Marburg virus. It also exhibited antiviral activity towards a negative-sense RNA virus from the rhabdovirus family, vesicular stomatitis virus, and a positive-sense RNA virus, Zika virus. Overall, these data demonstrate the potential of the EBOV MGA to identify anti-EBOV compounds and identifies the benzoquinoline series as a broad-spectrum antiviral lead.


Assuntos
Antivirais/farmacologia , Ebolavirus/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Quinolinas/farmacologia , Replicação Viral/efeitos dos fármacos , Antivirais/química , Relação Dose-Resposta a Droga , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Ebolavirus/genética , Humanos , Quinolinas/química , Bibliotecas de Moléculas Pequenas
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