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2.
Nat Commun ; 12(1): 4407, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315870

RESUMO

Alcohol Use Disorder (AUD) affects a large portion of the population. Unfortunately, efficacious medications to treat the disease are limited. Studies in rodents suggest that mTORC1 plays a crucial role in mechanisms underlying phenotypes such as heavy alcohol intake, habit, and relapse. Thus, mTORC1 inhibitors, which are used in the clinic, are promising therapeutic agents to treat AUD. However, chronic inhibition of mTORC1 in the periphery produces undesirable side effects, which limit their potential use for the treatment of AUD. To overcome these limitations, we designed a binary drug strategy in which male mice were treated with the mTORC1 inhibitor RapaLink-1 together with a small molecule (RapaBlock) to protect mTORC1 activity in the periphery. We show that whereas RapaLink-1 administration blocked mTORC1 activation in the liver, RapaBlock abolished the inhibitory action of Rapalink-1. RapaBlock also prevented the adverse side effects produced by chronic inhibition of mTORC1. Importantly, co-administration of RapaLink-1 and RapaBlock inhibited alcohol-dependent mTORC1 activation in the nucleus accumbens and attenuated alcohol seeking and drinking.


Assuntos
Consumo de Bebidas Alcoólicas/patologia , Encéfalo/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Animais , Intolerância à Glucose/complicações , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Endogâmicos C57BL , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Especificidade de Órgãos , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Perda de Peso/efeitos dos fármacos
3.
Science ; 373(6554): 541-547, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34326236

RESUMO

Repurposing drugs as treatments for COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has drawn much attention. Beginning with sigma receptor ligands and expanding to other drugs from screening in the field, we became concerned that phospholipidosis was a shared mechanism underlying the antiviral activity of many repurposed drugs. For all of the 23 cationic amphiphilic drugs we tested, including hydroxychloroquine, azithromycin, amiodarone, and four others already in clinical trials, phospholipidosis was monotonically correlated with antiviral efficacy. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the physicochemical properties of drugs and does not reflect specific target-based activities-rather, it may be considered a toxic confound in early drug discovery. Early detection of phospholipidosis could eliminate these artifacts, enabling a focus on molecules with therapeutic potential.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Reposicionamento de Medicamentos , Lipidoses/induzido quimicamente , Fosfolipídeos/metabolismo , SARS-CoV-2/efeitos dos fármacos , Células A549 , Animais , Antivirais/química , Antivirais/uso terapêutico , Antivirais/toxicidade , COVID-19/virologia , Cátions , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Feminino , Humanos , Camundongos , Testes de Sensibilidade Microbiana , SARS-CoV-2/fisiologia , Tensoativos/química , Tensoativos/farmacologia , Tensoativos/toxicidade , Células Vero , Replicação Viral/efeitos dos fármacos
4.
bioRxiv ; 2021 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-33501437

RESUMO

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths worldwide and massive societal and economic burden. Recently, a new variant of SARS-CoV-2, known as B.1.1.7, was first detected in the United Kingdom and is spreading in several other countries, heightening public health concern and raising questions as to the resulting effectiveness of vaccines and therapeutic interventions. We and others previously identified host-directed therapies with antiviral efficacy against SARS-CoV-2 infection. Less prone to the development of therapy resistance, host-directed drugs represent promising therapeutic options to combat emerging viral variants as host genes possess a lower propensity to mutate compared to viral genes. Here, in the first study of the full-length B.1.1.7 variant virus , we find two host-directed drugs, plitidepsin (aplidin; inhibits translation elongation factor eEF1A) and ralimetinib (inhibits p38 MAP kinase cascade), as well as remdesivir, to possess similar antiviral activity against both the early-lineage SARS-CoV-2 and the B.1.1.7 variant, evaluated in both human gastrointestinal and lung epithelial cell lines. We find that plitidepsin is over an order of magnitude more potent than remdesivir against both viruses. These results highlight the importance of continued development of host-directed therapeutics to combat current and future coronavirus variant outbreaks.

5.
Cancer Res ; 81(7): 1627-1632, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33509943

RESUMO

Effective treatment of pediatric solid tumors has been hampered by the predominance of currently "undruggable" driver transcription factors. Improving outcomes while decreasing the toxicity of treatment necessitates the development of novel agents that can directly inhibit or degrade these elusive targets. MYCN in pediatric neural-derived tumors, including neuroblastoma and medulloblastoma, is a paradigmatic example of this problem. Attempts to directly and specifically target MYCN have failed due to its similarity to MYC, the unstructured nature of MYC family proteins in their monomeric form, the lack of an understanding of MYCN-interacting proteins and ability to test their relevance in vivo, the inability to obtain structural information on MYCN protein complexes, and the challenges of using traditional small molecules to inhibit protein-protein or protein-DNA interactions. However, there is now promise for directly targeting MYCN based on scientific and technological advances on all of these fronts. Here, we discuss prior challenges and the reasons for renewed optimism in directly targeting this "undruggable" transcription factor, which we hope will lead to improved outcomes for patients with pediatric cancer and create a framework for targeting driver oncoproteins regulating gene transcription.


Assuntos
Antineoplásicos/isolamento & purificação , Resistencia a Medicamentos Antineoplásicos , Proteína Proto-Oncogênica N-Myc/fisiologia , Neoplasias/tratamento farmacológico , Terapias em Estudo , Idade de Início , Antineoplásicos/história , Antineoplásicos/uso terapêutico , Criança , Descoberta de Drogas/história , Descoberta de Drogas/métodos , Descoberta de Drogas/tendências , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais/história , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Ensaios de Seleção de Medicamentos Antitumorais/tendências , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , História do Século XX , História do Século XXI , Humanos , Proteína Proto-Oncogênica N-Myc/antagonistas & inibidores , Proteína Proto-Oncogênica N-Myc/genética , Neoplasias/epidemiologia , Neoplasias/genética , Terapias em Estudo/história , Terapias em Estudo/métodos , Terapias em Estudo/tendências
6.
Science ; 371(6532): 926-931, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33495306

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins interact with the eukaryotic translation machinery, and inhibitors of translation have potent antiviral effects. We found that the drug plitidepsin (aplidin), which has limited clinical approval, possesses antiviral activity (90% inhibitory concentration = 0.88 nM) that is more potent than remdesivir against SARS-CoV-2 in vitro by a factor of 27.5, with limited toxicity in cell culture. Through the use of a drug-resistant mutant, we show that the antiviral activity of plitidepsin against SARS-CoV-2 is mediated through inhibition of the known target eEF1A (eukaryotic translation elongation factor 1A). We demonstrate the in vivo efficacy of plitidepsin treatment in two mouse models of SARS-CoV-2 infection with a reduction of viral replication in the lungs by two orders of magnitude using prophylactic treatment. Our results indicate that plitidepsin is a promising therapeutic candidate for COVID-19.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Depsipeptídeos/farmacologia , Fator 1 de Elongação de Peptídeos/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Alanina/análogos & derivados , Alanina/farmacologia , Alanina/uso terapêutico , Animais , Antivirais/uso terapêutico , COVID-19/prevenção & controle , COVID-19/virologia , Proteínas do Nucleocapsídeo de Coronavírus/biossíntese , Proteínas do Nucleocapsídeo de Coronavírus/genética , Depsipeptídeos/administração & dosagem , Depsipeptídeos/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Feminino , Células HEK293 , Humanos , Pulmão/virologia , Camundongos Endogâmicos C57BL , Mutação , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , RNA Viral/biossíntese , RNA Viral/genética , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Replicação Viral/efeitos dos fármacos
7.
ACS Cent Sci ; 6(10): 1753-1761, 2020 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-33145412

RESUMO

We report the identification of three cyclic peptide ligands of K-Ras(G12D) using an integrated in vitro translation-mRNA display selection platform. These cyclic peptides show preferential binding to the GTP-bound state of K-Ras(G12D) over the GDP-bound state and block Ras-Raf interaction. A co-crystal structure of peptide KD2 with K-Ras(G12D)·GppNHp reveals that this peptide binds in the Switch II groove region with concomitant opening of the Switch II loop and a 40° rotation of the α2 helix, and that a threonine residue (Thr10) on KD2 has direct access to the mutant aspartate (Asp12) on K-Ras. Replacing this threonine with non-natural amino acids afforded peptides with improved potency at inhibiting the interaction between Raf1-RBD and K-Ras(G12D) but not wildtype K-Ras. The union of G12D over wildtype selectivity and GTP state/GDP state selectivity is particularly desirable, considering that oncogenic K-Ras(G12D) exists predominantly in the GTP state in cancer cells, and wildtype K-Ras signaling is important for the maintenance of healthy cells.

8.
Bioorg Med Chem ; 28(20): 115712, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33069070

RESUMO

Alternative splicing of the androgen receptor (AR) is frequently observed in castration resistant prostate cancer (CRPC). One AR isoform, the AR-V7 splice variant, is a constitutively active transcription factor which lacks a ligand binding domain and is therefore undruggable. AR-V7 expression correlates with resistance to androgen receptor signaling inhibitors (ARSi) and poor clinical prognoses. The occurrence of the AR-V7 splice variant is driven by alternative splicing of AR pre-mRNA by the spliceosome, however the mechanistic details are poorly understood. We demonstrate that the splicing factor RBM39 is critical for alternative splicing of the AR-V7 splice variant mRNA transcripts from AR pre-mRNA, and that the anti-cancer drug, indisulam, reduces AR-V7 mRNA levels by degrading RBM39. We report that indisulam effectively reduces AR-V7 in in vitro and in vivo models.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/genética , Sulfonamidas/farmacologia , Processamento Alternativo/efeitos dos fármacos , Processamento Alternativo/genética , Animais , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Androgênicos/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/química , Células Tumorais Cultivadas
9.
Cancer Cell ; 38(1): 129-143.e7, 2020 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-32531271

RESUMO

Using unbiased kinase profiling, we identified protein kinase A (PKA) as an active kinase in small cell lung cancer (SCLC). Inhibition of PKA activity genetically, or pharmacologically by activation of the PP2A phosphatase, suppresses SCLC expansion in culture and in vivo. Conversely, GNAS (G-protein α subunit), a PKA activator that is genetically activated in a small subset of human SCLC, promotes SCLC development. Phosphoproteomic analyses identified many PKA substrates and mechanisms of action. In particular, PKA activity is required for the propagation of SCLC stem cells in transplantation studies. Broad proteomic analysis of recalcitrant cancers has the potential to uncover targetable signaling networks, such as the GNAS/PKA/PP2A axis in SCLC.


Assuntos
Cromograninas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Neoplasias Pulmonares/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteína Fosfatase 2/metabolismo , Proteômica/métodos , Carcinoma de Pequenas Células do Pulmão/metabolismo , Células A549 , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cromograninas/genética , Cisplatino/administração & dosagem , Cisplatino/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Proteína Fosfatase 2/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
10.
Cancer Res ; 80(4): 709-718, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31806641

RESUMO

The mTOR signaling is dysregulated prominently in human cancers including glioblastoma, suggesting mTOR as a robust target for therapy. Inhibitors of mTOR have had limited success clinically, however, in part because their mechanism of action is cytostatic rather than cytotoxic. Here, we tested three distinct mTOR kinase inhibitors (TORKi) PP242, KU-0063794, and sapanisertib against glioblastoma cells. All agents similarly decreased proliferation of glioblastoma cells, whereas PP242 uniquely induced apoptosis. Apoptosis induced by PP242 resulted from off-target cooperative inhibition of JAK2 and protein kinase C alpha (PKCα). Induction of apoptosis was also decreased by additional on-target inhibition of mTOR, due to induction of autophagy. As EGFR inhibitors can block PKCα, EGFR inhibitors erlotinib and osimertinib were tested separately in combination with the JAK2 inhibitor AZD1480. Combination therapy induced apoptosis of glioblastoma tumors in both flank and in patient-derived orthotopic xenograft models, providing a preclinical rationale to test analogous combinations in patients. SIGNIFICANCE: These findings identify PKCα and JAK2 as targets that drive apoptosis in glioblastoma, potentially representing a clinically translatable approach for glioblastoma.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Acrilamidas/farmacologia , Acrilamidas/uso terapêutico , Compostos de Anilina/farmacologia , Compostos de Anilina/uso terapêutico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Autofagia/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Cloridrato de Erlotinib/farmacologia , Cloridrato de Erlotinib/uso terapêutico , Feminino , Glioblastoma/patologia , Humanos , Indóis/farmacologia , Indóis/uso terapêutico , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Camundongos , Morfolinas/farmacologia , Morfolinas/uso terapêutico , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-alfa/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Purinas/farmacologia , Purinas/uso terapêutico , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Neuro Oncol ; 22(4): 457-469, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-31678994

RESUMO

BACKGROUND: The transcription factor signal transducer and activator of transcription 3 (STAT3) drives progression in glioblastoma (GBM), suggesting STAT3 as a therapeutic target. Surprisingly however, GBM cells generally show primary resistance to STAT3 blockade. METHODS: Human glioblastoma cell lines LN229, U87, SF767, and U373, and patient-derived xenografts (PDXs) GBM8 and GBM43 were used to evaluate epidermal growth factor receptor (EGFR) activation during STAT3 inhibition. Protein and gene expression experiments, protein stability assays, cytokine arrays, phospho-tyrosine arrays and EGFR-ligand protein arrays were performed on STAT3 inhibitor-treated cells. To evaluate antitumor activity, we administered a betacellulin (BTC)-neutralizing antibody alone and in combination with STAT3 inhibition. BTC is an EGFR ligand. We therefore treated mice with orthotopic xenografts using the third-generation EGFR inhibitor osimertinib, with or without STAT3 knockdown. RESULTS: We demonstrate that both small-molecule inhibitors and knockdown of STAT3 led to expression and secretion of the EGFR ligand BTC, resulting in activation of EGFR and subsequent downstream phosphorylation of nuclear factor-kappaB (NF-κB). Neutralizing antibody against BTC abrogated activation of both EGFR and NF-κB in response to inhibition of STAT3; with combinatorial blockade of STAT3 and BTC inducing apoptosis in GBM cells. Blocking EGFR and STAT3 together inhibited tumor growth, improving survival in mice bearing orthotopic GBM PDXs in vivo. CONCLUSION: These data reveal a feedback loop among STAT3, EGFR, and NF-κB that mediates primary resistance to STAT3 blockade and suggest strategies for therapeutic intervention.


Assuntos
Glioblastoma , Animais , Betacelulina , Linhagem Celular Tumoral , Glioblastoma/tratamento farmacológico , Camundongos , NF-kappa B/metabolismo , Fosforilação , Fator de Transcrição STAT3/metabolismo
12.
Science ; 366(6471)2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31831640

RESUMO

The p27 protein is a canonical negative regulator of cell proliferation and acts primarily by inhibiting cyclin-dependent kinases (CDKs). Under some circumstances, p27 is associated with active CDK4, but no mechanism for activation has been described. We found that p27, when phosphorylated by tyrosine kinases, allosterically activated CDK4 in complex with cyclin D1 (CDK4-CycD1). Structural and biochemical data revealed that binding of phosphorylated p27 (phosp27) to CDK4 altered the kinase adenosine triphosphate site to promote phosphorylation of the retinoblastoma tumor suppressor protein (Rb) and other substrates. Surprisingly, purified and endogenous phosp27-CDK4-CycD1 complexes were insensitive to the CDK4-targeting drug palbociclib. Palbociclib instead primarily targeted monomeric CDK4 and CDK6 (CDK4/6) in breast tumor cells. Our data characterize phosp27-CDK4-CycD1 as an active Rb kinase that is refractory to clinically relevant CDK4/6 inhibitors.


Assuntos
Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Regulação Alostérica , Antineoplásicos/farmacologia , Biocatálise , Linhagem Celular Tumoral , Cristalografia por Raios X , Ciclina D1/química , Quinase 4 Dependente de Ciclina/química , Inibidor de Quinase Dependente de Ciclina p27/química , Ativação Enzimática , Humanos , Fosforilação , Conformação Proteica , Proteína do Retinoblastoma/metabolismo
13.
FASEB J ; 33(12): 13131-13144, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31638431

RESUMO

Despite the high and preferential expression of p38γ MAPK in the myocardium, little is known about its function in the heart. The aim of the current study was to elucidate the physiologic and biochemical roles of p38γ in the heart. Expression and subcellular localization of p38 isoforms was determined in mouse hearts. Comparisons of the cardiac function and structure of wild-type and p38γ knockout (KO) mice at baseline and after abdominal aortic banding demonstrated that KO mice developed less ventricular hypertrophy and that contractile function is better preserved. To identify potential substrates of p38γ, we generated an analog-sensitive mutant to affinity tag endogenous myocardial proteins. Among other proteins, this technique identified calpastatin as a direct p38γ substrate. Moreover, phosphorylation of calpastatin by p38γ impaired its ability to inhibit the protease, calpain. We have identified p38γ as an important determinant of the progression of pathologic cardiac hypertrophy after aortic banding in mice. In addition, we have identified calpastatin, among other substrates, as a novel direct target of p38γ that may contribute to the protection observed in p38γKO mice.-Loonat, A. A., Martin, E. D., Sarafraz-Shekary, N., Tilgner, K., Hertz, N. T., Levin, R., Shokat, K. M., Burlingame, A. L., Arabacilar, P., Uddin, S., Thomas, M., Marber, M. S., Clark, J. E. p38γ MAPK contributes to left ventricular remodeling after pathologic stress and disinhibits calpain through phosphorylation of calpastatin.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Calpaína/metabolismo , Proteína Quinase 12 Ativada por Mitógeno/metabolismo , Remodelação Ventricular/fisiologia , Animais , Calpaína/genética , Ecocardiografia , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Proteína Quinase 12 Ativada por Mitógeno/genética , Fosforilação , Isoformas de Proteínas , Espectrometria de Massas em Tandem , Remodelação Ventricular/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
14.
Angew Chem Int Ed Engl ; 58(45): 16314-16319, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31557383

RESUMO

Here we report the design, synthesis, and characterization of bifunctional chemical ligands that induce the association of Ras with ubiquitously expressed immunophilin proteins such as FKBP12 and cyclophilin A. We show this approach is applicable to two distinct Ras ligand scaffolds, and that both the identity of the immunophilin ligand and the linker chemistry affect compound efficacy in biochemical and cellular contexts. These ligands bind to Ras in an immunophilin-dependent fashion and mediate the formation of tripartite complexes of Ras, immunophilin, and the ligand. The recruitment of cyclophilin A to GTP-bound Ras blocks its interaction with B-Raf in biochemical assays. Our study demonstrates the feasibility of ligand-induced association of Ras with intracellular proteins and suggests it as a promising therapeutic strategy for Ras-driven cancers.


Assuntos
Ciclofilinas/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Proteína 1A de Ligação a Tacrolimo/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Ciclofilinas/química , Humanos , Ligantes , Conformação Proteica , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas p21(ras)/química , Bibliotecas de Moléculas Pequenas/química , Proteína 1A de Ligação a Tacrolimo/química
16.
Sci Signal ; 12(583)2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138768

RESUMO

Inhibitors targeting KRASG12C, a mutant form of the guanosine triphosphatase (GTPase) KRAS, are a promising new class of oncogene-specific therapeutics for the treatment of tumors driven by the mutant protein. These inhibitors react with the mutant cysteine residue by binding covalently to the switch-II pocket (S-IIP) that is present only in the inactive guanosine diphosphate (GDP)-bound form of KRASG12C, sparing the wild-type protein. We used a genome-scale CRISPR interference (CRISPRi) functional genomics platform to systematically identify genetic interactions with a KRASG12C inhibitor in cellular models of KRASG12C mutant lung and pancreatic cancer. Our data revealed genes that were selectively essential in this oncogenic driver-limited cell state, meaning that their loss enhanced cellular susceptibility to direct KRASG12C inhibition. We termed such genes "collateral dependencies" (CDs) and identified two classes of combination therapies targeting these CDs that increased KRASG12C target engagement or blocked residual survival pathways in cells and in vivo. From our findings, we propose a framework for assessing genetic dependencies induced by oncogene inhibition.


Assuntos
Neoplasias Pulmonares/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisteína/genética , Feminino , Genômica , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Camundongos , Camundongos Nus , Mutação , Transplante de Neoplasias , Oncogenes , Neoplasias Pancreáticas/genética , Ligação Proteica , Proteômica , Proteínas Proto-Oncogênicas p21(ras)/genética , Análise de Sequência de RNA , Transdução de Sinais/efeitos dos fármacos
17.
Exp Mol Med ; 51(4): 1-17, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30992425

RESUMO

CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that forms an active complex with cyclin Y (CCNY). Although both proteins have been recently implicated in cancer pathogenesis, it is still unclear how the CDK16/CCNY complex exerts its biological activity. To understand the CDK16/CCNY network, we used complementary proteomic approaches to identify potential substrates of this complex. We identified several candidates implicating the CDK16/CCNY complex in cytoskeletal dynamics, and we focused on the microtubule-associated protein regulator of cytokinesis (PRC1), an essential protein for cell division that organizes antiparallel microtubules and whose deregulation may drive genomic instability in cancer. Using analog-sensitive (AS) CDK16 generated by CRISPR-Cas9 mutagenesis in 293T cells, we found that specific inhibition of CDK16 induces PRC1 dephosphorylation at Thr481 and delocalization to the nucleus during interphase. The observation that CDK16 inhibition and PRC1 downregulation exhibit epistatic effects on cell viability confirms that these proteins can act through a single pathway. In conclusion, we identified PRC1 as the first substrate of the CDK16/CCNY complex and demonstrated that the proliferative function of CDK16 is mediated by PRC1 phosphorylation. As CDK16 is emerging as a critical node in cancer, our study reveals novel potential therapeutic targets.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Ciclo Celular/genética , Divisão Celular/genética , Divisão Celular/fisiologia , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Quinases Ciclina-Dependentes/genética , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Fosforilação , Ligação Proteica/genética , Ligação Proteica/fisiologia
18.
Cell Host Microbe ; 25(3): 454-462.e6, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30827827

RESUMO

Legionella pneumophila (L.p.), the microbe responsible for Legionnaires' disease, secretes ∼300 bacterial proteins into the host cell cytosol. A subset of these proteins affects a wide range of post-translational modifications (PTMs) to disrupt host cellular pathways. L.p. has 5 conserved eukaryotic-like Ser/Thr effector kinases, LegK1-4 and LegK7, which are translocated during infection. Using a chemical genetic screen, we identified the Hsp70 chaperone family as a direct host target of LegK4. Phosphorylation of Hsp70s at T495 in the substrate-binding domain disrupted Hsp70's ATPase activity and greatly inhibited its protein folding capacity. Phosphorylation of cytosolic Hsp70 by LegK4 resulted in global translation inhibition and an increase in the amount of Hsp70 on highly translating polysomes. LegK4's ability to inhibit host translation via a single PTM uncovers a role for Hsp70 in protein synthesis and directly links it to the cellular translational machinery.


Assuntos
Células Eucarióticas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Interações Hospedeiro-Patógeno , Legionella pneumophila/enzimologia , Fosfotransferases/metabolismo , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Células Eucarióticas/microbiologia , Doença dos Legionários/microbiologia , Fosforilação , Fatores de Virulência/metabolismo
19.
Sci Signal ; 12(570)2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808819

RESUMO

Tumors comprise cancer stem cells (CSCs) and their heterogeneous progeny within a stromal microenvironment. In response to transforming growth factor-ß (TGF-ß), epithelial and carcinoma cells undergo a partial or complete epithelial-mesenchymal transition (EMT), which contributes to cancer progression. This process is seen as reversible because cells revert to an epithelial phenotype upon TGF-ß removal. However, we found that prolonged TGF-ß exposure, mimicking the state of in vivo carcinomas, promotes stable EMT in mammary epithelial and carcinoma cells, in contrast to the reversible EMT induced by a shorter exposure. The stabilized EMT was accompanied by stably enhanced stem cell generation and anticancer drug resistance. Furthermore, prolonged TGF-ß exposure enhanced mammalian target of rapamycin (mTOR) signaling. A bitopic mTOR inhibitor repressed CSC generation, anchorage independence, cell survival, and chemoresistance and efficiently inhibited tumorigenesis in mice. These results reveal a role for mTOR in the stabilization of stemness and drug resistance of breast cancer cells and position mTOR inhibition as a treatment strategy to target CSCs.


Assuntos
Antineoplásicos/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Animais , Benzamidas/farmacologia , Linhagem Celular Transformada , Transformação Celular Neoplásica/genética , Células Cultivadas , Dioxóis/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
20.
Protein Sci ; 28(3): 654-662, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30636329

RESUMO

The proteomic mapping of enzyme-substrate interactions is challenged by their transient nature. A method to capture interacting protein kinases in complexes with a single substrate of interest would provide a new tool for mapping kinase signaling networks. Here, we describe a nucleotide-based substrate analog capable of reprogramming the wild-type phosphoryl-transfer reaction to produce a kinase-acrylamide-based thioether crosslink to mutant substrates with a cysteine nucleophile substituted at the native phosphorylation site. A previously reported ATP-based methacrylate crosslinker (ATP-MA) was capable of mediating kinase crosslinking to short peptides but not protein substrates. Exploration of structural variants of ATP-MA to enable crosslinking of protein substrates to kinases led to the discovery that an ADP-based methacrylate (ADP-MA) crosslinker was superior to the ATP scaffold at crosslinking in vitro. The improved efficiency of ADP-MA over ATP-MA is due to reduced inhibition of the second step of the kinase-substrate crosslinking reaction by the product of the first step of the reaction. The new probe, ADP-MA, demonstrated enhanced in vitro crosslinking between the Src tyrosine kinase and its substrate Cortactin in a phosphorylation site-specific manner. The kinase-substrate crosslinking reaction can be carried out in a complex mammalian cell lysate setting, although the low abundance of endogenous kinases remains a significant challenge for efficient capture.


Assuntos
Cortactina/metabolismo , Reagentes para Ligações Cruzadas/química , Quinases da Família src/química , Quinases da Família src/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Galinhas , Cisteína/química , Cisteína/metabolismo , Células HEK293 , Humanos , Cinética , Metacrilatos/química , Modelos Moleculares , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato
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