RESUMO
Carfilzomib (CFZ) is a second-generation proteasome inhibitor that is Food and Drug Administration and European Commission approved for the treatment of relapsed or refractory multiple myeloma. CFZ is an epoxomicin derivative with an epoxyketone electrophilic warhead that irreversibly adducts the catalytic threonine residue of the ß5 subunit of the proteasome. Although CFZ produces a highly potent, sustained inactivation of the proteasome, the electrophilic nature of the drug could potentially produce off-target protein adduction. To address this possibility, we synthesized an alkynyl analog of CFZ and investigated protein adduction by this analog in HepG2 cells. Using click chemistry coupled with streptavidin based IP and shotgun tandem mass spectrometry (MS/MS), we identified two off-target proteins, cytochrome P450 27A1 (CYP27A1) and glutathione S-transferase omega 1 (GSTO1), as targets of the alkynyl CFZ probe. We confirmed the adduction of CYP27A1 and GSTO1 by streptavidin capture and immunoblotting methodology and then site-specifically mapped the adducts with targeted MS/MS methods. Although CFZ adduction of CYP27A1 and GSTO1 in vitro decreased the activities of these enzymes, the small fraction of these proteins modified by CFZ in intact cells should limit the impact of these off-target modifications. The data support the high selectivity of CFZ for covalent modification of its therapeutic targets, despite the presence of a reactive electrophile. The approach we describe offers a generalizable method to evaluate the safety profile of covalent protein-modifying therapeutics.
Assuntos
Colestanotriol 26-Mono-Oxigenase/metabolismo , Glutationa Transferase/metabolismo , Oligopeptídeos/química , Inibidores de Proteassoma/síntese química , Linhagem Celular Tumoral , Química Click , Células Hep G2 , Humanos , Estrutura Molecular , Inibidores de Proteassoma/química , Inibidores de Proteassoma/farmacologia , Espectrometria de Massas em TandemRESUMO
Oprozomib is an oral proteasome inhibitor currently under investigation in patients with hematologic malignancies or solid tumors. Oprozomib elicits potent pharmacological actions by forming a covalent bond with the active site N-terminal threonine of the 20S proteasome. Oprozomib has a short half-life across preclinical species and in patients due to systemic clearance via metabolism. Potential for drug-drug interactions (DDIs) could alter the exposure of this potent therapeutic; therefore, a thorough investigation of pathways responsible for metabolism is required. In the present study, the major drug-metabolizing enzyme responsible for oprozomib metabolism was identified in vitro. A diol of oprozomib was found to be the predominant metabolite in human hepatocytes, which formed via direct epoxide hydrolysis. Using recombinant epoxide hydrolases (EHs) and selective EH inhibitors in liver microsomes, microsomal EH (mEH) but not soluble EH (sEH) was found to be responsible for oprozomib diol formation. Coincubation with 2-nonylsulfanyl-propionamide, a selective mEH inhibitor, resulted in a significant decrease in oprozomib disappearance (>80%) with concurrent complete blockage of diol formation in human hepatocytes. On the contrary, a selective sEH inhibitor did not affect oprozomib metabolism. Pretreatment of hepatocytes with the pan-cytochrome P450 (P450) inhibitor 1-aminobenzotriazole resulted in a modest reduction (â¼20%) of oprozomib metabolism. These findings indicated that mEH plays a predominant role in oprozomib metabolism. Further studies may be warranted to determine whether drugs that are mEH inhibitors cause clinically significant DDIs with oprozomib. On the other hand, pharmacokinetics of oprozomib is unlikely to be affected by coadministered P450 and sEH inhibitors and/or inducers.
Assuntos
Clorpromazina/análogos & derivados , Sistema Enzimático do Citocromo P-450/metabolismo , Epóxido Hidrolases/metabolismo , Inibidores de Proteassoma/metabolismo , Administração Oral , Adulto , Clorpromazina/metabolismo , Interações Medicamentosas/fisiologia , Feminino , Meia-Vida , Hepatócitos/metabolismo , Humanos , Masculino , Microssomos Hepáticos/metabolismo , Pessoa de Meia-Idade , Oxirredução , Proteínas Recombinantes/metabolismo , Triazóis/metabolismo , Adulto JovemRESUMO
BACKGROUND: Elevated immune activation persists during treated human immunodeficiency virus (HIV) infection and is associated with blunted CD4 recovery and premature mortality, but its causes remain incompletely characterized. We hypothesized that asymptomatic cytomegalovirus (CMV) replication might contribute to immune activation in this setting. METHODS: Thirty antiretroviral therapy-treated HIV-infected CMV-seropositive participants with CD4 counts <350 cells/mm(3) were randomized to receive valganciclovir 900 mg daily or placebo for 8 weeks, followed by an additional 4-week observation period. The primary outcome was the week 8 change in percentage of activated (CD38(+) HLA-DR(+)) CD8(+) T cells. RESULTS: Fourteen participants were randomized to valganciclovir and 16 to placebo. Most participants (21 [70%] of 30) had plasma HIV RNA levels <75 copies/mL. The median CD4 count was 190 (IQR: 134-232) cells/mm(3), and 12 (40%) of 30 had detectable CMV DNA in saliva, plasma, or semen at baseline. CMV DNA continued to be detectable at weeks 4-12 in 7 (44%) of 16 placebo-treated participants, but in none of the valganciclovir-treated participants (P = .007). Valganciclovir-treated participants had significantly greater reductions in CD8 activation at weeks 8 (P = .03) and 12 (P = .02) than did placebo-treated participants. These trends were significant even among those with undetectable plasma HIV RNA levels. CONCLUSIONS: CMV (and/or other herpesvirus) replication is a significant cause of immune activation in HIV-infected individuals with incomplete antiretroviral therapy-mediated CD4(+) T cell recovery. CLINICAL TRIALS REGISTRATION: NCT00264290.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Linfócitos T CD4-Positivos/fisiologia , Ganciclovir/análogos & derivados , Infecções por HIV/tratamento farmacológico , Ativação Linfocitária/efeitos dos fármacos , Fármacos Anti-HIV/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/complicações , Infecções por Citomegalovirus/imunologia , DNA Viral/análise , DNA Viral/sangue , Ganciclovir/farmacologia , Ganciclovir/uso terapêutico , Infecções por HIV/complicações , Infecções por HIV/imunologia , Humanos , Saliva/virologia , Sêmen/virologia , Valganciclovir , Replicação ViralRESUMO
BACKGROUND: A new method for detecting circulating Ewing sarcoma cells using flow cytometry is described. This strategy exploits the nearly universal expression of CD99 and the lack of expression of CD45 by Ewing sarcoma cells. PROCEDURE: Ewing sarcoma cell line A673, peripheral blood mononuclear cells (PBMCs), and bone marrow mononuclear cells (BMMCs) were stained for CD99 and CD45 in order to detect CD99+CD45- cells by flow cytometry. Known quantities of A673 Ewing sarcoma cells were spiked into control PBMCs to test the accuracy of this method. Control PBMCs were evaluated to assess the level of background staining. RESULTS: Flow cytometry was accurate at frequencies as low as one A673 cell per 500,000 PBMCs. The background rate of CD99+CD45- cell detection was low in PBMCs from nine healthy volunteers (median 0.0001% of total cells; range 0-0.00046%) and was further reduced by incorporating stains to exclude dead cells, progenitor cells, and monocytes. In one subject with newly diagnosed localized Ewing sarcoma, CD99+CD45- cells were detected in both blood (0.0021%) and bone marrow (0.048%). CONCLUSIONS: Multicolor flow cytometry for CD99+CD45- cells provides a new strategy for detecting circulating Ewing sarcoma cells. Clinical evaluation and validation of this method is ongoing.
Assuntos
Neoplasias Ósseas/diagnóstico , Citometria de Fluxo , Células Neoplásicas Circulantes/patologia , Sarcoma de Ewing/diagnóstico , Antígeno 12E7 , Antígenos CD/imunologia , Biomarcadores Tumorais/imunologia , Medula Óssea/imunologia , Estudos de Casos e Controles , Moléculas de Adesão Celular/imunologia , Humanos , Antígenos Comuns de Leucócito/imunologia , Células Neoplásicas Circulantes/imunologia , PrognósticoRESUMO
This communication describes the discovery of a novel series of Aurora kinase inhibitors. Key SAR and critical binding elements are discussed. Some of the more advanced analogues potently inhibit cellular proliferation and induce phenotypes consistent with Aurora kinase inhibition. In particular, compound 21 (SNS-314) is a potent and selective Aurora kinase inhibitor that exhibits significant activity in pre-clinical in vivo tumor models.
Assuntos
Neoplasias Experimentais/tratamento farmacológico , Compostos de Fenilureia/química , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Quinazolinas/farmacologia , Tiazóis/química , Tiazóis/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Aurora Quinases , Ensaios de Seleção de Medicamentos Antitumorais , Células HCT116 , Humanos , Camundongos , Transplante de Neoplasias , Neoplasias Experimentais/enzimologia , Quinazolinas/química , Relação Estrutura-AtividadeRESUMO
Inflammation has increasingly been recognized as a critical component influencing tumor growth. Recent reports have revealed conflicting evidence for the role of Toll-like receptors (TLR) in modulating tumorigenesis. In our study, we implicate TLR3 in mediating immune surveillance with increased growth of implanted transgenic adenocarcinoma of the mouse prostate (TRAMP) tumors in TLR3(-/-) compared with TLR3(+/+) mice. Activation of TLR3 by polyinosinic-polycytidylic acid (polyI:C) leads to induction of multiple inflammatory pathways, including NF-kappaB, mitogen-activated protein kinases, and interferon (IFN) regulatory factors. We explored the potential of TLR3 stimulation in prostate cancer immunotherapy and showed that treatment with polyI:C can strongly suppress both s.c. implanted TRAMP tumors in syngenic mice as well as orthotopic prostate cancers in TRAMP C57Bl6 x FvB F1 Tg(+/-) transgenic mice. Treated tumors remained well differentiated to moderately differentiated with increased infiltration of T lymphocytes and natural killer (NK) cells compared with poorly differentiated adenocarcinoma observed in untreated tumors. Like TLR3(-/-) mice, IFN-alpha receptor 1 (IFNAR1)(-/-) mice exhibited reduced tumor surveillance and impaired tumor suppression following polyI:C treatment. We observed that type I IFN-dependent induction of cytokines was responsible for NK activation, with depletion of NK cells leading to increased tumor growth as well as expansion of CD4(+)CD25(+)Foxp3(+) T regulatory lymphocytes. Our study therefore delineates the importance of IFNAR-dependent functions in TLR3-mediated tumor suppression and supports the use of TLR3 agonists for prostate cancer immune-based therapies.
Assuntos
Interferon Tipo I/imunologia , Neoplasias da Próstata/imunologia , Receptor 3 Toll-Like/imunologia , Animais , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/imunologia , Linhagem Celular Tumoral , Tolerância Imunológica , Indutores de Interferon/farmacologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Células Matadoras Naturais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Poli I-C/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Receptor de Interferon alfa e beta/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
PURPOSE: The Aurora family of serine/threonine kinases (Aurora-A, Aurora-B, and Aurora-C) plays a key role in cells orderly progression through mitosis. Elevated expression levels of Aurora kinases have been detected in a high percentage of melanoma, colon, breast, ovarian, gastric, and pancreatic tumors. We characterized the biological and pharmacological properties of SNS-314, an ATP-competitive, selective, and potent inhibitor of Aurora kinases. METHODS: We studied the biochemical potency and selectivity of SNS-314 to inhibit Aurora kinases A, B, and C. The inhibition of cellular proliferation induced by SNS-314 was evaluated in a broad range of tumor cell lines and correlated to inhibition of histone H3 phosphorylation, inhibition of cell-cycle progression, increase in nuclear content and cell size, loss of viability, and induction of apoptosis. The dose and administration schedule of SNS-314 was optimized for in vivo efficacy in mouse xenograft models of human cancer. RESULTS: In the HCT116 human colon cancer xenograft model, administration of 50 and 100 mg/kg SNS-314 led to dose-dependent inhibition of histone H3 phosphorylation for at least 10 h, indicating effective Aurora-B inhibition in vivo. HCT116 tumors from animals treated with SNS-314 showed potent and sustained responses including reduction of phosphorylated histone H3 levels, increased caspase-3 and appearance of increased nuclear size. The compound showed significant tumor growth inhibition in a dose-dependent manner under a variety of dosing schedules including weekly, bi-weekly, and 5 days on/9 days off. CONCLUSIONS: SNS-314 is a potent small-molecule inhibitor of Aurora kinases developed as a novel anti-cancer therapeutic agent for the treatment of diverse human malignancies.