RESUMO
A series of pyridoxine hydroxamic acid analog bearing a 5-aryl-spacers were synthesized. Evaluation of these novel HIV integrase complex inhibitors revealed compounds with high potency against wild-type HIV virus.
Assuntos
Inibidores de Integrase de HIV/química , Integrase de HIV/química , HIV-1/fisiologia , Ácidos Hidroxâmicos/química , Animais , Domínio Catalítico , Feminino , Integrase de HIV/metabolismo , Inibidores de Integrase de HIV/síntese química , Inibidores de Integrase de HIV/farmacocinética , Meia-Vida , Humanos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/farmacocinética , Concentração Inibidora 50 , Piridoxina/química , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
A new class of biosensors, fluorogen activating proteins (FAPs), has been successfully used to track receptor trafficking in live cells. Unlike the traditional fluorescent proteins (FPs), FAPs do not fluoresce unless bound to their specific small-molecule fluorogens, and thus FAP-based assays are highly sensitive. Application of the FAP-based assay for protein trafficking in high-throughput flow cytometry resulted in the discovery of a new class of compounds that interferes with the binding between fluorogens and FAP, thus blocking the fluorescence signal. These compounds are high-affinity, nonfluorescent analogs of fluorogens with little or no toxicity to the tested cells and no apparent interference with the normal function of FAP-tagged receptors. The most potent compound among these, N,4-dimethyl-N-(2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)benzenesulfonamide (ML342), has been investigated in detail. X-ray crystallographic analysis revealed that ML342 competes with the fluorogen, sulfonated thiazole orange coupled to diethylene glycol diamine (TO1-2p), for the same binding site on a FAP, AM2.2. Kinetic analysis shows that the FAP-fluorogen interaction is more complex than a homogeneous one-site binding process, with multiple conformational states of the fluorogen and/or the FAP, and possible dimerization of the FAP moiety involved in the process.
Assuntos
Ligação Proteica/efeitos dos fármacos , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Bioensaio/métodos , Técnicas Biossensoriais , Linhagem Celular Tumoral , Fluorescência , Corantes Fluorescentes/metabolismo , Humanos , Cinética , Transporte Proteico/efeitos dos fármacos , Células U937RESUMO
Overactive GTPases have often been linked to human diseases. The available inhibitors are limited and have not progressed far in clinical trials. We report here a first-in-class small molecule pan-GTPase inhibitor discovered from a high throughput screening campaign. The compound CID1067700 inhibits multiple GTPases in biochemical, cellular protein and protein interaction, as well as cellular functional assays. In the biochemical and protein interaction assays, representative GTPases from Rho, Ras, and Rab, the three most generic subfamilies of the GTPases, were probed, while in the functional assays, physiological processes regulated by each of the three subfamilies of the GTPases were examined. The chemical functionalities essential for the activity of the compound were identified through structural derivatization. The compound is validated as a useful molecular probe upon which GTPase-targeting inhibitors with drug potentials might be developed.
Assuntos
Inibidores Enzimáticos/química , GTP Fosfo-Hidrolases/antagonistas & inibidores , Compostos Heterocíclicos com 2 Anéis/química , Sondas Moleculares/química , Tioureia/análogos & derivados , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Receptores ErbB/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Compostos Heterocíclicos com 2 Anéis/síntese química , Compostos Heterocíclicos com 2 Anéis/farmacologia , Humanos , Integrinas/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Sondas Moleculares/metabolismo , Sondas Moleculares/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Tioureia/síntese química , Tioureia/química , Tioureia/farmacologia , Células U937 , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7 , Proteínas ras/metabolismoRESUMO
The University of New Mexico Center for Molecular Discovery (UNMCMD) is an academic research center that specializes in discovery using high throughput flow cytometry (HTFC) integrated with virtual screening, as well as knowledge mining and drug informatics. With a primary focus on identifying small molecules that can be used as chemical probes and as leads for drug discovery, it is a central core resource for research and translational activities at UNM that supports implementation and management of funded screening projects as well as "up-front" services such as consulting for project design and implementation, assistance in assay development and generation of preliminary data for pilot projects in support of competitive grant applications. The HTFC platform in current use represents advanced, proprietary technology developed at UNM that is now routinely capable of processing bioassays arrayed in 96-, 384- and 1536-well formats at throughputs of 60,000 or more wells per day. Key programs at UNMCMD include screening of research targets submitted by the international community through NIH's Molecular Libraries Program; a multi-year effort involving translational partnerships at UNM directed towards drug repurposing - identifying new uses for clinically approved drugs; and a recently established personalized medicine initiative for advancing cancer therapy by the application of "smart" oncology drugs in selected patients based on response patterns of their cancer cells in vitro. UNMCMD discoveries, innovation, and translation have contributed to a wealth of inventions, patents, licenses and publications, as well as startup companies, clinical trials and a multiplicity of domestic and international collaborative partnerships to further the research enterprise.
Assuntos
Descoberta de Drogas , Citometria de Fluxo/métodos , Ensaios de Triagem em Larga Escala/métodos , Universidades/organização & administração , Alergia e Imunologia/organização & administração , Doenças Transmissíveis/tratamento farmacológico , Doenças Transmissíveis/imunologia , Reposicionamento de Medicamentos , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Neoplasias/tratamento farmacológico , New Mexico , Medicina de Precisão , Pesquisa Translacional Biomédica , Interface Usuário-Computador , Fluxo de TrabalhoRESUMO
Chemotherapeutics tumor resistance is a principal reason for treatment failure, and clinical and experimental data indicate that multidrug transporters such as ATP-binding cassette (ABC) B1 and ABCG2 play a leading role by preventing cytotoxic intracellular drug concentrations. Functional efflux inhibition of existing chemotherapeutics by these pumps continues to present a promising approach for treatment. A contributing factor to the failure of existing inhibitors in clinical applications is limited understanding of specific substrate/inhibitor/pump interactions. We have identified selective efflux inhibitors by profiling multiple ABC transporters against a library of small molecules to find molecular probes to further explore such interactions. In our primary screening protocol using JC-1 as a dual-pump fluorescent reporter substrate, we identified a piperazine-substituted pyrazolo[1,5-a]pyrimidine substructure with promise for selective efflux inhibition. As a result of a focused structure-activity relationship (SAR)-driven chemistry effort, we describe compound 1 (CID44640177), an efflux inhibitor with selectivity toward ABCG2 over ABCB1. Compound 1 is also shown to potentiate the activity of mitoxantrone in vitro as well as preliminarily in vivo in an ABCG2-overexpressing tumor model. At least two analogues significantly reduce tumor size in combination with the chemotherapeutic topotecan. To our knowledge, low nanomolar chemoreversal activity coupled with direct evidence of efflux inhibition for ABCG2 is unprecedented.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Antineoplásicos/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Linhagem Celular , Resistencia a Medicamentos Antineoplásicos , Citometria de Fluxo , Ensaios de Triagem em Larga Escala , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos SCID , Proteínas de Neoplasias/metabolismo , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Person-to-person transmission of influenza viruses occurs by contact (direct and fomites) and non-contact (droplet and small particle aerosol) routes, but the quantitative dynamics and relative contributions of these routes are incompletely understood. The transmissibility of influenza strains estimated from secondary attack rates in closed human populations is confounded by large variations in population susceptibilities. An experimental method to phenotype strains for transmissibility in an animal model could provide relative efficiencies of transmission. We developed an experimental method to detect exhaled viral aerosol transmission between unanesthetized infected and susceptible ferrets, measured aerosol particle size and number, and quantified the viral genomic RNA in the exhaled aerosol. During brief 3-hour exposures to exhaled viral aerosols in airflow-controlled chambers, three strains of pandemic 2009 H1N1 strains were frequently transmitted to susceptible ferrets. In contrast one seasonal H1N1 strain was not transmitted in spite of higher levels of viral RNA in the exhaled aerosol. Among three pandemic strains, the two strains causing weight loss and illness in the intranasally infected 'donor' ferrets were transmitted less efficiently from the donor than the strain causing no detectable illness, suggesting that the mucosal inflammatory response may attenuate viable exhaled virus. Although exhaled viral RNA remained constant, transmission efficiency diminished from day 1 to day 5 after donor infection. Thus, aerosol transmission between ferrets may be dependent on at least four characteristics of virus-host relationships including the level of exhaled virus, infectious particle size, mucosal inflammation, and viral replication efficiency in susceptible mucosa.
Assuntos
Expiração , Furões/virologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Infecções por Orthomyxoviridae/transmissão , Pandemias , Estações do Ano , Aerossóis , Animais , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H1N1/fisiologia , Pulmão/virologia , Masculino , Nebulizadores e Vaporizadores , Pandemias/prevenção & controle , RNA Viral/metabolismo , Especificidade da Espécie , Fatores de Tempo , Carga ViralRESUMO
Influenza virus is a major cause of morbidity and mortality worldwide, yet little quantitative understanding of transmission is available to guide evidence-based public health practice. Recent studies of influenza non-contact transmission between ferrets and guinea pigs have provided insights into the relative transmission efficiencies of pandemic and seasonal strains, but the infecting dose and subsequent contagion has not been quantified for most strains. In order to measure the aerosol infectious dose for 50% (aID(50)) of seronegative ferrets, seasonal influenza virus was nebulized into an exposure chamber with controlled airflow limiting inhalation to airborne particles less than 5 µm diameter. Airborne virus was collected by liquid impinger and Teflon filters during nebulization of varying doses of aerosolized virus. Since culturable virus was accurately captured on filters only up to 20 minutes, airborne viral RNA collected during 1-hour exposures was quantified by two assays, a high-throughput RT-PCR/mass spectrometry assay detecting 6 genome segments (Ibis T5000™ Biosensor system) and a standard real time RT-qPCR assay. Using the more sensitive T5000 assay, the aID(50) for A/New Caledonia/20/99 (H1N1) was approximately 4 infectious virus particles under the exposure conditions used. Although seroconversion and sustained levels of viral RNA in upper airway secretions suggested established mucosal infection, viral cultures were almost always negative. Thus after inhalation, this seasonal H1N1 virus may replicate less efficiently than H3N2 virus after mucosal deposition and exhibit less contagion after aerosol exposure.
Assuntos
Furões , Vírus da Influenza A Subtipo H1N1/patogenicidade , Infecções por Orthomyxoviridae/transmissão , Aerossóis , Ar , Animais , Linhagem Celular , Embrião de Galinha , Técnicas de Cultura , Cães , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Nebulizadores e Vaporizadores , Reação em Cadeia da Polimerase , Politetrafluoretileno/química , Fatores de Tempo , Vírion/genética , Vírion/isolamento & purificaçãoRESUMO
The transmembrane ubiquitin ligase K5/MIR2 of Kaposi sarcoma herpesvirus (KSHV) mediates internalization and lysosomal degradation of glycoproteins involved in antigen presentation and co-stimulation. In endothelial cells (ECs), K5 additionally reduced expression of CD31/platelet-endothelial cell adhesion molecule (PECAM), an adhesion molecule regulating cell-cell interactions of ECs, platelets, monocytes, and T cells. K5 also reduced EC migration, a CD31-dependent process. Unlike other K5 substrates, both newly synthesized and pre-existing CD31 molecules were targeted by K5. K5 was transported to the cell surface and ubiquitinated pre-existing CD31, resulting in endocytosis and lysosomal degradation. In the endoplasmic reticulum, newly synthesized CD31 was degraded by proteasomes, which required binding of phosphofurin acidic cluster sorting protein-2 (PACS-2) to acidic residues in the carboxyterminal tail of K5. Thus, CD31, a novel target of K5, is efficiently removed from ECs by a dual degradation mechanism that is regulated by the subcellular sorting of the ubiquitin ligase. K5-mediated degradation of CD31 is likely to affect EC function in KS tumors.
Assuntos
Células Endoteliais/imunologia , Células Endoteliais/virologia , Herpesvirus Humano 8/enzimologia , Herpesvirus Humano 8/patogenicidade , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular , Células Cultivadas , Regulação para Baixo , Células Endoteliais/citologia , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Sarcoma de Kaposi/enzimologia , Sarcoma de Kaposi/imunologia , Sarcoma de Kaposi/virologia , Especificidade por Substrato , Proteínas de Transporte Vesicular , Proteínas Virais/metabolismoRESUMO
The poxviral RING protein p28 is a virulence factor whose molecular function is unknown. Many cellular RING-containing proteins act as ubiquitin ligases (RING-E3s) connecting selected substrate proteins to the ubiquitination machinery. Here we demonstrate that vaccinia virus p28 and its homologue in myxoma virus, M143R, can mediate the formation of polyubiquitin conjugates, while RING mutants of both p28 and M143R cannot. Furthermore, p28 is ubiquitinated in vivo and ubiquitin colocalizes with p28 to virus factories independently of an intact RING domain. These results implicate the ubiquitin system in poxviral virulence.
Assuntos
Ubiquitina-Proteína Ligases/metabolismo , Vaccinia virus/enzimologia , Vaccinia virus/patogenicidade , Proteínas Virais/genética , Linhagem Celular , Células HeLa , Humanos , Mutação , Myxoma virus , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas Virais/metabolismo , Virulência , Replicação ViralRESUMO
Poxviruses and gamma-2 herpesviruses share the K3 family of viral immune evasion proteins that inhibit the surface expression of glycoproteins such as major histocompatibility complex class I (MHC-I), B7.2, ICAM-1, and CD95(Fas). K3 family proteins contain an amino-terminal PHD/LAP or RING-CH domain followed by two transmembrane domains. To examine whether human homologues are functionally related to the viral immunoevasins, we studied seven membrane-associated RING-CH (MARCH) proteins. All MARCH proteins located to subcellular membranes, and several MARCH proteins reduced surface levels of known substrates of the viral K3 family. Two closely related proteins, MARCH-IV and MARCH-IX, reduced surface expression of MHC-I molecules. In the presence of MARCH-IV or MARCH-IX, MHC-I was ubiquitinated and rapidly internalized by endocytosis, whereas MHC-I molecules lacking lysines in their cytoplasmic tail were resistant to downregulation. The amino-terminal regions containing the RING-CH domain of several MARCH proteins examined catalyzed multiubiquitin formation in vitro, suggesting that MARCH proteins are ubiquitin ligases. The functional similarity of the MARCH family and the K3 family suggests that the viral immune evasion proteins were derived from MARCH proteins, a novel family of transmembrane ubiquitin ligases that seems to target glycoproteins for lysosomal destruction via ubiquitination of the cytoplasmic tail.
Assuntos
Regulação para Baixo , Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Ligases/metabolismo , Ubiquitina/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Células HeLa , Humanos , Proteínas Imediatamente Precoces/genética , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Transfecção , Proteínas Virais/genéticaRESUMO
The genomes of several poxviruses contain open reading frames with homology to the K3 and K5 genes of Kaposi's sarcoma-associated herpesvirus (KSHV) and the K3 gene of murine gammaherpesvirus 68, which target major histocompatibility complex class I (MHC-I) as well as costimulatory molecules for proteasomal or lysosomal degradation. The homologous gene product of myxomavirus (MV), M153R, was recently shown to reduce the cell surface expression of MHC-I. In addition, normal MHC-I surface expression was observed in cells infected with MV lacking M153R (J. L. Guerin, J. Gelfi, S. Boullier, M. Delverdier, F. A. Bellanger, S. Bertagnoli, I. Drexler, G. Sutter, and F. Messud-Petit, J. Virol. 76:2912-2923, 2002). Here, we show that M153R also downregulates the T-cell coreceptor CD4 and we study the molecular mechanism by which M153R achieves the downregulation of CD4 and MHC-I. Upon M153R expression, CD4 was rapidly internalized and degraded in lysosomes, whereas deletion of M153R from the genome of MV restored CD4 expression. The downregulation of both CD4 and MHC-I was dependent on the presence of lysine residues in their cytoplasmic tails. Increased ubiquitination of CD4 was observed upon coexpression with M153R in the presence of inhibitors of lysosomal acidification. Surface expression of CD4 was restored upon overexpression of Hrs, a ubiquitin interaction motif-containing protein that sorts ubiquitinated proteins into endosomes. Moreover, the purified PHD/LAP zinc finger of M153R catalyzed the formation of multiubiquitin adducts in vitro. Our data suggest that M153R acts as a membrane-bound ubiquitin ligase that conjugates ubiquitin to the cytoplasmic domain of substrate glycoproteins, with ubiquitin serving as a lysosomal targeting signal. Since a similar mechanism was recently proposed for KSHV K5, it seems that members of the unrelated families of gamma-2 herpesviruses and poxviruses share a common immune evasion mechanism that targets host cell immune receptors.
Assuntos
Antígenos CD4/metabolismo , Endocitose , Ligases/metabolismo , Poxviridae/enzimologia , Ubiquitina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , DNA Viral , Regulação para Baixo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Humanos , Dados de Sequência MolecularRESUMO
Many viruses have developed mechanisms to escape the cellular immune response by inhibiting antigen presentation from major histocompatibility complex (MHC) molecules. Most of these immune escape mechanisms are highly host adapted and specific to a given virus species or family. Recent observations however, suggest that a conserved family of viral proteins is used by both gamma-2 herpesviruses and by poxviruses to downregulate MHC class I. In addition, other cell surface molecules involved in immune recognition by T cells and NK cells are also downregulated. Two open reading frames (ORFs), K3 and K5, of Kaposi's sarcoma associated virus (KSHV) and one ORFs, K3, of murine gamma herpesvirus 68 (MHV 68) inhibit surface expression of MHC I molecules. In cells transfected with KSHV-K3 and KSHV-K5, MHC I is rapidly endocytosed and degraded in lysosomes whereas in MHV 68-K3 transfected cells, MHC I is targeted for proteasomal degradation. The K3 and K5 genes display a characteristic conserved domain structure of an amino-terminal plant homeo domain/leukemia associated protein-zinc finger domain followed by two carboxyterminal transmembrane domains. Related proteins are not only found in other gamma-2 herpesviruses, but also in several poxviruses. Moreover, recent data suggest that the K3-related protein of myxoma virus also downregulates MHC I. The presence of similar genes in eukaryotic genomes further indicates that the viral ORFs were originally derived from host genes of as yet unknown function. The molecular mechanism of MHC I downregulation by this novel gene family is only poorly understood at present. However, several lines of evidence suggest that they might function as ubiquitin ligases that regulate the intracellular transport of transmembrane proteins through ubiquitination.