RESUMO
Classic Galactosemia is a rare inborn error of metabolism that is caused by deficiency of galactose-1-phosphate uridyltransferase (GALT), an enzyme within the Leloir pathway that is responsible for the conversion of galactose-1-phosphate (gal-1-p) and UDP-glucose to glucose-1-phosphate and UDP-galactose. This deficiency results in elevated intracellular concentrations of its substrate, gal-1-p, and this increased concentration is believed to be the major pathogenic mechanism in Classic Galactosemia. Galactokinase (GALK) is an upstream enzyme of GALT in the Leloir pathway and is responsible for conversion of galactose and ATP to gal-1-p and ADP. Therefore, it was hypothesized that the identification of a small-molecule inhibitor of human GALK would act to prevent the accumulation of gal-1-p and offer a novel entry therapy for this disorder. Herein we describe a quantitative high-throughput screening campaign that identified a single chemotype that was optimized and validated as a GALK inhibitor.
Assuntos
Galactoquinase/antagonistas & inibidores , Animais , Benzoxazóis/síntese química , Benzoxazóis/química , Benzoxazóis/metabolismo , Cristalografia por Raios X , Galactoquinase/genética , Galactoquinase/metabolismo , Galactosefosfatos/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Cinética , Camundongos , Microssomos Hepáticos/metabolismo , Conformação Molecular , Ligação Proteica , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Compostos de Espiro/química , Relação Estrutura-AtividadeRESUMO
Little has been published on the demographic composition of the clinical and translational science research workforce within the Clinical and Translational Science Awards (CTSA) Program despite the well-documented need for greater diversity in the biomedical research workforce. Analyses of workforce demographic reveal that women and members of underrepresented groups remain persistently underrepresented in the CTSA hub and training components principal investigators. In contrast, in the CTSA Program career development and training programs, females have greater representation as participants, and non-Whites were better represented in training programs.
RESUMO
BACKGROUND: The recently approved anti-AIDS drug rilpivirine (TMC278, Edurant) is a nonnucleoside inhibitor (NNRTI) that binds to reverse transcriptase (RT) and allosterically blocks the chemical step of DNA synthesis. In contrast to earlier NNRTIs, rilpivirine retains potency against well-characterized, clinically relevant RT mutants. Many structural analogues of rilpivirine are described in the patent literature, but detailed analyses of their antiviral activities have not been published. This work addresses the ability of several of these analogues to inhibit the replication of wild-type (WT) and drug-resistant HIV-1. RESULTS: We used a combination of structure activity relationships and X-ray crystallography to examine NNRTIs that are structurally related to rilpivirine to determine their ability to inhibit WT RT and several clinically relevant RT mutants. Several analogues showed broad activity with only modest losses of potency when challenged with drug-resistant viruses. Structural analyses (crystallography or modeling) of several analogues whose potencies were reduced by RT mutations provide insight into why these compounds were less effective. CONCLUSIONS: Subtle variations between compounds can lead to profound differences in their activities and resistance profiles. Compounds with larger substitutions replacing the pyrimidine and benzonitrile groups of rilpivirine, which reorient pocket residues, tend to lose more activity against the mutants we tested. These results provide a deeper understanding of how rilpivirine and related compounds interact with the NNRTI binding pocket and should facilitate development of novel inhibitors.
Assuntos
Infecções por HIV/virologia , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Nitrilas/farmacologia , Pirimidinas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Linhagem Celular , Cristalografia , Infecções por HIV/tratamento farmacológico , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/metabolismo , HIV-1/genética , HIV-1/fisiologia , Humanos , Modelos Moleculares , Estrutura Molecular , Mutação , Nitrilas/síntese química , Nitrilas/química , Pirimidinas/síntese química , Pirimidinas/química , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/química , RilpivirinaRESUMO
Introduction: Identifying the most effective ways to support career development of early stage investigators in clinical and translational science should yield benefits for the biomedical research community. Institutions with Clinical and Translational Science Awards (CTSA) offer KL2 programs to facilitate career development; however, the sustained impact has not been widely assessed. Methods: A survey comprised of quantitative and qualitative questions was sent to 2144 individuals that had previously received support through CTSA KL2 mechanisms. The 547 responses were analyzed with identifying information redacted. Results: Respondents held MD (47%), PhD (36%), and MD/PhD (13%) degrees. After KL2 support was completed, physicians' time was divided 50% to research and 30% to patient care, whereas PhD respondents devoted 70% time to research. Funded research effort averaged 60% for the cohort. Respondents were satisfied with their career progression. More than 95% thought their current job was meaningful. Two-thirds felt confident or very confident in their ability to sustain a career in clinical and translational research. Factors cited as contributing to career success included protected time, mentoring, and collaborations. Conclusion: This first large systematic survey of KL2 alumni provides valuable insight into the group's perceptions of the program and outcome information. Former scholars are largely satisfied with their career choice and direction, national recognition of their expertise, and impact of their work. Importantly, they identified training activities that contributed to success. Our results and future analysis of the survey data should inform the framework for developing platforms to launch sustaining careers of translational scientists.
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Two new marine-derived sesquiterpene benzoquinones which we designate as neopetrosiquinones A (1) and B (2), have been isolated from a deep-water sponge of the family Petrosiidae. The structures were elucidated on the basis of their spectroscopic data. Compounds 1 and 2 inhibit the in vitro proliferation of the DLD-1 human colorectal adenocarcinoma cell line with IC(50) values of 3.7 and 9.8 µM, respectively, and the PANC-1 human pancreatic carcinoma cell line with IC(50) values of 6.1 and 13.8 µM, respectively. Neopetrosiquinone A (1) also inhibited the in vitro proliferation of the AsPC-1 human pancreatic carcinoma cell line with an IC(50) value of 6.1 µM. The compounds are structurally related to alisiaquinone A, cyclozonarone, and xestoquinone.
Assuntos
Benzoquinonas/química , Poríferos/química , Sesquiterpenos/química , Animais , Benzoquinonas/isolamento & purificação , Benzoquinonas/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/farmacologiaRESUMO
Four new depsipeptides, mirabamides E-H (1-4), and the known depsipeptide mirabamide C (5) have been isolated from the sponge Stelletta clavosa, collected from the Torres Strait. The planar structures were determined on the basis of extensive 1D and 2D NMR and HRESIMS. The absolute configurations were established by the advanced Marfey's method, NMR, and GC-MS. The four new compounds all showed strong inhibition of HIV-1 in a neutralization assay with IC(50) values of 121, 62, 68, and 41 nM, respectively.
Assuntos
Fármacos Anti-HIV/isolamento & purificação , Fármacos Anti-HIV/farmacologia , Depsipeptídeos/isolamento & purificação , Depsipeptídeos/farmacologia , Poríferos/química , Animais , Fármacos Anti-HIV/química , Depsipeptídeos/química , HIV-1/efeitos dos fármacos , Concentração Inibidora 50 , Estrutura Molecular , Ressonância Magnética Nuclear BiomolecularRESUMO
Six new depsipeptides belonging to two different structural classes, termed celebesides A-C and theopapuamides B-D, have been isolated from the marine sponge Siliquariaspongia mirabilis. Their structures were determined using extensive 2D NMR and ESI-MS/MS techniques. Celebesides are unusual cyclic depsipeptides that comprise a polyketide moiety and five amino acid residues, including an uncommon 3-carbamoyl threonine, and a phosphoserine residue in celebesides A and B. Theopapuamides B-D are undecapeptides with an N-terminal fatty acid moiety containing two previously unreported amino acids, 3-acetamido-2-aminopropanoic acid and 4-amino-2,3-dihydroxy-5-methylhexanoic acid. The relative configuration of the polyketide moiety in celebesides was resolved by J-based analysis and quantum mechanical calculations, the results of which were self-consistent. Celebeside A neutralized HIV-1 in a single-round infectivity assay with an IC(50) value of 1.9 +/- 0.4 microg/mL while the nonphosphorylated analog celebeside C was inactive at concentrations as high as 50 microg/mL. Theopapuamides A-C showed cytotoxicity against human colon carcinoma (HCT-116) cells with IC(50) values between 2.1 and 4.0 microg/mL and exhibited strong antifungal activity against wildtype and amphotericin B-resistant strains of Candida albicans at loads of 1-5 microg/disk.
Assuntos
Depsipeptídeos/isolamento & purificação , Depsipeptídeos/farmacologia , HIV-1/fisiologia , Poríferos/química , Internalização do Vírus/efeitos dos fármacos , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Depsipeptídeos/química , Fungos/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Teoria Quântica , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
4'-Phosphopantetheinyl transferases (PPTases) catalyze a post-translational modification essential to bacterial cell viability and virulence. We present the discovery and medicinal chemistry optimization of 2-pyridinyl-N-(4-aryl)piperazine-1-carbothioamides, which exhibit submicromolar inhibition of bacterial Sfp-PPTase with no activity toward the human orthologue. Moreover, compounds within this class possess antibacterial activity in the absence of a rapid cytotoxic response in human cells. An advanced analogue of this series, ML267 (55), was found to attenuate production of an Sfp-PPTase-dependent metabolite when applied to Bacillus subtilis at sublethal doses. Additional testing revealed antibacterial activity against methicillin-resistant Staphylococcus aureus , and chemical genetic studies implicated efflux as a mechanism for resistance in Escherichia coli . Additionally, we highlight the in vitro absorption, distribution, metabolism, and excretion and in vivo pharmacokinetic profiles of compound 55 to further demonstrate the potential utility of this small-molecule inhibitor.
Assuntos
Antibacterianos/síntese química , Proteínas de Bactérias/antagonistas & inibidores , Piridinas/síntese química , Tioureia/análogos & derivados , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Animais , Antibacterianos/farmacocinética , Antibacterianos/farmacologia , Dipeptídeos/farmacologia , Farmacorresistência Bacteriana , Sinergismo Farmacológico , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/metabolismo , Humanos , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Microssomos Hepáticos/metabolismo , Piridinas/farmacocinética , Piridinas/farmacologia , Metabolismo Secundário , Relação Estrutura-Atividade , Tioureia/síntese química , Tioureia/farmacocinética , Tioureia/farmacologiaRESUMO
Nuclear protein import in eukaryotic cells is mediated by karyopherin proteins, which bind to specific nuclear localization signals on substrate proteins and transport them across the nuclear envelope and into the nucleus. Replication protein A (RPA) is a nuclear protein comprised of three subunits (termed Rfa1, Rfa2, and Rfa3 in Saccharomyces cerevisiae) that binds single-stranded DNA and is essential for DNA replication, recombination, and repair. RPA associates with two different karyopherins in yeast, Kap95, and Msn5/Kap142. However, it is unclear which of these karyopherins is responsible for RPA nuclear import. We have generated GFP fusion proteins with each of the RPA subunits and demonstrate that these Rfa-GFP chimeras are functional in yeast cells. The intracellular localization of the RPA proteins in live cells is similar in wild-type and msn5Δ deletion strains but becomes primarily cytoplasmic in cells lacking functional Kap95. Truncating the C-terminus of any of the RPA subunits results in mislocalization of the proteins to the cytoplasm and a loss of protein-protein interactions between the subunits. Our data indicate that Kap95 is likely the primary karyopherin responsible for RPA nuclear import in yeast and that the C-terminal regions of Rfa1, Rfa2, and Rfa3 are essential for efficient nucleocytoplasmic transport of each RPA subunit.
Assuntos
Transporte Ativo do Núcleo Celular/genética , Proteína de Replicação A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Western Blotting , Primers do DNA/genética , Proteínas de Fluorescência Verde/metabolismo , Imunoprecipitação , Microscopia de Fluorescência , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína de Replicação A/genética , Proteínas de Saccharomyces cerevisiae/genética , Especificidade da EspécieRESUMO
A new highly unsaturated macrolide lactam, termed mirabilin ( 1), was isolated from the aqueous extract of the marine sponge Siliquariaspongia mirabilis. Mirabilin is characterized by the presence of a 35-membered macrolide lactam ring bearing a pentadiene conjugated system and a tetrasubstituted tetrahydropyran ring. A linear polyketide moiety is attached to the macrocyclic ring through an amide linkage. The structure of mirabilin was determined using extensive 2D NMR and ESIMS and tandem MS techniques. Mirabilin inhibits the growth of the tumor cell line HCT-116 with an IC 50 value of 0.27 +/- 0.09 microM and is noncytotoxic to several other cell lines.
Assuntos
Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Macrolídeos/isolamento & purificação , Macrolídeos/farmacologia , Ácido Oleanólico/análogos & derivados , Poríferos/química , Saponinas/isolamento & purificação , Saponinas/farmacologia , Animais , Antineoplásicos/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Concentração Inibidora 50 , Macrolídeos/química , Biologia Marinha , Micronésia , Estrutura Molecular , Ácido Oleanólico/química , Ácido Oleanólico/isolamento & purificação , Ácido Oleanólico/farmacologia , Saponinas/químicaRESUMO
Four new cyclic depsipeptides termed mirabamides A-D (1-4) have been isolated from the marine sponge Siliquariaspongia mirabilis and shown to potently inhibit HIV-1 fusion. Their structures were elucidated by NMR and ESIMS, and absolute stereochemistry of the amino acids was determined using advanced Marfey's methods and NMR. Mirabamides contain two new entities, including 4-chlorohomoproline in 1-3 and an unusual glycosylated amino acid, beta-methoxytyrosine 4'-O-alpha-L-rhamnopyranoside (in 1, 2, and 4), along with a rare N-terminal aliphatic hydroxy acid. These elements proved to be useful for anti-HIV structure-activity relationship studies. Mirabamide A inhibited HIV-1 in neutralization and fusion assays with IC50 values between 40 and 140 nM, as did mirabamides C and D (IC50 values between 140 nM and 1.3 microM for 3 and 190 nM and 3.9 microM for 4), indicating that these peptides can act at the early stages of HIV-1 entry. The potent activity of depsipeptides containing the glycosylated beta-OMe Tyr unit demonstrates that beta-OMe Tyr itself is not critical for activity. Mirabamides A-C inhibited the growth of B. subtilis and C. albicans at 1-5 microg/disk in disk diffusion assays.