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1.
Infection ; 2024 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-39117931

RESUMO

PURPOSE: Sepsis causes significant worldwide morbidity and mortality. Inability to clear an infection and secondary infections are known complications in severe sepsis and likely result in worsened outcomes. We sought to characterize risk factors of these complications. METHODS: We performed a secondary analysis of clinical data from 401 subjects enrolled in the PHENOtyping sepsis-induced Multiple organ failure Study. We examined factors associated with prolonged infection, defined as infection that continued to be identified 7 days or more from initial identification, and secondary infection, defined as new infections identified ≥ 3 days from presentation. Multivariable adjustment was performed to examine laboratory markers of immune depression, with immunocompromised and immunocompetent subjects analyzed separately. RESULTS: Illness severity, immunocompromised status, invasive procedures, and site of infection were associated with secondary infection and/or prolonged infection. Persistent lymphopenia, defined as an absolute lymphocyte count (ALC) < 1000 cells/µL twice in the first five days, and persistent neutropenia, defined as absolute neutrophil count (ANC) < 1000 cells/µL twice in the first five days, were associated with secondary and prolonged infections. When adjusted in multivariable analysis, persistent lymphopenia remained associated with secondary infection in both immunocompromised (aOR = 14.19, 95% CI [2.69, 262.22] and immunocompetent subjects (aOR = 2.09, 95% CI [1.03, 4.17]). Persistent neutropenia was independently associated with secondary infection in immunocompromised subjects (aOR = 5.34, 95% CI [1.92, 15.84]). Secondary and prolonged infections were associated with worse outcomes, including death. CONCLUSIONS: Laboratory markers of immune suppression can be used to predict secondary infection. Lymphopenia is an independent risk factor in immunocompromised and immunocompetent patients for secondary infection.

2.
Bioorg Med Chem Lett ; 21(2): 849-52, 2011 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-21185721

RESUMO

S6K1 (p70 S6 kinase-1) is thought to play a critical role in the development of obesity and insulin resistance, thus making it an attractive target in developing medicines for the treatment of these disorders. We describe a novel thiophene urea class of S6K inhibitors. The lead matter for the development of these inhibitors came from mining the literature for reports of weak off-target S6K activity. These optimized inhibitors exhibit good potency and excellent selectivity for S6K over a panel of 43 kinases.


Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Tiofenos/química , Tiofenos/farmacologia , Humanos , Microssomos Hepáticos/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Relação Estrutura-Atividade , Tiofenos/metabolismo , Ureia/química , Ureia/metabolismo , Ureia/farmacologia
3.
J Nucleic Acids ; 2018: 8247935, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30009048

RESUMO

Alpha-1-antitrypsin (AAT) deficiency is a genetic disorder that produces inactive/defective AAT due to mutations in the SERPINA1 gene encoding AAT. This disease is associated with decreased activity of AAT in the lungs and deposition of excessive defective AAT protein in the liver. Currently there is no specific treatment for liver disease associated with AAT deficiency. AAT lung disease is often treated with one of several serum protein replacement products; however, long-term studies of the effectiveness of SerpinA1 replacement therapy are not available, and it does not reduce liver damage in AAT deficiency. mRNA therapy could potentially target both the liver and lungs of AAT deficient patients. AAT patient fibroblasts and AAT patient fibroblast-derived hepatocytes were transfected with SERPINA1-encoding mRNA and cell culture media were tested for SerpinA1 expression. Our data demonstrates increased SerpinA1 protein in culture media from treated AAT patient fibroblasts and AAT patient fibroblast-derived hepatocytes. In vivo studies in wild type mice demonstrate SERPINA1 mRNA biodistribution in liver and lungs, as well as SerpinA1 protein expression in these two target organs which are critically affected in AAT deficiency. Taken together, our data suggests that SerpinA1 mRNA therapy has the potential to benefit patients suffering from AAT deficiency.

4.
Sci Rep ; 8(1): 7859, 2018 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-29777164

RESUMO

Thrombotic thrombocytopenic purpura (TTP) is primarily caused by deficiency of ADAMTS13 within the blood stream due to either genetic defects or presence of inhibitory autoantibodies. Preclinical and clinical studies suggest that enzyme replacement therapy with recombinant human ADAMTS13 protein (rhADAMTS13) is effective and safe in treatment of TTP. However, frequent dosing would be required due to the relatively short half-life of rhADAMTS13 in circulation as well as the presence of inhibitory autoantibodies that collectively result in the poor pharmacological profile of rhADAMTS13. With technical breakthroughs in exploring mRNA as therapeutics, we hypothesized that restoration of ADAMTS13 activity for a prolonged duration of time can be achieved through systemic dosing of mRNA, wherein the dosed mRNA would utilize hepatic cells as bioreactors for continuous production of ADAMTS13. To test this hypothesis, mRNA encoding human ADAMTS13 WT or an ADAMTS13 variant, that had demonstrated resistance to predominant clinical TTP autoantibodies, was formulated in lipid nano-particles for liver-targeted delivery. In both ADAMTS13-sufficient and -deficient mice, a single dose of the formulated mRNAs at 1 mg/kg resulted in expression of hADAMTS13 at or above therapeutically relevant levels in mice for up to five days. This proof-of-concept study suggests that mRNA therapy could provide a novel approach for TTP treatment.


Assuntos
Proteína ADAMTS13/genética , Terapia Genética/métodos , RNA Mensageiro/genética , Proteína ADAMTS13/sangue , Proteína ADAMTS13/metabolismo , Animais , Autoanticorpos/sangue , Portadores de Fármacos/química , Células HEK293 , Humanos , Lipídeos/química , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Knockout , Mutagênese , Nanopartículas/química , Púrpura Trombocitopênica Trombótica/terapia , Púrpura Trombocitopênica Trombótica/veterinária , RNA Mensageiro/sangue , RNA Mensageiro/química , RNA Mensageiro/uso terapêutico
5.
Chem Biol Drug Des ; 73(2): 179-88, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19207420

RESUMO

Aberrant activation of the phosphoinositide 3-kinase pathway because of genetic mutations of essential signalling proteins has been associated with human diseases including cancer and diabetes. The pivotal role of 3-phosphoinositide-dependent kinase-1 in the PI3K signalling cascade has made it an attractive target for therapeutic intervention. The N-terminal lobe of the 3-phosphoinositide-dependent kinase-1 catalytic domain contains a docking site which recognizes the non-catalytic C-terminal hydrophobic motifs of certain substrate kinases. The binding of substrate in this so-called PDK1 Interacting Fragment pocket allows interaction with 3-phosphoinositide-dependent kinase-1 and enhanced phosphorylation of downstream kinases. NMR spectroscopy was used to a screen 3-phosphoinositide-dependent kinase-1 domain construct against a library of chemically diverse fragments in order to identify small, ligand-efficient fragments that might interact at either the ATP site or the allosteric PDK1 Interacting Fragment pocket. While majority of the fragment hits were determined to be ATP-site binders, several fragments appeared to interact with the PDK1 Interacting Fragment pocket. Ligand-induced changes in 1H-15N TROSY spectra acquired using uniformly 15N-enriched PDK1 provided evidence to distinguish ATP-site from PDK1 Interacting Fragment-site binding. Caliper assay data and 19F NMR assay data on the PDK1 Interacting Fragment pocket fragments and structurally related compounds identified them as potential allosteric activators of PDK1 function.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Proteínas Serina-Treonina Quinases/química , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Sítio Alostérico , Domínio Catalítico , Simulação por Computador , Humanos , Hidrogênio/química , Ligantes , Nitrogênio/química , Conformação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína
6.
Anal Chim Acta ; 627(1): 105-11, 2008 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-18790133

RESUMO

Several recent reports suggest that stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme in monounsaturated fatty acid synthesis, plays an important role in regulating lipid homeostasis and lipid oxidation in metabolically active tissues. As several manifestations of type 2 diabetes and related metabolic disorders are associated with alterations in intracellular lipid partitioning, pharmacological manipulation of SCD1 activity might be of benefit in the treatment of these disease states. In an effort to identify small molecule inhibitors of SCD1, we have developed a mass spectrometry based high-throughput screening (HTS) assay using deuterium labeled stearoyl-CoA substrate and induced rat liver microsomes. The methodology developed allows the use of a nonradioactive substrate which avoids interference by the endogenous SCD1 substrate and/or product that exist in the non-purified enzyme source. Throughput of the assay was up to twenty 384-well assay plates per day. The assay was linear with protein concentration and time, and was saturable for stearoyl-CoA substrate (K(m)=10.5 microM). The assay was highly reproducible with an average Z' value=0.6. Conjugated linoleic acid and sterculic acid, known inhibitors of SCD1, exhibited IC(50) values of 0.88 and 0.12 microM, respectively. High-throughput mass spectrometry screening of over 1.7 million compounds in compressed format demonstrated that the enzyme target is druggable. A total of 2515 hits were identified (0.1% hit rate), and 346 were confirmed active (>40% inhibition of total SCD activity at 20 microM--14% conformation rate). Of the confirmed hits 172 had IC(50) values of <10 microM, including 111 <1 microM and 48 <100 nM. A large number of potent drug-like (MW<450) hits representing six different chemical series were identified. The application of mass spectrometry to high-throughput screening permitted the development of a high-quality screening protocol for an otherwise intractable target, SCD1. Further medicinal chemistry and characterization of SCD inhibitors should lead to the development of reagents to treat metabolic disorders.


Assuntos
Acil Coenzima A/metabolismo , Deutério/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Microssomos Hepáticos/enzimologia , Estearoil-CoA Dessaturase/antagonistas & inibidores , Animais , Ciclopropanos/farmacologia , Citocromo-B(5) Redutase/metabolismo , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/enzimologia , Ácidos Graxos Monoinsaturados/farmacologia , Humanos , Modelos Lineares , Ácidos Linoleicos Conjugados/farmacologia , Masculino , Espectrometria de Massas , Microssomos Hepáticos/efeitos dos fármacos , Ratos , Bibliotecas de Moléculas Pequenas/farmacologia , Coloração e Rotulagem , Estearoil-CoA Dessaturase/metabolismo , Especificidade por Substrato , Fatores de Tempo
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