RESUMO
Although the initial outbreaks of the deadly coronavirus that causes severe acute respiratory syndrome (SARS-CoV) were controlled by public health measures, the development of vaccines and antiviral agents for SARS-CoV is essential for improving control and treatment of future outbreaks. One potential target for SARS-CoV antiviral drug development is the 3C-like protease (3CLpro). This enzyme is an attractive target since it is essential for viral replication, and since there are now a number of high resolution X-ray structures of SARS-CoV 3CLpro available making structure-based drug-design possible. As a result, SARS-CoV 3CLpro has become the focus of numerous drug discovery efforts worldwide, but as a consequence, a variety of different 3CLpro expression constructs and kinetic assays have been independently developed making evaluation and comparison between potential inhibitors problematic. Here, we review the literature focusing on different SARS-CoV 3CLpro expression constructs and assays used to measure enzymatic activity. Moreover, we provide experimental evidence showing that the activity of 3CLpro enzymatic is significantly reduced when non-native sequences or affinity-tags are added to the N- or C-termini of the enzyme, or when the enzyme used in assays is at concentrations below the equilibrium dissociation constant of the 3CLpro dimer. We demonstrate for the first time the utility of a highly sensitive and novel Alexa488-QSY7 FRET-based peptide substrate designed for routine analysis and high-throughput screening, and show that kinetic constants determined from FRET-based assays that are uncorrected for inner-filter effects can lead to artifacts. Finally, we evaluated the effects of common assay components including DTT, NaCl, EDTA and DMSO on enzymatic activity, and we recommend standardized assay conditions and constructs for routine SARS-CoV 3CLpro assays to facilitate direct comparisons between SARS-CoV 3CLpro inhibitors under development worldwide.
Assuntos
Cisteína Endopeptidases/análise , Cisteína Endopeptidases/metabolismo , Avaliação Pré-Clínica de Medicamentos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Sequência de Aminoácidos , Proteases 3C de Coronavírus , Cristalização , Cisteína Endopeptidases/genética , Inibidores Enzimáticos/farmacologia , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/metabolismo , Histidina/genética , Histidina/metabolismo , Humanos , Cinética , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Especificidade por SubstratoRESUMO
Ibrutinib is a novel oral therapy that has shown significant efficacy as initial treatment of chronic lymphocytic leukemia (CLL). It is a high-cost continuous therapy differing from other regimens that are given for much shorter courses. Our objective was to evaluate the cost-effectiveness of ibrutinib for first-line treatment of CLL in patients older than age 65 years without a 17p deletion. We developed a semi-Markov model to analyze the cost-effectiveness of ibrutinib vs a comparator therapy from a US Medicare perspective. No direct comparison between ibrutinib and the best available treatment alternative, obinutuzumab plus chlorambucil (chemoimmunotherapy), exists. Therefore, we compared ibrutinib to a theoretical treatment alternative, which was modeled to confer the effectiveness of an inferior treatment (chlorambucil alone) and the costs and adverse events of chemoimmunotherapy, which would provide ibrutinib with the best chance of being cost-effective. Even so, the incremental cost-effectiveness ratio of ibrutinib vs the modeled comparator was $189 000 per quality-adjusted life-year (QALY) gained. To reach a willingness-to-pay threshold (WTP) of $150 000 per QALY, the monthly cost of ibrutinib would have to be at most $6800, $1700 less than the modeled cost of $8500 per month (a reduction of $20 400 per year). When the comparator efficacy is increased to more closely match that seen in trials evaluating chemoimmunotherapy, ibrutinib costs more than $262 000 per QALY gained, and the monthly cost of ibrutinib would need to be lowered to less than $5000 per month to be cost-effective. Ibrutinib is not cost-effective as initial therapy at a WTP threshold of $150 000 per QALY gained.
Assuntos
Leucemia Linfocítica Crônica de Células B , Modelos Econômicos , Pirazóis , Pirimidinas , Adenina/análogos & derivados , Idoso , Deleção Cromossômica , Cromossomos Humanos Par 17 , Custos e Análise de Custo , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/economia , Masculino , Piperidinas , Pirazóis/administração & dosagem , Pirazóis/economia , Pirimidinas/administração & dosagem , Pirimidinas/economia , Síndrome de Smith-MagenisRESUMO
Acquired lymphedema is a cancer sequela and a global health problem currently lacking pharmacologic therapy. We have previously demonstrated that ketoprofen, an anti-inflammatory agent with dual 5-lipoxygenase and cyclooxygenase inhibitory properties, effectively reverses histopathology in experimental lymphedema. We show that the therapeutic benefit of ketoprofen is specifically attributable to its inhibition of the 5-lipoxygenase metabolite leukotriene B4 (LTB4). LTB4 antagonism reversed edema, improved lymphatic function, and restored lymphatic architecture in the murine tail model of lymphedema. In vitro, LTB4 was functionally bimodal: Lower LTB4 concentrations promoted human lymphatic endothelial cell sprouting and growth, but higher concentrations inhibited lymphangiogenesis and induced apoptosis. During lymphedema progression, lymphatic fluid LTB4 concentrations rose from initial prolymphangiogenic concentrations into an antilymphangiogenic range. LTB4 biosynthesis was similarly elevated in lymphedema patients. Low concentrations of LTB4 stimulated, whereas high concentrations of LTB4 inhibited, vascular endothelial growth factor receptor 3 and Notch pathways in cultured human lymphatic endothelial cells. Lymphatic-specific Notch1-/- mice were refractory to the beneficial effects of LTB4 antagonism, suggesting that LTB4 suppression of Notch signaling is an important mechanism in disease maintenance. In summary, we found that LTB4 was harmful to lymphatic repair at the concentrations observed in established disease. Our findings suggest that LTB4 is a promising drug target for the treatment of acquired lymphedema.
Assuntos
Leucotrieno B4/antagonistas & inibidores , Linfedema/tratamento farmacológico , Animais , Anti-Inflamatórios/uso terapêutico , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Cetoprofeno/uso terapêutico , Leucotrieno B4/metabolismo , Linfedema/metabolismo , Camundongos , Transdução de Sinais/efeitos dos fármacosRESUMO
Cro proteins from different lambdoid bacteriophages are extremely variable in their target consensus DNA sequences and constitute an excellent model for evolution of transcription factor specificity. We experimentally tested a bioinformatically derived evolutionary code relating switches between pairs of amino acids at three recognition helix sites in Cro proteins to switches between pairs of nucleotide bases in the cognate consensus DNA half-sites. We generated all eight possible code variants of bacteriophage λ Cro and used electrophoretic mobility shift assays to compare binding of each variant to its own putative cognate site and to the wild-type cognate site; we also tested the wild-type protein against all eight DNA sites. Each code variant showed stronger binding to its putative cognate site than to the wild-type site, except some variants containing proline at position 27; each also bound its cognate site better than wild-type Cro bound the same site. Most code variants, however, displayed poorer affinity and specificity than wild-type λ Cro. Fluorescence anisotropy assays on λ Cro and the triple code variant (PSQ) against the two cognate sites confirmed the switch in specificity and showed larger apparent effects on binding affinity and specificity. Bacterial one-hybrid assays of λ Cro and PSQ against libraries of sequences with a single randomized half-site showed the expected switches in specificity at two of three coded positions and no clear switches in specificity at noncoded positions. With a few caveats, these results confirm that the proposed Cro evolutionary code can be used to reengineer Cro specificity.
Assuntos
Bacteriófago lambda/genética , Proteínas de Ligação a DNA/genética , DNA/genética , Evolução Molecular , Código Genético , Proteínas Repressoras/genética , Proteínas Virais Reguladoras e Acessórias/genética , Dicroísmo Circular , Ensaio de Desvio de Mobilidade Eletroforética , Polarização de Fluorescência , Engenharia Genética , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Especificidade por Substrato , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
Peloruside A is a microtubule-stabilizing macrolide that binds to beta tubulin at a site distinct from the taxol site. The site was previously identified by H-D exchange mapping and molecular docking as a region close to the outer surface of the microtubule and confined in a cavity surrounded by a continuous loop of protein folded so as to center on Y340. We have isolated a series of peloruside A-resistant lines of the human ovarian carcinoma cell line A2780(1A9) to better characterize this binding site and the consequences of altering residues in it. Four resistant lines (Pel A-D) are described with single-base mutations in class I ß-tubulin that result in the following substitutions: R306H, Y340S, N337D, and A296S in various combinations. The mutations are localized to peptides previously identified by Hydrogen-Deuterium exchange mapping, and center on a cleft in which the drug side chain appears to dock. The Pel lines are 10-15-fold resistant to peloruside A and show cross resistance to laulimalide but not to any other microtubule stabilizers. They show no cross-sensitivity to any microtubule destabilizers, nor to two drugs with targets unrelated to microtubules. Peloruside A induces G2/M arrest in the Pel cell lines at concentrations 10-15 times that required in the parental line. The cells show notable changes in morphology compared to the parental line.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Lactonas/metabolismo , Mutação , Tubulina (Proteína)/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Medição da Troca de Deutério , Pontos de Checagem da Fase G2 do Ciclo Celular , Humanos , Microtúbulos/metabolismo , Estrutura Terciária de Proteína , Tubulina (Proteína)/genéticaRESUMO
Ligand binding can induce shifts in protein conformation. In the case of tubulin, these drug-induced confirmational changes can prevent or stabilize microtubule polymerization. 5',5'-Dithiobis(2-nitrobenzoate) (DTNB) reacts with free and accessible sulfhydryls and stoichiometrically produces a detectable product, which allows an exact measurement of reacted thiols. Since binding of small ligands may alter conformational dynamics, it may also affect the reactivity of thiols on tubulin. Differences in DTNB reactivity with thiols upon ligand binding can therefore be used to deduce binding characteristics. We will describe two methods that use tubulin cysteine reactivity with DTNB in the presence of drug to define ligand-binding characteristics.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Reagentes de Sulfidrila/farmacologia , Moduladores de Tubulina/análise , Moduladores de Tubulina/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Avaliação Pré-Clínica de Medicamentos/instrumentação , Humanos , Ligantes , Ligação Proteica , Compostos de Sulfidrila/metabolismo , Reagentes de Sulfidrila/química , Tubulina (Proteína)/análise , Tubulina (Proteína)/química , Moduladores de Tubulina/químicaRESUMO
A stereoselective total synthesis of the cytotoxic natural products tubulysin U, tubulysin V, and its unnatural epimer epi-tubulysin V, is reported. Simplified analogues containing N,N-dimethyl-D-alanine as a replacement for the N-terminal N-Me-pipecolinic acid residue of the tubulysins are also disclosed. Biological evaluation of these natural products and analogues provided key information with regard to structural and stereochemical requirements for antiproliferative activity and tubulin polymerization inhibition.