Cyclic amide bioisosterism: strategic application to the design and synthesis of HCV NS5B polymerase inhibitors.

Conformational modeling has been successfully applied to the design of cyclic bioisosteres used to replace a conformationally rigid amide bond in a series of thiophene carboxylate inhibitors of HCV NS5B polymerase. Select compounds were equipotent with the original amide series. Single-point mutant binding studies, in combination with inhibition structure-activity relationships, suggest this new series interacts at the Thumb-II domain of NS5B. Inhibitor binding at the Thumb-II site was ultimately confirmed by solving a crystal structure of 8b complexed with NS5B.

Discovery and SAR of potent, orally available 2,8-diaryl-quinoxalines as a new class of JAK2 inhibitors.

We have designed and synthesized a novel series of 2,8-diaryl-quinoxalines as Janus kinase 2 inhibitors. Many of the inhibitors show low nanomolar activity against JAK2 and potently suppress proliferation of SET-2 cells in vitro. In addition, compounds from this series have favorable rat pharmacokinetic properties suitable for in vivo efficacy evaluation.

Modulation of activation-loop phosphorylation by JAK inhibitors is binding mode dependent.

Janus kinase (JAK) inhibitors are being developed for the treatment of rheumatoid arthritis, psoriasis, myeloproliferative neoplasms, and leukemias. Most of these drugs target the ATP-binding pocket and stabilize the active conformation of the JAK kinases. This type I binding mode can lead to an increase in JAK activation loop phosphorylation, despite blockade of kinase function. Here we report that stabilizing the inactive state via type II inhibition acts in the opposite manner, leading to a loss of activation loop phosphorylation. We used X-ray crystallography to corroborate the binding mode and report for the first time the crystal structure of the JAK2 kinase domain in an inactive conformation. Importantly, JAK inhibitor-induced activation loop phosphorylation requires receptor interaction, as well as intact kinase and pseudokinase domains. Hence, depending on the respective conformation stabilized by a JAK inhibitor, hyperphosphorylation of the activation loop may or may not be elicited.

Identification of a potent Janus kinase 3 inhibitor with high selectivity within the Janus kinase family.

We describe a synthetic approach toward the rapid modification of phenyl-indolyl maleimides and the discovery of potent Jak3 inhibitor 1 with high selectivity within the Jak kinase family. We provide a rationale for this unprecedented selectivity based on the X-ray crystal structure of an analogue of 1 bound to the ATP-binding site of Jak3. While equally potent compared to the Pfizer pan Jak inhibitor CP-690,550 (2) in an enzymatic Jak3 assay, compound 1 was found to be 20-fold less potent in cellular assays measuring cytokine-triggered signaling through cytokine receptors containing the common γ chain (γC). Contrary to compound 1, compound 2 inhibited Jak1 in addition to Jak3. Permeability and cellular concentrations of compounds 1 and 2 were similar. As Jak3 always cooperates with Jak1 for signaling, we speculate that specific inhibition of Jak3 is not sufficient to efficiently block γC cytokine signal transduction required for strong immunosuppression.

Potent and selective inhibition of polycythemia by the quinoxaline JAK2 inhibitor NVP-BSK805.

The recent discovery of an acquired activating point mutation in JAK2, substituting valine at amino acid position 617 for phenylalanine, has greatly improved our understanding of the molecular mechanism underlying chronic myeloproliferative neoplasms. Strikingly, the JAK2(V617F) mutation is found in nearly all patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia and primary myelofibrosis. Thus, JAK2 represents a promising target for the treatment of myeloproliferative neoplasms and considerable efforts are ongoing to discover and develop inhibitors of the kinase. Here, we report potent inhibition of JAK2(V617F) and JAK2 wild-type enzymes by a novel substituted quinoxaline, NVP-BSK805, which acts in an ATP-competitive manner. Within the JAK family, NVP-BSK805 displays more than 20-fold selectivity towards JAK2 in vitro, as well as excellent selectivity in broader kinase profiling. The compound blunts constitutive STAT5 phosphorylation in JAK2(V617F)-bearing cells, with concomitant suppression of cell proliferation and induction of apoptosis. In vivo, NVP-BSK805 exhibited good oral bioavailability and a long half-life. The inhibitor was efficacious in suppressing leukemic cell spreading and splenomegaly in a Ba/F3 JAK2(V617F) cell-driven mouse mechanistic model. Furthermore, NVP-BSK805 potently suppressed recombinant human erythropoietin-induced polycythemia and extramedullary erythropoiesis in mice and rats.

Crystal Structures of Human MdmX (HdmX) in Complex with p53 Peptide Analogues Reveal Surprising Conformational Changes.

p53 tumor suppressor activity is negatively regulated through binding to the oncogenic proteins Hdm2 and HdmX. The p53 residues Leu(26), Trp(23), and Phe(19) are crucial to mediate these interactions. Inhibiting p53 binding to both Hdm2 and HdmX should be a promising clinical approach to reactivate p53 in the cancer setting, but previous studies have suggested that the discovery of dual Hdm2/HdmX inhibitors will be difficult. We have determined the crystal structures at 1.3 A of the N-terminal domain of HdmX bound to two p53 peptidomimetics without and with a 6-chlorine substituent on the indole (which binds in the same subpocket as Trp(23) of p53). The latter compound is the most potent peptide-based antagonist of the p53-Hdm2 interaction yet to be described. The x-ray structures revealed surprising conformational changes of the binding cleft of HdmX, including an "open conformation" of Tyr(99) and unexpected "cross-talk" between the Trp and Leu pockets. Notably, the 6-chloro p53 peptidomimetic bound with high affinity to both HdmX and Hdm2 (K(d) values of 36 and 7 nm, respectively). Our results suggest that the development of potent dual inhibitors for HdmX and Hdm2 should be feasible. They also reveal possible conformational states of HdmX, which should lead to a better prediction of its interactions with potential biological partners.

Selection and characterization of replicon variants dually resistant to thumb- and palm-binding nonnucleoside polymerase inhibitors of the hepatitis C virus.

Multiple nonnucleoside inhibitor binding sites have been identified within the hepatitis C virus (HCV) polymerase, including in the palm and thumb domains. After a single treatment with a thumb site inhibitor (thiophene-2-carboxylic acid NNI-1), resistant HCV replicon variants emerged that contained mutations at residues Leu419, Met423, and Ile482 in the polymerase thumb domain. Binding studies using wild-type (WT) and mutant enzymes and structure-based modeling showed that the mechanism of resistance is through the reduced binding of the inhibitor to the mutant enzymes. Combined treatment with a thumb- and a palm-binding polymerase inhibitor had a dramatic impact on the number of replicon colonies able to replicate in the presence of both inhibitors. A more exact characterization through molecular cloning showed that 97.7% of replicons contained amino acid substitutions that conferred resistance to either of the inhibitors. Of those, 65% contained simultaneously multiple amino acid substitutions that conferred resistance to both inhibitors. Double-mutant replicons Met414Leu and Met423Thr were predominantly selected, which showed reduced replication capacity compared to the WT replicon. These findings demonstrate the selection of replicon variants dually resistant to two NS5B polymerase inhibitors binding to different sites of the enzyme. Additionally, these findings provide initial insights into the in vitro mutational threshold of the HCV NS5B polymerase and the potential impact of viral fitness on the selection of multiple-resistant mutants.

Kinetic analysis of a high-affinity antibody/antigen interaction performed by multiple Biacore users.

To explore the reliability of Biacore-based assays, 22 study participants measured the binding of prostate-specific antigen (PSA) to a monoclonal antibody (mAb). Each participant was provided with the same reagents and a detailed experimental protocol. The mAb was immobilized on the sensor chip at three different densities and a two-step assay was used to determine the kinetic and affinity parameters of the PSA/mAb complex. First, PSA was tested over a concentration range of 2.5-600 nM to obtain k(a) information. Second, to define the k(d) of this stable antigen/antibody complex accurately, the highest PSA concentration was retested with the dissociation phase of each binding cycle monitored for 1h. All participants collected data that could be analyzed to obtain kinetic parameters for the interaction. The association and the extended-dissociation data derived from the three antibody surfaces were globally fit using a simple 1:1 interaction model. The average k(a) and k(d) for the PSA/mAb interaction as calculated from the 22 analyses were (4.1+/-0.6) x 10(4) M(-1) s(-1) and (4.5+/-0.6) x 10(-5) s(-1), respectively. Overall, the experimental standard errors in the rate constants were only approximately 14%. Based on the kinetic rate constants, the affinity (K(D)) of the PSA/mAb interaction was 1.1+/-0.2 nM.

Amino acid sequence and tertiary structure of Cratylia mollis seed lectin.

Carbohydrate-protein interactions play a key role in many biological processes. Cramoll is a lectin purified from Cratylia mollis seeds that is taxonomically related to concanavalin A (Con A). Although Cramoll and Con A have the same monosaccharide specificity, they have different glycoprotein binding profiles. We report the primary structure of Cramoll, determined by Edman degradation and mass spectrometry and its 1.77 A crystallographic structure and compare it with the three-dimensional structure of Con A in an attempt to understand how differential binding can be achieved by similar or nearly identical structures. We report here that Cramoll consists of 236 residues, with 82% identity with Con A, and that its topological architecture is essentially identical to Con A, because the Calpha positional differences are below 3.5 A. Cramoll and Con A have identical binding sites for MealphaMan, Mn2+, and Ca2+. However, we observed six substitutions in a groove adjacent to the extended binding site and two in the extended binding site that may explain the differences in binding of oligosaccharides and glycoproteins between Cramoll and Con A.

Composição química e digestibilidade de partes e subprodutos de aves nas formas crua e cozida para cães / Chemical Composition and digestibility of parts and chicken by products in the raw forms and cooked for dogs

Foram realizados dois experimentos para determinar a composição e a digestibilidade de partes e subprodutos de aves em cães. Todos os ingredientes foram avaliados nas formas crua e cozida. No primeiro experimento foram estudados o pescoço, dorso e pé, e no segundo experimento cabeça, resíduo de CMS e fígado. Cada alimento, na forma crua ou cozida, foi fornecido a quatro animais da raça Terrier brasileiro, dois machos e duas fêmeas. Cada ensaio de digestibilidade foi composto por 5 dias de adaptação às condições experimentais e 5 dias de coleta de material. Os animais foram pesados antes e depois do período experimental. Foram alimentados uma vez ao dia, com livre acesso ao alimento por uma hora. Durante o período experimental o alimento foi pesado antes e após o período de consumo e cada animal recebeu aproximadamente 50g alimento/Kg de PV. As fezes foram coletadas diariamente, pesadas e congeladas. Após o período de coleta, todo o material armazenado foi descongelado, homogeneizado e pré-seco em estufa ventilada a 600C, sendo então realizadas as análises laboratoriais. Quanto à composição química dos ingredientes, no primeiro experimento o pé apresentou os maiores valores de PB e MM, enquanto que o pescoço apresentou o maior valor de EB e o dorso, os maiores valores de MS, MO e EE. No segundo experimento, o fígado apresentou os maiores valores de MO, PB e EB, enquanto o resíduo de CMS apresentou os maiores valores de MM e MS, e a cabeça o maior valor para o EE. O cozimento não determinou variações marcantes na composição de nenhum dos alimentos estudados, entretanto, de uma forma geral, os alimentos cozidos quando comparados a forma crua, apresentaram teores menores em PB e maiores em EE. Com relação à digestibilidade os coeficientes de digestibilidade da MO e da PB do pescoço aumentaram significativamente quando este foi cozido, porém os valores de EM e ED reduziram. Quanto ao dorso, o cozimento determinou uma redução significativa nos valores de EM e ED. Os coeficientes de digestibilidade da MS, MO e PB e a ED e EM do pé foram significativamente maiores para o pé cozido em relação ao pé cru. Para o alimento cabeça, apenas o coeficiente de digestibilidade da PB apresentou um aumento significativo em relação ao alimento cru. Os coeficientes de digestibilidade da MS, MO e PB e a ED e EM aumentaram significativamente quando o resíduo de CMS foi cozido. Já o valor do coeficiente de digestibilidade da PB e a ED e EM do fígado, apresentaram uma redução significativa quando este foi cozido. Em relação à ingestão diária de EM no segundo experimento, os grupos de animais que consumiram cabeça crua e cabeça cozida apresentaram valores significativamente maiores do que aqueles que consumiram resíduo de CMS e fígado nas formas crua e cozida. Concluiu-se que é possível a inclusão de todos os ingredientes estudados nas rações de cães, desde que considerados os seus reais valores nutricionais; a inclusão do resíduo de CMS merece atenção especial, uma vez que apresentou baixos valores em EB e EM e os menores valores de coeficientes de digestibilidade da MS e PB, especialmente na forma crua; o cozimento melhorou de forma significativa a digestibilidade dos alimentos pescoço, pé, cabeça e resíduo de CMS; o cozimento do fígado resultou em diminuição na digestibilidade da proteína e dos níveis de ED e EM do mesmo

Two experiments were conducted to determine the composition and the digestibility of chicken by products by dogs. The ingredients were evaluated raw and cooked. The ingredients studied were chicken head, neck, back, feet, liver and residue of mechanically separated chicken meat (MSM). In the first experiment neck, back and feet were evaluated and in the second one head, MSM and liver. Each ingredient was offered raw or cooked to measure the digestibility coefficient in four brazilian terrier dogs, two of each gender. Each digestibility assay had a five day adaptation period and five days to collect the material. The animals were weighted before and after the experimental period, and the food was served once a day. During the experiments, the food was weighted before and after being offered. Each animal ate approximately 50g food/Kg body weight. The feces were collected daily, and freezed at - 40C. After the feces collection fase all the freezed material was defrosted, homogenized and pre-dryed in a ventilated stove at 600C, so the bromatological analyses of food and feces were done. In the first experiment the feet had the highest values for CP and MC, and the back, the highest values for DM, OM and fat. Among the tested ingredients in the second experiment, the liver had the highest values for OM, CP, and gross energy (GE), and the MSM had the highest values for MM and DM. The head had the highest values for fat content. Cooking had no significant effect on the ingredients tested, but, the general observation was that the cooked ingredients had lower values for CP and higher values for fat content. Considering the digestibility coefficient the OM and CP of neck had a significant increase when it was used in the cooked form, but, the ME and the digestible energy (DE) values decreased considerably. Considering the back, the heat treatment resulted in a significant reduction of ME and DE and in the feet, the digestibility coefficients of DM, OM, CP, ME and DE were higher using the same treatment. Considering the head, only the CP digestibility coefficient had a significant improvement relative to the raw component. The digestibility coefficients of DM, OM, CP, DE,and ME had a significant increase when the MSM was cooked. Yet, when the liver was cooked, the CP, DE and ME digestility coefficients had a significant reduction. In relation to the ME ingestion on the second experiment, the groups of animals that ingested raw and cooked head had a significant higher values when compared to the dogs fed with liver and MSM in the raw and cooked form. It was concluded that it is possible to use all the ingredients tested in dog foods formulas, but it must be considered their real nutritional values. The inclusion of MSM deserves special attention, since it presented the lowest values for GE and ME as well as the lowest digestibility coefficients for DM and CP specially when raw. The heat treatment influenced positively the digestibility of neck, feet, head and MSM, while cooking the liver had a negative effect on the digestibility of protein and DE and ME.

Epidemiologia dos portadores de lúpus eritematoso sistêmico atendidos no hospital Ofir Loyola no período de 1985 a 2000 / Epidemiology of the bearers of sistemic eritematose Lupus researched at Ofir Loyola Hospital from 1985 a 200

Introdução: O Lúpus Eritematoso Sistêmico (LES) é uma doença de origem auto-imune, de etiologia desconhecida, podendo afetar um ou mais órgãos. Apresenta incidência relativamente comum. Objetivo: O trabalho aborda os aspectos epidemiologicos de pacientes lúpicos,demonstrando a variedade das manifestações clínicas e o comportamento dessa patologia em nossa região. Métodos: Nos 104 pacientes internados no Hospital Ofir Loyola (HOL) no período de 1986 a 2000, foram analisados os seguintes dados; idade, sexo, raça, critérios diagnósticados do LES, manifestações clínicas, motivo das internações, tempo de doença e causas de óbito. Resultado: Foi observado a maior incidência em mulheres na idade fértil e nos miscigenados, a frequência do comprometimento osteoarticular, a importância do envolvimento renal e a elevada mortalidade na segunda década de vida. Conclusão: A ocorrência dos variados aspectos pesquisados, é semelhante as encontradas na literatura

Amino acid sequence and three-dimensional structure of the Tn-specific isolectin B4 from Vicia villosa

The partial amino acid sequence of the tetrametric isolectin B4 from Vivia villosa seeds has been determined by peptide analysis, and its three-dimensional structure solved by molecular replacement techniques and refined at 2.9 A resolution to a crystallographic R-factor of 21 per cent. Each sub unit displays the thirteen-stranded B-barrel topology characteristic of legume lectins. The amino acid residues involved in metal-and sugar-binding are similar to those of other GalNAc-specific lectins, indicating thar residues outside the carbohydrate-binding pocket modulate the affinity for the Tn glycopeptide. Isolectin B4 displays an unusual quaternary, probably due to protein glycosylation