Open tubular columns containing the immobilized ligand binding domain of peroxisome proliferator-activated receptors α and γ for dual agonists characterization by frontal affinity chromatography with mass spectrometry detection.

The peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily. In the last years novel PPARs ligands have been identified and these include PPARα/γ dual agonists. To rapidly identify novel PPARs dual ligands, a robust binding assay amenable to high-throughput screening toward PPAR isoforms would be desirable. In this work we describe a parallel assay based on the principles of frontal affinity chromatography coupled to mass spectrometry (FAC-MS) that can be used to characterize dual agonists. For this purpose the ligand binding domain of PPARα receptor was immobilized onto the surface of open tubular capillaries to create new PPAR-alpha-OT columns to be used in parallel with PPAR-gamma-OT columns. The two biochromatographic systems were used in both ranking and Kd experiments toward new ureidofibrate-like dual agonists for subtype selectivity ratio determination. In order to validate the system, the Kd values determined by frontal analysis chromatography were compared to the affinity constants obtained by ITC experiments. The results of this study strongly demonstrate the specific nature of the interaction of the ligands with the two immobilized receptor subtypes.

Pathology of nasal infection caused by Conidiobolus lamprauges and Pythium insidiosum in sheep.

Conidiobolomycosis and pythiosis are important diseases of sheep in midwestern Brazil. Veterinary practitioners consider it difficult to differentiate between these diseases because they have similar clinical features. In this study, 186 sheep were subjected to necropsy examination over a 6-year period. Thirty (16.1%) cases of rhinitis in sheep that were caused by Conidiobolus lamprauges (n = 15) or Pythium insidiosum (n = 15) were investigated further. The lesions of C. lamprauges infection were mainly rhinopharyngeal (86.7%), localized to the ethmoidal region and associated with exophthalmos. The lesions appear as a white to yellow, firm mass that microscopically appears as a granulomatous inflammatory reaction with numerous giant cells. In contrast, P. insidiosum infection is associated with rhinofacial (93.3%) lesions that mainly involve the frontal region and hard palate and appear as an irregular, friable, yellow to red mass. Microscopically, pythiosis presents as diffuse necrotizing eosinophilic rhinitis. Immunohistochemistry using polyclonal antisera raised specifically against the two organisms was used to confirm the identity of the infectious agent in each disease. This study reports the first immunohistochemical diagnosis of conidiobolomycosis and the first description of a rhinopharyngeal lesion caused by P. insidiosum in sheep.

Intoxicação aguda com sementes de Crotalaria spectabilis (Leg. Papilionoideae) em suínos / Acute poisoning with Crotalaria spectabilis (Leg. Papilionoideae) seeds in pigs

Relata-se necrose hepatocelular em suínos após consumo de ração que continha grãos de sorgo-granífero (Sorghum bicolor) acidentalmente contaminado com sementes de Crotalaria spectabilis. Morreram 76 suínos em quatro propriedades no município de Juscimeira, MT. Os sinais clínicos iniciaram-se 24-48 horas após o consumo da ração contaminada e foram caracterizados por depressão, letargia, apatia, inapetência, vômito, mucosas ictéricas ou pálidas, ascite, decúbito esternal, decúbito lateral com movimentos de pedalagem e convulsões, a evolução clínica foi de 48-60 horas seguida de morte. As Principais alterações macroscópicas foram fígado aumentado de tamanho com evidenciação do padrão lobular, ascite e hidrotórax com líquido de coloração amarelo avermelhado contendo filamentos com aspecto de fibrina, linfonodos aumentados e edema pulmonar interlobular. A doença foi reproduzida utilizando-se 16 suínos divididos em seis grupos que receberam sementes de C. spectabilis em diferentes doses. Necrose hepatocelular ocorreu em sete suínos, sendo dois que receberam doses diárias 2,5g/kg e cinco que receberam doses únicas de 5,0 e 9,5g/kg. Dez doses diárias de 0,5 e 1,25g/kg causaram fibrose hepática.

Hepatocellular necrosis are reported in swine after consumption of diets containing grains of "sorgo-granífero" (Sorghum bicolor) accidentally contaminated with Crotalaria spectabilis seeds in the municipality of Juscimeira, MT. Clinical signs began 24-48 hours after consumption of contaminated ration and were characterized by depression, lethargy, apathy, loss of appetite, vomiting, pale or jaundiced mucous membranes, ascites, lateral recumbency, the lateral position with paddling and convulsions, clinical outcome was 48-60 hours followed by death. 76 pigs died in four properties. The main gross lesions were liver increased in size and lobular illustration with red-brown central areas interspersed with yellowish areas, ascites and hydrothorax with reddish-yellow liquid containing filaments with aspect of fibrin, enlarged lymph nodes and interlobular pulmonary edema. The disease was experimentally reproduced with 16 pigs divided into six groups that received seeds of C. spectabilis in different doses. Hepatocellular necrosis occurred in seven pigs, two of which received daily doses of 2.5g/kg and five who received single doses of 5 and 9.5g/ kg. Ten daily doses of 0.5 and 1.25g/kg caused liver fibrosis.

Modulation of penicillin acylase properties via immobilization techniques: one-pot chemoenzymatic synthesis of Cephamandole from Cephalosporin C.

The modulation of penicillin G acylase (PGA) properties via immobilization techniques has been performed studying the acylation of 7-aminocephalosporanic acid with R-mandelic acid methyl ester. PGA from Escherichia coli, immobilized onto agarose activated with glycidol (glyoxyl-agarose), has been used for the design of a novel one-pot synthesis of Cephamandole in aqueous medium and without isolation of intermediates, through three consecutive biotransformations catalyzed by D-amino acid oxidase, glutaryl acylase and PGA.

Potentiation of inhibition of wild-type and mutant human immunodeficiency virus type 1 reverse transcriptases by combinations of nonnucleoside inhibitors and d- and L-(beta)-dideoxynucleoside triphosphate analogs.

Combinations of reverse transcriptase (RT) inhibitors are currently used in anti-human immunodeficiency virus therapy in order to prevent or delay the emergence of resistant virus and to improve the efficacy against viral enzymes carrying resistance mutations. Drug-drug interactions can result in either positive (additive or synergistic inhibition) or adverse (antagonistic interaction, synergistic toxicity) effects. Elucidation of the nature of drug interaction would help to rationalize the choice of antiretroviral agents to be used in combination. In this study, different combinations of nucleoside and nonnucleoside inhibitors, including D- and L-(beta)-deoxy- and -dideoxynucleoside triphosphate analogues, have been tested in in vitro RT assays against either recombinant wild-type RT or RT bearing clinically relevant nonnucleoside inhibitor resistance mutations (L100I, K103N, Y181I), and the nature of the interaction (either synergistic or antagonistic) of these associations was evaluated. The results showed that (i) synergy of a combination was not always equally influenced by the individual agents utilized, (ii) a synergistic combination could improve the sensitivity profile of a drug-resistant mutant enzyme to the single agents utilized, (iii) L-(beta)-enantiomers of nucleoside RT inhibitors were synergistic when combined with nonnucleoside RT inhibitors, and (iv) inter- and intracombination comparisons of the relative potencies of each drug could be used to highlight the different contributions of each drug to the observed synergy.

Novel nonsubstrate inhibitors of human thymidine phosphorylase, a potential target for tumor-dependent angiogenesis.

Thymidine phosphorylase/platelet-derived endothelial cell growth factor (TP/PD-ECGF) is an enzyme involved in thymidine metabolism and homeostasis, and its catalytic activity appears to play an important role in angiogenesis. Here we describe the cloning and expression of a His-tagged human TP/PD-ECGF and its assay with uracil and thymine analogues. We present the design, synthesis, and biological evaluation of novel 6-(phenylalkylamino)uracil derivatives which, at micromolar concentrations, inhibit both catabolic and anabolic reactions of human TP in vitro. These base analogues are not converted by the enzyme into the nucleoside form, thus representing pure nonsubstrate inhibitors of the enzyme.

Selective interaction of the human immunodeficiency virus type 1 reverse transcriptase nonnucleoside inhibitor efavirenz and its thio-substituted analog with different enzyme-substrate complexes.

Accumulating data have brought the nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) into the forefront of antiretroviral therapy. Among the emerging compounds in this class, a particularly attractive one is efavirenz (Sustiva), recently approved for clinical use by the U.S. Food and Drug Administration. In the present study, the equilibrium dissociation constants for efavirenz binding to the different catalytic forms of human immunodeficiency virus type 1 RT as well as the association and dissociation rates have been determined using a steady-state kinetic approach. In addition, the same enzymological analysis has been extended to the thio-substituted analog, sefavirenz, which showed comparable activity in vitro against RT. Both compounds have been found to act as purely uncompetitive inhibitors at low drug concentrations (5 to 50 nM) and as mixed noncompetitive inhibitors at higher doses (50 to 500 nM). This behavior can be interpreted in terms of the relative affinities for the different catalytic forms of the enzyme. Both efavirenz and sefavirenz showed increasing affinities for the different forms of RT in the following order: free enzyme < (i.e., bound with lower affinity) binary RT-template-primer (TP) complex < ternary RT-TP-deoxynucleoside triphosphate (dNTP) complex. The rate of binding of the two inhibitors to the different enzyme-substrate complexes was well below the diffusion limit (on the order of 10(4) M(-1) s(-1)); however, both inhibitors, when bound to the ternary RT-TP-dNTP complex, showed very low dissociation rates, on the order of 10(-4) s(-1) for both compounds, typical of tightly binding inhibitors. Thus, efavirenz and its thio-substituted derivative sefavirenz appear to be peculiar in their mechanism of action, being selective tightly binding inhibitors of the ternary RT-TP-dNTP complex. Efavirenz is the first clinically approved NNRTI to show this property.

Synthesis and in vitro anti-mycobacterium activity of N-alkyl-1, 2-dihydro-2-thioxo-3-pyridinecarbothioamides. Preliminary toxicity and pharmacokinetic evaluation.

Disseminated infections with Mycobacterium tuberculosis (MT) and Mycobacterium avium complex (MAC) are increasingly opportunistic diseases in patients with advanced acquired human immunodeficiency syndrome (AIDS). A series of N-alkyl-1, 2-dihydro-2-thioxo-3-pyridinecarbothioamides has been synthesized, and MICs for MT and MAC strains, either standard or isolated from infected patients, have been determined. Preliminary tests show a good activity and a very low toxicity for some derivatives. Pharmacokinetic studies in the rat show a very rapid elimination from the body after intravenous administration and a poor absorption after oral administration.

3H-[1,2]Dithiolo[3,4-b]pyridine-3-thione and its derivatives synthesis and antimicrobial activity.

A series of 2-substituted isothiazolo[5,4-b]pyridine-3(2H)-thiones, isothiazolo[5,4-b]pyridin-3(2H)-ones, N-substituted 2-sulfanylnicotinamides and the corresponding carbothioamide derivatives were synthesized and evaluated for their antimicrobial activity against several strains of Gram+ and Gram- bacteria and fungi. Chemical syntheses were resumed into a comprehensive cyclic route that enables the reversible conversion for each derivative of the series considered. Among the tested compounds the N-(aralkyl)-2-sulfanylnicotinamides show the highest fungitoxicity (MIC = 1.25-5 microg/ml). The best activity towards Gram-positive bacteria was in the range of 2.5-5 microg/ml. Activity against Gram-negative bacteria was generally very poor for all compounds.

Synthesis and molecular modeling of novel HSV1 uracil-DNA glycosylase inhibitors.

In a recent paper the first selective inhibitors of HSV1 uracil-DNA glycosylase (UDG) acting in the micromolar range have been reported. A 28.5 kDa catalytic fragment of HSV1 UDG has been crystallized in the presence of uracil, and the structure was recently solved. Starting with the optimized model of binding between 6-(4'-n-octylanilino)uracil (octAU) and UDG some new derivatives have been predicted to be active. In vitro studies with the novel synthetized compounds confirm the plausibility of the model and define the structure features for UDG inhibitors.

Molecular modeling and synthesis of inhibitors of herpes simplex virus type 1 uracil-DNA glycosylase.

We recently reported the properties of the first selective inhibitors of herpes simplex virus type 1 (HSV1) uracil-DNA glycosylase (UDG), an enzyme of DNA repair that has been proposed to be required for reactivation of the virus from latency. 6-(4-Octylanilino)uracil (octAU) was the most potent inhibitor among a series of 6-(4-alkylanilino)uracils, acting in the micromolar range and without effect against human UDG. A 28.5-kDa catalytic fragment of HSV1 UDG has been crystallized in the presence of uracil, and the structure was recently solved. We have used the coordinates of this structure in order to study interaction of our inhibitors with the enzyme, and a model of binding between octAU and UDG has been derived. Starting with the optimized model, the activity of several octAU analogues was predicted, and the values compared favorably with experimental results found for the synthetic compounds. Several hydrophilic derivatives were predicted and found to be active as UDG inhibitors. These compounds will be useful to determine if UDG, like the viral thymidine kinase, is required for reactivation of HSV1 from latency in nerve cells.