Using a Fragment-Based Approach to Identify Alternative Chemical Scaffolds Targeting Dihydrofolate Reductase from Mycobacterium tuberculosis.

Dihydrofolate reductase (DHFR), a key enzyme involved in folate metabolism, is a widely explored target in the treatment of cancer, immune diseases, bacteria, and protozoa infections. Although several antifolates have proved successful in the treatment of infectious diseases, they have been underexplored to combat tuberculosis, despite the essentiality of M. tuberculosis DHFR (MtDHFR). Herein, we describe an integrated fragment-based drug discovery approach to target MtDHFR that has identified hits with scaffolds not yet explored in any previous drug design campaign for this enzyme. The application of a SAR by catalog strategy of an in house library for one of the identified fragments has led to a series of molecules that bind to MtDHFR with low micromolar affinities. Crystal structures of MtDHFR in complex with compounds of this series demonstrated a novel binding mode that considerably differs from other DHFR antifolates, thus opening perspectives for the development of relevant MtDHFR inhibitors.

Folate biosynthesis pathway: mechanisms and insights into drug design for infectious diseases.

Folate pathway is a key target for the development of new drugs against infectious diseases since the discovery of sulfa drugs and trimethoprim. The knowledge about this pathway has increased in the last years and the catalytic mechanism and structures of all enzymes of the pathway are fairly understood. In addition, differences among enzymes from prokaryotes and eukaryotes could be used for the design of specific inhibitors. In this review, we show a panorama of progress that has been achieved within the folate pathway obtained in the last years. We explored the structure and mechanism of enzymes, several genetic features, strategies, and approaches used in the design of new inhibitors that have been used as targets in pathogen chemotherapy.

Cloning, expression, purification and biophysical analysis of two putative halogenases from the glycopeptide A47,934 gene cluster of Streptomyces toyocaensis.

Glycopeptides are an important class of antibiotics used in the treatment of several infections, including those caused by methicillin resistant Staphylococcus aureus. Glycopeptides are biosynthesized by a Non Ribosomal Peptide Synthase (NRPS) and the resulting peptide precursors are decorated by several tailoring enzymes, such as halogenases and glycosyltransferases. These enzymes are important targets of protein engineering to produce new derivatives of known antibiotics. Herein we show the production of two putative halogenases, denominated StaI and StaK, involved in the biosynthesis of the glycopeptide A47,934 in Streptomyces toyocaensis NRRL 15,009. This antibiotic together with the compound UK-68,597 are the unique glycopeptides which have two putative halogenases identified in their gene clusters and three chloride substituent atoms attached to their aglycones. StaI and StaK were successfully produced in E. coli in the soluble fraction with high purity using the wild type gene for StaI and a synthetic codon optimized gene for StaK. We have purified both enzymes by two chromatographic steps and a good yield was obtained. These putative halogenases were co-purified with the co-factor FAD, which are differently reduced by the enzyme SsuE in vitro. We have further confirmed that these putative halogenases are monomeric using a calibrated gel filtration column and through circular dichroism, we confirmed that both enzymes are folded with a predominance of α-helices. Molecular models for StaI and StaK were generated and together with sequence and phylogenetic analysis, we could infer some structural insights of StaI and StaK from the biosynthesis of compound A47,934.

High anion gap metabolic acidosis secondary to pyroglutamic aciduria (5-oxoprolinuria): association with prescription drugs and malnutrition.

Two cases of High Anion Gap Metabolic Acidosis (HAGMA) due to pyroglutamic acid (5-oxoproline) are described. In both cases the HAGMA developed during an episode of hospital treatment, in conjunction with paracetamol and antibiotic prescription, and the surviving patient made an uneventful recovery after the drugs were withdrawn. Clinicians need to be aware of this cause for metabolic acidosis because it may be a more common metabolic disturbance in compromised patients than would be expected, and the discontinuation of drugs implicated in the aetiology is therapeutic.

Tissue oxygenation and perfusion in patients with systemic sepsis.

OBJECTIVE: Multiple organ dysfunction is associated with systemic sepsis. To investigate whether this is attributable to peripheral tissue hypoperfusion and/or cellular hypoxia, simultaneous measurements of tissue perfusion and oxygenation were made in patients with severe sepsis and in controls. DESIGN: Prospective, observational study. SETTING: Adult intensive care unit, tertiary referral center. PATIENTS: Volunteers (group C, n = 7), patients undergoing cardiopulmonary bypass (group B, n = 6), and patients with severe sepsis (group S, n = 6). INTERVENTIONS: Limb ischemia and reperfusion. MEASUREMENTS AND MAIN RESULTS: Tissue oxygenation and microvascular flow were measured by using microelectrodes inserted into brachoradialis muscle and overlying subcutaneous tissue. Forearm cutaneous red cell flux and regional blood flow were measured simultaneously. Responses to 20 mins of limb ischemia and subsequent reperfusion were observed. Baseline muscle tissue oxygenation was greater in sepsis (1.7 +/- 0.2, 1.5 +/- 0.7, and 4.4 +/- 0.6 kPa for groups C, B, and S, respectively, mean +/- sem, p <.05), although baseline subcutaneous tissue oxygenation did not vary between groups. During ischemia tissue oxygenation, values decreased in muscle (to 1.3 +/- 0.2, 1.0 +/- 0.4, and 1.5 +/- 0.4 kPa for groups C, B, and S, respectively) and subcutaneous tissue (to 2.0 +/- 0.3, 1.7 +/- 0.5, and 2.3 +/- 0.2 kPa for groups C, B, and S, respectively). Decline in tissue oxygen tension was initially more rapid in septic muscle compared with controls (25% decrease, 68 +/- 23 vs. 176 +/- 38 for group S vs. group C, p <.05, and 50% decrease, 126 +/- 34 vs. 398 +/- 72 secs for group S vs. group C, p <.01). However, overall rate of tissue decline was similar (95% decrease, 444 +/- 122 vs. 614 +/- 96 for group S vs. group C, p >.05). After reperfusion, significant differences in muscle tissue oxygenation reappeared between groups (2.0 +/- 0.3, 1.5 +/- 0.7, and 4.0 +/- 0.4 kPa for groups C, B, and S, respectively, p <.05). There were no differences in time to 25%, 50%, or 95% tissue oxygen recovery. Whole limb reperfusion was significantly less in patient groups compared with controls (10.6 +/- 0.9, 4.5 +/- 1.2, and 4.3 +/- 1.6 mL x 100 mL(-1) x min(-1) for groups C, B, and S, respectively, p <.05). CONCLUSIONS: Significant differences in tissue oxygenation distribution between muscle and subcutaneous tissues occur in patients with severe sepsis. High baseline muscle tissue oxygen levels are accompanied by rapid extraction of oxygen during stagnant ischemia.

Abnormal tissue oxygenation and cardiovascular changes in endotoxemia.

Experimental sepsis induces disturbances in microcirculatory flow and nutrient exchange that may result in impaired tissue oxygenation. Volume resuscitation is a principal clinical intervention in patients with sepsis. Nitric oxide (NO) has been implicated in the pathophysiology of endotoxemia, but few data exist concerning the effects of either NO synthase inhibition (NOSi) or volume resuscitation on microvascular regulation and tissue oxygenation. Amperometric measurements were made of skeletal muscle (tissue) oxygen tension (PtO2) and its response to changes in fraction of inspired oxygen (FIO2) in rats rendered endotoxemic. Simultaneous measurements were made of systemic hemodynamic indices and arterial blood gas tensions. At normal PaO2, PtO2 in endotoxemic animals was significantly lower than in control animals, with marked attenuation of the response to increasing FIO2. These changes were associated with significant metabolic acidemia. In volume-resuscitated endotoxemic rats, PtO2 and blood pH were unchanged. A significant reduction in the PtO2 response to hyperoxia was observed in animals treated with the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME), an effect not reversed by fluid resuscitation. These data suggest that significant tissue hypoxia and abnormal microvascular control occur in endotoxemia. Volume resuscitation can reverse the changes in PtO2, whereas nitric oxide synthase (NOS) inhibition has deleterious effects on muscle PtO2 in both control and endotoxemic animals.

Tissue oxygenation and perfusion in endotoxemia.

Sepsis is believed to induce disturbances in microcirculatory flow and nutrient exchange, which may result in impaired tissue oxygenation. With the use of an established rat model of endotoxemia, voltametric measurements were made of skeletal muscle (tissue) oxygen tension (PtO2) and its response to inspired oxygen concentration (FIO2). Steady-state nutritive flow and the response of endotoxemic muscle to ischemia-reperfusion were also measured. In the presence of a normal arterial PO2, mean muscle PtO2 in the endotoxemic group was significantly lower than controls (52 +/- 9 vs. 24 +/- 4 Torr, P < 0.01; +/- SE). Endotoxemic muscle PtO2 values showed less heterogeneity than control groups and significant attenuation of the response to increasing FIO2 to 0.95 (mean rise in PtO2 +/- SE; 27 +/- 7 vs. 80 +/- 11 Torr for endotoxemic and control groups, respectively; P < 0.01). No steady-state differences in tissue perfusion or response to ligation-induced ischemia-reperfusion could be demonstrated between endotoxemic and control rats. These data suggest that there is significant tissue hypoxia and abnormal microvascular control of oxygenation in endotoxemia, even in the presence of normal microcirculatory perfusion.