Leishmania trypanothione synthetase-amidase structure reveals a basis for regulation of conflicting synthetic and hydrolytic activities

The bifunctional trypanothione synthetase-amidase catalyzes biosynthesis and hydrolysis of the glutathione-spermidine adduct trypanothione, the principal intracellular thiol-redox metabolite in parasitic trypanosomatids. These parasites are unique with regard to their reliance on trypanothione to determine intracellular thiol-redox balance in defense against oxidative and chemical stress and to regulate polyamine levels. Enzymes involved in trypanothione biosynthesis provide essential biological activities, and those absent from humans or for which orthologues are sufficiently distinct are attractive targets to underpin anti-parasitic drug discovery. The structure of Leishmania major trypanothione synthetase-amidase, determined in three crystal forms, reveals two catalytic domains. The N-terminal domain, a cysteine, histidine-dependent amidohydrolase/peptidase amidase, is a papain-like cysteine protease, and the C-terminal synthetase domain displays an ATP-grasp family fold common to C:N ligases. Modeling of substrates into each active site provides insight into the specificity and reactivity of this unusual enzyme, which is able to catalyze four reactions. The domain orientation is distinct from that observed in a related bacterial glutathionylspermidine synthetase. In trypanothione synthetase-amidase, the interactions formed by the C terminus, binding in and restricting access to the amidase active site, suggest that the balance of ligation and hydrolytic activity is directly influenced by the alignment of the domains with respect to each other and implicate conformational changes with amidase activity. The potential inhibitory role of the C terminus provides a mechanism to control relative levels of the critical metabolites, trypanothione, glutathionylspermidine, and spermidine in Leishmania.     

Vasopressin stimulates urinary kallikrein excretion in the isolated erythrocyte-perfused rat kidney

We have found that arginine vasopressin (AVP) (10 pg/ml) stimulates urinary kallikrein in the isolated erythrocyte perfused rat kidney. (In this model, perfusate flow rate approximates blood flow rates in vivo and morphology is normal.) Urinary kallikrein excretion rose from 6.9 +/- 0.8 to 14.9 +/- 2.4 ng/min 20 min after the addition of AVP to the perfusate, and then fell towards baseline levels over the next 30 min. 1-Desamino-8-D-AVP (8 pg/ml) caused a comparable increase in kallikrein excretion. Prostaglandin synthesis inhibition with indomethacin did not alter the stimulatory effect of AVP on kallikrein excretion. Parathyroid hormone 1-34 (144 ng/ml) and calcitonin (102 ng/ml) also increased urinary kallikrein. Kallikrein excretion rose from 9.1 +/- 2.0 to 24 +/- 4.5 ng/min in response to calcitonin and from 8.3 +/- 1.6 to 43.7 +/- 3.4 ng/min following the addition of parathyroid hormone to the perfusate. Kallikrein was found to accumulate in the perfusate in a linear fashion. Based on the slope of the relationship between perfusate kallikrein and time, the rate of release of kallikrein into the perfusate was estimated to be 0.79 ng/min in control kidneys. The rate of release of kallikrein into the perfusate in kidneys treated with AVP was the same (0.74 ng/min). Thus while kallikrein is released into the perfusate, this process is not influenced by AVP. In conclusion, AVP stimulates release of kallikrein into the urine (but not the perfusate) independently of systemic events. The effect of AVP is not mediated by prostaglandins. This effect of AVP is mediated via stimulation of the V2 receptor and also occurs in response to two other hormones (calcitonin and parathyroid hormone) that are known to stimulate adenyl cyclase in the rat distal nephron.     

Bis(glutathionyl)spermine and other novel trypanothione analogues in Trypanosoma cruzi

Trypanosomatids differ from other cells in their ability to conjugate glutathione with the polyamine spermidine to form the antioxidant metabolite trypanothione (N1,N8-bis(glutathionyl)spermidine). In Trypanosoma cruzi, trypanothione is synthesized by an unusual trypanothione synthetase/amidase (TcTryS) that forms both glutathionylspermidine and trypanothione. Because T. cruzi is unable to synthesize putrescine and is dependent on uptake of exogenous polyamines by high affinity transporters, synthesis of trypanothione may be circumstantially limited by lack of spermidine. Here, we show that the parasite is able to circumvent the potential shortage of spermidine by conjugating glutathione with other physiological polyamine substrates from exogenous sources (spermine, N8-acetylspermidine, and N-acetylspermine). Novel thiols were purified from epimastigotes, and structures were determined by matrix-assisted laser desorption ionization time-of-flight analysis to be N1,N12-bis(glutathionyl)spermine, N1-glutathionyl-N8-acetylspermidine, and N1-glutathionyl-N12-acetylspermine, respectively. Structures were confirmed by enzymatic synthesis with recombinant TcTryS, which catalyzes formation of these compounds with kinetic parameters equivalent to or better than those of spermidine. Despite containing similar amounts of spermine and spermidine, the epimastigotes, trypomastigotes, and amastigotes of T. cruzi preferentially synthesized trypanothione. Bis(glutathionyl)spermine disulfide is a physiological substrate of recombinant trypanothione reductase, comparable to trypanothione and homotrypanothione disulfides. The broad substrate specificity of TcTryS could be exploited in the design of polyamine-based inhibitors of trypanothione metabolism.     

Development of isoform selective PI3-kinase inhibitors as pharmacological tools for elucidating the PI3K pathway

Using a parallel synthesis approach to target a non-conserved region of the PI3K catalytic domain a pan-PI3K inhibitor 1 was elaborated to provide alpha, delta and gamma isoform selective Class I PI3K inhibitors 21, 24, 26 and 27. The compounds had good cellular activity and were selective against protein kinases and other members of the PI3K superfamily including mTOR and DNA-PK.     

Synthesis of 2-,4,-6-, and/or 7-substituted quinoline derivatives as human dihydroorotate dehydrogenase (hDHODH) inhibitors and anticancer agents: 3D QSAR-assisted design

Following our research for human dihydroorotate dehydrogenase (hDHODH) inhibitors as anticancer agents, herein we describe 3D QSAR-based design, synthesis and in vitro screening of 2-,4,-6-, and/or 7-substituted quinoline derivatives as hDHODH inhibitors and anticancer agents. We have designed 2-,4,-6-, and/or 7-substituted quinoline derivatives and predicted their hDHODH inhibitory activity based on 3D QSAR study on 45 substituted quinoline derivatives as hDHODH inhibitors, and also predicted toxicity. Designed compounds were docked into the binding site of hDHODH. Designed compounds which showed good predictive activity, no toxicity, and good docking score were selected for the synthesis, and in vitro screening as hDHODH inhibitors in an enzyme inhibition assay, and anticancer agents in MTT assay against cancer cell lines (HT-29 and MDA-MB-231). Synthesized compounds 7 and 14 demonstrated IC₅₀ value of 1.56?µM and 1.22?µM, against hDHODH, respectively, and these are our lead compounds for the development of new hDHODH inhibitors and anticancer agents.     

A single enzyme catalyses formation of Trypanothione from glutathione and spermidine in Trypanosoma cruzi

Protozoa of the order Kinetoplastida differ from other organisms in their ability to conjugate glutathione (l-gamma-glutamyl-cysteinyl-glycine) and spermidine to form trypanothione [N(1),N(8)-bis(glutathionyl)spermidine], a metabolite involved in defense against chemical and oxidant stress and other biosynthetic functions. In Crithidia fasciculata, trypanothione is synthesized from GSH and spermidine via the intermediate glutathionylspermidine in two distinct ATP-dependent reactions catalyzed by glutathionylspermidine synthetase (GspS; EC ) and trypanothione synthetase (TryS; EC ), respectively. Here we have cloned a single copy gene (TcTryS) from Trypanosoma cruzi encoding a protein with 61% sequence identity with CfTryS but only 31% with CfGspS. Saccharomyces cerevisiae transformed with TcTryS were able to synthesize glutathionylspermidine and trypanothione, suggesting that this enzyme is able to catalyze both biosynthetic steps, unlike CfTryS. When cultures were supplemented with aminopropylcadaverine, yeast transformants contained glutathionylaminopropylcadaverine and homotrypanothione [N(1),N(9)-bis(glutathionyl)aminopropylcadaverine], metabolites that have been previously identified in T. cruzi, but not in C. fasciculata. Kinetic studies on recombinant TcTryS purified from Escherichia coli revealed that the enzyme displays high-substrate inhibition with glutathione (K(m) and K(i) of 0.57 and 1.2 mm, respectively, and k(cat) of 3.4 s(-1)), but obeys Michaelis-Menten kinetics with spermidine, aminopropylcadaverine, glutathionylspermidine, and MgATP as variable substrate. The recombinant enzyme possesses weak amidase activity and can hydrolyze trypanothione, homotrypanothione, or glutathionylspermidine to glutathione and the corresponding polyamine.     

Quinazolinone fungal efflux pump inhibitors. Part 2: In vitro structure-activity relationships of (N-methyl-piperazinyl)-containing derivatives

Structure-activity relationships of a novel series of fungal efflux pump inhibitors with respect to potentiation of the activity of fluconazole against strains of Candida albicans and Candida glabrata over-expressing ABC-type efflux pumps are systematically explored.     

1-Thiazol-2-yl-N-3-methyl-1H-pyrozole-5-carboxylic acid derivatives as antitumor agents

A class of substituted 1-thiazol-2-yl-N-3-methyl-1H-pyrozole-5-carboxylic acid derivatives was found to have potent anti-proliferative activity against a broad range of tumor cell lines. A compound from this class (14) was profiled across a broad panel of hematologic and solid tumor cancer cell lines demonstrating cell cycle arrest at the G0/G1 interphase and has potent anti-proliferative activity against a distinct and select set of cancer cell types with no observed effects on normal human cells. An example is the selective inhibition of human B-cell lymphoma cell line (BJAB). Compound 14 was orally bioavailable and tolerated well in mice. Synthesis and structure activity relationships (SAR) in this series of compounds are discussed.