Louise L. Major

Lipidomic analysis of bloodstream and procyclic form Trypanosoma brucei

The biological membranes of Trypanosoma brucei contain a complex array of phospholipids that are synthesized de novo from precursors obtained either directly from the host, or as catabolised endocytosed lipids. This paper describes the use of nanoflow electrospray tandem mass spectrometry and high resolution mass spectrometry in both positive and negative ion modes, allowing the identification of approximately 500 individual molecular phospholipids species from total lipid extracts of cultured bloodstream and procyclic form T. brucei. Various molecular species of all of the major subclasses of glycerophospholipids were identified including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol as well as phosphatidic acid, phosphatidylglycerol and cardolipin, and the sphingolipids sphingomyelin, inositol phosphoceramide and ethanolamine phosphoceramide. The lipidomic data obtained in this study will aid future biochemical phenotyping of either genetically or chemically manipulated commonly used bloodstream and procyclic strains of Trypanosoma brucei. Hopefully this will allow a greater understanding of the bizarre world of lipids in this important human pathogen.

High-Resolution Structures of RmlC from Streptococcus suis in Complex with Substrate Analogs Locate the Active Site of This Class of Enzyme

Nature achieves the epimerization of carbohydrates by a variety of chemical routes. One common route is that performed by the class of enzyme defined by dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase (RmlC) from the rhamnose pathway. Earlier studies failed to identify the key residues in catalysis. We report the 1.3 A structure of RmlC from Streptococcus suis type 2 and its complexes with dTDP-D-glucose and dTDP-D-xylose. The streptococcal RmlC enzymes belong to a separate subgroup, sharing only 25% identity with RmlC from other bacteria, yet the S. suis enzyme has similar kinetic properties and structure to other RmlC enzymes. Structure, sequence alignment, and mutational analysis have now allowed reliable identification of the catalytic residues and their roles.

Synthesis and in vitro/in vivo Evaluation of the Antitrypanosomal Activity of 3-Bromoacivicin, a Potent CTP Synthetase Inhibitor

The first convenient synthesis of enantiomerically pure (αS,5S)‐α‐amino‐3‐bromo‐4,5‐dihydroisoxazol‐5‐yl acetic acid (3‐bromoacivicin) is described. We demonstrate that 3‐bromoacivicin is a CTP synthetase inhibitor three times as potent as its 3‐chloro analogue, the natural antibiotic acivicin. Because CTP synthetase was suggested to be a potential drug target in African trypanosomes, the in vitro/in vivo antitrypanosomal activity of 3‐bromoacivicin was assessed in comparison with acivicin. Beyond expectation, we observed a 12‐fold enhancement in the in vitro antitrypanosomal activity, while toxicity against mammalian cells remained unaffected. Despite its good in vitro activity and selectivity, 3‐bromoacivicin proved to be trypanostatic and failed to completely eradicate the infection when tested in vivo at its maximum tolerable dose.

Synthesis and biological evaluation of CTP synthetase inhibitors as potential agents for the treatment of African trypanosomiasis.

Acivicin analogues with an increased affinity for CTP synthetase (CTPS) were designed as potential new trypanocidal agents. The inhibitory activity against CTPS can be improved by increasing molecular complexity, by inserting groups able to establish additional interactions with the binding pocket of the enzyme. This strategy has been pursued with the synthesis of α‐amino‐substituted analogues of Acivicin and N1‐substituted pyrazoline derivatives. In general, there is direct correlation between the enzymatic activity and the in vitro anti‐trypanosomal efficacy of the derivatives studied here. However, this cannot be taken as a general rule, as other important factors may play a role, notably the ability of uptake/diffusion of the molecules into the trypanosomes.

Trypanosoma brucei Bloodstream Forms Depend upon Uptake of myo-Inositol for Golgi Complex Phosphatidylinositol Synthesis and Normal Cell Growth

ABSTRACT myo-Inositol is a building block for all inositol-containing phospholipids in eukaryotes. It can be synthesized de novo from glucose-6-phosphate in the cytosol and endoplasmic reticulum. Alternatively, it can be taken up from the environment via Na+- or H+-linked myo-inositol transporters. While Na+-coupled myo-inositol transporters are found exclusively in the plasma membrane, H+-linked myo-inositol transporters are detected in intracellular organelles. In Trypanosoma brucei, the causative agent of human African sleeping sickness, myo-inositol metabolism is compartmentalized. De novo-synthesized myo-inositol is used for glycosylphosphatidylinositol production in the endoplasmic reticulum, whereas the myo-inositol taken up from the environment is used for bulk phosphatidylinositol synthesis in the Golgi complex. We now provide evidence that the Golgi complex-localized T. brucei H+-linked myo-inositol transporter (TbHMIT) is essential in bloodstream-form T. brucei. Downregulation of TbHMIT expression by RNA interference blocked phosphatidylinositol production and inhibited growth of parasites in culture. Characterization of the transporter in a heterologous expression system demonstrated a remarkable selectivity of TbHMIT for myo-inositol. It tolerates only a single modification on the inositol ring, such as the removal of a hydroxyl group or the inversion of stereochemistry at a single hydroxyl group relative to myo-inositol.

The essential roles of cytidine diphosphate‐diacylglycerol synthase in bloodstream form Trypanosoma brucei

Lipid metabolism in Trypanosoma brucei, the causative agent of African sleeping sickness, differs from its human host in several fundamental ways. This has lead to the validation of a plethora of novel drug targets, giving hope of novel chemical intervention against this neglected disease. Cytidine diphosphate diacylglycerol (CDP‐DAG) is a central lipid intermediate for several pathways in both prokaryotes and eukaryotes, being produced by CDP‐DAG synthase (CDS). However, nothing is known about the single T. brucei CDS gene (Tb927.7.220/EC 2.7.7.41) or its activity. In this study we show TbCDS is functional by complementation of a non‐viable yeast CDS null strain and that it is essential in the bloodstream form of the parasite via a conditional knockout. The TbCDS conditional knockout showed morphological changes including a cell‐cycle arrest due in part to kinetoplast segregation defects. Biochemical phenotyping of TbCDS conditional knockout showed drastically altered lipid metabolism where reducing levels of phosphatidylinositol detrimentally impacted on glycoylphosphatidylinositol biosynthesis. These studies also suggest that phosphatidylglycerol synthesized via the phosphatidylglycerol‐phosphate synthase is not synthesized from CDP‐DAG, as was previously thought. TbCDS was shown to localized the ER and Golgi, probably to provide CDP‐DAG for the phosphatidylinositol synthases.

Screening the MayBridge Rule of 3 Fragment Library for Compounds That Interact with the Trypanosoma brucei myo-Inositol-3-Phosphate Synthase and/or Show Trypanocidal Activity

Inositol-3-phosphate synthase (INO1) has previously been genetically validated as a drug target against Trypanosoma brucei, the causative agent of African sleeping sickness. Chemical intervention of this essential enzyme could lead to new therapeutic agents. Unfortunately, no potent inhibitors of INO1 from any organism have been reported, so a screen for potential novel inhibitors of T. brucei INO1was undertaken. Detection of inhibition of T. brucei INO1 is problematic due to the nature of the reaction. Direct detection requires differentiation between glucose-6-phosphate and inositol-3-phosphate. Coupled enzyme assays could give false positives as potentially they could inhibit the coupling enzyme. Thus, an alternative approach of differential scanning fluorimetry to identify compounds that interact with T. brucei INO1 was employed to screen ~670 compounds from the MayBridge Rule of 3 Fragment Library. This approach identified 38 compounds, which significantly altered the Tm of TbINO1. Four compounds showed trypanocidal activity with ED50s in the tens of micromolar range, with 2 having a selectivity index in excess of 250. The trypanocidal and general cytotoxicity activities of all of the compounds in the library are also reported, with the best having ED50S of ~20 μM against T. brucei.

Coupled Enzyme Activity and Thermal Shift Screening of the Maybridge Rule of 3 Fragment Library Against Trypanosoma brucei Choline Kinase; A Genetically Validated Drug Target

Infectious diseases caused by parasitic protozoa affect approximately 15% of the global pop‐ ulation, and more than 65% of the population in the Third and developing world, yet cur‐ rent drug therapies for protozoal infections are woefully inadequate. As protozoal infections take their toll predominantly in the developing world, market forces are insufficient to pro‐ mote the development of novel anti-protozoal drugs. In 2000, only ca. 0.1% of global invest‐ ment in health research was spent on drug discovery for tropical diseases [1].

Inhibitors of Trypanosoma cruzi Sir2 related protein 1 as potential drugs against Chagas disease.

Chagas disease remains one of the most neglected diseases in the world despite being the most important parasitic disease in Latin America. The characteristic chronic manifestation of chagasic cardiomyopathy is the region’s leading cause of heart-related illness, causing significant mortality and morbidity. Due to the limited available therapeutic options, new drugs are urgently needed to control the disease. Sirtuins, also called Silent information regulator 2 (Sir2) proteins have long been suggested as interesting targets to treat different diseases, including parasitic infections. Recent studies on Trypanosoma cruzi sirtuins have hinted at the possibility to exploit these enzymes as a possible drug targets. In the present work, the T. cruzi Sir2 related protein 1 (TcSir2rp1) is genetically validated as a drug target and biochemically characterized for its NAD+-dependent deacetylase activity and its inhibition by the classic sirtuin inhibitor nicotinamide, as well as by bisnaphthalimidopropyl (BNIP) derivatives, a class of parasite sirtuin inhibitors. BNIPs ability to inhibit TcSir2rp1, and anti-parasitic activity against T. cruzi amastigotes in vitro were investigated. The compound BNIP Spermidine (BNIPSpd) (9), was found to be the most potent inhibitor of TcSir2rp1. Moreover, this compound showed altered trypanocidal activity against TcSir2rp1 overexpressing epimastigotes and anti-parasitic activity similar to the reference drug benznidazole against the medically important amastigotes, while having the highest selectivity index amongst the compounds tested. Unfortunately, BNIPSpd failed to treat a mouse model of Chagas disease, possibly due to its pharmacokinetic profile. Medicinal chemistry modifications of the compound, as well as alternative formulations may improve activity and pharmacokinetics in the future. Additionally, an initial TcSIR2rp1 model in complex with p53 peptide substrate was obtained from low resolution X-ray data (3.5 Å) to gain insight into the potential specificity of the interaction with the BNIP compounds. In conclusion, the search for TcSir2rp1 specific inhibitors may represent a valuable strategy for drug discovery against T. cruzi.