**2.1. Materials**

used for chemotherapy of early stages of the disease, as is pentamidine. The arsenical melar‐ soprol is extremely toxic, with death for ~1 in 20 of cases and treatment failures as high as 30% in certain areas [4, 6]. Treatment of the second stage of the disease, where the parasites cross the blood-brain barrier and invade the central nervous system, is limited to melarso‐ prol and eflornithine [7].The WHO as a desperate measure recently introduced nifurtimoxeflornithine combination therapy for the treatment of HAT. This is despite nifurtimox, a drug often used to treat Chagas' disease (caused by the related protozoan the South Ameri‐

Hence there is an urgent need for new, more effective, less toxic, cheap and easy to adminis‐ ter therapeutic agents to treat African sleeping sickness and other closely related parasitic diseases, e.g. Chagas' disease and Leishmaniasis, whose current treatments suffer from simi‐

*T. brucei* is able to survive and multiply in the harsh environment of a mammalian hosts' bloodstream. This is due to the parasite's dense cell-surface coat of the glycosylphosphatidy‐ linositol anchored variant surface glycoprotein (5 X 106 dimers/cell) [9-11], which protects the parasite in two ways. Firstly by acting as a diffusion barrier, such that complement is unable to reach and attack the plasma membrane of *T. brucei*. Secondly *T. brucei* is able to undergo antigenic variation, where by it is able to express a new variant surface glycopro‐ tein from a repertoire of more than 1000 different genes, before the hosts' innate immune system is able to catch up [12, 13]. This antigenic variation is why a vaccine against this par‐

Phospholipids account for ~80% of total lipids in *T. brucei* with a significant proportion of these containing a choline-phosphate headgroup; phosphatidylcholine (PC) (~48%) and sphingomyelin (~15%) [14,15]. Sphingomyelin is made from PC via the sphingomyelin syn‐ thases transferring the choline-phosphate headgroup from PC to a ceramide lipid moiety [16]. These lipids contribute to the structural integrity of the membrane and in addition de‐ termine membrane fluidity and cell surface charge. Unsurprisingly, the biosynthesis and utilisation of these choline-containing molecules are implicated in a variety of cellular proc‐ esses, including signaling, intracellular cellular protein sorting and transport [reviewed in 16]. Phosphocholine has been reported to be a required mitogen for DNA synthesis induced by growth factors [17]. Recently we have shown that the essential *T. brucei* neutral sphingo‐ myelinase is actively involved in post Golgi sorting of the glycosylphosphatidylinositol

Most eukaryotes have three alternative pathways by which PC can be synthesised [19 and reviewed in 20]. The first two pathways both involve three consecutive methylations of PE by S-adenosyl-L-methionemethyltransferases [20]. The PE can be derived from two alterna‐ tive pathways, either from the concerted actions of the CDP-DAG dependantphosphatidyl‐ serine synthase and phosphatidylserine decarboxylase, or via the CDP-ethanolamine branch of the Kennedy pathway. This involves phosphorylation of ethanolamine by an ethanola‐ mine kinase, its activation to CDP-ethanolamine by an ethanolamine-phosphate cytidyl‐ transferase and its transfer to diacylglycerol by an ethanolamine phosphotransferase. The

can *Trypanosoma cruzi*), having low efficacy against HAT [8].

anchored variant surface glycoprotein mentioned earlier [18].

lar limitations.

414 Drug Discovery

asite is not a viable option as a therapy.

All materials unless stated were purchased either from Sigma/Aldrich or Invitrogen. An in house Maybridge Rule of 3 Fragment Library kept in master plates at 200 mM in DMSO (100%), was transferred into working plates with compounds occupying the central 80 wells of a 96-well plate, at 10 mM in 5% DMSO, allowing the two outside columns for positive and negative controls.

absorbance at 341 nm was monitored for 10 min at room temperature. For testing inhibition of the coupling enzymes (PK/LDH), standard buffer conditions were used but the assay con‐ tained 1 mM PEP, 0.1 mM ADP and 0.5 mM NADH. The PK/LDH was titrated to give a change in absorbance of approximately 0.05 absorbance units/min in the absence of inhibi‐

Coupled Enzyme Activity and Thermal Shift Screening of the Maybridge Rule of 3 Fragment Library...

http://dx.doi.org/10.5772/52668

417

Differential scanning fluorimetry was set up in 96 well PCR plates using a reaction volume of 100 µL. Samples contained 2.1 µM *Tb*CK, 6 mM MgCl2, 50 mM HEPES pH 8.0, 80 mMNaCl, 5.25% glycerol (v/v) and 1.4 x Sypro Orange (Invitrogen), Maybridge Ro3 com‐ pounds were screened at 1 mM concentration with a final DMSO concentration of 0.5% (v/ v).Two controls with eight repetitions per plate were used for the thermal shift experiments:

Differential fluorimetric scans were performed in a realtime PCR machine (Stratagene Mx3005P with software MxPro v 4.01) using a temperature scan from 25°C to 95°C at 0.5°C min-1.Data were then exported to Excel for analysis using "DSF analysis" modified from the template provided by Niesen et al. [38].Tm values were calculated by non-linear regression, fitting the Boltzmann equation to the denaturation curves using GraFit. *Tb*CK Tm, in the presence of 6 mM MgCl2 and 0.5% DMSO, 41.21 ± 0.03°C (n > 60), Tm for *Tb*CK and 0.5 mM

Screening for inhibitors of the genetically validated drug target *Tb*CK is problematic due to the difficulty in following the reaction either continuously or directly. A direct choline kin‐ ase activity assay assessing the production of phosphocholine, utilising a modified method of Kim *et. al.* [39], using *Tb*CK and radiolabelled choline has been performed previously [23]. However this is not suitable for screening purposes, so choline kinase activity was measured by a spectrophotometric coupled assay (Figure 1). This coupled enzyme assay utilises regen‐ eration of ATP from the ADP by-product of the choline kinase by pyruvate kinase, and sub‐ sequent oxidation of NADH as the resulting pyruvate is converted to lactate, by lactate dehydrogenase. This assay using coupled enzymes is also problematic, as a compound

An alternative approach for screening is differential scanning fluorimetry (Figure 2), allow‐ ing identification of compounds that interact with the *Tb*CK protein, either to stabilise or de‐

Initially *Tb*CK was subjected to differential scanning fluorimetry to ascertain if this approach was possible. Known components required for enzyme activity were tested to see if thermal shifts were observed.In the presence of 6 mM MgCl2, a Tm of 41.2°C was obtained (Figure 1C, solid dark line). The addition of 0.5 mM ATP resulted in a > 3°C Tm shift for *Tb*CK(Figure 1C,

could potentially inhibit the coupled enzymes giving rise to a false positive.

stabilise it, therefore influencing the protein's Tm (melting point) [38-40].

**2.4. Differential scanning fluorimetry with** *Tb***CK**

0.5% DMSO; 0.5 mM ATP, 0.5% DMSO.

ATP = 44.46 ± 0.05°C (n > 60).

**3. Results and discussion**

tor.
