**1. Introduction**

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].

One such neglected parasitic disease is Human African Trypanosomiasis (HAT) or African sleeping sickness, which is caused by the protozoan parasite *Trypanosoma brucei* and is trans‐ mitted by the bite of the Tsetse fly. The WHO estimates that HAT constitutes a serious health risk to 60 million people in sub-Saharan Africa, 300,000-500,000 of whom become in‐ fected each year, with an estimated 10,000 fatalities. The related disease in cattle, Cattle Try‐ panosomiasis or Nagana, also represents a major health concern due to its devastating economic, social and nutritional impact on African families, estimated by the WHO as an annual economic loss of ~US\$ 4 billion. As such, the total burden of Trypanosomiasis trans‐ lates into 1,598,000 Disability-Adjusted Life Years, this is on a par with big killers such as *Mycobacterium tuberculosis* and Malaria [2, 3].

Treatment of HAT is dependent upon four drugs: suramin, melarsoprol, pentamidine and eflornithine.These therapies are often toxic, difficult to administer and increasingly have an acquired drug resistance [4, 5].Developed before the 1950s suramin and melarsoprol are

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

presence of this branch of the Kennedy pathway was demonstrated in *T. brucei* [22], howev‐

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

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

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The trypanosomal genomes have revealed that *T. brucei* does not contain homologues for any methyltransferase(s) required to convert PE to PC [24] (neither does *T. cruzi*, but *Leish‐ mania* do). *Plasmodium falciparum* have an alternative single plant-like S-adenosyl-L-methio‐ nemethyltransferase [25-27], responsible for phosphoethanolamine conversion to phosphocholine, however there are no trypanosomatid homologues. This rather surprising absence of PE to PC methylation has been confirmed by *in vivo* labellings by ourselves and

The third alternative pathway for *de novo* synthesis of PC, and the only pathway by which *T. brucei* can *de novo* synthesise PC, utilises the CDP-choline branch of the Kennedy pathway [19, 29-33]. This involves the phosphorylation of choline by a choline kinase, its activation to CDP-choline by a choline-phosphate cytidyltransferase and its transfer to diacylglycerol by a choline phosphotransferase. Biochemical characterisation of the two choline/ethanolamine kinases involved in the initial steps of the Kennedy pathway show that unusually amongst

Collectively this evidence of an absence of redundancy of *de novo* PC synthesis in *T. brucei*, compared with other organisms (including humans), suggests *T. brucei* has a vulnerability to inhibition of their only way to synthesise PC, i.e. the Kennedy pathway. Recently we have exploited this fact by genetically validating the only *T. brucei* choline kinase (*Tb*CK) as a drug target both in culture and in an animal model [34]. Chemical intervention of the *Tb*CK enzyme activity is likely to interfere with the parasite's biology in multiple ways and *Tb*CK

In this study we interrogate ~630 compounds of the Maybridge Rule of 3 Fragment Library for compounds that interact with, and inhibit *Tb*CK.The Maybridge Rule of 3 Fragment Li‐ brary is a small collection of quantifiable diverse [35, 36], pharmacophoric rich, chemical en‐ tities that comply with the following criteria; MW ≤ 300, cLogP ≤ 3, H-Bond Acceptors ≤ 3, H-Bond Donors ≤ 3, Rotatable bonds (Flexibility Index) ≤ 3, Polar Surface Area ≤ 60 Å2 and aqueous solubility ≥ 1 mM using LogS and high purity (≥ 95%). Comparisons between two different screening methods, a coupled enzyme activity assay and differential scanning fluo‐ rimetry,has allowed identification of compounds that interact and inhibit the *T. brucei* chol‐

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

er only recently have the constituent enzymes been characterized [23].

eukaryotes only one of the kinases is able to phosphorylate choline [23].

is therefore of interest as a target for novel chemotherapeutics.

ine kinase, several of which possess selective trypanocidal activity.

others [15, 22, 28].

**2. Experimental**

**2.1. Materials**

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‐ lar limitations.

*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‐ asite is not a viable option as a therapy.

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 anchored variant surface glycoprotein mentioned earlier [18].

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 presence of this branch of the Kennedy pathway was demonstrated in *T. brucei* [22], howev‐ er only recently have the constituent enzymes been characterized [23].

The trypanosomal genomes have revealed that *T. brucei* does not contain homologues for any methyltransferase(s) required to convert PE to PC [24] (neither does *T. cruzi*, but *Leish‐ mania* do). *Plasmodium falciparum* have an alternative single plant-like S-adenosyl-L-methio‐ nemethyltransferase [25-27], responsible for phosphoethanolamine conversion to phosphocholine, however there are no trypanosomatid homologues. This rather surprising absence of PE to PC methylation has been confirmed by *in vivo* labellings by ourselves and others [15, 22, 28].

The third alternative pathway for *de novo* synthesis of PC, and the only pathway by which *T. brucei* can *de novo* synthesise PC, utilises the CDP-choline branch of the Kennedy pathway [19, 29-33]. This involves the phosphorylation of choline by a choline kinase, its activation to CDP-choline by a choline-phosphate cytidyltransferase and its transfer to diacylglycerol by a choline phosphotransferase. Biochemical characterisation of the two choline/ethanolamine kinases involved in the initial steps of the Kennedy pathway show that unusually amongst eukaryotes only one of the kinases is able to phosphorylate choline [23].

Collectively this evidence of an absence of redundancy of *de novo* PC synthesis in *T. brucei*, compared with other organisms (including humans), suggests *T. brucei* has a vulnerability to inhibition of their only way to synthesise PC, i.e. the Kennedy pathway. Recently we have exploited this fact by genetically validating the only *T. brucei* choline kinase (*Tb*CK) as a drug target both in culture and in an animal model [34]. Chemical intervention of the *Tb*CK enzyme activity is likely to interfere with the parasite's biology in multiple ways and *Tb*CK is therefore of interest as a target for novel chemotherapeutics.

In this study we interrogate ~630 compounds of the Maybridge Rule of 3 Fragment Library for compounds that interact with, and inhibit *Tb*CK.The Maybridge Rule of 3 Fragment Li‐ brary is a small collection of quantifiable diverse [35, 36], pharmacophoric rich, chemical en‐ tities that comply with the following criteria; MW ≤ 300, cLogP ≤ 3, H-Bond Acceptors ≤ 3, H-Bond Donors ≤ 3, Rotatable bonds (Flexibility Index) ≤ 3, Polar Surface Area ≤ 60 Å2 and aqueous solubility ≥ 1 mM using LogS and high purity (≥ 95%). Comparisons between two different screening methods, a coupled enzyme activity assay and differential scanning fluo‐ rimetry,has allowed identification of compounds that interact and inhibit the *T. brucei* chol‐ ine kinase, several of which possess selective trypanocidal activity.
