T. brucei

African sleeping sickness is a parasitic disease of increasing importance, with an estimated 300,000-500,000 cases annually. The etiological agents, T. brucei gambiense and T. brucei rhodesiense, are transmitted to humans by the bite of Tsetse flies. 

Melarsoprol, a trivalent organic melaminophenyl arsenic compound, kills intracerebral parasites of both T. brucei gambiense and T. brucei rhodesiense. Melarsoprol accumulates via an adenosine/adenine transporter in trypanosomes and is believed to inhibit glycolytic enzymes. Melarsoprol leads to a rapid lysis of trypanosomes. Melarsoprol is highly toxic to humans. 

Indeed, a bDNA assay for diagnosis of African trypanosomiasis was developed and compared with buffy coat microscopy for detection of T brucei in human blood samples (Harris etal., 1996). Two repetitive DNA sequences found only in the T. brucei complex, a 177-bp satellite repeat and the ribosomal mobile element, were selected as targets in the bDNA assay. The assay used the standard bDNA components capture probes, target probes, amplifier molecules, and alkaline phosphatase-labeled probes. Various blood fractions and sample preparation methods were examined. Ultimately, buffy coat samples resulted in the highest sensitivity. Although typanosomes do not infect leukocytes, they cosediment with them. 

The limit of detection of the assay was estimated to be 200 parasites/ml of blood. The detection limit is well within the range of sensitivity needed to diagnosis trypanosomiasis, as the parasitemia may vary from 5000 to 1,500,000 parasites/ml (Vickerman, 1974). The bDNA assay was compared with buffy coat microscopy for detection of T brucei in 56 blood samples (36 buffy coat positive and 20 buffy coat negative by microscopy). There was complete concordance between the results of the two tests in terms of identifying specimens as positive of negative. However, the numbers of parasites observed by microscopy were lower overall than those calculated with the bDNA assay. The authors suggested that the excess of leukocytes in the buffy coat could interfere with the microscopic detection of typanosomes, resulting in lower apparent parasitemia than the true value. 

Biosynthesis of polyamines is essential for growth and multiplication of T. brucei, hence discovery of drug candidates that inhibit enzymes in the polyamine biosynthesis pathway represent an attractive approach to development of trypanocides. The consequences of gene knockout of ornithine decarboxylase (ODC), the target of eflornithine (3), have been further characterized and suggest that new inhibitors of this enzyme may be particularly effective [18]. 

A third enzyme in the polyamine biosynthesis pathway, trypanothione synthetase-amidase (TRYS), has been shown to be essential by both genetic and chemical methods [24]. Specifically, the indazole analog 24 has been identified in a TRYS screen (IC50 = 140 nM) and shown to inhibit growth of wild-type (IC50 = 5.1 pM) and TRYS-dKO (IC50 = 0.46 mM) T. brucei parasites in culture [25]. 

One of the most compelling targets in the polyamine biosynthesis pathway has been S-adenosylmethionine decarboxylase (SAM-DC). This target was chemically validated with the discovery of trypanocidal activity of MDL-73811 nearly two decades ago. Work to understand the unique kinetics for inhibition of this enzyme in T. brucei has shown that a catalytically... 

Inhibitors of several enzymes in the glycolytic pathway, upon which survival of T. brucei is dependent, have been described. Lonidamine (29) has been shown to inhibit T. brucei hexokinase (IC50 = 850 pM) and be toxic to the parasite (T.b. LD50 = 50 pM) in culture [31]. A series of mannitol derivatives have been discovered, which inhibit T. brucei phos-phofructokinase (TbPFK) [32]. The most potent compound (30) within this series exhibits an IC50 = 23 pM in a recombinant enzyme assay and inhibits parasite growth in vitro (IC50 = 30 pM). 

As in all eukaryotic cells, protein kinases play an important role in the life cycle of the kinetoplastids and, as such, are attractive targets. Recent efforts, predominantly through genetic (RNAi) means, have validated a number of kinases as essential for survival of T. brucei, but few have been explored with chemical probes [33-35]. 

T. brucei is unable to synthesize purines de novo and, as such, is dependent upon salvage mechanisms from the host. A number of transporters and enzymes are used by T. brucei to accomplish this task, and inhibition of these targets offers promise for development of trypanocides [39]. This strategy has been validated by demonstration that cordycepin (34), a substrate for T. brucei adenosine kinase (TbAK), which terminates RNA synthesis and parasite growth, can cure stage 2 HAT infections in mice when coadministered with deoxycoformycin (35), an adenosine deaminase inhibitor [40]. 

It is a sulfated napthylamine and used as first line therapy for early hemolymphatic African trypanosomiasis (caused by T. brucei gambiense). It has very tight protein binding and having short initial half life but terminal half life is about 50 days and is excreted by kidney. It is also used for chemoprophylaxis against African trypanosomiasis. 

It is effective against advanced T. brucei gambiense infection. 

II. Three Glycolytic Enzymes Of T. Brucei Molecular Biology, Biochemistry, and X-Ray Crystallography... 

Glyceraldehyde-3-phosphate dehydrogenase is a homotetramer that carries out the oxidative phosphorylation of glyceraldehyde-3-phosphate into 1,3-bisphos- phoglycerate. During this reaction NADH is formed. Each subunit of the enzyme consists of two domains and has an NAD+ binding site. The N-terminal domain anchors the adenosine portion of the cofactor while the nicotinamide portion is involved in the catalytic reaction at the C-terminal domain. T brucei... 

Exploitable structural differences between T.brucei(full) and human (dashed) TIM. The inhibitor 2-phosphoglycolate as observed in the structure of the human enzyme indicates the location of the active site. Drug design targets are the T.brucei AlalOO-TyrlOl, which are considerably different from their human counterpart His-Val. (From Ref.25. Copyright 1994 by Cambridge University Press.)... 

Two-dimensional structure of SPADNS, a micromolar inhibitor of T.brucei PGK. 

Since the four yeast PGK inhibitors are commercially available it was logical to test them for T. brucei PGK inhibition. The first three compounds were active in the millimolar range. However, SPADNS exhibited a K of 10.0 pMin these in preliminary tests [88]. Moreover, when assayed against a commercially available rabbit muscle PGK, SPADNS had no influence on the enzyme kinetics up to a concentration of 250 pM[88], In conclusion, SPADNS appears to be an excellent lead because of its potency and selectivity. Crystallographic experiments to determine its binding mode to I brucei VGK are underway. 

From the selectivity point of view the adenosine binding site of GAPDH is attractive for drug design, as we explained in Section II.B. Unfortunately, inhibition studies on T brucei and L. mexicana GAPDH revealed the poor affinity of our natural lead adenosine with IC50 values of 100 mMand 50 mM, respectively. Moreover, adenosine is an antiselective lead because the IC50... 

C2-subst C8-subst C2 -subst T. brucei L. mexicana human... 

Predicted binding mode of 2 -deoxy-2 -(3-methoxybenzamido)adenosine to T.brucei GAPDH. (From Ref. 13. Copyright 1994 by the American Chemical Society.)... 

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