Structure-based Design of Drugs for Trypanosomiasis and Leishmaniasis
Current groups collaborating on the project:

  • Dr. Paul Michels: Molecular Biology
  • Dr. Wim Hol: Crystallography
  • Dr. Christophe Verlinde: Drug Design
  • Dr. Mike Gelb: Synthesis & Enzyme Inhibition Studies
  • Dr. Wes van Voorhis and Dr. Fred Buckner: Parasitology
  • Dr. Fred Opperdoes: Parasitology
Diseases
African trypanosomiasis

The World Health Organization estimates that 400,000 people are infected with African trypanosomiasis, with 60 million living in endemic areas. The disease, caused by the protozoon Trypanosoma brucei and transmitted by the tse-tse fly, is always fatal without treatment. There is no vaccine, and current drugs are higly toxic, require administration in a hospital setting, and are not always effective due to resistance. New drugs are desperately needed.
More info at WHO: African trypanosomiasis.

Chagas' disease aka. American trypanosomiasis

The World Health Organization estimates that 18 million people are infected with Chagas' disease, with 100 million living in endemic areas. The disease, caused by the protozoon Trypanosoma cruzi and transmitted by reduviid bugs or via bloodtransfusion, is characterized by an acute transient stage followed after several years by a chronic stage in a third of the cases. During the chronic disease irreversible damage to the heart, oesophagus and colon develops, invariably leading to death, and there are no drugs to stop this process.
More info at WHO: Chagas' disease.

Leishmaniasis

Leishmaniasis, caused by eleven species of Leishmania, currently affects some 12 million people with 350 million at risk, according to the World Health Organization. There are four forms of the disease. Visceral leishmaniasis is the most serious form and fatal if untreated. Other forms are cutaneous, mucocutaneous, and diffuse cutaneous leishmaniasis. All are accompanied by some of the most horrible tissue destruction and scars known to mankind.
More info at WHO: Leishmaniasis.
Targets
In the human host African trypanosomes rely on glycolysis to the stage of pyruvate as a sole source of energy. Hence, enzymes of glycolysis are potential drug targets provided that differ sufficiently form their human equivalents. Calculations on the glycolytic flux in trypanosomes by Dr. Hans Westerhoff suggest that aldolase (ALD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), and glycerol-3-phosphate dehydrogenase (GDH) exert considerable control. Also, evidence is mounting that in the human host Trypanosoma cruzi relies on glycolysis as its major energy source. Because of the high degree of sequence similarity with the equivalent glycolytic enzymes of Leishmania we have also included leishmanial enzymes in our studies.
Structures
Thus far the structures of the following targets were determined by the group of Dr. Wim Hol.

  • Aldolase from T. brucei
    • Chudzik D.M., Michels P.A., de Walque S., Hol W.G.J.. (2000). Structures of Type 2 Peroxisomal Targeting Signals in Two Trypanosomatid Aldolases. J Mol Biol. 300, 697-707.
  • Triosephosphate isomerase from T. brucei
    • Wierenga R.K., Noble M.E., Vriend G., Nauche S., Hol W.G.J. (1991). Refined 1.83 A structure of trypanosomal triosephosphate isomerase crystallized in the presence of 2.4 M-ammonium sulphate. A comparison with the structure of the trypanosomal triosephosphate isomerase-glycerol-3-phosphate complex. J Mol Biol. 220, 995-1015.
  • Glyceraldehyde-3-phosphate dehydrogenase from T. brucei and L. mexicana
    • Vellieux F.M.D., Hajdu J., Verlinde C.L.M.J., Groendijk H., Read R.J., Greenhough T.J., Campbell J.W., Kalk K.H., Littlechild J.A., Watson H.C., Hol W.G.J. (1993). Structure of glycosomal glyceraldehyde-3- phosphate dehydrogenase from Trypanosoma brucei determined from Laue data. Proc. Natl. Acad. Sci. USA 90, 2355-2359. [MEDLINE: 8460146].
    • Kim H., Feil I.K., Verlinde C.L.M.J., Petra P.H., Hol W.G.J. (1995). Crystal structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase: from Leishmania mexicana: Implications for structure- based drug design and a new position for the inorganic phosphate binding site. Biochemistry 34, 14975-14986. [MEDLINE: 7578111]
  • Phosphoglycerate kinase from T. brucei
    • Bernstein BE, Michels PAM, Hol WGJ (1997). Synergistic effects of substrate- induced conformational changes in phosphoglycerate kinase activation. Nature 385, 275-278.
    • Bernstein BE, Williams DM, Bressi JC, Kuhn P, Gelb MH, Blackburn GM, Hol WGJ (1998). A bisubstrate analog induces unexpected conformational changes in phosphoglycerate kinase from Trypanosoma brucei. J Mol Biol 279, 1137-1148.
  • Glycerol-3-phosphate dehydrogenase from L. mexicana
    • Suresh S., Turley S., Opperdoes F.R., Michels P.A., Hol W.G.J. (2000). A potential target enzyme for trypanocidal drugs revealed by the crystal structure of NAD-dependent glycerol-3-phosphate dehydrogenase from Leishmania mexicana. Structure Fold Des. 8, 541-52.
Design and Results
Most progress has been made with GAPDH. Because of significant structural differences in the vicinity of the NAD adenosine pockets between the human and the parasite enzymes adenosine was chosen as a lead for drug design. It inhibits both parasite and human GAPDH very weakly at ~50 mM, but provides the opportunity to reach a cleft which only exists on the parasite enzyme surface. Thus, by replacing the adenosine ribosyl's 2'-hydroxyl with a 2'-benzamido group selectivivity was obtained. Subsequently, extra affinity was achieved with an appropriate N6-substituent designed to fit in a nearby hydrophobic slot. The resulting compound 2'-deoxy- 2'-(3-methoxybenzamido)-N6-(1-naphthalenemethyl)adenosine was shown to be a competitive inhibitor with respect to NAD of T. brucei GAPDH and L. mexicana GAPDH with IC50 values of 2 µM and 200 nM, respectively. This compound does not affect human GAPDH at concentrations up to its solubility limit (0.04 mM). Thus, a selective inhibitor with 5 orders of magnitude potency improvement over the lead was designed.


Fig. 1: Predicted binding mode of designed inhibitor.

The inhibitor has also been tested on cultures of live trypanosomes. In vitro it blocks the growth of bloodstream form T. brucei with an ED50 of about 30 µM. In addition the compound was tested for its ability to inhibit the growth of T.cruzi amastigotes in T3T fibroblast host cells. Similar effects as in T.brucei were seen, and the compound appeared to be harmless to mammalian cells at concentrations up to 0.05 mM.


Fig. 2: Optical microscopy showing T3T fibroblasts infected with T.cruzi in absence of inhibitor (pannel 1), in presence of 16 µM(pannel 2), and of 40 µM inhibitor (pannel 3).
References
  • Verlinde C.L.M.J., Callens M., Van Calenbergh S., Van Aerschot A., Herdewijn P., Hannaert V., Michels P.A.M., Opperdoes F.R., Hol W.G.J. (1994). Selective inhibition of trypanosomal glyceraldehyde-3- phosphate dehydrogenase by protein structure-based design: Towards new drugs for the treatment of sleeping sickness. J. Med. Chem. 37, 3605-3613. [MEDLINE: 7932587]
  • Kim H., Feil I.K., Verlinde C.L.M.J., Petra P.H., Hol W.G.J. (1995). Crystal structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase: from Leishmania mexicana: Implications for structure- based drug design and a new position for the inorganic phosphate binding site. Biochemistry 34, 14975-14986. [MEDLINE: 7578111]
  • Buckner F.S., Verlinde C.L.M.J., La Flamme A.C. & Van Voorhis W.C. (1996). Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing beta-galactosidase. Antimicrob. Agents Chemother. 40, 2592-2597. [MEDLINE: 8913471]
  • Verlinde C.L.M.J., Kim H., Bernstein B.E., Mande S.C., Hol W.G.J. (1997). Anti-trypanosomiasis drug development based on structures of glycolytic enzymes. In 'Structure-based drug design', Ed. Veerapandian. Marcel Dekker, NY, pp. 365-394.
  • Verlinde C.L.M.J. (1998). Five Orders of Magnitude Affinity Gain in Anti-trypanosomal Drug Development. On-line Proceedings of the 5th Internet World Congress on Biomedical Sciences '98 at McMaster University, Canada (URL: http://www.mcmaster.ca/inabis98/harmtox/verlinde0900)
  • Aronov A.M., Buckner F.S., Van Voorhis W.C., Verlinde C.L.M.J., Opperdoes F.R., Hol W.G.J., Gelb M.H. (1999). Structure-based design of sub-micromolar, biologically active inhibitors of trypanosomatid glyceraldehyde- 3-phosphate dehydrogenase. Proc. Natl. Acad. Sci. USA 96, 4273-4278. [MEDLINE: 10200252]
    		•