Novel Inhibitors of Cruzain and Trypanosoma brucei Cathpesin B as Potential Drug Candidates for the Treatment of African Trypanosomiasis and Chagas Disease - PROJECT SUMMARY
With neither vaccines nor well-tolerated therapies available, the genus Trypanosoma causes
debilitating human diseases of high morbidity for which effective drugs are urgently needed.
Trypanosoma cruzi causes Chagas Disease (CD) in Central and South America, resulting in
50,000 annual deaths, with >40 recent cases reported in Texas. Trypanosoma brucei rhodesiense
and T. brucei gambiense cause African sleeping sickness (HAT), affecting over 60,000 people
world-wide. Both CD and HAT comprise unmet medical needs for which effective medicines do
not exist. The establishment and maintenance of human infection by both T. cruzi and T. brucei
requires, respectively, the related cysteine proteases cruzain and TbCat B. The objective of this
proposal is to identify novel reversible-covalent inhibitors (RCIs) of cruzain and TbCatB, that exert
potent and selective enzyme inhibition and trypanocidal activity in cell cultures of T. cruzi and T.
brucei spp., and are curative in murine models of trypanosomiasis. Our approach builds on
precedent compounds such as K11777, a tri-peptide analogue that contains an electrophilic vinyl-
sulfone moiety (Fig. 1 – SPECIFIC AIMS). K11777 is an irreversible covalent inactivator of
cruzain and TbCatB which forms a stable covalent adduct with the active-site cysteines of
protozoal cysteine proteases. K11777 cures Chagas disease in animal models, and has
progressed to human clinical trials. However, recent focus has shifted from irreversible enzyme
inactivators to reversible covalent inhibitors (RCIs), as the latter have fewer toxicity issues and
are more selective for their intended targets. We will examine the introduction of a 2-vinyl-
heterocyclic group in a selective peptide scaffold, replacing the vinyl-sulfone “warhead” of
K11777, as we believe such a moiety would provide suitable RCIs for cruzain and TbCatB. To
date we have made peptidomimetic 2-vinyl heterocycle inhibitors (P2VHIs) which include a
pyrimidine, oxazole or thiazole group conjugated to the vinyl group. These P2VHIs exhibit time-
dependent, reversible inhibition of cruzain (Ki = 7-20 nM), and kill trypanosomes in cell culture.
We propose that (a) the electrophilicity of the vinyl group may be “tuned” by substitution of the
heterocyclic group, and that (b) addition of an enzymatic cysteine residue on the vinyl group will
be readily reversible. In the R21 phase of this proposal, we will advance this concept and produce
a series of P2VHIs that exhibit (a) improved potency for cruzain, (b) inhibition of TbCatB, (c)
contain peptide scaffolds which are selective for protozoan proteases, and (d) be trypanocidal at
low micromolar concentrations. We will determine if a reversible covalent adduct is formed with
the active-site cysteines of these targets. In the R33 phase, we will develop a chemical model for
the 2-vinyl heterocycles that allows us to probe and optimize the reversible reactivity of thiols
toward P2VHIs. We will produce a series of second-generation P2VHIs with the physicochemical
properties, toxicity and safety profiles of drug candidates. We will evaluate these compounds in
cellular models of human infection, followed by proof-of-concept assessment in murine models of
CD and HAT.
