Browsing by Author "McKerrow, James H."

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    Early Invasion of Brain Parenchyma by African Trypanosomes
    Frevert, Ute; Movila, Alexandru; Nikolskaia, Olga V.; Raper, Jayne; Mackey, Zachary B.; Abdulla, Maha; McKerrow, James H.; Grab, Dennis J. (PLOS, 2012-08-31)
    Human African trypanosomiasis or sleeping sickness is a vector-borne parasitic disease that has a major impact on human health and welfare in sub-Saharan countries. Based mostly on data from animal models, it is currently thought that trypanosome entry into the brain occurs by initial infection of the choroid plexus and the circumventricular organs followed days to weeks later by entry into the brain parenchyma. However, Trypanosoma brucei bloodstream forms rapidly cross human brain microvascular endothelial cells in vitro and appear to be able to enter the murine brain without inflicting cerebral injury. Using a murine model and intravital brain imaging, we show that bloodstream forms of T. b. brucei and T. b. rhodesiense enter the brain parenchyma within hours, before a significant level of microvascular inflammation is detectable. Extravascular bloodstream forms were viable as indicated by motility and cell division, and remained detectable for at least 3 days post infection suggesting the potential for parasite survival in the brain parenchyma. Vascular inflammation, as reflected by leukocyte recruitment and emigration from cortical microvessels, became apparent only with increasing parasitemia at later stages of the infection, but was not associated with neurological signs. Extravascular trypanosomes were predominantly associated with postcapillary venules suggesting that early brain infection occurs by parasite passage across the neuroimmunological blood brain barrier. Thus, trypanosomes can invade the murine brain parenchyma during the early stages of the disease before meningoencephalitis is fully established. Whether individual trypanosomes can act alone or require the interaction from a quorum of parasites remains to be shown. The significance of these findings for disease development is now testable.
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    Mining a Cathepsin Inhibitor Library for New Antiparasitic Drug Leads
    Ang, Kenny K. H.; Ratnam, Joseline; Gut, Jiri; Legac, Jennifer; Hansell, Elizabeth; Mackey, Zachary B.; Skrzypczynska, Katarzyna M.; Debnath, Anjan; Engel, Juan C.; Rosenthal, Philip J.; McKerrow, James H.; Arkin, Michelle R.; Renslo, Adam R. (PLOS, 2011-05-01)
    The targeting of parasite cysteine proteases with small molecules is emerging as a possible approach to treat tropical parasitic diseases such as sleeping sickness, Chagas’ disease, and malaria. The homology of parasite cysteine proteases to the human cathepsins suggests that inhibitors originally developed for the latter may be a source of promising lead compounds for the former. We describe here the screening of a unique ,2,100-member cathepsin inhibitor library against five parasite cysteine proteases thought to be relevant in tropical parasitic diseases. Compounds active against parasite enzymes were subsequently screened against cultured Plasmodium falciparum, Trypanosoma brucei brucei and/or Trypanosoma cruzi parasites and evaluated for cytotoxicity to mammalian cells. The end products of this effort include the identification of sub-micromolar cell-active leads as well as the elucidation of structure-activity trends that can guide further optimization efforts.
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    RNA Interference of Trypanosoma brucei Cathepsin B and L Affects Disease Progression in a Mouse Model
    Abdulla, Maha-Hamadien; O'Brien, Theresa C.; Mackey, Zachary B.; Sajid, Mohamed; Grab, Dennis J.; McKerrow, James H. (PLOS, 2008-09-01)
    We investigated the roles played by the cysteine proteases cathepsin B and cathepsin L (brucipain) in the pathogenesis of Trypansoma brucei brucei in both an in vivo mouse model and an in vitro model of the blood–brain barrier. Doxycycline induction of RNAi targeting cathepsin B led to parasite clearance from the bloodstream and prevent a lethal infection in the mice. In contrast, all mice infected with T. brucei containing the uninduced Trypanosoma brucei cathepsin B (TbCatB) RNA construct died by day 13. Induction of RNAi against brucipain did not cure mice from infection; however, 50% of these mice survived 60 days longer than uninduced controls. The ability of T. b. brucei to cross an in vitro model of the human blood–brain barrier was also reduced by brucipain RNAi induction. Taken together, the data suggest that while TbCatB is the more likely target for the development of new chemotherapy, a possible role for brucipain is in facilitating parasite entry into the brain.
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    Trypanosoma cruzi CYP51 Inhibitor Derived from a Mycobacterium tuberculosis Screen Hit
    Chen, Chiung-Kuang; Doyle, Patricia S.; Yermalitskaya, Ludmila V.; Mackey, Zachary B.; Ang, Kenny K. H.; McKerrow, James H.; Podust, L.arissa M. (PLOS, 2009-02-01)
    Background: The two front-line drugs for chronic Trypanosoma cruzi infections are limited by adverse side-effects and declining efficacy. One potential new target for Chagas’ disease chemotherapy is sterol 14a-demethylase (CYP51), a cytochrome P450 enzyme involved in biosynthesis of membrane sterols. Methodology/Principal Finding: In a screening effort targeting Mycobacterium tuberculosis CYP51 (CYP51Mt), we previously identified the N-[4-pyridyl]-formamide moiety as a building block capable of delivering a variety of chemotypes into the CYP51 active site. In that work, the binding modes of several second generation compounds carrying this scaffold were determined by high-resolution co-crystal structures with CYP51Mt. Subsequent assays against the CYP51 orthologue in T. cruzi, CYP51Tc, demonstrated that two of the compounds tested in the earlier effort bound tightly to this enzyme. Both were tested in vitro for inhibitory effects against T. cruzi and the related protozoan parasite Trypanosoma brucei, the causative agent of African sleeping sickness. One of the compounds had potent, selective anti–T. cruzi activity in infected mouse macrophages. Cure of treated host cells was confirmed by prolonged incubation in the absence of the inhibiting compound. Discrimination between T. cruzi and T. brucei CYP51 by the inhibitor was largely based on the variability (phenylalanine versus isoleucine) of a single residue at a critical position in the active site. Conclusions/Significance: CYP51Mt-based crystal structure analysis revealed that the functional groups of the two tightly bound compounds are likely to occupy different spaces in the CYP51 active site, suggesting the possibility of combining the beneficial features of both inhibitors in a third generation of compounds to achieve more potent and selective inhibition of CYP51Tc.