Crystal Structures of TbCatB and Rhodesain, Potential Chemotherapeutic Targets and Major Cysteine Proteases of Trypanosoma brucei

Christian Tschudi,Iain D Kerr,Zachary B Mackey,Peng Wu,Linda S Brinen,Rachael Marion-Tsukamaki

doi:10.1371/journal.pntd.0000701

Abstract

Background Trypanosoma brucei is the etiological agent of Human African Trypanosomiasis, an endemic parasitic disease of sub-Saharan Africa. TbCatB and rhodesain are the sole Clan CA papain-like cysteine proteases produced by the parasite during infection of the mammalian host and are implicated in the progression of disease. Of considerable interest is the exploration of these two enzymes as targets for cysteine protease inhibitors that are effective against T. brucei.Methods and FindingsWe have determined, by X-ray crystallography, the first reported structure of TbCatB in complex with the cathepsin B selective inhibitor CA074. In addition we report the structure of rhodesain in complex with the vinyl-sulfone K11002.ConclusionsThe mature domain of our TbCat•CA074 structure contains unique features for a cathepsin B-like enzyme including an elongated N-terminus extending 16 residues past the predicted maturation cleavage site. N-terminal Edman sequencing reveals an even longer extension than is observed amongst the ordered portions of the crystal structure. The TbCat•CA074 structure confirms that the occluding loop, which is an essential part of the substrate-binding site, creates a larger prime side pocket in the active site cleft than is found in mammalian cathepsin B-small molecule structures. Our data further highlight enhanced flexibility in the occluding loop main chain and structural deviations from mammalian cathepsin B enzymes that may affect activity and inhibitor design. Comparisons with the rhodesain•K11002 structure highlight key differences that may impact the design of cysteine protease inhibitors as anti-trypanosomal drugs.

Highlights

IntroductionThe protozoan parasite Trypanosoma brucei is the cause of Human African Trypanosomiasis (HAT, sleeping sickness) in humans and nagana in domestic livestock [1,2,3]
The TbCatNCA074 structure confirms that the occluding loop, which is an essential part of the substrate-binding site, creates a larger prime side pocket in the active site cleft than is found in mammalian cathepsin B-small molecule structures
Comparisons with the rhodesainNK11002 structure highlight key differences that may impact the design of cysteine protease inhibitors as anti-trypanosomal drugs

Summary

Introduction

The protozoan parasite Trypanosoma brucei is the cause of Human African Trypanosomiasis (HAT, sleeping sickness) in humans and nagana in domestic livestock [1,2,3]. With over 60 million people at risk and 50,000–70,000 infected, new drugs are required to control the spread of disease and associated mortality. Clan CA cysteine proteases play central roles during the lifecycle of many parasitic organisms [11] and have been established as effective drug targets in treating many parasitic diseases [12,13,14,15]. Bloodstream T. brucei parasites express two papain family cysteine proteases, rhodesain (brucipain, trypanopain), a cathepsin L-like enzyme and TbCatB, a cathepsin B-like enzyme. TbCatB and rhodesain are the sole Clan CA papain-like cysteine proteases produced by the parasite during infection of the mammalian host and are implicated in the progression of disease. Of considerable interest is the exploration of these two enzymes as targets for cysteine protease inhibitors that are effective against T. brucei

Methods

Results

Discussion

Conclusion