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bioRxiv · 10.64898/2025.12.17.694860

Structural basis for the inhibition of Trypanosoma brucei enolase by a camelid single-domain antibody

Li, Z.·Smiejkowska, N.·Vansevenant, J.·Mertens, J.·Van Wielendaele, P.·Pinto Torres, J. E.·Magez, S.·Sterckx, Y. G.
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Abstract

Trypanosoma brucei is an extracellular protozoan that causes neglected tropical diseases in both humans and livestock. The parasite has a bipartite life cycle involving an insect vector and a mammalian host. Within the latter, it mainly thrives as a blood-borne parasite that relies on glycolysis to support its energy metabolism. It is for this reason that trypanosomal glycolytic enzymes have been investigated as potential targets for the development of trypanosome-killing drugs. Recent work from our lab has shown that they are also interesting biomarkers for the detection of active trypanosome infections. T. brucei enolase (TbrENO) is a trypanosomal glycolytic enzyme that has gathered interest in both drug and diagnostics development. In this paper, we report the discovery of a camelid single domain antibody (sdAb aka nanobody) that specifically recognises and inhibits TbrENO. The sdAbs inhibitory mechanism is unraveled through a combination of protein biochemistry, biophysics, and structural biology. Author summaryTrypanosoma brucei is a unicellular parasite that lives in the bloodstream of humans and animals, where it causes serious but often overlooked diseases. Because it depends heavily on breaking down glucose to produce energy, the parasites glucose-processing proteins (called glycolytic enzymes) have become important targets for both new treatments and improved diagnostic tools. One of these proteins, called T. brucei enolase (TbrENO), has recently drawn attention for its potential in drug development and disease detection. In this study, we discovered a special type of antibody, known as a camelid single-domain antibody (sdAb aka nanobody), that can specifically recognize and block the activity of TbrENO. We employed a combination of various laboratory techniques to understand exactly how the sdAb binds to and inhibits the enzyme. Our findings provide new insight into how TbrENO can be inhibited in a way that does not require active site binding and highlight the value of sdAbs as precise tools for targeting key parasite proteins.

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Li, Z., Smiejkowska, N., Vansevenant, J., Mertens, J., Van Wielendaele, P., Pinto Torres, J. E., Magez, S., Sterckx, Y. G.. 2025-12-18. Structural basis for the inhibition of Trypanosoma brucei enolase by a camelid single-domain antibody. https://doi.org/10.64898/2025.12.17.694860

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