X-ray crystallography uncovers new therapeutic targets in parasitic worm microbiota

Microbiota research is of paramount importance because of its significant impact on human health, agriculture and the environment. The trillions of microorganisms in our bodies play crucial roles in maintaining the immune system and absorbing and synthesizing essential nutrients. Indeed, changes in the microbiome are linked to many human diseases.

Like humans, other organisms, including pathogens or disease vectors, rely on their microbiomes. Disrupting these pathogens’ microbiota is therefore an approach with vast therapeutic potential, although it remains largely underexploited. 

A remarkable example is Wolbachia – a bacterial microbiota found in arthropods including mosquitos and roundworms. Wolbachia is essential for the survival of parasitic worms that cause lymphatic filariasis (elephantiasis) and onchocerciasis (river blindness), both devastating but neglected diseases that affect about 120 million people worldwide.

There is no rapid cure for these infections, and current treatments are contraindicated for children under 9 and pregnant women, so more effective treatments are urgently needed. Previous clinical studies have shown promising results for antibiotics targeting Wolbachia for treating human filarial diseases [1]. However, identifying new drug targets for Wolbachia is a significant challenge.

Benzoxaboroles, a family of boron-based compounds, target Leucyl-tRNA synthetase (LeuRS), an enzyme involved in protein synthesis. These compounds have demonstrated potent antimicrobial activities, exemplified by Tavaborole (an FDA-approved anti-fungal), Epetraborole, and Ganfeborole, which are currently in phase II clinical trials for anti-Gram-negative and anti-tubercular uses, respectively. 

This study aimed to detect active compounds against Wolbachia by screening a library of benzoxaboroles using an infection model of Wolbachia. Scientists identified the most effective molecules by observing Wolbachia depletion inside mosquito cells via fluorescence, as shown in Figure 1. This strategy, combined with activity experiments, yielded potent inhibitors with in vitro and in cellulo efficacy in the low nanomolar range.
 

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Fig. 1: Depletion of Wolbachia bacteria in mosquito-derived cells after treatment with Benzoxaboroles. a) C6/36 insect cells (A. albopictus) stably infected with Wolbachia were analyzed by fluorescence. Single-cell image analysis shows the nucleus (fluorescent large shapes) in the centre surrounded by colonizing Wolbachia (small fluorescent dots) in the cytoplasm. b) Reduction of Wolbachia load in host cells treated with Benzoxaboroles at 9 μM for 7 days. 

Microcalorimetry and thermal shift assays confirmed that the molecular target was Wolbachia’s Leucyl-tRNA synthetase (LeuRS), with the best compound showing single-digit nanomolar binding affinity. The most promising candidates were further analysed using nuclear magnetic resonance (NMR) and X-ray crystallography at the ESRF, to understand the inhibition mechanism in detail.

Crystal structures of the LeuRS-ligand complex, with or without tRNA, obtained at beamlines ID29, ID23-1, ID23-2 and ID30A-1, revealed that benzoxaboroles bind to the editing site of LeuRS, forming a covalent adduct with the ribose hydroxyl groups of either AMP or tRNA^Leu terminal adenosine (Figure 2), and thus trapping the enzyme in an unproductive conformation that blocks protein synthesis.

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Fig. 2: Model of Wolbachia LeuRS built up by using as templates the crystal structures of the Wolbachia LeuRS editing domain and of the full-length E. coli LeuRS–tRNA^Leu-Cmpd9, both determined at the ESRF beamlines. The zoomed-in view shows the adduct formed by the tRNA^Leu terminal adenosine with the oxaborole group of compound 9.

Interestingly, microcalorimetry showed no binding of the benzoxaboroles to LeuRS in the absence of AMP, while NMR experiments demonstrated that the boron-based compound could directly interact with AMP, ATP or tRNA^Leu to form the covalent adduct. Thus, similar to the anti-tubercular Ganfeborole, benzoxaboroles are prodrugs capable of self-activating with adenosine-based molecules in a LeuRS-independent manner [2].

The high-resolution crystal structures of Wolbachia and E. coli (used as a model) LeuRS revealed a deep network of interactions in the drug-binding pocket, including the coordination of a positively charged hydroxonium ion with the benzoxaborole’s negatively-charged boron atom, explaining the selectivity of these inhibitors. Additionally, two cysteines specific to the Wolbachia genus were found approximately 4 Å away from the active molecule. These insights could enable the development of narrow-spectrum anti-infectives targeting Wolbachia, a crucial property for developing new antimicrobials that minimize dysbiosis in patients.

In conclusion, this study highlights the benefits of selectively disrupting bacterial microbiota that are in strong symbiosis with host pathogens causing human disease. As research continues to uncover the complexities of the microbiota, transformative breakthroughs are anticipated that will revolutionize the control of infectious diseases.

Principal publication and authors
Targeting a microbiota Wolbachian aminoacyl-tRNA synthetase to block its pathogenic host, G. Hoffmann (a), M. Lukarska (a), R.H. Clare (b), E.K.G. Masters (b), K.L. Johnston (b), L. Ford (b), J.D. Turner (b), S.A. Ward (b), M.J. Taylor (b), M.R. Jensen (c), A. Palencia (a), Sci. Adv. 10, eado1453 (2024); https://doi.org/10.1126/sciadv.ado1453
(a) Institute for Advanced Biosciences (IAB), Grenoble (France)
(b) Liverpool School of Tropical Medicine, Liverpool (UK)
(c) Institut de Biologie Structurale (IBS), Grenoble (France)

References
[1] M.J. Taylor et al., The Lancet 365, 2116-2121 (2005).
[2] G. Hoffmann et al., JACS 145, 800-810 (2023).