Inhibiting Protein Prenylation with Benzoxaboroles to Target Fungal Plant Pathogens

Abstract

Fungal pathogens pose an increasing threat to global food security through devastating effects on staple crops and contamination of food supplies with carcinogenic toxins. Widespread deployment of agricultural fungicides has increased crop yields but is driving increasingly frequent resistance to available agents and creating environmental reservoirs of drug-resistant fungi that can also infect susceptible human populations. To uncover non-cross-resistant modes of antifungal action, we leveraged the unique chemical properties of boron chemistry to synthesize novel 6-thiocarbamate benzoxaboroles with broad spectrum activity against diverse fungal plant pathogens. Through whole genome sequencing of Saccharomyces cerevisiae isolates selected for stable resistance to these compounds, we identified mutations in the protein prenylation-related genes, CDC43 and ERG20. Alleleswapping experiments confirmed that point mutations in CDC43, which encodes an essential catalytic subunit within geranylgeranyl transferase I (GGTase I) complex, were sufficient to confer resistance to the benzoxaboroles. Mutations in ERG20, which encodes an upstream farnesyl pyrophosphate synthase in the geranylgeranylation pathway, also conferred resistance. Consistent with impairment of protein prenylation, the compounds disrupted membrane localization of the classical geranylgeranylation substrate Cdc42. Guided by molecular docking predictions, which favored Cdc43 as the most likely direct target, we overexpressed and purified functional GGTase I complex to demonstrate direct binding of benzoxaboroles to it and concentration-dependent inhibition of its transferase activity. Further development of the boron-containing scaffold described here offers a promising path to the development of GGTase I inhibitors as a mechanistically distinct broad spectrum fungicide class with reduced potential for cross-resistance to antifungals in current use.

Probing the Molecular Function of Novel Boron-containing Compounds

“…With the advancements in boron neutron capture therapy and the approval of the bortezomib (Velcade®), the first boron-containing therapeutic approved by the U.S. Food and Drug Administration (FDA), the interest in the potential applications of BCCs has exponentially increased in the last two decades. Boron containing compounds (BCC) can exhibit an extensive range of biological effects, including anticancer 21 , antiviral 14,22 , antifungal 23,24 , antibacterial 14,25 , antiprotozoal [26][27][28] , antibiotic 29 , anticancerogenic 30 , antimutagenic 31 , anti-inflammatory 32,33 , anti-oxidative 34,35 as well as antiangiogenic 36 . This is due to their stability, broad range of reactivity and low toxicity.…”