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bioRxiv · 10.64898/2026.09.27.751175

Natural product-derived azaphilones are selective scaffolds for covalent lysine liganding in live cells

Ding, W.·Brameyer, S.·Kirsch, S. H.·Sandargo, B.·Surup, F.·Hacker, S. M.·Jung, K.·Sieber, S.
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Abstract

Lysine-directed covalent liganding offers a promising strategy to expand the covalently ligandable proteome beyond cysteine. However, broadly applicable and selective lysine-reactive scaffolds remain limited. Here, we designed SCL-alkyne, a simplified azaphilone probe derived from the natural product sclerotiorin (SCL), for the global profiling of lysine reactivity in bacteria. SCL-alkyne displayed high intrinsic selectivity for lysine in vitro and in live Escherichia coli cells. In situ proteome profiling revealed that 84% of engaged sites belong to lysine residues and the protein N-terminus. Reactivity mapping provided an inventory of highly reactive sites showing that only a limited subset of reactive lysines is located in functional regions of proteins. Analysis of proteins enriched via the SCL-alkyne probe unraveled bacterial chemotaxis as a major target pathway which was confirmed by an impaired chemotactic response upon treatment of bacteria with SCL-alkyne. Competitive profiling of three structurally related azaphilone natural products identified 60 protein targets belonging to pathways related to iron-sulfur cluster assembly and antioxidant function. This study not only highlights the diverse modes-of-action of natural product azaphilones, but also establishes its core scaffold as effective lysine-reactive chemotype for identifying ligandable lysines.

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Ding, W., Brameyer, S., Kirsch, S. H., Sandargo, B., Surup, F., Hacker, S. M., Jung, K., Sieber, S.. 2026-09-28. Natural product-derived azaphilones are selective scaffolds for covalent lysine liganding in live cells. https://doi.org/10.64898/2026.09.27.751175

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