Ribosome-Templated Azide–Alkyne Cycloadditions Using Resistant Bacteria as Reaction Vessels: in Cellulo Click Chemistry

Abstract

click chemistry has been a powerful method for fragment-based drug design since its discovery in 2002. Recently, we demonstrated that the bacterial ribosome can template the azide-alkyne cycloaddition reaction to expedite the discovery of novel antibiotics. We now report this process can be performed in an antibiotic-resistant bacterial cell. The corresponding triazole products formed are potent antibiotics that inhibit bacterial growth; moreover, the potency of each cycloadduct can be visualized using the traditional MIC assay in a 96-well plate format. We characterized the clicked products by independent chemical synthesis and LC-MS analysis, which showed that mass count percent increase was directly proportional to 1/MIC. In other words, potent compounds detected by MIC were formed in greater amounts. Control experiments unambiguously showed the ribosome was responsible for templating triazole formation. Significantly, our method (1) obviates the need to isolate bacterial ribosomes; (2) could be applied to different bacterial strains, which broadens the scope and facilitates the discovery of narrow-spectrum antibiotics; and (3) does not require the knowledge of mode-of-action and thus could uncover novel antibiotic targets. We believe this method could be expanded and implemented as a novel approach for antibiotic drug discovery.

From Screening to Targeted Degradation: Strategies for the Discovery and Optimization of Small Molecule Ligands for PCSK9

“…Protein-templated in situ click chemistry has been shown to lower the activation barrier required for an azide and alkyne to irreversibly form a 1,2,3-triazole without the use of metal catalysis (Mamidyala and Finn, 2010). In addition, this type of proximity-driven protein-templated reactivity has been applied to the templatedriven optimization of inhibitors for multiple target classes, to rapidly discover selective ligands with high affinity for their target protein (Bhardwaj et al, 2017;Jin et al, 2018;Millward et al, 2012;Thirumurugan et al, 2013). Thus, we used a target-guided ligand optimization approach wherein we coupled proximity-driven in situ click chemistry with AS/MS to directly detect ligands bound to PCSK9, without the need for additional denaturation or purification steps (Figure 2A).…”

Ribosome‐targeting antibacterial agents: Advances, challenges, and opportunities

“…This method has three advantages: (1) Compared with other in situ click chemistry strategies 140,141 and in vitro transcription/translation systems, this method couples chemical synthesis and activity evaluation, which greatly improved the efficiency of lead compound discovery. (2) This system employs crude bacterial ribosome extracts for target‐oriented synthesis and activity tests.…”

Kinetic Target-Guided Synthesis: Reaching the Age of Maturity

“…[32][33] More than 12% of warhead-bearing reagents for enzyme-templated KTGS are inspired by the substrate or co-factor, like biotin 34,35 or NAD 28 . A few warhead-bearing reagents were directly derived from drugs (9.4%) that are exclusively natural compounds: kanamycin 36 and solithromycin 37 . Among the reagents inspired by previous SAR (62.5%) some were highly inspired by drugs or clinical candidates like celecoxib 30 for COX2 inhibitors, rupintrivir (AG7088) 38 for enteroviral protease inhibitors or navitoclax (ABT-263) 21 for Bcl inhibitors.…”