Promysalin Elicits Species-Selective Inhibition of Pseudomonas aeruginosa by Targeting Succinate Dehydrogenase

Abstract

Natural products have served as an inspiration to scientists both for their complex three-dimensional architecture and exquisite biological activity. Promysalin is one such Pseudomonad secondary metabolite that exhibits narrow-spectrum antibacterial activity, originally isolated from the rhizosphere. We herein utilize affinity-based protein profiling (AfBPP) to identify succinate dehydrogenase (Sdh) as the biological target of the natural product. The target was further validated in silico, in vitro, in vivo, and through the selection, and sequencing, of a resistant mutant. Succinate dehydrogenase plays an essential role in primary metabolism of Pseudomonas aeruginosa as the only enzyme that is involved both in the tricarboxylic acid cycle (TCA) and in respiration via the electron transport chain. These findings add credence to other studies that suggest that the TCA cycle is an understudied target in the development of novel therapeutics to combat P. aeruginosa, a significant pathogen in clinical settings.

Thinking Outside the Box—Novel Antibacterials To Tackle the Resistance Crisis

“…After binding to cellular protein targets and cell lysis,t he alkyne group allows conjugation with azide-functionalized fluorophore or affinity tags by the copper-catalyzed click reaction. [127] Promysalin is an effective antibacterial compound in P. aeurigonsa;however, its mode of action was poorly understood. Additional functionalization with photo-cross-linking moieties (such as benzophenones,a ryl azides,o rd iazirines) has extended the application to noncovalent parent compounds and is known as affinity-based protein profiling (AfBPP).…”

“…Additional functionalization with photo-cross-linking moieties (such as benzophenones,a ryl azides,o rd iazirines) has extended the application to noncovalent parent compounds and is known as affinity-based protein profiling (AfBPP). [127] In addition to small molecules,A(f)BPP has also been applied for the target elucidation of antibacterial peptides by means of modified amino acids. [119] There are numerous examples in which A(f)BPP revealed the molecular targets of small molecules in eukaryotic and prokaryotic cells.…”

From General to Specific: Can Pseudomonas Primary Metabolism Be Exploited for Narrow‐Spectrum Antibiotics?

“…Controlled by the Gac regulatory system, P. putida produces this secondary metabolite, which promotes its own swarming and biofilm formation and exhibits species‐specific growth inhibitory activity against other Pseudomonads, including human‐pathogenic P. aeruginosa (IC 50 =67 n m against PA14; IC 50 =4.1 μ m against PA01) . The initial hypothesis that the mechanism of action was through chelation of iron, preventing P. aeruginosa from sequestering sufficient iron for growth, was refuted when promysalin's potency was seemingly unaffected by varying the iron concentration . Ultimately, the succinate dehydrogenase C‐subunit (SdhC) was identified as the biological target through an affinity‐based protein profiling approach; this was further validated by computational molecular docking as well as genome sequencing of a promysalin‐resistant PA14 mutant (Scheme ) …”

Über bisherige Denkweisen hinaus – neue Wirkstoffe zur Überwindung der Antibiotika‐Krise

“…B. einen Alkin-Tag, zur späteren bioorthogonalen Modifikation verfügen. [127] Promysalin ist ein effektiver antibakterieller Wirkstoff gegen P. aeruginosa,d essen molekularer Wirkmechanismus jedoch kaum verstanden war.D ad as Molekülk einerlei offensichtlich kovalente Gruppen aufweist, wurde ein Derivat mit einer photovernetzenden Gruppe und einem Alkin-Tag [128] synthetisiert. [122,123] Bei Konjugation mit einem Fluorophor-Azid werden die Zielproteine anschließend mit-tels SDS-PAGEa ufgetrennt und durch Fluoreszenz-Imaging des Gels visualisiert.…”

“…Im Anschluss an die Bindung der zellulären Zielproteine und die Zelllyse kann die Alkin-Einheit zur Konjugation an Azid-modifizierte Fluorophore oder Affinitäts-Tags mittels Kupfer-katalysierter Klick-Reaktion verwendet werden. [127] Im Allgemeinen kann A(f)BPP nicht nur zur Zielprotein-Identifizierung von niedermolekularen Verbindungen verwendet werden, sondern auch fürdie Target-Analyse von antibakteriellen Peptiden, in die modifizierte Aminosäuren eingebracht wurden. Sofern ein Affinitäts-Tag (z.…”