Synergetic Antimicrobial Activity and Mechanism of Clotrimazole-Linked CO-Releasing Molecules

This work was financially supported by Fundação para a Ciência e Tecnologia (Portugal) through fellowship PD/BD/ 148006/2019 (SSM), PTDC/SAU-INF/29313/2017 grant, and R&D unit LISBOA-01-0145-FEDER007660 (MostMicro) cofounded by FCT/MCTES and FEDER funds under the PT2020 Partnership Agreement. The NMR data was acquired at CERMAX, Instituto de Tecnologia Quıḿ ica e Bioloǵ ica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal, with equipment funded by FCT, project AAC 01/ SAICT/2016. This work was partially supported by the PPBIPortuguese Platform of BioImaging (PPBI-POCI-01- 0145-FEDER-022122) cofunded by national funds from OE “Orçamento de Estado” and by European funds from FEDER“Fundo Europeu de Desenvolvimento Regional”. LMS and SSM acknowledge funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no. 810856. H.B.-O., T.S., C.M., F.O., J.B., and M.A. gratefully acknowledge funding by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), project ID 398967434 (TRR 261, projects A01, A06, A10, and Z02). A.B. appreciates funding by the German Federal Ministry for Education and Research (project Gramneg. Design).Several metal-based carbon monoxide-releasing molecules (CORMs) are active CO donors with established antibacterial activity. Among them, CORM conjugates with azole antibiotics of type [Mn(CO)3(2,2′-bipyridyl)(azole)]+ display important synergies against several microbes. We carried out a structure-activity relationship study based upon the lead structure of [Mn(CO)3(Bpy)(Ctz)]+ by producing clotrimazole (Ctz) conjugates with varying metal and ligands. We concluded that the nature of the bidentate ligand strongly influences the bactericidal activity, with the substitution of bipyridyl by small bicyclic ligands leading to highly active clotrimazole conjugates. On the contrary, the metal did not influence the activity. We found that conjugate [Re(CO)3(Bpy)(Ctz)]+ is more than the sum of its parts: while precursor [Re(CO)3(Bpy)Br] has no antibacterial activity and clotrimazole shows only moderate minimal inhibitory concentrations, the potency of [Re(CO)3(Bpy)(Ctz)]+ is one order of magnitude higher than that of clotrimazole, and the spectrum of bacterial target species includes Gram-positive and Gram-negative bacteria. The addition of [Re(CO)3(Bpy)(Ctz)]+ to Staphylococcus aureus causes a general impact on the membrane topology, has inhibitory effects on peptidoglycan biosynthesis, and affects energy functions. The mechanism of action of this kind of CORM conjugates involves a sequence of events initiated by membrane insertion, followed by membrane disorganization, inhibition of peptidoglycan synthesis, CO release, and break down of the membrane potential. These results suggest that conjugation of CORMs to known antibiotics may produce useful structures with synergistic effects that increase the conjugate's activity relative to that of the antibiotic alone.publishersversionpublishe