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Isocyanides inhibit bacterial pathogents by covalent targeting of essential metabolic enzymes

ABSTRACT: Isonitrile (also termed isocyanide) natural products exhibit potent antimicrobial activities, but little is known about their molecular targets. Here, we focus on the so far neglected group of monoisonitriles to gain further insights into their antimicrobial mode of action (MoA). Screening a focused monoisonitrile library revealed a potent S. aureus growth inhibitor with a different MoA compared to previously described diisonitrile antibiotics. Chemical proteomics via competitive cysteine reactivity profiling, unraveled covalent modifications of two essential metabolic enzymes involved in the fatty acid biosynthetic process (FabF) and the hexosamine pathway (GlmS) at their active site cysteines. In-depth studies with the recombinant enzymes demonstrated concentration-dependent labeling, covalent binding to the catalytic site and corresponding functional inhibition by the isocyanide. Thermal proteome profiling and full proteome studies of compound-treated S. aureus further highlighted the destabilization and dysregulation of proteins related to the targeted pathways. The here described novel, covalent isocyanide MoA highlights the versatility of the functional group, making it a great tool and out-of-the-box starting point for the development of innovative antibiotics.

INSTRUMENT(S): Orbitrap Fusion, Q Exactive

ORGANISM(S): Staphylococcus Aureus

SUBMITTER: Nina Bach

LAB HEAD: Stephan Axel Sieber

PROVIDER: PXD050437 | Pride | 2024-06-26

REPOSITORIES: Pride

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			Action	DRS
	20220614_AG_NCTC8325_0_39I16_exp_1.raw	Raw
	20220614_AG_NCTC8325_0_39I16_exp_2.raw	Raw
	20220614_AG_NCTC8325_0_39I16_exp_3.raw	Raw
	20220614_AG_NCTC8325_0_39I16_exp_wt.raw	Raw
	20220614_AG_NCTC8325_0_39I16_stat_1.raw	Raw

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Project description:Drug-resistant bacterial pathogens still cause high levels mortality annually despite the availability of many antibiotics. Staphylococcus aureus (MRSA) is especially problematic and the rise in resistance to front line treatments like vancomycin and linezolid calls for new chemical modalities to treat chronic and relapsing S, aureus infections. Halogenated N-(1,3,4-oxadiazol-2-yl)benzamides are interesting class of antimicrobial agents, which have been described by multiple groups to be effective against different bacterial pathogens. The modes of action of a few N-(1,3,4-oxadiazol-2-yl)benzamides have been elucidated. For example, oxadiazoles KKL-35 and MBX-4132, have been described as inhibitors of trans-translation (a ribosome rescue pathway) while HSGN-94 was shown to inhibit lipoteichoic acid. However other similarly halogenated N-(1,3,4-oxadiazol-2-yl)benzamides neither inhibit trans-translation nor LTA but are potent antimicrobial agents. For example, HSGN-220, -218, and -144 are N-(1,3,4-oxadiazol-2-yl)benzamides that are modified with OCF3, SCF3 or SF5, and have remarkable minimum inhibitory concentrations (MICs) ranging from 1 to 0.06 microgram per mL against MRSA clinical isolates and show a low propensity to resistance to MRSA over 30 days. The mechanism of action (MOA) of these highly potent oxadiazoles is however unknown. To provide insights into how these halogenated N-(1,3,4-oxadiazol-2-yl)benzamides inhibit bacterial growth, we performed global proteomics and RNA expression analysis of some essential genes of S. aureus treated with HSGN-220, -218, and -144. These studies revealed that the oxadiazoles HSGN-220, -218, and -144 are multi-action antibiotics that regulate menaquinone biosynthesis and other essential proteins like DnaX, Pol IIIC, BirA, LexA, and DnaC. In addition, these halogenated N-(1,3,4-oxadiazol-2-yl)benzamides were able to depolarize bacterial membranes and regulate siderophore biosynthesis and heme regulation. Iron starvation appears to be part of the MOA that led to bacterial killing. This study demonstrates that N-(1,3,4-oxadiazol-2-yl)benzamides are indeed privileged scaffolds for the development of antibacterial agents and that subtle modifications lead to changes to MOA.

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Isocyanides inhibit bacterial pathogents by covalent targeting of essential metabolic enzymes

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