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In view of the relentless increase in antibiotic resistance in human pathogens, efforts are needed to safeguard our future therapeutic options against infectious diseases. In addition to regulatory changes in our antibiotic use, this will have to include the development of new therapeutic compounds. One area that has received growing attention in recent years is the possibility to treat or prevent infections by targeting the virulence mechanisms that render bacteria pathogenic. Antivirulence targets include bacterial adherence, secretion of toxic effector molecules, bacterial persistence through biofilm formation, quorum sensing and immune evasion. Effective small-molecule compounds have already been identified that suppress such processes. In this review, we discuss the susceptibility of such compounds to the development of resistance, by comparison with known resistance mechanisms observed for classical bacteriostatic or bacteriolytic antibiotics, and by review of available experimental case studies. Unfortunately, appearance of resistance mechanisms has already been demonstrated for some, showing that the quest of new, lasting drugs remains complicated.

In this review we discuss the ideas behind antivirulence therapies, the progress in the field, and more particular the anticipated susceptibility of antivirulence therapies to drug resistance mechanisms. For this analysis, we first review mechanisms of antibacterial resistance known from classical, on-the-market antibacterials, and than discuss their relevance with respect to two broad groups of antivirulence therapeutics: quorum sensing inhibitors and bacterial adherence inhibitors in Gram-negative pathogens.

Large substituents are commonly seen as entirely repulsive through steric hindrance. Such groups have additional attractive effects arising from weak London dispersion forces between the neutral atoms. Steric interactions are recognized to have a strong influence on isomerization processes, such as in azobenzene-based molecular switches. Textbooks indicate that steric hindrance destabilizes the Z isomers. Herein, we demonstrate that increasing the bulkiness of electronically equal substituents in the meta-position decreases the thermal reaction rates from the Z to the E isomers. DFT computations revealed that attractive dispersion forces essentially lower the energy of the Z isomers.

London’s calling: It is generally accepted that large alkyl substituents destabilize the Z isomer of double bonds. In the case of meta-substituted azobenzenes, it could be shown that the stability of the Z isomer increases as the size of the substituent grows. Computations show that the enhancement in stability is based on intramolecular London dispersion forces, which compete with steric hindrance in the Z isomers.

Quaternary ammonium compounds (QACs) are a vital class of antiseptics. Recent investigations into their construction are uncovering novel and potent multicationic variants. Based on a trisQAC precedent, we have implemented a scaffold-hopping approach to develop alternative QAC architectures that display 1–3 long alkyl chains in specific projections from cyclic and branched core structures bearing 3–4 nitrogen atoms. The preparation of 30 QAC structures allowed for correlation of scaffold structure with antimicrobial activity. We identified QACs with limited conformational flexibility that have improved bioactivity against planktonic bacteria as compared to their linear counterparts. We also confirmed that resistance, as evidenced by an increased minimum inhibitory concentration (MIC) for methicillin-resistant Staphylococcus aureus (MRSA) compared to methicillin-susceptible Staphylococcus aureus (MSSA), can reduce efficacy up to 64-fold for monocationic QACs. Differentiation of antimicrobial and anti-biofilm activity, however, was not observed, suggesting that these compounds utilize a non-specific mode of eradication.

Hopping along: Bacteria resistant to quaternary ammonium compounds (QACs) present significant health threats, but novel multicationic variants (multiQACs) show promise for addressing this issue. Using a scaffold-hopping approach, we have efficiently prepared alternate architectures of multiQACs that show excellent potency against methicillin-resistant Staphylococcus aureus (MRSA) and biofilms.

Transmembrane proteins are critical for signaling, transport, and metabolism, yet their reconstitution in synthetic membranes is often challenging. Non-enzymatic and chemoselective methods to generate phospholipid membranes in situ would be powerful tools for the incorporation of membrane proteins. Herein, the spontaneous reconstitution of functional integral membrane proteins during the de novo synthesis of biomimetic phospholipid bilayers is described. The approach takes advantage of bioorthogonal coupling reactions to generate proteoliposomes from micelle-solubilized proteins. This method was successfully used to reconstitute three different transmembrane proteins into synthetic membranes. This is the first example of the use of non-enzymatic chemical synthesis of phospholipids to prepare proteoliposomes.

Membrane Proteins Inc.: The spontaneous reconstitution of functional integral membrane proteins (gray) during the de novo synthesis of biomimetic phospholipid bilayers is described. The method takes advantage of bioorthogonal coupling reactions for the non-enzymatic generation of proteoliposomes from micelle-solubilized proteins. This chemoselective approach results in a fast and clean reconstitution without the need for dialysis to remove excess detergent.

Vertebrate glycans constitute a large, important, and dynamic set of post-translational modifications that are notoriously difficult to manipulate and image. Although the chemical reporter strategy has been used in conjunction with bioorthogonal chemistry to image the external glycosylation state of live zebrafish and detect tumor-associated glycans in mice, the ability to image glycans systemically within a live organism has remained elusive. Here, we report a method that combines the metabolic incorporation of a cyclooctyne-functionalized sialic acid derivative with a ligation reaction of a fluorogenic tetrazine, allowing for the imaging of sialylated glycoconjugates within live zebrafish embryos.

How exSiating! Systemic fluorescence imaging of cell-surface glycans inside a live animal is described for the first time. Metabolic incorporation of a cyclooctyne-functionalized sialic acid into glycans during zebrafish embryogenesis followed by ligation with a fluorogenic tetrazine enables the visualization of sialoglycoconjugates.

Glycosylphosphatidylinositol (GPI) anchoring of proteins to the cell surface is important for various biological processes, but GPI-anchored proteins are difficult to study. An effective strategy was developed for the metabolic engineering of cell-surface GPIs and GPI-anchored proteins by using inositol derivatives carrying an azido group. The azide-labeled GPIs and GPI-anchored proteins were then tagged with biotin on live cells through a click reaction, which allows further elaboration with streptavidin-conjugated dyes or other molecules. The strategy can be used to label GPI-anchored proteins with various tags for biological studies.

Sweet and sticky: An effective strategy was developed for the metabolic engineering of cell-surface glycosylphosphatidylinositols (GPIs) and GPI-anchored proteins by using azido-modified inositol (blue hexagon) derivatives. This strategy can be used to label GPI-anchored proteins with various tags through click chemistry in live cells for biological studies.