Quinolone antibiotics such as ciprofloxacin inhibit DNA gyrase, leading to DNA double-strand breaks that result in rapid bacterial killing and induction of the mutagenic SOS DNA repair response. By contrast, the ciprofloxacin analogue IMP-1700 inhibits ciprofloxacin-induced SOS, suggesting a novel mechanism of action. Here, we provide evidence that IMP-1700 targets the quinolone binding domain of DNA gyrase but triggers a significantly higher frequency of division septa in S. aureus compared with other DNA gyrase targeting antibiotics, including ciprofloxacin. In keeping with this finding, the lipopeptide antibiotic daptomycin, which targets the division septum, bound more strongly to IMP-1700-treated cells relative to S. aureus exposed to other DNA gyrase inhibitors, leading to increased bacterial killing. This finding extended to a panel of paired daptomycin susceptible and resistant clinical isolates. We conclude that the ciprofloxacin analogue IMP-1700 has distinct effects on the cell envelope of S. aureus, despite appearing to share the same target as the parent drug, which result in the resensitisation of daptomycin resistant bacteria to the lipopeptide antibiotic.

Necroptosis is a form of programmed cell death that, when dysregulated, is associated with cancer and inflammatory and neurodegenerative diseases. Here, starting from hits identified from a phenotypic high-throughput screen for inhibitors of necroptosis, we synthesized a library of compounds containing a 7-phenylquinoline motif and validated their anti-necroptotic activity in a novel live-cell assay. Based on these data, we designed an optimized photoaffinity probe for target engagement studies and through biochemical and cell-based assays established receptor-interacting kinase 1 (RIPK1) as the cellular target, with inhibition of necroptosis arising from the prevention of RIPK1 autophosphorylation and activation. X-ray crystallography and mass spectrometry revealed that these compounds bind at the hinge region of the active conformation of RIPK1, establishing them as type I kinase inhibitors. In addition, we demonstrated in vitro synergy with type III kinase inhibitors, such as necrostatin-1 and found that lead compounds protected mice against acute inflammation in necroptosis models in vivo. Overall, we present a novel pharmacophore for inhibition of human RIPK1, a key protein involved in necroptosis, and provide a photoaffinity probe to explore RIPK1 target engagement in cells.

Flaviviruses, including Zika virus (ZIKV), are a significant global health concern, yet no licensed antivirals exist to treat disease. The small membrane (M) protein plays well-defined roles during viral egress and remains within virion membranes following release and maturation. However, it is unclear whether M plays a functional role in this setting. Here, we show that M forms oligomeric membrane-permeabilising channels in vitro, with increased activity at acidic pH and sensitivity to the prototypic channel-blocker, rimantadine. Accordingly, rimantadine blocked an early stage of ZIKV cell culture infection. Structure-based channel models, comprising hexameric arrangements of two trans-membrane domain protomers were shown to comprise more stable assemblages than other oligomers using molecular dynamics simulations. Models contained a predicted lumenal rimantadine-binding site, as well as a second druggable target region on the membrane-exposed periphery. In silico screening enriched for repurposed drugs/compounds predicted to bind to either one site or the other. Hits displayed superior potency in vitro and in cell culture compared with rimantadine, with efficacy demonstrably linked to virion-resident channels. Finally, rimantadine effectively blocked ZIKV viraemia in preclinical models, supporting that M constitutes a physiologically relevant target. This could be explored by repurposing rimantadine, or development of new M-targeted therapies.

The design, synthesis, and conformational analysis of a novel aromatic oligoester helix mimetic scaffold is reported. A range of amino acid-type side-chain functionality can be readily incorporated into monomer building blocks over three facile synthetic steps. Analysis of representative dimers revealed a stable conformer capable of effective mimicry of a canonical α-helix and the scaffold was found to be surprisingly stable to degradation in aqueous solutions at acidic and neutral pH.

A core skill for practicing organic chemists is the ability to apply organic chemistry to design experiments. In this article we describe an activity to help students along the pathway toward developing into practicing organic chemists. The workshop teaches students how to use organic chemistry to design synthetic organic chemistry experiments by connecting theory to the choices of various reaction conditions, which the students had to choose for their target molecules. Students who undertook this activity were able to design procedures yielding their target molecules and evidenced the development of experimental design skills.

Inhibition of inflammasome and pyroptotic pathways are promising strategies for clinical treatment of autoimmune and inflammatory disorders. MCC950, a potent inhibitor of the NLR-family inflammasome pyrin domain-containing 3 (NLRP3) protein, has shown encouraging results in animal models for a range of conditions; however, until now, no off-targets have been identified. Herein, we report the design, synthesis, and application of a novel photoaffinity alkyne-tagged probe for MCC950 (IMP2070) which shows direct engagement with NLRP3 and inhibition of inflammasome activation in macrophages. Affinity-based chemical proteomics in live macrophages identified several potential off-targets, including carbonic anhydrase 2 (CA2) as a specific target of IMP2070, and independent cellular thermal proteomic profiling revealed stabilization of CA2 by MCC950. MCC950 displayed noncompetitive inhibition of CA2 activity, confirming carbonic anhydrase as an off-target class for this compound. These data highlight potential biological mechanisms through which MCC950 and derivatives may exhibit off-target effects in preclinical or clinical studies.

A set of web-based stereochemistry tools which help students use virtual 3D models to translate Newman projections and chair conformations and assign R/S labels to stereocenters from 2D dashed-wedged structures is described. These tools are unique in the way they link 2D dashed-wedged structures to 3D models to help students develop visual/spatial skills. They also provide feedback on the individual parts of a student's attempt to switch between structural representations and apply the Cahn-Ingold-Prelog rules. The incorporation of these stereochemistry resources into an organic chemistry course led to improvements in students' ability to draw accurate Newman projections and chair conformations and assign R and S labels to stereocenters.