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Bacterial DNA gyrase is considered one of the validated targets for antibacterial drug discovery. Benzopyrones have been reported as promising derivatives that inhibit bacterial DNA gyrase B through competitive binding into the ATP binding site of the B subunit. In this study, we designed and synthesized twenty-two benzopyrone-based derivatives with different chemical features to assess their antimicrobial and photosensitizing activities. The antimicrobial activity was evaluated against B. subtilis, S. aureus, E. coli, and C. albicans. Compounds 6a and 6b (rigid tetracyclic-based derivatives), 7a-7f (flexible-linker containing benzopyrones), and 8a-8f (rigid tricyclic-based compounds) exhibited promising results against B. subtilis, S. aureus, and E. coli strains. Additionally, these compounds demonstrated photosensitizing activities against the B. subtilis strain. Both in silico molecular docking and in vitro DNA gyrase supercoiling inhibitory assays were performed to study their potential mechanisms of action. Compounds 8a-8f exhibited the most favorable binding interactions, engaging with key regions within the ATP binding site of the DNA gyrase B domain. Moreover, compound 8d displayed the most potent IC50 value (0.76 μM) compared to reference compounds (novobiocin = 0.41 μM and ciprofloxacin = 2.72 μM). These results establish a foundation for structure-based optimization targeting DNA gyrase inhibition with antibacterial activity.

The inhibitory-kappaB kinases (IKKs) IKKα and IKKβ play central roles in regulating the non-canonical and canonical NF-κB signalling pathways. Whilst the proteins that transduce the signals of each pathway have been extensively characterised, the clear dissection of the functional roles of IKKα-mediated non-canonical NF-κB signalling versus IKKβ-driven canonical signalling remains to be fully elucidated. Progress has relied upon complementary molecular and pharmacological tools; however, the lack of highly potent and selective IKKα inhibitors has limited advances. Herein, we report the development of an aminoindazole-pyrrolo[2,3-b]pyridine scaffold into a novel series of IKKα inhibitors. We demonstrate high potency and selectivity against IKKα over IKKβ in vitro and explain the structure–activity relationships using structure-based molecular modelling. We show selective target engagement with IKKα in the non-canonical NF-κB pathway for both U2OS osteosarcoma and PC-3M prostate cancer cells by employing isoform-related pharmacodynamic markers from both pathways. Two compounds (SU1261 [IKKα Ki = 10 nM; IKKβ Ki = 680 nM] and SU1349 [IKKα Ki = 16 nM; IKKβ Ki = 3352 nM]) represent the first selective and potent pharmacological tools that can be used to interrogate the different signalling functions of IKKα and IKKβ in cells. Our understanding of the regulatory role of IKKα in various inflammatory-based conditions will be advanced using these pharmacological agents.