Two-component signal transduction systems (TCSs) are widespread types of protein machinery, typically consisting of a histidine kinase membrane sensor and a cytoplasmic transcriptional regulator that can sense and respond to environmental signals. TCSs are responsible for modulating genes involved in a multitude of bacterial functions, including cell division, motility, differentiation, biofilm formation, antibiotic resistance, and virulence. Pathogenic bacteria exploit the capabilities of TCSs to reprogram gene expression according to the different niches they encounter during host infection. This review focuses on the role of TCSs in regulating the virulence phenotype of Shigella, an intracellular pathogen responsible for severe human enteric syndrome. The pathogenicity of Shigella is the result of the complex action of a wide number of virulence determinants located on the chromosome and on a large virulence plasmid. In particular, we will discuss how five TCSs, EnvZ/OmpR, CpxA/CpxR, ArcB/ArcA, PhoQ/PhoP, and EvgS/EvgA, contribute to linking environmental stimuli to the expression of genes related to virulence and fitness within the host. Considering the relevance of TCSs in the expression of virulence in pathogenic bacteria, the identification of drugs that inhibit TCS function may represent a promising approach to combat bacterial infections.

Waldiomycin is an inhibitor of histidine kinases (HKs). Although most HK inhibitors target the ATP-binding region, waldiomycin binds to the intracellular dimerization domain (DHp domain) with its naphthoquinone moiety presumed to interact with the conserved H-box region. To further develop inhibitors targeting the H-box, various 2-aminonaphthoquinones with cyclic, aliphatic, or aromatic amino groups and naphtho [2,3-d] isoxazole-4,9-diones were synthesized. These compounds were tested for their inhibitory activity (IC₅₀) against WalK, an essential HK for Bacillus subtilis growth, and their minimum inhibitory concentrations (MIC) against B. subtilis. As a result, 11 novel HK inhibitors were obtained as naphthoquinone derivatives (IC₅₀: 12.6–305 µM, MIC: 0.5–128 µg ml⁻¹). The effect of representative compounds on the expression of WalK/WalR regulated genes in B. subtilis was investigated. Four naphthoquinone derivatives induced the expression of iseA (formerly yoeB), whose expression is negatively regulated by the WalK/WalR system. This suggests that these compounds inhibit WalK in B. subtilis cells, resulting in antibacterial activity. Affinity selection/mass spectrometry analysis was performed to identify whether these naphthoquinone derivatives interact with WalK in a manner similar to waldiomycin. Three compounds were found to competitively inhibit the binding of waldiomycin to WalK, suggesting that they bind to the H-box region conserved in HKs and inhibit HK activity.

Waldiomycin is a natural product originally isolated from actinomycete extracts. It inhibits histidine kinases (HKs), which are essential components of bacterial two-component signal transduction systems, by targeting the conserved H-box region and suppressing autophosphorylation. Here, we identified novel HK inhibitors using amino acid mapping (AAM)-based virtual screening. We compared the AAM descriptor of the naphthoquinone moiety of waldiomycin, which directly interacts with the H-box region, with those of approximately 12 million commercially available compounds and identified 714 candidates with similar descriptor values. From these candidates, we selected 9-hydroxy- 4H -pyrido[1,2- a ]pyrimidin-4-one as a promising and structurally distinct scaffold and synthesized 15 derivatives. The inhibitory activities of these derivatives were evaluated against the HKs WalK, VicK, and EnvZ. Three 4 H -pyrido[1,2- a ]pyrimidin-4-one derivatives ( FF-054 , FF-055 , and FF-060 ) exhibited potent inhibitory activity, with IC ₅₀ values ranging from 4.39 to 259 µM. Notably, the IC ₅₀ values of these compounds increased three- to six-fold against the EnvZ S242A mutant, in which the conserved H-box residue is substituted. Furthermore, nuclear magnetic resonance (NMR) analysis using the EnvZ DHp domain (residues 223–289, encompassing the H-box region) confirmed direct interaction with this domain. Taken together, these results demonstrate that pyrido[1,2- a ]pyrimidin-4-one derivatives FF-054 , FF-055 , and FF-060 are novel HK inhibitors that target the H-box region. This study represents the first successful application of AAM-based screening to identify HK inhibitors targeting this highly conserved region.