To find small molecules with the broad anti-HK activity, Wilke et al. (2015) developed a fluorescence polarization displacement assay-based high-throughput screening (HTS) method and performed the screen of ∼53,000 diverse small molecules for their competitive binding to the ATP site of HKs. In this work, the researchers employed three conserved HKs (HK853 from Thermotoga maritima, VicK from S. pneumoniae, and CheA from E. coli) representing the 11 subfamilies of HKs as enzymatic inhibition targets and used ADP-BODIPY, a fluorescent nonhydrolyzable adenosine diphosphate (ADP) probe, as an indicator for ATP-competitive inhibitors of HKs in the screening. The natural product luteolin (3’,4’,5,7-tetrahydroxyflavone, also referred to as LUT) was discovered, which is a widely distributed flavonoid compound derived from vegetables, fruits, and herbs with a range of proposed mechanisms, including antioxidant, and anti-inflammatory activities.

LUT is structurally similar to the flavonoid genistein, which was previously reported to inhibit a yeast HK but only showed a slight inhibition on HK853 activity in follow-up assays (Huang et al., 1992; Wilke et al., 2015). In contrast to the flavonoid genistein, LUT inhibited HK853, VicK, and CheA with the half-maximal inhibitory concentrations (IC₅₀s) of 15.1, 216, and 111 μM, respectively, and inhibited the growth of E. coli DC2 with a minimum inhibitory concentration (MIC) of 8 μg/ml, but showed no activity against B. subtilis 3610 (MIC >128 μg/ml), without significant cytotoxicity on Vero 76 cells (Wilke et al., 2015). Zhou et al. (2019) , by combining with Nuclear Magnetic Resonance (NMR), isothermal titration calorimetry (ITC), and molecular docking studies, it was predicted that LUT inhibits the autophosphorylation activity of HK853 from Thermotoga maritime (T. maritime) by occupying the binding pocket of ADP by forming hydrogen bonds and π-π stacking interaction. As the analysis of binding interactions was only based on molecular docking and has not been confirmed by any experimental data, in the current review, we do not include such information.

However, LUT is a well-known multiple targets binding antagonist with inhibition of fatty acid synthase, angiotensin, and vascular endothelial growth factor receptor. It also can bind to a Ser/Thr protein kinase named casein kinase 2(CK₂) (Lolli et al., 2012; National Center for Biotechnology Information, 2022). Thus, the poor selectivity of LUT for HKs has limited its further development as a specific HK inhibitor.

In the same study in which LUT was screened out, two benzothiazole compounds, compounds 11 and 12, were also obtained through the HTS method based on the fluorescence polarization displacement assay by Wilke et al. (2015). To distinguish the cited compounds in this review, they have been renamed by using the surname of the first author plus their original IDs. For example, the compounds 11 and 12 in the original paper were renamed as Wilke-11 and 12, respectively. All the compounds in this review, except those with specific names, are renamed according to this rule. Wilke-11 showed activities against HK853 and VicK, with IC₅₀ values of 1.21 and 75 μM, respectively, and Wilke-12, the known US Food and Drug Administration (FDA)- approved medicine named riluzole for amyotrophic lateral sclerosis, was found to be active against HK853, VicK, and CheA, with IC₅₀s of 7.15, 618, and 1,340 μM, respectively. Compared with Wilke-12, Wilke-11 exhibited increased HK inhibitory potency, suggesting that the aminobenzothiazole is possible to be an important scaffold in the development of HK inhibitors.

Wilke-11 inhibited the growth of B. subtilis 3610 and E. coli DC2, with MICs ranging from 49 μg/ml to 64 μg/ml and from 32 μg/ml to 64 μg/ml respectively, which is lower than that of Wilke-12 against the same bacteria (MIC = 293 and 128 μg/ml, respectively). However, these compounds were cytotoxic against Vero 76 cells at concentrations exhibiting antibacterial effects, which should be improved in further structural optimizations (Wilke et al., 2015).

In general, Wilke’s compounds provide a possible starting point for the design of broad-spectrum HK inhibitors and the aminobenzothiazole group is widely treated as a general scaffold for the following structural optimization in numerous bacterial species.

Subsequently, based on the leading compounds of Wilke-11 and 12, Goswami et al. (2018) generated a small library of compounds containing the benzothiazole as the centre core for HTS screening, and Rilu-4 and -12 were identified as the most promising compounds. Similar to Wilke-11, Rilu-12 contains a rigid tricyclic structure with two amino moieties, which performed an inhibitory effect with IC₅₀ as 1.56 μM against HK853. Rilu-4 contains an electron-withdrawing group −NO₂, and performed an IC₅₀ of 8.3 μM against HK853. Moreover, Rilu’s compounds significantly reduced the production of virulence factors, such as the Pseudomonas quinolone signals (PQS) and toxins (e.g., pyocyanin, phenazine-1-carboxamide, and phenazine-1-carboxylic acid) which were involved in QS and redox-balancing mechanisms. They severely affected the motility behaviors of P. aeruginosa PA14 (A strain obtained from a burn wound, which can encode a large number of TCSs in its genome), thereby decreasing the ability to swarm and attach to surfaces. These benzothiazole derivatives provide the possibilities of applications as potential antivirulence agents, which is a viable alternative to traditional antibacterial therapy for the treatment of infections caused by antibiotic-resistant organisms, either alone or in combination with antibiotic treatment. At present, researchers are aiming to improve the efficacy and explore the key inhibition mechanisms of Rilu-inhibitors.

Qin et al. (2006) firstly used a structure-based virtual screening (SBVS) method to discover potential inhibitors of the S. epidermidis WalK from a small molecular leading compound library, and among 76 candidates targeting the WalK ATP-binding domain. Seven compounds displayed significant growth inhibitory activity against S. epidermidis. Among them, Qin-2, -5, and -7 were thiazolidione analogs. Qin-2 and -5 were effective against S. aureus, S. pyogenes, and S. mutans, and Qin-7 was only effective against the non-biofilm-forming S. epidermidis ATCC 12228. All compounds inhibited the ATPase activity of WalK protein, with IC₅₀s ranging from 6.5 to 29 μM.

Pan et al. (2010) designed and synthesized a series of new 2-arylimino-3-aryl-thiazolidine-4-ones compounds based on the thiazolidione core structure of Qin-2 to acquire more effective and less toxic HK inhibitors, and six derivatives (Pan-10, Pan-12, Pan-20, Pan-27, Pan-28, and Huang-29, Figure 5 ) were synthesized by modifying the functional groups through cyclisation, aldol condensation, substitution, and hydrolysis reactions Huang et al. (2012). All six derivatives showed concentration-dependent inhibition of the autophosphorylation activity of WalK, with IC₅₀s of 88.35, 61.15, 34.83, 66.68, 22.15, and 82.51 μM, respectively, which are comparable to Qin-2 (IC₅₀ = 47.9 μM).

The bactericidal and biofilm-killing activities of Pan’s derivatives were significantly improved by comparing to Qin-2. It was observed that Pan-12, -20, -28, -27, and Huang-29 showed significant antibacterial activities against S. epidermidis and S. aureus with MICs ranging from 1.5 μg/ml to 6.6 μg/ml, and the antibacterial activity of Pan-10 was lower with MICs of 12.2 and 24.3 μg/ml against S. epidermidis and S. aureus respectively. All derivatives showed strong bactericidal activities against S. epidermidis ATCC35984 both in immature (6 h-old) biofilms and mature (24 h-old) biofilms, and eliminated proliferation of the immature biofilm. None of the derivatives displayed significant effects on the growth of E. coli strain ATCC 25922, cytotoxicity on Vero cells, or hemolysis induction in human erythrocytes (Huang et al., 2012).

To improve the antimicrobial activity and reduce the toxicity of Qin-2, another six analogs (Liu-38, Liu-39, Liu-57, Liu-60, Liu-74, and Liu-81, Figure 6) of Qin-2 were designed and synthesized by modifying functional groups. They showed inhibitory activities on the autophosphorylation of WalK, with IC₅₀ values ranging from 24.2 to 71.2 μM. These compounds displayed good antibacterial activities against S. epidermidis and S. aureus (MICs of 1.5–6.3 μM), including clinical methicillin-resistant S. epidermis (MRSE) and MRSA, which were significantly improved by comparing to Qin-2. All derivatives displayed antibiofilm activities against both the immature and mature biofilms of S. epidermidis ATCC 35984, and no significant cytotoxicity or hemolytic activities were observed at the concentration of 200 mM, indicating higher safety of these derivatives than the prototype compound Qin-2. Liu-74, and Liu-81 were further evaluated in vivo in the rabbit subcutaneous biofilm infection model, and the reduced bacterial viabilities were observed, indicating the potential efficacies against clinical biofilm infections in vivo (Liu et al., 2014).

By introducing chlorine and fluorine substitutions, Zhao et al. (2014) . designed a series of thiazolo [3, 2-a] pyrimidine-3-one derivatives based on Qin-5 as the leading compound. The majority of these compounds showed significant activities against S. epidermidis and S. aureus. Among them, compound Zhao-4e, which contains four Cl substituents, exhibited a high-level inhibitory activity against WalK (IC₅₀ of 12.65 μM). Additionally, Zhao-4e showed stronger antibacterial activities against S. epidermidis and S. aureus than cefazolin. The MICs of Zhao-4e against S. epidermidis ATCC35984, S. epidermidis ATCC12228, and S. aureus ATCC25923 were 1.143, 2.289, and 4.578 μg/ml, respectively. Same as the other thiazolidione derivatives, Zhao-4e didn’t show antibacterial activity against gram-negative strains including E. coli and S. flexneri. Zhao-4e presented a promising antibiofilm activity against S. epidermidis ATCC 35984 at 36.6 μg/ml and showed no hemolytic activity on human erythrocytes at the same concentration.

Lv et al. (2017) reported another series of Qin-5’s analogs, represented by seven derivatives (Lv-23, Lv-24, Lv-25, Lv-32, Lv-33, Lv-34, and Lv-35, Figure 7). They exhibited concentration-dependent inhibitions on the autophosphorylation activity of WalK, with IC₅₀ values ranging from 61.94 to 83.91 µM.

All seven derivatives displayed higher antibacterial and antibiofilm activities against S. epidermidis and S. aureus (with MICs ranging from 1.56 to 6.25 µM) than that of Qin-5, and they showed bactericidal activities with the minimum bactericidal concentration (MBC, defined as the minimum concentration of an antimicrobial drug that is bactericidal.) values ranging from 12.5 to 100 µM (4× to 32×MIC), respectively. Lv-32, Lv-33, Lv-34, and Lv-35 exhibited a rapidly bactericidal activity against S. epidermidis with a 3-log reduction (CFU/ml) within 1 h, while vancomycin only caused a decrease of about 1 log after 1 h. All the derivatives reduced the proportion of viable cells in mature biofilms and showed no cytotoxicity on Vero cells or obvious hemolysis to human erythrocytes. However, Lv-34 and Lv-35 performed limited aqueous solubilities, which may hinder their further development as potential drug candidates.

In summary, the newly designed thiazolidione derivatives we mentioned above get some improvements on their antibacterial activities, but further structural modifications are still needed to improve the drug-like properties, and increase their antibiofilm and antibacterial properties for the treatment of biofilm-associated infections and multidrug-resistant bacterial infections.

Based on the crystal structure of the ATP pocket of WalK, by using structure-based drug design, a series of thiophene derivatives were synthesized through the palladium-catalyzed Suzuki-Miyaura cross-coupling reaction (Boibessot et al., 2016). Among these inhibitors, eight compounds (Boibessot-6c, -6d, -6e, -6h, -6i, -6k, -6s, and -7c, Figure 8) were discovered to inhibit the autophosphorylation activity of the HKs WalK, PhoR, and ResE from B. subtilis in vitro, with IC₅₀ ranges of 52.81–196.9, 1.63–122.6, and 20.3–243.9 μM, respectively. Moreover, these compounds did not inhibit the DNA-gyrase in E. coli, which contains a structurally related CA domain with HKs (Boibessot et al., 2016).

Among these eight compounds, Boibessot-6d and -6e exhibited broad-spectrum antimicrobial activities against both gram-positive and gram-negative bacteria including some important human pathogens, with MICs ranging from 7 μg/ml to 32 μg/ml, while Boibessot-6d was slightly more potent than Boibessot-6e. The MIC of both compounds against B. subtilis and Bacillus anthracis was 7 μg/ml, which was lower than that against S. aureus, S. pyogenes, Listeria monocytogenes, S. enterica, and E. coli (ranging from 10 μg/ml to 32 μg/ml). However, Boibessot-6d and -6e showed moderate or poor antimicrobial activities against E. faecalis, with MICs of 32 and >64 μg/ml, respectively. Researchers observed that Boibessot-6d could induce slow bacterial lysis over a time course of 9 h which is similar to vancomycin, but Boibessot-6e induced a more rapid cell lysis process, which further indicated that Boibessot-6d and -6e exhibit bactericidal activity rather than bacteriostatic. Additionally, Boibessot-6d could be used as an adjuvant at the concentration of 25 μg/ml combined with amoxicillin or cefotaxime for all the tested E. faecalis and S. aureus isolates and restored the sensitivity of antibiotic-resistant isolates. Unspecific lytic activity was not observed because both compounds did not exhibit hemolytic activities on sheep red blood cells (Boibessot et al., 2016). Compounds of this series can be considered as potent leads for the development of broadly effective antibacterial agents.

The ATP-binding domain of HK is highly conserved with the ATPase domain of eukaryotic Hsp90 molecular chaperones, suggesting that Hsp90 inhibitors may also function as HKs inhibitors. Vo et al. (2017) screened the ATP-competitive Hsp90 inhibitors against CckA, which is essential for virulence in Caulobacter crescentus, and the diaryl pyrazole was identified as a promising scaffold for HK inhibitors. A small library of diaryl pyrazole compounds was synthesized, and among them, compound Vo-5b was identified as one of the most potent HK inhibitors. Moreover, the structural activity relationship (SAR) study indicated the 4-chlororescorcinol group was an important pharmacophore for the inhibitor’s potency.

By using an ATPase-coupled enzyme assay with CckA-ΔTM, a construct of CckA spanning amino acids 70–691 containing the whole cytosolic portion, Vo-5b and other diaryl pyrazole derivatives were discovered as ATP competitive inhibitors, indicating that they may directly bind to the ATP active site. Inhibition assays confirmed that Vo-5b showed more potent inhibitory activity against CckA (IC₅₀s of 28 μM) than PhoQ (IC₅₀s of 328 μM), an HK in Salmonella that is essential for virulence, and showed no inhibitory activity against DivJ, an HK in Caulobacter crescentus that regulates cellular differentiation (Vo et al., 2017). Vo-5b showed in vitro inhibitory effects against C. crescentus, B. subtilis, and E. coli with MICs of 74.4, 49.6–74.4, and 12.4–24.8 μg/ml, respectively (Vo et al., 2017).

Velikova et al. (2016) reported the identification of putative HK autophosphorylation inhibitors by using a combination of in silico and in vitro fragment-based screening by differential scanning fluorimetry. Velikova-13 was discovered as the most potent compound, inhibiting the autophosphorylation of HK in a concentration-dependent manner, with IC₅₀s against S. aureus and E. coli HK PhoR of 212 and 16 μM, respectively.

Velikova-13 mainly showed antibacterial activity against gram-positive pathogens and could inhibit the growth of tested S. aureus, including methicillin-resistant S. aureus (MRSA), S. epidermidis, and S. pneumoniae (MICs of 8–16, 1–16, and 16 μg/ml, respectively). Velikova-13 was bactericidal for the tested gram-positive strains, including S. aureus DSM 20231 (MBC = 33 μg/ml), MRSA (MBC ≤16 μg/ml) and S. epidermidis DSM 20044 (MBC = 8 μg/ml), while Velikova-13 showed no effects against the tested gram-negative strains including K. pneumoniae and P. aeruginosa (MIC > 500 μg/ml) (Velikova et al., 2016). However, the MICs against S. aureus and other gram-positive bacteria were significantly lower than the IC₅₀s against S. aureus PhoR, which indicated that antibacterial activities of Velikova-13 may be caused by other unknown mechanisms.

The result of native polyacrylamide gel electrophoresis suggested that inhibitory activities of Velikova-13 and other compounds are unrelated to the protein aggregation. The molecular docking analysis (without experimental confirmation) predicted that Velikova-13 may bind to the ATP-binding site in the CA domain (Velikova et al., 2016).

Targeting the ATPase domain of VicK, by using the structure-based virtual screening on a natural Traditional Chinese Medicine (TCM) monomers library (Li et al., 2009; Zhang et al., 2015), five TCM monomers, Zhang-1, -2, -3, -4, -5, were identified by Zhang et al. (2015), which specifically inhibited the autophosphorylation of VicK in dose-dependent manners, showing IC₅₀ values of 3.8, 5.4, 15.4, 4.6, and 9.1 µM, respectively.

The antibacterial results showed that the MICs of Zhang-1, -2, -3, -4, and -5 against S. pneumoniae were 37.1, 38.5, 17, 68.5, and 21 μg/ml, respectively, and all of these five compounds showed antimicrobial effects against penicillin-resistant S. pneumoniae, especially Zhang-3 and Zhang-5 showed moderate antibacterial activities with 50% minimum inhibitory concentration (MIC₅₀, defined as the minimum inhibitory concentration required to inhibit the growth of 50% of organisms.) values of 17 and 42 μg/ml, respectively. Besides, these five compounds also showed antibacterial activities against S. mutans, S. pyogenes, S.mitis, and S. pseudopneumoniae. Importantly, Zhang-3 and Zhang-5 also displayed inhibitory activities against MRSA, with MIC of 34–68 and 84.4–336 μg/ml, respectively. Zhang-3 and -5 showed significantly synergic antimicrobial activity with penicillin in vivo and in vitro, and effectively reduced nasopharyngeal and lung colonization caused by different penicillin-resistant pneumococcal serotypes, and they also performed synergic antimicrobial activities with erythromycin and tetracycline. In vivo experiments showed that the survival time of mice infected by S. pneumoniae NCTC7466 could be prolonged by the treatment of these five compounds. The time-killing assays showed that the compounds elicited bactericidal effects against S. pneumoniae D39, which led to a 6-log reduction in colony-forming units after exposure to compounds at 4 × MICs for 24 h. The crystal violet staining revealed that the five compounds at MIC or sub-MIC could inhibit the formation of S. pneumoniae biofilm, without significant toxicity on Vero cells, which reflects a good safety (Zhang et al., 2015). The present study provides evidence of the antibacterial efficacies of the five leading compounds against pneumococcus and other gram-positive bacteria, and they may serve as an alternative strategy for the treatment of bacterial infections.

By screening over 10,000 Streptomyces extracts with sensitive differential growth assays, signermycin B was discovered by Watanabe et al. as the first antibiotic targeting at the WalK dimerization domain according to surface plasmon resonance (SPR) experiments (Watanabe et al., 2003; Watanabe et al., 2012).

Signermycin B exhibited the WalK inhibitory activities (including S. aureus, E. faecalis, B. subtilis, and S. mutans, with IC₅₀s ranging from 37 to 62 μM) and well antimicrobial activities (including MRSA and vancomycin-resistant E. faecalis, with MIC values ranging from 3.13 μg/ml to 6.25 μg/ml) against gram-positive bacteria (Watanabe et al., 2012). It can be noticed that the MICs against the tested gram-positive bacteria were significantly lower than the IC₅₀s against WalK, which indicated that WalK may not be the only target of signermycin B and there could be some other molecular mechanisms for Signermycin B’s antibacterial activities.

In the WalK expression experiment, the antibacterial activities of signermycin B were significantly decreased against WalK-induced cells, but such phenomenon was not observed in the WalR-overexpression cells, which indicated that signermycin B could specifically target at WalK. Following SPR experiment and competition assay with ATP indicated that signermycin B binds directly to the dimerization domain of WalK of B. subtilis and showed no ATP competition effects, which suggested that signermycin B contains a different mechanism, unlike most HK inhibitors targeting the ATP binding site. Protein cross-linking was performed in the presence of the cross-linker glutaraldehyde to explore the mechanism of action of signermycin B, which interfered with the cross-linking of WalK dimers rather than causing the protein aggregation. Moreover, for B. subtilis and S. aureus, signermycin B preferentially down-regulated the expression of WalR regulon genes including yocH, yvCE, yoeB, yjeA, isaA, and ssaA in vivo, thereby it can inhibit the cell division (Watanabe et al., 2012). The above findings suggested that the dimerization domain of WalK could be considered as a potential binding site in the discovery of novel antibacterial agents, and supported the further development of signermycin B as an antimicrobial against drug-resistant bacteria.

Focusing on the discovery of new antibacterial agents, Igarashi et al. (2013) screened metabolites from Streptomyces sp. MK844-mF10 to isolate HK inhibitors by using a differential growth assay they previously reported (Okada et al., 2007), and the waldiomycin was isolated as a WalK inhibitor, composing of 1,3-dioxolane-2-carboxylic acid linked to an angucyclic polyketide via a tetraene linker and tetrahydropyran, which belongs to angucycline antibiotics and structurally relates to dioxamycin (Sawa et al., 1991).

Waldiomycin showed moderate antibacterial activity against S. aureus and B. subtilis, including methicillin-resistant strains, with MICs ranging from 8 μg/ml to 16 μg/ml, but showed no activity against E. faecalis, M. smegmatis, and all the tested gram-negative bacteria (Igarashi et al., 2013). Among the tested strains, the antimicrobial activity of Waldiomycin against S. aureus at a comparable level with other angucyclinone antibiotics, such as aquayamycin (MIC = 6.25–12.5 μg/ml) (Sezaki et al., 1968), rabelomycin (MIC = 6.3 μg/ml) (Liu et al., 1970), and sakyomicin A (MIC = 9.38 μg/ml) (Nagasawa et al., 1984).

Waldiomycin can inhibit the autophosphorylation activities of WalK from B. subtilis, S. aureus, E. faecalis, and S. mutans, with IC₅₀s of 10.2, 8.8, 9.2, and 25.8 µM, respectively (Fakhruzzaman et al., 2015). The qRT-PCR analysis of WalR regulon genes suggested that waldiomycin repressed the WalK/WalR system in B. subtilis and S. aureus thereby inhibiting the expression of several cell wall metabolism genes. The morphology of S. aureus cells treated with waldiomycin displayed an increased aggregation, rather than the proper cellular dissemination. The autolysis experiment showed that the waldiomycin-treated S. aureus cells showed high resistance to Triton X-100-induced and lysostaphin-induced lysis, and these cell lysis phenotypes are consistent with those of cells starved for the WalK/WalR. Since the WalK/WalR system can regulate autolysis, these results further indicated that waldiomycin could inhibit WalK. Furthermore, it was confirmed that waldiomycin inhibited the WalK autophosphorylation in vivo by observing the phosphorylated WalK ratio in cells using Phos-tag sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Fakhruzzaman et al., 2015). Further research showed that waldiomycin not only inhibited WalK of gram-positive bacteria but also blocked the activities of most class-I HKs in gram-negative bacteria, including E. coli QseC, EnvZ, and PhoQ with IC₅₀s of 15.1, 22.4, 12.5 µM, which suggested that waldiomycin was a general inhibitor of class-I HKs (Eguchi et al., 2017).

A series of experiments showed that waldiomycin inhibited HK in an ATP-non-competitive manner, suggesting that the binding site of waldiomycin may not be located in the ATP active site in the CA domain. Additionally, more detailed experiments, including nuclear magnetic resonance (NMR) spectroscopy and protein amino acid mutation, indicated that waldiomycin may bind to the H-box region of the DHp domain, which contains the conserved His residue, to inhibit the class-I HKs and suppress the autophosphorylations (Eguchi et al., 2017; Kato et al., 2017). The broad-spectrum HK inhibition activities of waldiomycin showed that the H-box could be a promising target for the design of novel broad-spectrum HK inhibitors especially those HKs with the WalK-type H-box region. Besides, the binding interactions between waldiomycin and S. aureus WalK were analysed by Radwan and Mahrous (2020) through molecular docking studies and molecular dynamics simulations, which indicated that the amino acid residue Lys100 was the key residue for hydrogen bonding with waldiomycin at the binding site and possibly important for the enzyme activity. Studies also showed that when the carboxylic acid of waldiomycin was replaced by the methyl ester group, the antibiotic activity disappeared (Radwan and Mahrous, 2020; King and Blackledge, 2021).

By screening a molecular library containing 420 FDA-approved drugs based on the TCS-dependent biofilm effect, maprotiline, an FDA-approved tetracyclic antidepressant drug, was discovered with the potential to combat Francisella infection (Dean and van Hoek, 2015).

Maprotiline effectively reduces the biofilm formation by affecting Francisella QseC. Although the exact molecular mechanisms still need to be elucidated, based on computational modeling, researchers predicted the periplasmic sensor domain of QseC might be the binding site of maprotiline (Dean and van Hoek, 2015). Moreover, maprotiline could significantly downregulate the expression of IglC, which is a known Francisella virulence factor, in a manner of QseC dependence, as the same decrease in IglC expression was not observed in the qseC mutant. Further in vivo studies have demonstrated that maprotiline can prolong the time of disease onset and survival in F. novicida-infected mice and waxworm, which indicated that maprotiline may not only disrupt TCS signaling in vitro to decrease the formation of biofilm, but also decrease the virulence of Francisella in vivo through disrupting the expression of TCS-dependent virulence factor (Dean and van Hoek, 2015).

Maprotiline showed strong antivirulence activity without direct inhibition on the growth of bacteria, which indicated maprotiline was less likely to confer bacterial resistance. Such an antivirulence approach could be a new direction for the drug discovery against antibiotic-resistant pathogens or could be used in conjunction with other known antibiotics to potentiate the antibacterial activities. Further investigations on maprotiline as a potential drug candidate to treat infections caused by Francisella and other pathogens would be expected to generate novel anti-bacterial drugs (Dean and van Hoek, 2015; Feldmann and Cavanagh, 2015).

LED209 was identified by Rasko et al. (2008) from HTS of a library containing 150,000 small organic compounds. It selectively inhibited the binding of the signaling molecules to QseC from different gram-negative pathogenic bacteria. Studies showed that LED209 at very low level of concentrations (e.g., 5 pM in vitro) could block the autophosphorylation of QseC triggered by both the host stress hormones (norepinephrine or epinephrine) and the bacterial signal AI-3, and the subsequent virulence gene expression, but did not influence the bacterial growth. The latest research has revealed the unique action mode of LED209 as a prodrug, by which it loses an aniline group and releases a warhead of isothiocyanate OM188 (Figure 9). With this unique mechanism, OM188 can avoid being metabolized and/or degraded. The warhead OM188 allosterically modifies QseC by convalently binding to Lysines (K256 and K427) of the HK, impairs the function of QseC, and prevents the activation of the virulence program of several gram-negative pathogens both in vitro and during the treatment of Salmonella typhimurium and Francisella tularensis murine infection. Besides, LED209 can decrease the formation of biofilm in the enteroaggregative E. coli O104:H4 and in several multidrug-resistant clinical isolates of recurrent urinary tract infections. Pharmacokinetics and toxicology studies have shown that LED209 had desirable pharmacokinetics and safety in vitro and in rodents. However, the plasma exposures with LED209 were high, which indicated the limited penetration of the blood-brain barrier (Curtis et al., 2014).

Recently, researches focused on the combination or conjugation of LED209 with other antibacterial molecules open an area for broader applications of LED209. Poly (amidoamine) (PAMAM), which was reported as a broad-spectrum antimicrobial agent, has limitations as its cytotoxicity to mammalian cells and poor selectivity. Xue et al. (2015) conjugated G3 PAMAM with LED209 to generate a multifunctional agent PAMAM-LED209 which showed higher selectivity on gram-negative bacteria and lower cytotoxicity to mammalian cells due to the reduced positive charges in G3 PAMAM surface than using PAMAM alone. The conjugate not only remained the strong antibacterial activity, but also inhibited virulence gene expression of gram-negative bacteria without inducing resistance development of the pathogens. A subsequently obtained chitosan (CS)-LED209 was also reported to have a higher selectively antimicrobial and anti-adhesion activity against MDR-E. coli as well as the lower cytotoxicity to the mammalian cell than CS (Zhou et al., 2019). An all D-amino acid analogue of Tachyplesin I (TPAD) which belongs to cationic β-hairpin antimicrobial peptides with broad-spectrum antimicrobial activity was reported, but it was found that TPAD tends to induce bacterial resistance by activating the QseC/B TCS. The combination of TPAD with LED209 could significantly enhance the bactericidal effect against MDR gram-negative bacteria such as Pseudoalteromonas and S. maltophilia , which opens an avenue to improve the antimicrobial efficiency of antimicrobial peptides (Yu et al., 2020). Moreover, Zhou et al. (2021) covalently modified cellulose membrane (CM) with LED209 via click reaction to produce an antibacterial material (LED209-CM). LED209-CM exhibited marked anti-adhesion ability to enterohemorrhagic E. coli, and significantly reduced the formation of bacterial biofilm. LED209-CM was also able to repress the expression of virulence genes in enterohemorrhagic E. coli and indicated no cytotoxicity to mammalian cells. This construction strategy of antibacterial surface expanded the application potential of LED209 in implant-related infections.

Zhang et al. (2019) constructed an artificial proteoliposome-based system containing an artificial cell membrane with full-length AgrC, which contains a high similarity with the actual cellular environment. The model was used to screen inhibitors for AgrC among a molecular library containing 14 traditional Chinese medicine monomers by incorporating AgrC into liposomes via a detergent-mediated method. Among these monomers, two TCM monomers, rhein, and aloeemodin were screened out. Rhein and aloeemodin can inhibit the autophosphorylation of AgrC, with IC₅₀ values of 13.7 and 62.2 μM, respectively. Rhein and aloeemodin showed inhibitory activities on the growth of S. aureus in a concentration-dependent manner, with MICs of 32 and 64 μg/ml, respectively. Biofilm formation decreased significantly approximately 20.0% and 33.3% respectively when S. aureus was treated with rhein and aloeemodin at the concentrations of 1/2 × MICs. Subinhibitory concentrations of these compounds can significantly reduce the expression of three virulence factors (hla, clfA, and clpP) in S. aureus which was regulated by the agr system. The results preliminarily confirmed these two traditional Chinese medicine monomers can be the targeting inhibitors of AgrC.

The PhoP/PhoQ system modulates the Shigella flexneri virulence and stress responses of Mg²⁺, pH, and antibacterial peptides (Lin et al., 2017). Using HTS and enzymatic activity-coupled assays, Cai et al. (2011) reported four compounds Cai-1, -2, -3, and -4 as potential PhoQ inhibitors.

These four compounds inhibited the autophosphorylation activity of S. flexneri PhoQc and displayed high binding affinities to the S. flexneri PhoQc protein in the SPR response (K_D = 4.50, 10.6, 7.56, and 9.40 μM, respectively). Luminescent kinase assays showed that the IC₅₀ values of Cai-1, -2, -3, and -4 were 69.37, 48.9, 7.99, and 27.2 μM, respectively. Additionally, the growth curve of S. flexneri 9380 showed that the four potential PhoQ inhibitors had no obvious effect on the growth of S. flexneri 9380 at 200 μM in vitro because the PhoQ/PhoP signaling system did not regulate the cell growth directly. Cai-1, -2, and -3 inhibited the activity of S. flexneri 9380 to invade HeLa cells. Mice inoculated with S. flexneri 9380 pretreated with four compounds displayed no inflammation, whereas mice inoculated with S. flexneri 9380 alone displayed a severe keratoconjunctival inflammation. At the effective concentrations, four inhibitors exhibited the low cytotoxicity and hemolysis of mammalian cells. Thus, these studies demonstrated that all four potential PhoQ inhibitors could reduce the virulence of Shigella. The compound structures need to be further modified to increase the efficacy and the stage of infection which is inhibited by these inhibitor candidates needs to be identified (Cai et al., 2011).

The PrrBA TCS, one of the four conserved TCSs presented in all mycobacterial species, is critical for the viability of M. tuberculosis and is necessary for the initial stage of M. tuberculosis infection in macrophages (Tyagi and Sharma, 2003; Haydel et al., 2012). Bellale et al. (2014) screened a representative set (100,000) of the AstraZeneca’s molecular library against M. tuberculosis H37Rv, and the MIC, MBC, selectivity index (the ratio of IC₅₀ against human A549 cell line to MIC against M. tuberculosis), and confirmation of activity by resynthesis were evaluated.

A group of diarylthiazole compounds was identified that contained potent antimycobacterial activities against M. tuberculosis. Subsequently, over 40 diarylthiazole derivatives were synthesized, and most of them showed desirable physicochemical properties and potent MICs against M. tuberculosis (MIC ≤ 1 μg/ml), such as Bellale-31 and -48, which exhibited significant antimycobacterial activities (M. tuberculosis MIC = 0.4 and 0.25 μg/ml, MBC = 0.8 and 0.5 μg/ml, respectively). Moreover, it was observed in the whole genome sequencing of the representative resistant mutant strains that point mutations in prrb gene imparted the resistance to the tested diarylthiazole compounds, suggesting that the sensor kinase PrrB may be a target of diarylthiazole (Bellale et al., 2014), but the specific mechanisms of diarylthiazole still need to be clarified.

In S. aureus, SaeRS is composed of the HK SaeS, RR SaeR, and two auxiliary proteins SaeP and SaeQ. The TCS is regarded as a master virulence regulator because it controls the production of over 20 virulence factors, such as hemolysins, leukocidins, superantigens, surface proteins, and proteases (Liu et al., 2016; Mizar et al., 2018).

Xanthoangelol B was screened out by Mizar et al. (2018) using a GFP (Green fluorescent protein) reporter system they developed previously to screen plant-derived SaeRS inhibitors (Yeo et al., 2018). The compound has been synthesized at present, and a derivative named PM-56 was obtained during the synthesis.

Xanthoangelol B and PM-56 showed excellent inhibitory activities against SaeRS, with IC₅₀s against the SaeRS GFP reporter at 2.1 and 4.3 μM, respectively. Significant transcriptional suppression of four downstream virulence genes [α-hemolysin (hla), aureolysin (aur), γ-hemolysin, and staphylokinase] were observed, which further implied the action of both compounds on the SaeRS pathway and their possibility of development as antivirulence agents (Mizar et al., 2018).

In vitro, neither compound suppressed the growth of S. aureus at IC₉₀ (against SaeRS TCS), especially PM-56, which displayed a low effect on bacterial growth and is therefore considered a desirable antivirulence agent. In an infection model of G. mellonella larvae, both xanthoangelol B and PM-56 provided excellent protection to the insects from lethal S. aureus infection and reduced bacterial burdens in a dose-dependent manner (Mizar et al., 2018).

The direct binding of xanthoangelol B and PM-56 to SaeS was determined using an intrinsic fluorescence quenching assay, by which the Tyr fluorescence of SaeS at 306 nm was monitored before and after binding of the compounds and the Kd values were calculated according to the percent fluorescence quenching upon ligand binding, and the Kd values of 40 and 6.8 μM for the two compounds to the HK were identified, respectively. Based on the measurements from SDS-PAGE autoradiography and the effects on phosphotransferase (PT) activity of SaeS, both compounds displayed a specific inhibition on HK activity of SaeS, with IC₅₀ values of 220 and 160 μM for xanthoangelol B and PM-56, respectively. In additon, Xanthoangelol B and PM-56 could also inhibit another HK, AgrC, with IC₅₀ of 339 and 140 μM, respectively, indicating the possibility that xanthoangelol B scaffold could be further developed as a broad-spectrum HK inhibitor despite the IC₅₀ values higher than expected (Mizar et al., 2018).

DosS-DosT/DosR(DosRST) is composed of two heme-based histidine sensor kinases, DosS, and DosT, and a response regulator DosR (Roberts et al., 2004). Zheng et al. (2017) used a DosRST-dependent fluorescent M. tuberculosis reporter strain to identify new DosRST inhibitors by using a whole-cell phenotypic HTS to screen a small-molecule library containing ∼540,000 compounds.

Two compounds, Zheng-103A and Zheng-102A, were identified and contained inhibition abilities on the triacylglycerol synthesis, survival, and isoniazid tolerance under the condition of hypoxia, which is associated with the physiological processes of M. tuberculosis persistence. Recombinant DosS and DosT proteins were used to conduct biochemical studies to determine the mechanism of action of Zheng-102A and Zheng-103A. Zheng-103A can inhibit the autophosphorylation activity of both DosS and DosT with IC₅₀s of 0.5 and 5 μM, respectively, while Zheng-102A only inhibited the autophosphorylation of DosS with IC₅₀ of 1.9 μM (Zheng et al., 2017). Their research confirmed that only DosR-controlled pathways are targeted by Zheng-102A, while 13 genes were downregulated in the ΔdosR mutant treated with Zheng-103A, which indicated that although Zheng-103A is highly specific for the DosRST pathway, other targets exist (Zheng et al., 2017). Besides, the synergistic interactions of inhibitors showed that the artemisinin, a compound targeting at the sensing domain of DosST heme, was synergistic with Zheng-102A and Zheng-103A, which indicated that the inhibitors’ function by distinct mechanisms could be combined to improve the potency (Zheng et al., 2020). Further studies will be required to evaluate the antibacterial activities and clinical significance of Zheng-103A and Zheng-102A in curing M. tuberculosis infection.