Bacterial Histidine Kinases as Novel Antibacterial Drug Targets

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Bacterial Histidine Kinases as Novel Antibacterial Drug Targets Agnieszka E. Bem, Nadya Velikova, M. Teresa Pellicer, Peter van Baarlen, Alberto Marina, and Jerry M. Wells ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/cb5007135 • Publication Date (Web): 01 Dec 2014 Downloaded from http://pubs.acs.org on December 8, 2014

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Bacterial Histidine Kinases as Novel Antibacterial Drug Targets Agnieszka E. Bem1, Nadya Velikova2, M. Teresa Pellicer3, Peter van Baarlen1, Alberto Marina2, 4, Jerry M. Wells1 corresponding author: [email protected] 1

Host-Microbe Interactomics, Wageningen University, De Elst 1, 6708 WD Wageningen, The Netherlands 2 Instituto de Biomedicina de Valencia-Consejo Superior de Investigaciones Cientificas (IBV-CSIC), Jaume Roig 11, 46010-Valencia, Spain 3 R&D Department Interquim, Ferrer HealthTech, Joan Buscalla 10, 08137-Sant Cugat del Valles Barcelona, Spain 4 Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBER-ISCIII), Jaume Roig 11, 46010-Valencia, Spain Abstract Bacterial histidine kinases (HKs) are promising targets for novel antibacterials. Bacterial HKs are part of bacterial two-component systems (TCSs), the main signal transduction pathways in bacteria, regulating various processes including virulence, secretion systems and antibiotic resistance. In this review, we discuss the biological importance of TCSs and bacterial HKs for the discovery of novel antibacterials, as well as published TCS and HK inhibitors that can be used as a starting point for structure-based approaches to develop novel antibacterials. The growing problem of antimicrobial drug resistance In 2004 the Infectious Diseases Society of America (IDSA) published an alarming report entitled “Bad bugs, no drugs” (1), discussing antimicrobial drug resistance (AMR) and the lack of therapeutic alternatives to current antibiotics that have become less effective because of AMR. Since then, infections caused by drug-resistant bacteria have increased worldwide. Unless action is taken to halt this problem we are facing the prospect of returning to a pre-antibiotic era (1). Nearly all classes of antibiotics were discovered in the 1950s. Resistance to these antibiotics was usually reported within a few years after their discovery, sometimes even years before their introduction to the market (2). Only two novel classes of antimicrobials, the oxazolidinones and cycliclipopeptides (3) have entered the market in the last 30 years. Concurrently, pharmaceutical companies appear to have closed antibiotics R&D programs and instead focus their financial and research efforts on pharmaceuticals for chronic pathologies such as cancer. Drug discovery projects on infectious diseases represent around 16% of all academic projects but comprise only 9% of pharmaceutical industry projects (4). In general, low investments in antibiotics research have been reported for Europe. During the period 2008-2013 less than 2% of the EU and UK total research budget was committed to microbiology research, and less than 1% was committed to antibiotics research (5). In the following sections, we outline

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which strategies have so far been used in antibiotics research, and emphasize how novel antibiotics may be targeted toward histidine kinases, with special reference to those HKs that are part of bacterial two-component systems (TCSs). Approaches to the discovery of novel antibacterial drugs For an antibacterial drug to be designated as novel it should fulfill at least one of the following criteria (6): 1) belong to a novel chemical class and act on a new target 2) work via new mechanism of action or binding site even if the drug target itself is not new 3) be biochemically modified to overcome bacterial resistance to the original drug 4) inhibit the same target as the original drug but have new physicochemical properties within its class permitting administration via a new route. Novel antibiotics ideally should be broad-spectrum and amenable to structural changes that allow for optimization of potency, specificity and absorption, distribution, metabolism, elimination and toxicity (ADMET) properties (7). The most successful phenotypic screens for inhibition of bacterial growth, usually determined via minimal inhibitory concentration (MIC) assays, have used chemical libraries of, or natural compounds from extracts from soil or marine ecosystems including bacterial secondary metabolites. Typically the putative inhibitory compounds, also known as lead compounds, are purified and followed by analytical chemistry and cytotoxicity assays. Chemical structure of the lead compound is optimized and ultimately, its mode of action is elucidated and its antibacterial spectrum determined. Nearly all antimicrobials on the market are based on (derivatives of) antibiotics produced by Streptomyces (8) or antibiotics from unculturable bacteria expressed in heterologous hosts such as S. lividans (9). The above screens have been supplemented with rational, knowledge-intensive in vitro and in silico screens. In structure-based virtual screening (SBVS), the 3D structure of the target protein is obtained experimentally or by homology modeling and analyzed in silico for potential physiochemical interactions of a virtual library of chemical compounds with the active site of the target (10). Selected ligands, or hit compounds, are then tested in vitro to confirm their target inhibitory capacity. Hit compounds that show biological activity (lead compounds) typically are further optimized by chemical modifications. In fragment-based drug discovery (FBDD), libraries of thousands of small chemical compounds, called fragments, are screened against purified protein representing the selected drug target. Fragments that bind weakly to different parts of the selected target site may be chemically linked to enhance selectivity and potency of inhibition (11). The advantage of this approach is that a collection of few thousands fragments covers a



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larger chemical space than HTS libraries of the same size due to possibility of pairwise combinations of fragments. Another approach exploited in antibacterial drug research is rational target based drug design (RTDD) using proteins that perform essential or conditionally essential functions required for bacterial survival (12). To identify inhibitors of a specific bacterial target in RTDD, high-throughput screening (HTS) is performed with libraries of natural compounds as well as small synthetic chemical molecules. In RTDD the ideal antibiotic targets should: 1) Have no human homologs or structurally similar proteins 2) Be highly conserved among various bacterial species to assure broad-spectrum of antibacterial action 3) Be strictly essential for bacterial viability such that their inhibition would lead to bacterial death but as targeting essentiality might increase resistance selection pressure, targeting virulence targets has been proposed as an alternative 4) Be ‘assayable’, i.e. should have easily measurable activity, in assays amenable to high-throughput screening (HTS) 5) Have available structural data on the target and genetic tools to validate the target in a key species (12). Bacterial HKs fulfill most of these requirements except that certain mammalian kinases (or ATPases) have a similar protein fold in the ATP domain (13–14). In the following sections we review progress towards the discovery of novel antibacterials targeting HKs of TCSs (15–16). Two-Component Systems TCSs are present in most bacteria and are the most common form of bacterial signal transduction. The number of TCSs found in bacterial species is correlated to the genome size and the range of environments in which the organism can grow and survive. TCSs are considered attractive antibacterial drug targets as multiple members are found in nearly all bacteria. Several TCSs have been shown to be essential for bacterial survival, and homologs have not been identified in mammals including humans (17). TCS signaling involves autophosphorylation of a membrane-bound histidine kinase (HK), phosphotransfer of the phosphoryl group to a cognate response regulator (RR), and ultimately modulation of the expression of target genes (Figure 1) (18). Appropriate phosphorylation levels of the RR are tightly regulated by the phosphatase activity of the HK, the RR or a partner protein (18–19). HK autophosphorylation is mediated via the catalytic and ATP-binding (CA) domain, which binds ATP and phosphorylates the HK at conserved histidine residue in the dimerization and histidine phosphotransfer (DHp) domain. The CA and DHp domains are conserved and present in all HKs, whereas the remaining sensor domains (periplasmic, PAS, GAF, HAMP) are variable and not present in all HKs (Figure 2) (18).


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The RR usually is a transcription factor that, upon phosphorylation, undergoes conformational changes that promote dimerization or higher order oligomerization states (20) favoring in the majority of cases higher affinity interaction between the RR and bacterial DNA and altered target gene expression (21). Alternatively, the RR may act as an enzyme such as a methylesterase (22) or an ATPase (23). TCSs signaling is terminated by dephosphorylation of the RR, which can be auto-induced or mediated by the cognate HK or by auxiliary proteins (24–25). Usually, each HK possesses one cognitive RR with strict partner specificity (26). TCSs exhibit linear signal transduction, which can be modulated by addition of extra modules to the conserved domain, as well as by addition of auxiliary proteins named “connector” proteins (18). TCS signaling can mediate rapid microbial adaptation in response to changes in the environment, including signals encountered in the environment of the host, by altering expression of specific genes (21) that may participate in virulence mechanisms, metabolic and developmental pathways, antibiotic resistance and regulation of type II/III/IV/VI secretion systems (27– 29) (Table ).

The link between TCS and antibiotics resistance TCSs may regulate antibiotic resistance by various mechanisms (Table 2). The VanRS or VraSR TCS mediates changes in bacterial cell wall metabolism causing resistance to vancomycin, an antibiotic targeting bacterial cell wall synthesis (30). Also single-nucleotide polymorphisms (SNPs) in the promoter region of WalKR, a TCS involved in cell wall synthesis, lead to increased vancomycin resistance (31). TCSs may also regulate the activity of efflux pumps and porins (32). Porins are proteins located in the membrane of Gram-negative bacteria that control outer membrane permeability of small molecules

including

carbapenem

antibiotics

(33),

tetracycline,

streptomycin

and

spectinomycin (34). Many efflux pumps, transporter membrane proteins that actively pump toxic compounds out of bacteria, are regulated by TCSs and are responsible for multi-drug resistance (MDR) of important human pathogens (Table 1). Efflux pumps recognize their substrate mainly by physicochemical properties such as hydrophobicity; due to their amphiphilic character several antibiotics are easily recognized and removed from bacterial cells by these pumps. TCS-mediated regulation of the efflux pumps of diverse important pathogens including K. pneumoniae (25) and A. baumannii (27) provides resistance to a wide range of antibiotics (Table 2) highlighting the importance of TCSs as attractive drug targets.



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TCS HKs are attractive antibacterial drug targets The conserved features of the histidine kinase CA domain and its essential role in TCS signal transduction make it an attractive target for structure-based virtual screening and phenotypic screening of biochemical inhibitors. Moreover, the high degree of sequence conservation in the CA catalytic site implies that inhibitors targeted against this site will possess broad-spectrum antibacterial activity (Figure 2). Altogether, this makes the CA domain an attractive HK target site for the discovery and development of broadspectrum

antibacterials.

polypharmacology) has

Simultaneous been proposed

inhibition as

a

of

strategy

multiple to

slow

targets down

(drug

resistance

development to drugs including novel antibacterials (16, 35–37). As bacteria possess multiple TCSs, inhibitors of the highly conserved CA domain are likely to shut down multiple signaling pathways compromising the ability of the bacteria to rapidly adapt to environmental changes including those encountered in the host during infection. For some bacteria TCS inhibition may not be bactericidal but it is likely to compromise efficient growth, thus reducing fitness. One potential downside to HKs is that the ATP-binding Bergerat fold that is present in the CA domain does also occur in several human protein families and is present in crucial proteins such as Hsp90. The Bergerat fold includes four conserved motifs; N, G1, G2 and G3 boxes in the HK CA domain corresponding to Motif I, Motif II, Motif III and Motif IV, respectively, in other members of the GHKL family (Figure 2 and 3) (18). The F box is a conserved motif preceding in the G2 box and is characteristic for HK CA domains. Presence of the Bergerat fold in microbial and human ATP-binding protein domains might lead to off-target inhibitory effects of HK autophosphorylation inhibitors (HKAIs) towards human ATP-binding domains and therefore, toxicity to mammalian cells. The similarity between the CA domain of bacterial TCS and eukaryotic proteins containing the Bergerat fold is exemplified by the demonstration that the eukaryotic Hsp90 inhibitor radicicol is an inhibitor of PhoQ autophosphorylation and was cocrystallized with the CA domain of PhoQ (38). However, other inhibitors with similar activity as radicicol could not be co-crystallized with PhoQ and did not inhibit PhoQ autophosphorylation (38). The differences between radicicol and the other Hsp90 inhibitors with respect to PhoQ inhibition were attributed to differences in the putative interaction with the ATP-lid and the ATP-lid conformation in the CA domain. The ATP-lid (Figure 3) is a variable loop that connects the G1 and G2 boxes or the corresponding motifs

in

other

GHKL

family

members.

The

ATP-lid

is

crucially

involved

in

autophosphorylation (18, 39) and contains the F box which is characteristic of bacterial HKs. Although HKs are structurally similar to some eukaryotic proteins, the biochemical study discussed above shows that it is possible to design inhibitors of microbial ATPbinding domains that have limited affinity for eukaryote ATP-binding protein domains and


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low toxicity to host cells (38). Indeed, despite sequence conservation of bacterial and eukaryote kinase domains, several research groups have focused on finding inhibitors for bacterial TCSs, including VanSR due to its role in antibiotic resistance, WalKR due to its essentiality, and QseCB and DosRST due to their role in virulence. Progress towards developing inhibitors of these TCSs is discussed in the remainder of this review.

Inhibition of the VanSR TCS, regulator of vancomycin resistance Vancomycin, an extracellular-acting glycopeptide antibiotic that inhibits bacterial cell wall synthesis, is now the only available antibiotic to treat methicillin-resistant Staphylococcus aureus (MRSA) (40). Vancomycin resistance has been observed in Enterococcus

faecalis

and

Enterococcus

faecium

(41–42).

The

genes

conferring

resistance to vancomycin are regulated by the VanSR TCS (43). It is suggested that VanS HK negatively regulates VanR RR function in the absence of vancomycin (32). Due to its clinical relevance, VanSR was considered an attractive drug target and several VanSR TCS inhibitors were discovered ( Table 3) that function by uncoupling energy required for ATP synthesis (44). Although these compounds could not be used readily as drugs due to their negative effect on mitochondrial respiration, they may provide a structural template for new inhibitors selective for VanSR TCSs during adjunct therapy (44). Inhibition of walKR, an essential TCS WalKR (syn. YycF/G, MicA/B, or VicK/R) is a highly conserved TCS in low-GC Grampositive bacteria. It is the most widely distributed and obligate essential TCS found in many pathogens belonging to the genera of Staphylococcus, Streptococcus, Enterococcus and Listeria. WalKR regulates genes responsible for cell wall metabolism and cell wall homeostasis (27, 45–46). Additionally, WalKR regulates genes involved in metabolism, stress response, virulence, host-microbe interactions, transport and regulatory pathways (Table 4) (28, 47). In most species, walR has been found to be essential for viability making it an attractive antibacterial target. Construction of inducible knock-outs of walR, as well as high structural conservation of WalR between species, led to the identification of a consensus WalR recognition sequence and also of the regulon controlled by this TCS in S. pneumoniae (47). WalKR activation has also been linked to vancomycin resistance in S. aureus (48– 49). It was shown that exposure to diverse antimicrobials induces vancomycin resistance phenotypes by either single or multiple mutations localized in the walKR operon (48, 50). Vancomycin resistance of S. aureus may be co-mediated by the VraRS and GraRS TCSs



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that are involved in the cellular response of S. aureus to cell wall damage (51) together with four other genes, a number of which are controlled by the WalKR TCS (50). Different drug discovery strategies have been used to identify inhibitors of WalK. New lead chemical compounds inhibiting the CA domain of WalK histidine kinase in Staphylococcus epidermidis (Figure 5) that were discovered using SBVS have been validated

experimentally

(Table

3)

(52).

In

another

study,

antibiotic-producing

Streptomyces strains were screened for their ability to produce potential inhibitors of Streptococcus mutans WalK (53). In this study, a new family of inhibitors termed walkmycins came out as promising lead compounds and are currently under preclinical development. Walkmycin C was shown to inhibit biofilm formation and competence in Streptococcus mutans (54). During this study, signermycin B emerged as a lead molecule targeting WalK dimerization (55).

Targeting virulence gene regulation by QseCB QseCB is a TCS involved in recognizing host-derived adrenergic signals and the bacterial quorum sensing signal autoinducer AI-3; AI-3 has been shown to trigger expression of virulence genes in several bacterial species (56). Homologs of QseC are present in at least 25 important human and plant pathogens, therefore, a QseC inhibitor is expected to be a promising drug for antivirulence therapy against a wide range of pathogenic bacteria (57). The QseC protein bears a periplasmic domain that recognizes both host adrenergic signals as well as the microbial AI-3 molecule (56). Drugs targeting the QseC periplasmic domain have an advantage compared to drugs with intracellular targets as they only need to pass the outer membrane to reach the target. Highthroughput screens to search for a compound that inhibits activation of QseC by AI-3 led to the identification of LED209 (Figure 5), an inhibitor of AI-3 binding to QseC from different Gram-negative pathogenic bacteria (Table 3) (57). LED209 inhibited QseC autophosphorylation and bacterial pathogenicity in vitro and in vivo, but not cell growth (57). Targeting M. tuberculosis persistence through inhibition of DosRST Tuberculosis (TB), the infectious disease caused by Mycobacterium tuberculosis, remains a major public health problem. In a study to identify mycobacterial targets, DosS (named after dormancy survival)came out as a target for dormant mycobacteria (58). It is worth mentioning that in this study DosS had been classified as a high-confidence target, as it had been identified after having excluded mycobacterial proteins with high similarity to commensal gut flora and host proteins. DosRST TCS

comprises the DosR response regulator and the DosS and DosT

histidine kinases that play an essential role in triggering and maintaining dormancy and


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in enabling future mycobacterial proliferation (59–61). In infected cells, hypoxia and toxic respiratory byproducts (i.e. NO, CO) trigger dormancy and metabolic adaptation of M. tuberculosis via DosR. DosR is activated by phosphorylation at D54 (62) and by phosphorylation at T198 and T205 by the serine-threonine kinase PknH. DosR phosphorylation induces its dimerization and promotes its binding to the promoter of the DosRST regulon (62). Several sets of small molecules have been assayed for their inhibition of DosR activity (63) (64). Several small molecules have been described as DosR inhibitors that bind DosR and inhibit its binding to its target DNA sequence (63). These molecules have also shown potential antibacterial activity against other mycobacteria (Table 3).

Challenges and future perspectives for the discovery and development of antibacterials targeting TCSs

TCSs are considered attractive drug targets and to date around a hundred TCS inhibitors belonging to different chemical classes have been described in the literature; most of these inhibitors target Gram-positive bacteria (Table 3). So far, two approaches have been used for TCS inhibitor discovery: 1) HTS assays with purified model TCSs and different types of compound libraries and 2) SBVS with different TCS structures or structural models. Of special relevance have been the screens for TCS inhibitors (mainly WalKR) following classical screening methods with secondary metabolites from soil Streptomyces spp. These screens have led to the discovery of walkmycin B, singermycin and waldiomycin. To this date, phenotypic screening of Streptomyces secondary metabolites has been the most successful. Additionally, one compound produced by Penicillium XR770 showed inhibition of the NRII (or NtrB) HK from E. coli but had no activity against whole cell bacteria (65). Alternative approaches to target-based screening with chemical or natural product libraries are structure based virtual screening (SBVS) and rational drug design following SBVS or fragment-based screening (Figure 4). SBVS has identified a range of TCSs inhibitors. In SBVS targeted against TCSs, the most successful screens have found inhibitors that target the conserved catalytic ATP-binding CA domain of HKs (66–67). The HK dimerization domains have also been proposed as good drug targets for TCSs inhibitors (67). Of particular interest are the thiazolidinone derivatives, which were targeted to the CA domain of WalKR and have antibacterial activity towards S. epidermidis (Figure 5). These hit compounds from SBVS were further modified with different halogen groups - six reduced the MICs for S. epidermidis and brought about lower toxicity for animal kidney cells (68).



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Although the TCSs inhibitors published to date still need to be improved and better characterized, several of them might be developed into more potent inhibitors (Figure 5; Table 3) for instance, hexapeptides (69). It is unfortunate that several inhibitors of HK autophosphorylation or phosphotransfer such as RWJ-49815, inhibit TCS through nonspecific protein aggregation (70). Some benzimidazole TCS inhibitors induce bacterial death mainly via membrane damage (71). TCS kinase inhibitors may also have cytotoxic or hemolytic activity in human cells, including RWJ-49815 (mentioned above) and cyclohexenes (70–71). Moreover, initial biochemical screens for TCS inhibitors may identify a high number of inhibitors acting through non-specific inhibitory mechanisms (71), yielding a large number of compounds that cannot be further developed into drugs. To develop inhibitors with more specificity for selected TCS such as WalKR we need more molecular structures of the HK domains from relevant pathogens (66). Already some chemical scaffolds that interact with conserved motifs of the CA domain of HKs have been proposed (66). For SBVS it is also important to include protein flexibility data and protein interactions with water molecules. SBVS has the advantage of screening much higher numbers of chemical compounds in shorter time than can be achieved by conventional methods. However, SBVS also results in many false positive hits, necessitating whole-cell phenotypic screening methods and biochemical assays to validate the mechanism of action of hit compounds. There are several ways to improve TCSs inhibitor discovery and drug design. First, solving the structure of model proteins would facilitate SBVS and as a consequence provide proteins for the library screening approach. These structures should preferably come from TCS targets in clinically important pathogens in order to facilitate the successful identification of specific inhibitors. Also structural data of HKs or RRs cocrystallized with inhibitors are of special interest, as they will lead to the discovery of the binding site of the inhibitor, as well as visualizing the chemical space, which might allow for a further refinement of target binding by novel inhibitors. Indeed, more structureactivity relationship data are needed to reduce toxicity of TCS inhibitors in eukaryote cells. All these steps will lead to a better understanding of the mechanism of action of TCSs inhibitors. Secondly, SBVS libraries of compounds should contain more diverse structures of potential inhibitors as proposed in general for antibacterial drug research (15). In general terms, a search for novel compounds should not strictly use the Lipinskirule-of-5 as most antibiotics and drugs that are used successfully to treat cancer or central nervous system disorders do not follow the Lipinski rule (15, 72). Libraries of compounds should be diversified and specifically prepared to be used in TCSs screening, and it may be useful to remove false-positives and analogous compounds from drug discovery libraries (70).


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Over the last decade, research aimed at finding novel TCSs inhibitors has dwindled. Shifting priorities in the pharmaceutical industry as a consequence of high financial risks and low return on long-term (10–15 years) investment compared to, for instance, anticancer drugs, and the high technical complexity in antibacterial drug discovery may have been responsible for the observed decrease in screening for TCSs inhibitors. With the prospect of returning to a pre-antibiotic era, many public-private partnerships have been initiated to aid TCSs inhibitor research (73–74). Based on research developments, we propose that TCSs remain promising antimicrobial drug targets. Around twenty independent research groups from both academia and industry have shown promising preliminary results and proof-of-concept studies. The role of TCSs in antimicrobial resistance and virulence regulation as well as their essentiality for bacterial growth and survival make them attractive topics for future collaborative research between academia and industry.

Acknowledgments AEB would like to thank J. Blaazer for fruitful discussions on the manuscript.



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Table 1 Pathways regulated by two-component systems in different bacterial pathogens.

Regulated pathway A. Nutrient acquisition of: Nitrogen Phosphorus B. Metabolism:

Regulating TCSs NtrBC PhoRB

Electron transport system Catabolic machinery

ArcAB BarA/UvrY, TcDE, CitAB, MalKR Nitrogen metabolism NarXL Glucose metabolism UphBA Carbon metabolism BarA/UvrY Cell wall metabolism YycFG, VraSR, DesKR C. Adaptation to changes in the environment: Osmolarity EnvZ/OmpR Light Cph/Rcp1, NblSR Chemotaxis CheYB Temperature walKR, DesKR Oxygen DevRS or DosRS D. Developmental pathways: Sporulation SpoA Biofilm formation KinAB Competence ComDE E. Virulence CiaRH, MicAB (WalKR), RR04-HK04, RR06-HK06, RitR AgrAC, SrrAB, SaeRS, AlRS, LytRS, PhoPQ, PmrAB, RscCYojN-RscB, OmpR-EnvZ, SsrBA, SirA-BarA, DegU, VirRS, AgrAC, LisRK, FigRS, ArsRS, VirRS F. Others: Flagellar assembly Secretion systems: a) Type II b) Type III

c)

Type IV

d)

Type VI

CheA, FlrBC a) TtsSR b) RhpRS, AlgZR, SpiR/SsrB, PhoPQ, OmpR/EnvZ, c) VirB/D4, PmrAB, CpxRA, LetAS, LqsRS d) EnvZ/OmpR


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Table 2 Regulation of antibiotic resistance by two-component systems in human and animal pathogens. Pathogen Staphylococcus aureus

Klebsiellapneum oniae Acinetobacterbau mannii Pseudomonas aeruginosa small colony variant (SCV) of Pseudomonas aeruginosa Mycobacterium tuberculosis Enterococcus faecalis Salmonella typhimurium Escherichia coli Stenotrophomon as maltophilia

Gained resistance fluoroquinolone bacitracin and nisin vancomycin cationic antimicrobial peptides tetracycline, nalidixic acid, tobramycin, streptomycin and spectinomycin chloramphenicol, erythromycin, nalidixic acid and trimethoprim ß-lactams and chloramphenicol aminoglycosides, fluoroquinolones, tetracycline, chloramphenicol, erythromycin, trimethoprim carbapenem

TCS implicated in resistance regulation ArlS/R (32) BraSR (75) VraRS,GraRS, VanSR (31) GraSR,Stk1/Stp1 (76) PhoBR (34) LysR (oxyRKP) (77) CpxAR (78) AdeSR (79) CzcRS (33)

aminoglycoside

PhoPQ (80)

multidrug

MtrAB (81)

ceftriaxone (ß-lactam)

CroRS (82)

antimicrobial peptide ciprofloxacin novobiocin and deoxycholate multidrug ß-lactams and novobiocin

PhoPQ,PmrAB BaeSR (83) BaeSR (84) (85) ArcBA (86) BaeR (87)

aminoglycosides, ß-lactams, fluoroquinolones

SmeSR (88)



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Table 3 Characteristics of published inhibitors of two-component systems. Inhibitor

Inhibited TwoComponent System Algr1/Algr2

Organisms tested

thiazole derivatives

VanR/S

E. faecium

a) bisamidineind ole (lead compound); b) amidinobenzi midazoles (23 compounds) tyramines derivative (RWJ-49815)

KinA/Spo0F

B. subtilis, S. aureus, E. faecalis, E. faecium, MRSA

KinA/Spo0F

B. subtilis, E. faecium, S. pneumoniae, MRSA

6-oxa isosteres of anacardic acids (11 compounds)

KinA/Spo0F, NRII/NRI

S. aureus, E. faecalis, E. faecium, MRSA

salicylanilides (closantel and 14 compounds)

KinA/Spo0F

B. subtilis

thienopyridin e, TEP (lead)

HpkA and DrrA

hexapeptides (N-acetylated C-amidated Daminoacid), 2 peptides

CheA

cyanoacetoac etamide (CAA)

HpkA77 (WalK, VanRS)

thiazole derivatives

P. aeruginosa

E. coli, S. aureus, S. saprophyticus, S. epidermidis, E. faecium, S. pneumoniae, S. pyogenes, S. vividans, E. coli, Moraxella catarrhalis, K. pneumoniae, Proteus mirabilis, P. aeruginosa S. pneumoniae, E. faecium

Drug discovery method HTS (natural and synthetic compounds) HTS (aliginate biosynthesis pathway targeted) HTS (TBDD; screening with purified proteins) and chemical synthesis HTS (TBDD; screening with purified proteins) Chemical synthesis of natural compounds with reported activity HTS (TBDD; screening with purified proteins) HTS (TBDD; screening with purified proteins) HTS of combinatori al chemistry library

HTS and SAR

Mechanism/commen ts

Ref

inhibition of a) phosphorylation/deph osphorylation of Algr2; b) DNA-binding activity of Algr2 inhibition of autophosporylation

(89)

a) - , b) reducing DNA binding by RR

(91)

(90)

(92)

(93)

negative effects on mitochondria

(44, 94)

competitive ATP inhibitor of HK autophosphorylation

(95)

inhibition of mammalian kinase protein C

(69)

inhibition of autophosporylation and phospotransfer,

(96)


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ethodin (ethacridine lactate) phenylocoum arin derivative NSC9608 (8 compounds) DrrA peptide

HpkA77

T. maritima

SBVS

DosRS

SBVS

PhoP/Q

M. tuberculosis (nonreplicating) S. enterica

HpkA77

T. maritima

-

a) thiazolidinone derivatives (3 compounds), b) benzamide derivatives (2 compounds), c) furan derivative (1 compound), d) pyrimidinone derivative (1 compound) sulfonoamide derivative, LED2009 diaryltriazolea nalogs (15 compounds)

WalK

S. epidermidis

SBVS (HTVS)

QseBC

E. coli

KinA/Spo0F

S. epidermidis, S. aureus, E. faecalis, E. faecium, VRE, MRSA MRSA

thioridazine

VraRS

Carolacton

PknB (not ComD/E, not VicK/R)

S. mutans

Walkmycin B

WalK/R

B. subtilis, S. aureus

Waldiomycin

WalK/R

B. subtilis, S. aureus

SBVS

non-competitve inhibitors with ATP; protein aggregation; strong serum binding activity protein agregation

(96)

(63) inhibition of PhoPDNA complex inhibition of autophosphorylation; protein aggregation binds to HK and inhibits its autophosphorylation

(97) (96)

(98)

(57)

Chemical modifications of closantel

re-use of known neuroleptic antipsychoti c drug

Secondary metabolite from Streptomyc es sp. Secondary metabolite from Streptomyc es sp. Secondary metabolite from Streptomyc es sp.

affected membrane fluidity, which disturbed signal transduction; thioridazine can reverse resistance to oxacilin (methicilin analogue)

(99)

(100– 101)

(102– 103)

inhibition of autophosphorylation by binding to HK cytoplasmic domain


(53)

(67)


Table 4. Categories of genes that are regulated by WalKR in S. aureus and S. pneumoniae

Staphylococcus aureus

Cell wall metabolism atlA, lytM, ssA (autolysins; AtlA role in biofilm formation), isaA, cceD (proteins with transglycosylase domain, autolytic activity), fmtB(methicilin resistance determinant FmtB protein)

prsA (transport protein), opp3CDF (oligopeptide transport)

Streptococcus pneumoniae

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Page 16 of 28

Cell wall metabolism pscB (extracellular cell wall hydrolase; role in cell division), lytB (cell wall hydrolase), lytN, fabK (trans-2-enoyl-ACP reductase II), pspA (pneumococcal surface protein A), plaBCDA, yocH (autolysin), GbpB (extracellular cell wall hydrolase)

Transport

Transport pia system (iron transport system)

Metabolism pyrBC (aspartate carbamoyltransferases), carAB (carbamoyl-phosphate synthases), pyrEF (orotate phosphoribosyltransferases), ilvA1 (threonine dehydratase), argGH (urea cycle), pyr and pur operons (pyrimidine and purine metabolism), genes responsible for amino acids biosynthesis Virulence and Host-Microbe Interaction hla (alpha-hemolysin precursor), emp (fibrinogen-binding protein), splABCDEF (serine proteases), fnAB (fibronectin-binding protein A), coa (staphylocoagulase precursor), hlb (beta-hemolysin), sbi (immunoglobulin G-binding protein), chp (chemotaxis-inhibiting protein CHIPS), spa (Immunoglobulin G binding protein A precursor), can; efb (fibrinogen-binding protein), dltD ((D-alanine transfer protein), capDGGJMP (capsule biosynthesis) Metabolism fabDFGK (fatty acid metabolism), pspA (fatty acid metabolism), accABS (fatty acid metabolism), pur operon (purine synthesis), pyrD (pyrimidine synthesis), carB (carbamoyl-phosphate synthetase), nrdF (ribonucleotide-diphosphate reductase), mreC (degradation of the cell envelope) Virulence and Host-Microbe Interaction pspA (iron transporter)

Stress response grpE (heat shock protein), dnaJ (chaperone protein), radA (DNA repair protein)

Regulatory pathways seaPRSQ (TCS), hrcA (heat-inducible transcription repressor), sarST (staphylococcal accessory regulator-like protein, regulator T), icaR (ica operon transcription regulator)

Stress response grpE (chaperon, heat shock protein), dnaK (chaperon, heat shock protein), hrcA (heat-inducible transcription repressor), groEL (chaperonin)

Regulatory pathways n/a


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Figures

Figure 1 Two-Component System signaling pathway and inhibitors of TCS mediated signal transduction. TCSs represent the most common form of bacterial signal transduction, based on phosphotransfer. A phosphorus group is transferred from the CAdomain to a conserved His-residue of the histidine kinase and from there at a conserved Asp-residue of the RR. This leads to dimerization of the phosphorylated RR and activates it to induce the expression of its downstream target genes. Possible places of signal transduction are marked with stars.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2 Domain organization and conservation of HKs. A) Domain organisation of S. mutans WalK (PDB:4I5S). A HK consists of a periplasmic sensor domain (Sp, not shown) and cytoplasmic sensor domains (Sc), dimerization and phosphotransfer domain (DHp) and catalytic and ATP-binding domain (CA). Sc is variable and not present in all HKs, whereas DHp and CA are relatively more conserved than Sc and present in all HKs. B) Structure of the CA domain of T. maritima HK853 (PDB: 3DGE). The CA domain accommodates the nucleotide ligand (shown in sticks) in a well-defined binding pocket (shown as semi-transparent surface). The nucleotide binding-pocket includes the conserved N-, G1- , G2, G3-, and F-boxes (yellow, light green, dark green, pale green, and purple, respectively) and the variable ATP-lid (red). C) Sequence alignment of CA


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domains of WalK from different Gram-positive bacteria. Homologues of HKs present in different bacterial pathogens suggests broad-spectrum antibacterial effect of putative HKinhibitors D) Sequence alignment of CA domains of different E. coli HKs. Sequence similarities of the CA domain HKs suggest that a putative HK inhibitor would inhibit multiple targets (polypharmacology) which is expected to slow down antimicrobial resistance development.

Figure 3 The ATP-lid plays a critical role in ligand-binding and catalytic activity of members of GHKL-superfamily of proteins. The ATP-lid is a flexible loop variable in length and in sequence. Features of the ATP-lid can be exploited to design histidine-kinase autophosphorylation inhibitors with higher specificity to bacterial histidine kinases and reduced off-target effects to mammalian members of the GHKL-superfamily, i.e. with lower toxicity. Conservation scores were calculated using ConSurf. Ligands are shown in sticks.



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Figure

4.

Antibacterial drug

discovery

and

development

Page 20 of 28

pipeline.

Different

approaches can be used in antibacterial drug discovery (upper part of the scheme). Bottlenecks of drug discovery process are indicated as blue squares. Estimated amount of time of each phase of the antibacterial drug discovery process are indicated on the left side of the scheme. New business models for R&D to facilitate the development of novel antibacterials are listed in purple boxes. SBVS – Structural Based Virtual Screening, FBDD – Fragment Based Drug Discovery, HTS – High-Throughput Screening.


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A

CH3

B O HN HN

O

O

N

NH

C OH

O S

O

CH3

S HO CH3

O

H3C

O S O NH

N

O

H3C

HO OH

Figure 5. Specific TCSs inhibitors that can be used as a starting point for the design of more potent antibacterial drugs following structure-based drug design approaches. A) LED209 inhibits the binding of adrenergic signals to the periplasmic domain of E. coli QseC, preventing its autophosphorylation and consequently inhibiting QseC-mediated activation of virulence gene expression. B) Thiazolidione derivative was identified in a structure-based screening for ligands of the CA domain of S. epidermidis WalK. Thiazolidione derivative inhibited S. epidermidis WalK autophosphorylation (IC50 = 14 µM) and showed antibacterial effect against Gram-positive bacteria with MICs in the range of 2 – 6 µM. Discovery of thiazolidione derivates as TCSs inhibitors by SBVS demonstrated that this is a viable tool for discovery of HK inhibitors with antibacterial effects. C) NSC48630 inhibited the formation of S. enterica PhoP-DNA complex (IC50 = 3.6 µM). NSC4836 was identified as a putative PhoP ligand in a structure-based screening for ligands of the activated response regulator PhoP.



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Bacterial Histidine Kinases as Novel Antibacterial Drug Targets

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