Novel Antibacterial Compounds and their Drug Targets

Send Orders for Reprints to [email protected] Current Medicinal Chemistry, 2017, 24, 1-35

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REVIEW ARTICLE

Novel Antibacterial Compounds and their Drug Targets - Successes and Challenges Agnieszka A. Kaczor1,2,*, Andrzej Polski3, Karolina Sobótka-Polska4, Anna Pachuta-Stec4, Magdalena Makarska-Bialokoz5 and Monika Pitucha4,* 1

Department of Synthesis and Chemical Technology of Pharmaceutical Substances with Computer Modeling Lab, Faculty of Pharmacy with Division of Medical Analytics, Medical University of Lublin, 4A Chodzki St., PL-20093 Lublin, Poland; 2University of Eastern Finland, School of Pharmacy, Department of Pharmaceutical Chemistry, Yliopistonranta 1, P.O. Box 1627, FI-70211 Kuopio, Finland; 3Department of Applied Pharmacy, Faculty of Pharmacy with Division of Medical Analytics, Medical University of Lublin, 1 Chodzki St., PL-20093 Lublin, Poland; 4Department of Organic Chemistry, Faculty of Pharmacy with Division of Medical Analytics, 4A Chodzki St., PL-20093 Lublin, Poland; 5Department of Inorganic Chemistry, Faculty of Chemistry, M. Curie-Sklodowska University, M. Curie-Sklodowska Sq. 2, PL-20031 Lublin, Poland

ARTICLE HISTORY Received: August 23, 2016 Revised: October 12, 2016 Accepted: December 02, 2016 DOI: 10.2174/09298673236661612131 02127

Abstract: Infectious diseases are one of the most important and urgent health problems in the world. According to the World Health Organization (WHO) statistics, infectious and parasitic diseases are a cause of about 16% of all deaths worldwide and over 40% of deaths in Africa. A considerable progress that has been made during last hundred years in the fight against infectious diseases, in particular bacterial infections, can be attributed mainly to three factors: (1) the general improvement of living conditions, in particular sanitation; (2) development of vaccines and (3) development of efficient antibacterial drugs. Although considerable progress in reduction of the number of cases of bacterial infections, especially in lethal cases, has been made, continued cases and outbreaks of these diseases persist, which is caused by different contributing factors. Indeed, during last sixty years antibacterial drugs were used against various infectious diseases caused by bacterial pathogens with an undoubtable success. The most fruitful period for antibiotic development lasted from 40’s to 60’s of the last century and resulted in the majority of antibiotics currently on the market, which were obtained by screening actinomycetes derived from soil. Although the market for antibacterial drugs is nowadays greater than 25 billion US dollars per year, novel antibacterial drugs are still demanded due to developed resistance of many pathogenic bacteria against current antibiotics. In the last five years, one can observe a dramatic increase in cases of resistant bacteria strains (e.g. Klebsiella pneumoniae and E. coli) which are responsible for difficult to treat pneumonia and infections of urinary tract. The development of resistant bacteria strains is a side effect of antibiotic application for treatment: the infections become untreatable as a result of the existence of antibiotic-tolerant persisters. In this review, we discuss the challenges in antibacterial drug discovery, including the molecular basis of drug resistance, drug targets for novel antibacterial drugs, and new compounds (since year 2010) from different chemical classes with antibacterial activity, focusing on structure-activity relationships.

Keywords: Antibacterial activity, antibacterial drug target, antibiotics resistance, structure-activity relationship 1. INTRODUCTION The advance in application of antibiotics against the bacterial diseases has been regarded as the most prom-

ising idea in the previous century. Unfortunately, despite the possibility of applying more and more advanced and innovative technologies for antibiotic

_________________________________________________________ *Address correspondence to these authors at the Department of Synthesis and Chemical Technology of Pharmaceutical Substances with Computer Modeling Lab, Faculty of Pharmacy with Division of Medical Analytics, Medical University of Lublin, 4A Chodzki St., PL-20093 Lublin, Poland; University of Eastern Finland, School of Pharmacy, Department of Pharmaceutical Chemistry, Yliopistonranta 1, P.O. Box 1627, FI-70211 Kuopio, Finland; E-mail: [email protected] and Department of Organic Chemistry, Faculty of Pharmacy with Division of Medical Analytics, 4A Chodzki St., PL-20093 Lublin, Poland; E-mail: [email protected]

0929-8673/17 $58.00+.00

© 2017 Bentham Science Publishers

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treatment, the effectiveness of antibacterial drugs drastically declines. The excessive use of antibacterial and antifungal drugs has led to the occurrence of resistance to the overprescribed antibiotics, inducing consequently the re-emerging of many infectious diseases [13]. Nowadays, bacterial and fungal infections pose hazardous and difficult to solve health and social problems, inducing a considerable danger both for patients in the hospital and for the community [4]. Exemplification of such problems is the situation in the UK, where each year approximately twenty thousand cases of S. aureus infection occur. Unfortunately, 50% of infections are resistant to antibiotics [5]. Staphylococcus aureus, the pathogen causing a number of infections in animals and humans, is often found in multiple infections, including diseases of skin, lower respiratory tract and soft tissue. S. aureus, carried by around 30% of healthy people, is generally constantly ‘ready for the action’. Importantly, S. aureus contributes permanently to developing of antibiotic resistance. In addition, in the last years, quantity of methicillin-resistant S. aureus (MRSA) in hospital-acquired infections has risen by 10-15% in the well-developed countries of Western Europe, such as Germany and the UK, as well as in the USA [6]. Unfortunately, there is a risk that this tendency might be extending from hospitals to the community, because S. aureus is almost all over the human environment [5,7]. In recent years, the incidence and severity of other bacterial as well as fungal diseases have also increased, ranging from athlete’s foot, nails infections, dermatophytosis or candidiasis to such serious life-threatening disease as sepsis or histoplasmosis. The growing number of drug-resistant bacteria makes it necessary to search for novel antibacterial agents. Conventional drugs are only partially effective, predominantly because of the increasing occurrence of resistance to antibiotics, serious side effects like severe damage to the nervous and cardiovascular systems and the need of long period of treatment [8,9]. Even vancomycin, antibiotic which is perceived as the last chance, is increasingly insufficient, in terms of the fight with the drug-resistant infections [4]. Therefore, one of the main challenges in contemporary medicinal chemistry is the development of novel, effective, non-toxic, inexpensive and easily accessible antibacterial drugs interacting with novel molecular targets, different from the classical targets of antibiotics. The development of new antibacterial drugs can be achieved using one of the three fundamental methods: (i) the first one employs the drug originating from currently existing

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drugs, where innovative chemistry is used to optimize the compounds; (ii) the second one is based on the design of new molecules from known synthetic or natural compounds, and (iii) the third one needs the identification of novel target sites for the synthesis of the novel chemical entities [10,11]. Therefore it should be constantly consulted that the increasing availability of bioactivity databases, additionally with the use of computational approaches, give a chance to predict protein targets, which are the first step in computer-aided drug design, working on the basis of two major approaches: (i) structure-based drug screening (SBDS), used to identify novel chemicals from virtual compound libraries using molecular docking simulation tools, and (ii) ligand-based drug screening (LBDS), including pharmacophore modelling, the quantitative structureactivity relationship (QSAR) approach, and matched molecular pairs (MMP) search [12-14]. Recently, owning to the progress in modern analytical and computational methods some new targets have been identified. Among them an inhibition of cell wall biosynthesis has recently become a key target for antibacterial chemotherapy [15-17]. Although twenty years ago the attention of scientists was drawn to antibacterial drug development on Gram-positive bacteria, such as MRSA, and S. pneumoniae, the appearance of highly resistant Gram-negative strains of bacteria, like K. pneumoniae, P. aeruginosa, E. coli or A. baumannii emphasised the necessity of intensified study on targets in Gram-negative organisms. Successfully appeared that the structure of disaccharide-pentapeptide peptidoglycan is common both to Gram-positive, as well as Gram-negative bacteria, what predestines its biosynthesis to become attractive molecular target for the discovery of broad-spectrum drugs [18]. An alternate important target for newly developed antibacterial agents, like a porphyrin derivative XF-73, is the bacterial cell membrane [19]. Other antibacterial drug targets, like protein-protein interaction (PPI) networks (including both bacterial and host-pathogen protein complexes) [20,21], ribosomes [22] and many more have been also reported in literature [13,15]. There is no doubt that successful treatment of bacterial infections is strictly dependent on the availability of new drugs. Therefore in this review, we discuss the challenges in antibacterial drug discovery, including molecular basis of drug-resistance, proteins and ribosomes as drug targets for novel antibacterial drugs and new compounds with antibacterial activity (reported since the year 2010) acting on these targets with a focus on structure-activity relationships.

New Antibacterial Substances and their Drug Targets

2. HISTORICAL PERSPECTIVE Fungal and bacterial infections have been regarded as a serious threat frequently leading even to human deaths for centuries. Only two hundred years ago diseases such as tuberculosis, pneumonia, diphtheria and diarrhea were the dominant reasons of morbidity among adults and children [23]. In the late 19th century the correlation between the existence of microscopic pathogens and the development of various diseases was recognised, which led to the introduction of antiseptic procedures targeting high mortality related to postsurgical infections [24]. Furthermore, better hygiene and sanitation helped to reduce death rate arising from various diseases [24]. At last, appearance of antibacterial substances facilitated the treatment of multiple infectious diseases very effectively, i.e. the first drug with antimicrobial activity against syphilis elaborated by Erlich in 1911 [23]. The very first successful antibiotics discovery platform was elaborated by Waksman in the 1940s [25]. In this simple platform soil-derived streptomycetes were checked for their antibacterial activity against a susceptible test microorganism. That was done by detecting zones of growth inhibition on an overlay plate [25, 26]. That resembled the accidental discovery of penicillin (Chart 1) by Fleming in 1928 [27]. This was the first βlactam antibiotic, inhibiting cell wall biosynthesis and having a broad spectrum of activity. Waksman’s method of antibiotic search by application of a systematic screen was awarded a Nobel Prize. This method was used to screen streptomycetes which led to the discovery of streptomycin (Chart 1) in 1943 which was the first aminoglycoside and the first compound effective against tuberculosis (acting through binding of 30S ribosomal unit). The extensive application of ‘Waksman platform’ by the pharmaceutical industry led to the production of the major classes of antibiotics in the following 20 years [26]. The most important antibiotics from that era were (see Chart 1): (1) chloramphenicol discovered in 1946 with broad spectrum of activity, acting through binding of 50S ribosomal unit; (2) erythromycin, a macrolide discovered in 1948 with broad spectrum of activity, acting through binding of 50S ribosomal unit; (3) chlortetracycline, a tetracycline discovered in 1944 with broad spectrum of activity, acting through binding of 30S ribosomal unit; (4) vancomycin, a glycopeptide discovered in 1953, efficient against Gram-positive bacteria, acting through inhibition of cell wall biosynthesis; (5) rifampicin discovered

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in 1957, efficient against Gram-positive bacteria, acting through binding of RNA polymerase β-subunit [26]. Unquestionably, at first antibiotics discovery, and their later use in the middle of the last century were one of the biggest, or even the biggest achievements of humanity [28]. In further ten to fifteen years usual lifespan grew considerably. Moreover, many diseases caused by microorganisms were nearly vanished, whereas a number of viral diseases, as well as neoplastic have become easier to control [28]. Antibiotics therapy turned out efficient in fighting with fungal, bacterial, and protozoal infections as well as in treating of some physiological diseases (i.e. reducing cholesterol) [28]. Antibiotics became indispensable as food additives, veterinary use, as well as plant protection products (e.g. pesticides) [28]. Importantly, concurrently with the antibiotics application, the phenomenon of microorganisms resistance to antibiotics appeared. However, modifications of existing antibiotics generated active analogues [26]. The first synthetic antibiotics, the quinolones (e.g. ciprofloxacin, Chart 1) were developed in the 1960s - in the times when the speed of new drugs discovery appeared to outpace the dissemination of microbial resistance. Since the ‘golden age’ of the 1960s, there were ongoing changes in both science and medicine, while in 1990s economy became far more important [28]. However, the most worrying issues were a decrease in the new drug discovery rate, failure of efficiency of typical screening approaches and frequent cases of rediscovery. Furthermore, not a single broad-spectrum class of antimicrobial substances has been discovered since the 1960s [26]. The last clinically applicable antibiotic belonging to a new class was daptomycin (discovered in 1986 and efficient against Gram-positive bacteria, Chart 1), a lipopeptide that acts against the cell membrane of the bacteria, however it waited for approval up down to year 2003 [26]. Moreover, till 2012, when bedaquine (a new drug for tuberculosis) was approved (Chart 1), the streptogramins (e.g. streptogramin B, discovered in 1964, Chart 1) were the last discovered class of narrow-spectrum antibiotics (active against specific microbes). Yet even these substances awaited their introduction to the clinic for about 30 years [26]. Furthermore, linezolid (Chart 1), belonging to oxazolidinones was discovered in 1955 but approved in 2000; this antibiotic is efficient against Gram-positive bacteria and acts through binding of 50S ribosomal subunit [26].

4 Current Medicinal Chemistry, 2017, Vol. 24, No. 00 H2N

R

O

HN

N H

NH

HO H N CHO

H2N

O

NH

OH O

O

OH

OH Cl

S

OH HO

O

HN

N+

Cl O

O~

O

O

F

HO Streptomycin

O

O

O

HO

Penicillin

OH

NH

COOH

Kaczor et al.

HN

Chloramfenicol

OH

N

N

HN H2N

OH

HO

OH

N

OH O

HO

OH H

H

N

Ciprofloxacin

O

O

O

O

C 2H 5

Cl

HO

OH

O

OH OH

O NH

CONH2

O

O O OH

Erythromycin

H2N HO

HO

Chlorotetracycline

NH2 O

H N HOOC

O

H N

N H H

O

O

N H O

O O NH

N H

O HO

O HO O

O

O

H N O

N H

NHCH3

NH2

N OH O OH OH

O O

N

O O

O

OH

O

N N

OH OH

Vancomycin

H

N

NH

NH

Br HO

HN O

HO

HO H N

N H HN

Daptomycin

Cl

O O

O HOOC HOOC

N H

O

O

HO O

O

O

O

H N

O

OH O

N H

HO

HO O Cl

NH

O NH CONH2 O H N N H O O COOH

N H

N

N

N

O

N O

O O

O O O

O

NH H

O N

N O

HN O

N O

F

OH

Rifampicin

Bedaquiline

Streptogramin B

Linezolid

Chart (1).

At the end of previous century, it turned out obvious that the fight against microbes was lost. Unfortunately, the antibiotics resistance outstripped the discovery of new compounds with antimicrobial activity (a classic example is the growing rate of resistance to β-lactams [26]). 3. CHALLENGES IN DESIGN AND DISCOVERY OF ANTIBACTERIAL DRUGS 3.1. Antibacterial Resistance Shortly after the discovery and successful application of antimicrobial drugs, the resistance to antibiotics has become increasingly widespread. Despite the early triumph of the novel therapeutic agents, the emergence of resistant strains can be blamed on accumulated overuse and misuse of anti-infectious drugs [29-31]. Genetic mutations in bacteria and their acquisition of resistance genes changed them into much more dangerous and difficult to treat, leading to a greater risk of prolonged illness or even death. There are many difficulties in the treatment of patients infected with resistant bacterial strains, as a consequence of modified metabolism of such infected cells and high doses of drugs resulting often in serious side effects [30]. Therefore extensive understanding of molecular mechanisms de-

termining microbial antibiotic resistance as well as the design of novel smart antibacterial drugs with distinct action or multi-targeted combination treatment have been permanently required. The wide-spread problem of bacteria resistance forced an urgent need for emergency action for medicine, as well as to the whole scientific world. Nowadays, the epitome of crisis is the prevalence of ‘ESKAPE’ microbes (Staphylococcus aureus, Enterococcus spp., Enterobacter spp., Klebsiella spp., Pseudomonas aeruginosa, as well as Acinetobacter baumannii) exhibiting high multi-drug resistance. In fact, some strains of Gram-negative bacteria, including Acinetobacter baumannii, are already resistant to all presently known antimicrobial drugs. Recently, even such problems as childhood ear infections have become more frequent and difficult to treat [32]. The long list of drug-resistant bacteria, including sulfonamide-, penicillin-, methicillin-, macrolide-, vancomycin- and multidrug-resistant, was reported by Pelgrift and Friedman [33]. Different types of mechanisms are known to participate in the formation of bacterial resistance. As antibiotics action mechanism is either to shut down or to abolish key bacteria actions, the microbes resistance


mechanisms use all available strategies of stopping or hindering an active substance to hit the target cell or molecule. Therefore, the main kinds of clinically important resistance mechanisms involve (i) antibiotic destruction, (ii) modification of a target and (iii) restricted penetration and/or efflux of the compound, Fig. (1) [34]. Among the mechanisms mentioned above the bacteria efflux pump is one of the most significant. Efflux plays a vital action, not only in bacteria resistance against antibacterial drugs, but also at the same principle against antifungal, antimalarial and anticancer medicaments [35]. Efflux pumps consist of varied system collections, which are within and between the Gramnegative and Gram-positive divisions. They are simultaneously in charge of efflux of a number of toxic compounds, such as currently used antibacterial drugs [36]. Bacterial efflux pumps belong to two broad groups. The first one consists of the dedicated efflux pumps that recognize the specific antimicrobials, like in case of tetracyclines and macrolides. The second ones, more general multi-drug efflux pumps, are less discriminating in their choice of substrates than the first group and are represented by Pseudomonas aeruginosa [7]. Efflux results from the activity of membrane transporter proteins, which are known as multi-drug efflux


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systems (MES). 3.2. Other Challenges Besides the development of antibiotic resistances, other difficulties and challenges in the treatment of bacterial diseases and development of antibacterial drugs can be attributed to (i) human factors (irresponsible applications of antibiotics and underestimating of microbes), (ii) scientific failure (limitations of combinatorial synthesis and new HTS methods (highthroughput screening)), and (iii) unfavourable legal and economic factors such as increased costs, licensing, as well as strict regulations [28]. 3.2.1. Human Factors (Anthropocentric View) Due to uncontrolled usage of antibiotics the resistance genes have already been detected even at soil microorganisms. It is associated not only with the excessive application of antibiotics, but also they are often used in high concentrations and not specific ones. It is also assisted by uncontrolled, largely excessive use of antibiotics in agriculture, which contribute to the fast spread of antibiotic resistance. It therefore may lead to unpredictable and perilous consequences if the microorganisms induce full resistance [28].

Fig. (1). The mechanisms of antibacterial resistance. Reprinted by permission from Macmillan Publishers Ltd: [34] copyright 2002.


3.2.2. Scientific Failure 3.2.2.1. Limitations of High-Throughput Screening (HTS) and Computer-Assisted Drug Discovery (CADD) One of the main problems with antibacterial drug discovery is neglecting the limitations of current scientific approach which leads to too high expectations. This both concerns HTS methods and computerassisted drug discovery methods. It should be stressed that the main issue with target-based automated HTS approaches as well as virtual screening methods is the fact that identified substances (termed hits) although active on the target, are frequently ineffective in the host. The chemical compounds found in that way are often lacking therapeutic efficacy because of impediments to reaching the drug target. This is mainly related to permeability and efflux, which frequently prevent them from crossing cell-wall of the drug target [28]. Moreover, the hit-to-lead ratios, either in synthetic (0.001%) or natural products libraries are equally low [28]. It should be also emphasized that combinatorial chemistry itself can be not sufficient to obtain good results in research for new bioactive natural products leads. On the other hand, as the benefits of using the natural product libraries surpass chemical libraries, combinatorial chemistry is an excellent tool in looking for new natural product’s variations leads, which contribute to its complementary role in antibacterial drug discovery [28]. 3.2.2.2. Gram-Negative Bacteria Gram-negative bacteria are especially effective in keeping out drugs [26]. Their outer membrane plays a role of a barrier for amphipathic substances. However, all antibacterial compounds need to be amphipathic due to the requirement on their water solubility and, on the other hand, necessity to cross the cytoplasmic membrane. Importantly, the multidrug-resistant (MDR) pumps eject all drugs leaking in through the outer membrane. They mainly recognize chemically unrelated molecules on the basis of polarity. What is important, the more favorable ones are amphipathic molecules [26]. Hydrophilic substances penetration of the inner membrane is limited, which together with the outer membrane results in a perfect barrier. 3.2.2.3. Antibiotics and Guidelines of Lipinski’s ‘Rule of Five’ One of the most important failures in antibacterial drug discovery during the last fifty years reveals a bottleneck stemming from the small amount of drugs, which are able to break into the bacterial cell [26]. As it

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was already stated, HTS is capable of finding hits versus objectives both inside and outside human cells. However, these hits are often not useful as antibacterials as they are not able to overcome the bacterial membrane. It can be attributed to the tailoring of compound libraries which are typically limited to substances filtered with Lipinski’s rules based on the desired physicochemical properties, in order to increase the probability of oral bioavailability after administration [26]. Importantly, application of these principles does not always mean a success in finding of promising antibiotic lead substances as they shall fulfil various needs, e.g. the ability to penetrate into prokaryote cells [26]. However, it is ambiguous and rather unlikely that random libraries without any filters applied can perform better. Anyway, it is worth mentioning that these filters were not used in early screening libraries. Thus, there are still areas of chemistry with more matching properties for antibacterial drug discovery providing that we will be able to elaborate rules for effective bacterial penetration [26]. 3.2.2.4. Inherent Risk of Toxicity It should be kept in mind that the order of magnitude of micromolar concentrations at which antibacterial drugs are effective is two to three times greater than for drugs used on eukaryotic targets. Poor penetration is why this situation occurs, naturally when the rate of antibiotic binding to protein or ribosomal sites are similar to other medicines [26]. However, the necessity of supplying a sufficient amount of an antibiotic considerably increases toxicity, decreasing the chances of identification of good lead compounds [26]. 3.2.2.5. Clinical Trials It is noteworthy that in clinical trials testing of novel antibacterial substances takes place against severe infections lasting a few days only [26]. What makes it difficult to point out a clear-cut end points of a new substances during clinical trials is the fact that the majority of examined patients are infected by drugsusceptible bacteria. Furthermore, due to ethical reasons, placebos should not be given to ill patients [26]. 3.2.3. Economic Aspects The limitations described above, which science can overcome are accompanied by economic issues [26]. Antibacterial treatment usually takes a short time and generally lasts no longer than a week. Moreover, the inevitable development of resistance to any antibiotic further limits its time of usefulness. Contrary, patients suffering from any chronic disease need to be treated with their drugs on a daily basis, often till the end of


their lives. A good economic example is the comparison of annual sales of the top cholesterol-lowering drug atorvastatin - $12 billion during the duration of patent protection and only $2.5 billion for bringing the highest profit antibiotic - levofloxacin [26]. Anyway, an antibiotic market is still large enough to be attractive for the discovery of new antibiotics. 4. ANTIBACTERIAL DRUG TARGETS AND NOVEL CHEMICAL ENTITIES ACTING ON THEM Out of about two hundred bacteria conserving essential proteins, only a small fraction is nowadays considered as possible drug targets. Antimicrobial drugs disable bacteria by targeting necessary elements of microbial metabolism (Fig. 2). As an example, β-lactams, in particular cephalosporins or penicillin, successfully stop synthesis of bacterial cell-wall. Other important antibacterial drug targets are DNA gyrase (e.g. for quinolones), DNA-directed RNA polymerase (e.g. for rifampicin), protein synthesis (e.g. for chloramphenicol, macrolides, aminoglycosides, clindamycin, oxazolidinones and tetracyclines) and enzymes (e.g. for trimethoprim and sulphonamides) [34]. The most effective antibiotics hit only three pathways or targets: cell wall synthesis, the ribosome, as well as DNA topoisomerase and gyrase [26].


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are located in the inner and outer leaflets, respectively [37, 38]. Contrary, Gram-positive bacteria do not have the outer membrane so their peptidoglycan layer is much thicker in comparison to their Gram-negative counterparts [37, 38]. For antibacterial drugs the bacterial cell wall biosynthesis inhibition is one of the main molecular targets. Importantly, the peptidoglycan biosynthesis cycle is one of the most validated and well-researched targets for antibacterial treatment [39]. It consists of two complex stages. The first one is performed in the cytoplasm and involves creating a monomeric building block of N-acetylglucosamine-N-acetylmuramyl pentapeptide [39]. First the enolpyruvate from phosphoenolpyruvate is transferred to position 3 of UDP-N-acetylglucosamine which is catalysed by MurA [39]. The next step is formation of UDP-N-acetylmuramate which results from reduction of the enolpyruvate moiety to D-lactate catalysed by MurB. MurC, MurD, MurE and MurF, that are ATP-dependent amino acid ligases, catalyze the multi-step addition of the pentapeptide side-chain on the newly reduced D-lactyl group, which leads to UDP N-acetylmuramyl pentapeptide [39].

4.1. Cell Wall Biosynthesis Inhibition

Two globular domains of MurA are linked by a double-stranded connector (Fig. 3A) [39]. Similarly to both domains, the main-chain fold is constituted by three parallel internal helices, which are surrounded by three helices, as well as three four-stranded β-sheets [39]. The MurA catalytic site is placed in a deep cavity between the two globular domains [39]. The uridinyl ring of UDPN-acetylglucosamine is positioned between two hydrophobic surfaces formed by Pro-121 and Arg-120 on one face and by Leu-124 on the second one [39]. These interactions are accompanied by the hydrogen bonds formed by uridinyl base atoms O1 and N1, and supply binding specificity for the base [39]. MurA is the target of fosfomycin, which is a natural broad-spectrum antibacterial drug [40]. It is an epoxide and relatively poor antibiotic because an everincreasing number of bacteria is already resistant to fosfomycin [40]. Thus, more potent inhibitors are needed as derivative of 5-sulfonoxy-anthranilic acid shown in (Fig. 3A) [40] or others described in this chapter.

The first barrier for the antibacterial drug is the microbial cell wall [37,38]. Regarding the gram-negative bacteria, their outer membrane is the outermost barrier, however there are in addition the cytoplasmic membrane as well as the peptidoglycan layer. This outer membrane displays an asymmetric distribution of the lipids with phospholipids and lipopolysaccharide that

MurB, a mixed α+β protein, is built of three domains (Fig. 3B) [39]. Free enzyme structure in comparison to the substrate-bound one uncovers that the lack of UDP-N-acetylglucosamine enolpyruvate results in a rigid-body rotation of domain 3 in the direction of 1 and 2 domains [39]. As a consequence, when sugar substrate binds to MurB, it causes the shutdown of the

The efficacy of certain antibiotics, e.g. penicillin, is high only against a narrow spectrum of bacteria while some other antibiotics, e.g. ampicillin, inhibit not only a broad range of gram-negative, but also gram-positive bacteria. Importantly, the result of antibiotics action on bacteria may be different too. For example tetracycline which inhibits bacterial growth acts as a bacteriostatic agent. By contrast, penicillin kills bacteria and it is considered as a bactericidal agent. Treatment with a combination of antimicrobial drugs may be beneficial in comparison with the efficiency of each antimicrobial drug alone. This is the case of sulphamethozale and trimethoprim, which are synergistic. However, close attention should be paid to avoid antagonistic combinations of drugs [34].


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Fig. (2). Targets of antibiotics. The current number of exploited targets is very small, especially when compared to 200 conserved essential bacterial proteins. The most efficient antibiotics alone can hit maximally three targets or pathways: the ribosome (which consists of 50S and 30S subunits), cell wall synthesis and DNA topoisomerase or DNA gyrase. Reprinted by permission from Macmillan Publishers Ltd: [34] copyright 2002.

substrate binding channel over the substrate [39]. A single point mutation confirmed the key role of Ser-229 at the active site of the enzyme, serving as a general acid catalyst. [39,41]. Mur ligases (MurC-MurF) exhibit topological similarities to each other [42]. The enzymes consist of three structural domains (Fig. 3C): (i) an N-terminal domain primarily associated with binding the UDPMurNAc substrate, (ii) a central domain resembling the ATP binding domains of multiple ATPases and GTPases, and (iii) a C-terminal domain usually responsible for binding the incoming amino acid [42]. The enzymes have a modular multi-domain structure closely like other Mur family ATP-dependent amide-bond ligases [43]. The possible significant conformational changes, along with the acquirement of two N-terminal domains, which let this enzymes bind to a substrate that is the same at first end, while growing peptide tail is presented at the other. This tail ultimately becomes the rigid part of bacterial cell wall [43].

4.1.1. Inhibitors of MurA As it was already mentioned, there are certain pathogenic bacterial agents inherently resistant to fosfomycin which is an inhibitor of MurA. Moreover, fosfomycin resistance often occurs both as a result of its declined permeability, and different kinds of modifications, concerning the active site of an antibiotic, as well as enzymatic modification of this inhibitor alone [44]. It is why an urgent need exists to develop the structurally diversified inhibitors. Scholz et al. [45] worked on nitrovinyl derivatives which have been known as antibacterials for a long time. Nitrovinylfuran derivatives display broad spectrum of antibacterial and antifungal activities. Among this class of compounds derivatives (1) (Chart 2) have gained special attention. The compound with R=Br can potently suppress the activity of MurA enzymes, gaining the value of an IC50 equal 2.8 (±0.2) µM in case of the E. coli MurA enzyme [45]. Moreover, this compound is also a good inhibitor of E. coli MetAP (me-



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Fig. (3). Crystal structures of MurA-MurC. Proteins are shown in ribbon representation. Small molecules are shown as spheres with carbon atoms colored in cyan or magenta. A- Enterobacter cloacae MurA shown in complex with an inhibitor (PDB ID: 1YBG) [40]; B- E. coli MurB in complex with UDPN-acetylglucosamine enolpyruvate shown with carbons in magenta and FAD (flavin adenine dinucleotide) shown with carbons in cyan (PDB ID: 1UXY) [41]; C- E. coli MurC as an example of Mur ligases (PDB ID: 2F00) [43].

thionine aminopeptidase), albeit excluding the human MetAPs [45]. It was proposed that the nitrovinyl group in (1) and some its derivatives are involved in the reaction of Michael addition to nucleophilic structures located in the target proteins [45]. R Br

O

R NO2

O

R2

S

O

(1) HO

R1

(2)

R3

OH O O

O HO

(3)

O

O HO

HO

OH

HO (4)

Chart (2).

Miller et al. [46] synthesized benzothioxalone derivatives (2) (Chart 2) which were found to inhibit MurA of E. coli and MurA/MurZ from Staphylococcus aureus showing the IC50 values within range from 0.25 to 51 µM. Some representatives from this class suppress as well the activity of Staphylococcus aureus presenting MICs between 4 and 128 mg/L. Regarding the structure-activity relationship among these compounds, it was determined that the crucial factor influencing their inhibiting features was the location of an oxygen atom, favouring either C-5 or C-6 of the thiazolone ring - thiazolones missing these properties were established to be usually less efficient inhibitors of MurA and

MurZ [46]. The types of this substituent can be different, including hydroxyl groups. Furthermore, these compounds inhibited MurA in an irreversible manner. It was determined that a radiolabelled inhibitor belonging to this class of compounds bound stoichiometrically to the enzyme, which can be replaced by dithiothreitol [46]. This data is in accordance with the benzothioxalone inhibitors interacting with the enzymes through a covalent binding concerning the location analogous to C115 in E. coli MurA [46]. In order to validate the hypothesis about the binding manner of these inhibitors, molecular docking approach was employed. Docking of one of the most active derivatives with R, R1 and R3 = H and R2 =OH within the MurAUDP-GlcNAc co-crystal framework made it possible to conclude that this compound joins a pocket that places the carbonyl group of the thioxalone near the thiol of C115 , in accordance with the prerequisites for covalent adduct generation and privation of inhibiting properties against the mutant C115D MurA enzyme [46]. Shigetomi et al. reported antibacterial features of both tulipalin B and its analogs [47]. In particular, 6tuliposide B (3) and tulipalin B (4) (Chart 2) derivatives were obtained to examine their structure-activity relationship facing a wide spectrum of bacteria. On the bacterial MurA, which is the molecular target of 6tuliposide B, the (S)-3’,4’-dihydroxy-2’-methylenebutanoic acid functions comparably to cnicin. The structure-activity relationship analysis revealed that the structure of 3’,4’-dihydroxy-2’-methylenebutanoate can have liable antibacterial properties, as well as that the relation between the 6-tuliposide B glucose moiety (3) and antibacterial action is not explicit [47]. Notwithstanding the sugar group probably does not play significant role in antibacterial activity in vitro, while


the tuliposide presence with ability to convert enzymes in tulip tissues may clarify the enhanced defence mechanism addressed towards both microbial and insect attack [47]. The 6-Tuliposide B (3) displayed good activity on MRSA, excluding various MIC values found between 3’S- and 3’R-epimers, also tulipalin B (4) had a bit better antibacterial effect comparing to linear esters [47]. Thus, generation of tulipalin B (4) is recognized as essential for the inhibition process of microbes growth of 6-tuliposide B (3) [47]. 4.1.2. Inhibitors of MurB To our best knowledge, in the considered time range (2010-2014) no new MurB inhibitors were reported. Thus, it was decided to present the imidazolinone analogues (5) (Chart 3) which were described by Bronson et al. in 2003. These compounds are the primary examples of stereochemically discrete MurB inhibitors with proved antibacterial activity, exhibiting by definition a promising chemotype in the quest to innovative antibacterial agents [48]. Concerning the structure-activity relationship it was determined that the crucial role for MurB inhibitory action plays a lipophilic substituent on the nitrogen distal to the biphenyl ether moiety (N-1) [48]. Furthermore, it was concluded that it is not essential to have an amide linkage, however simply ‘capping’ the NH moiety with a methyl group is not adequate, as this leads to an activity decline. Moreover, it was concluded that a lipophilic substituent is required at the N-1 nitrogen atom [48]. O RCOO

N

N

NHR1 O

O (5)

Kaczor et al.

this compound did not present any antibacterial activity on a couple of different E. coli strains [49]. Shahul et al. [50] reported the discovery of pyrazolopyrimidines (7) (Chart 4) as new inhibitor agents of both the E. coli and Pseudomonas aeruginosa MurC. This was the first finding of a robust small molecule MurC inhibitor against gram-negative bacteria that proved antibacterial properties [50]. This study was a combination of medicinal chemistry efforts supported by molecular modelling and leading to the recognition of MurC inhibitors showing selectivity against a set of human kinases [50]. The conducted optimization made it possible to achieve subnanomolar enzyme inhibitors displaying considerable antibacterial activity towards efflux-deficient mutants of E. coli and Pseudomonas aeruginosa. The compounds did not exhibit the action against wild-type bacteria which can be connected with permeability and efflux barriers in the bacterial cell [50]. Tomašic et al. [51] carried out virtual screening for ATP-competitive inhibitors targeted at MurC, as well as MurD ligases, by the application of a consecutive hierarchical filters protocol. Several substances were submitted to investigation for blocking of MurC and MurD ligases, what prompted the identification of weak inhibitors with dual inhibitory activity [51]. Such obtained compounds are the representatives of new scaffolds for subsequent optimisation in order to multiple Mur ligase inhibitors possessing improved inhibitory potency (e.g., compounds (8) and (9) (Chart 4)) [51]. The group of Süssmuth identified a 13-mer peptide named feglymycin (10) (Chart 4), serving as a reversible inhibitor of MurA and MurC. The antibacterial activity studies did not display any effect against E. coli which can be attributed to difficulties referring to the diffusion of feglymycin across the outer membrane of bacteria (or a bacterium) [52].

Chart (3).

4.1.4. Inhibitors of MurD

4.1.3. Inhibitors of MurC

Sosič et al. modified the naphthalene sulphonamides which caused the replacement of the D-Glu moiety, with rigid cyclic counterparts, as it is presented for derivatives of compound (11) (Chart 5) [53]. It emerged that inclusion of rigid mimetics in exchange for D-Glu ameliorated the inhibitory action against MurD comparing to the parent derivative, what confirmed the utility of this conformational limitation [53]. The most potent inhibitors obtained were the [6-(4cyano-2-fluorobenzyloxy)-naphthalene-2-sulfonamide derivatives. As the authors stated, rigidisation is one of

Frlan et al. reported acyl-sulfonohydrazide-based compounds (6) (Chart 4) as potential MurC inhibitors [49]. This development was supported by an idea concerning an acyl-sulfonohydrazide group starring as a prospective diphosphate derivative. However, it turned out in the assays that the majority of these compounds were poor MurC inhibitors The best compound seems to have R1= 3-NO2 , R2 = iBu, R3=2-naphthyl which had IC50 towards E. coli MurC of 245 µM. Nonetheless,



N N R1

O S O N H

H N

R2 O S N O O H

R3

R

(6)

NH

R1

HN N

R1 NH N N

N N

N H

NH

H2N

NH

O

NH

O N

O

(9) OH

O NH

N

OH

O NH

NH

(8)

OH

O

S

R3 NH

R2

(7)

OH

11

O NH

NH

O NH

NH

O

O

O

O

O

HO

OH HO

OH HO

OH HO

OH HO

COOH

O NH

NH

NH

COOH

O OH

(10)

Chart (4).

the key aspects in the optimisation of medicinal chemistry procedure, most likely as a consequence of possible reduction of the entropic penalty upon binding of ligands to MurD. They also claimed that this procedure could conduce increased binding affinities and enhanced inhibitory properties of these derivatives [53]. In order to confirm the binding mode of one of derivatives of compound (11), the crystal structure of inhibitor-enzyme system was obtained as well. Important developments of MurD inhibitors have been accomplished by Tomašić et al. [54-56] and Zidar et al. [57,58] working within the research groups of Mašič and Kikelj. Tomašić et al. [54] obtained a number of hydroxy-substituted 5-benzylidenethiazolidin-4ones. Then they checked them for the inhibitory activity towards Mur ligases. The compound with the best potency (12) (Chart 5) against MurD-F presented IC50 from 2 to 6 µM, what made this compound an auspicious multi-target Mur ligases inhibitor. Moreover, they analyzed antimicrobial action against various strains, as well as inhibitory activity against protein kinases, mutagenicity and genotoxicity of (12) were investigated; the kinetic and NMR studies were performed as well [54]. This work was continued by Zidar et al. [57] who designed, synthesized, and assessed 5-benzylidenerhodanine- and 5-benzylidenethiazolidine-2,4-dionebased compounds (13) (Chart 5) as MurD inhibitors with E. coli IC50 values from 45 to 206 µM by variation of the linker between the two phenyl rings and substitution of the thiazolidine-2,4-dione ring by the rhodanine moiety. Furthermore, they also obtained the high-

resolution crystal structure of MurD in complex with (R,Z)-2-(3-[{4-([2,4-dioxothiazolidin-5-ylidene]methyl) phenylamino}methyl)enzamido) pentanedioic acid which exposed particularities of the binding mode of the inhibitor within the active site. Continuing the previous efforts, Tomašic et al. [55] formulated, synthesized and examined new D-glutamic acid-based inhibitors (14) (Chart 5) of E. coli MurD ligase which possess the 5-benzylidenethiazolidin-4one moiety [55]. The most active compounds with IC50 of 3-7 µM possessed an oxygen or sulfur atom as X group and had 0-2 carbon atom linker (n) and at the moment of the article publication were the most efficient MurD inhibitors. Furthermore, molecular docking experiment was performed to establish the binding mode of the investigated compounds. As an outcome of optimization of benzylidenethiazolidin-4-one series of MurD inhibitors, Zidar at al. designed, synthesized and evaluated four series of compounds (15) (Chart 5), and three of them exhibited good inhibitory potencies against MurD ligase from E. coli. The best compound had an IC50 of 28 µM and three other inhibitor agents had IC50 values below 50 µM. Moreover, one inhibitor displayed insufficient antibacterial activity against Gram-positive strains of Staphylococcus aureus and Enterococcus faecalis with MICs of 128 µg/mL. Additionally, the X-ray crystal structures of two inhibitors in complex with the MurD active site uncovered interactions with residues in the binding pocket [58]. Recently, Tomašić et al. [56] have formulated, synthesized, and assessed the first D-glutamic acid-


Kaczor et al.

O

O

S NHR O

O

NC

HO

OH OH

F

S

S

NH

N H

O

S

HOOC

(11)

HOOC O

COOH

O

(14)

O

NH

S

N

(13)

COOH

O

NH

HOOC

X [CH2]n COOH

X

N H

OH

S

O

OH O

O

NH

NH

S

X

NH

N H

X

R

(15)

COOH

HOOC

NH

S

O

O

O

O

NH

(12)

COOH

X

N H

R

O O

(17)

(16)

O O O

O

O S HN

HOOC

S

S

R

O

O

COOH

N H

O

NC

(18)

S

N

F

(19)

NH

N O O

(20)

N+

O~

Chart (5).

containing dual inhibitor (16) (Chart 5), of MurD and MurE ligases from E. coli, as well as S. aureus. These compounds exhibited IC50 from 6.4 to 180 µM and presented antimicrobial activity against gram-positive Staphylococcus aureus and its methicillin-resistant strain (MRSA) with MIC of 8 µg/mL [56]. The most promising compounds had a sulfur atom at the position of X in the tiazolone ring. Further developments of MurD inhibitors (17-19) (Chart 5) were reported by Barreteau et al. [59]. The SAR and co-crystal structures obtained for MurDEc became a standard for a design of the inhibitors derived from (17). The first series of glutamic acid-based inhibitors (17) selectively inhibited MurDEc. However, thiazolidin-4-one derivatives and sulphonamide inhibitors, in particular having aromatic surrogates for Dglutamic acid such as (18) and (19), inhibited almost all MurD orthologues with IC50 values in micromolar range. In particular, compound (18) seems to be a promising substance for subsequent design because it inhibits all MurD orthologues (IC50 values of 127-270 µM) [59]. Simćić et al. [60] synthesized N-(5-(5-nitro-2-oxo1,2-dihydro-3H-indol-3-ylidene)-4-oxo-2-thioxo-1,3thiazolidin-3-yl)nicotinamide (20) (Chart 5), a derivative of 2-oxoindolinylidene containing a new scaffold.

Next, they investigated its inhibition potency against the MurD enzyme from E. coli using an enzyme steady-state kinetics study. This derivative displayed competitive inhibition referring to UMA (UDPMurNAc-L-Ala), and diminished the growth of bacteria. Moreover, they isolated and purified 13C selectively labeled MurD enzyme from E. coli and investigated the interactions of novel inhibitors applying the 1H/13CHSQC 2D NMR technique. In addition, molecular dynamics simulations revealed that the complex of MurD with the inhibitor is stable. Interestingly, the mode of interactions of the new compounds was compared to the one of derivatives of naphthalene-N-sulfonamideD-Glu, transition state mimicking inhibitors, UMA and AMP-PCP, an ATP analog. It interacts with the UDP/ MurNAc binding region. Contrary to the transition state mimicking inhibitors, no interactions with Cterminal domain of the enzyme were discovered, which can be favorable for binding of the ligand [60]. 4.1.5. Inhibitors of MurE Shiu et al. [61] performed an evaluation of a new antibacterial natural product (21) (Chart 6) which has been lately isolated from the Hypericum olympicum L. cf. uniflorum. Importantly, MIC were evaluated for a series of microbes, such as: meticillin-resistant and



-susceptible strains of S. aureus, S. epidermidis and S. haemolyticus; vancomycin-resistant and -susceptible E. faecalis and E. faecium; penicillin-resistant and -susceptible S. pneumoniae; group A streptococci (S. pyogenes); and C. difficile [61]. MICs gained 2-8 mg/L for majority of Staphylococci spp. and all of Enterococci spp., however were equal ≥16 mg/L for S. haemolyticus and were >32 mg/L for all tested bacteria spp., when blood were presented. In addition, it was tested against Gram-negative bacteria, such as E. coli, P. aeruginosa and S. enterica but turned out to be inactive [61].

(21)

OH O

Chart (6).

4.1.6. Inhibitors of MurF Hrast and coworkers [62] developed a group of structurally similar derivatives (22) (Chart 7) using lately obtained cyanothiophene inhibitors of MurF from Streptococcus pneumoniae as a starting point and determined their inhibition properties towards different bacteria MurF enzymes [62]. Further optimization of the lead compound resulted in a group of nanomolar MurF inhibitors from Streptococcus pneumoniae and micromolar MurF inhibitors from E. coli and Staphylococcus aureus. Certain inhibitors also displayed antibacterial activity against Streptococcus pneumoniae R6. The compounds showed antibacterial activities against Streptococcus pneumoniae at concentrations from 16 µg/mL to 64 µg/mL [62]. These results, along with two new co-crystal structures, can be used for further compound modifications and creation of more active drugs. O

R

O

R1 S O HO Cl

S

X

Cl

CN

O

S

NH

NC

NH2 O

N H

N H

NH2+Cl~

Cl

(22)

(23)

Cl N Cl

N

N N

OH

(24)

Chart (7).

O

S NH

NC

N H

(25)

As a continuation of the efforts described above, Hrast et al. [63] synthesized and evaluated a second generation of 37 new MurF inhibitors with cyanothiophene scaffold (23) (Chart 7). It was found that the morpholinosulfonyl moiety and chlorine next to sulfonamide linker are important for efficient inhibition of MurFSp. Moreover, it can be also noticed in the cocrystal structure, where one of the sulfonyl oxygens of a derivative of (23) forms two hydrogen bonds with the enzyme and the chlorine forms hydrophobic interactions. Importantly, the most potent inhibitor has well balanced inhibitory properties against both MurF from Streptococcus pneumoniae and MurF from E. coli with IC50 values of 20 µM and 25 µM, respectively [63]. Sosič et al. obtained a series of 2,4,6-trisubstituted 1,3,5-triazines (24) (Chart 7), having a differentiated set of substituents (-OH, -SH, -OMe, -Cl, -HNR, -SR and amino acid groups) and tested their MurF inhibition [64]. One of the inhibitors displayed notable activity against MurF from E. coli [64].

O

HO

13

O

N

Cl

Turk et al. reported the expression, purification and biochemical depiction of MurF from S. pneumoniae [65]. Furthermore, they performed ligand-based virtual screening and effectively identified a novel hit having micromolar inhibitory activity. This compound (25) (Chart 7) showed micromolar inhibitory activities against MurFSp and MurFEc, with IC50 values of 126 µM and 56 µM, respectively [65]. 4.1.7. Inhibitors of MurI Basarab et al. [66] performed structure-activity relationships for a series of pyrazolopyrimidinediones (26) (Chart 8) which inhibit the growth of Helicobacter pylori by targeting glutamate racemase (MurF), an enzyme that provides D-glutamate for the construction of N-acetylglucosamine-N-acetylmuramic acid peptidoglycan subunits assimilated into the bacterial cell wall [66]. Application of a number of substituents helped in optimization of target activity, as well as pharmacokinetic stability in vivo. Interestingly, placing in the scaffold at the 7-position an imidazole ring leads to better potency owing to a hydrogen bonding network, which is present between 3-position nitrogen atom, side chains of Ser152 and Trp244 of this enzyme, as well as a bridging water molecule [66]. It was also demonstrated that the scaffold’s lipophilicity is significant to antimicrobial activity. It was also concluded that the imidazole moiety alkalinity can enhance solubility in water at lower pH values, which in turn could mean better oral bioavailability. The most active derivatives of (26) displayed selectivity for the MurI


Kaczor et al.

isozyme (IC50 of 1400, 26 and 34 nM, respectively) concerning the remaining MurI isozymes (from S. aureus, E. faecalis and E. coli), which IC50 is greater than tested maximum value of 400 µM. Furthermore, in vitro activity of Helicobacter pylori growth inhibition of the compounds presented MICs values from 0.5 to 8 µg/mL respectively [66]. O N

R1

O N R

N

N R2 X

(26)

Chart (8).

4.1.8. Multiple Inhibitors of the Mur Enzymes One of the most significant current trends in medicinal chemistry is development of multi-target drugs as it is thought that they may have better therapeutic effect comparing to the single-target drugs. However, the development of potential drugs that bind not only to one target is very challenging, involving both the necessity of the properly balanced affinities for the different targets, as well as the preservation of the drug-like properties [67]. In this context Perdih et al. [68], as a continuation of their earlier virtual screening campaign, designed and synthesized derivatives of benzene-1,3dicarboxylic acid 2,5-dimethylpyrrole (27, 28) (Chart 9) exhibiting dual MurD/MurE inhibition. Importantly, novel molecules containing benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole coupled with the five member-ring rhodanine moiety were found to be multiple inhibitors of the whole cascade of Mur ligases (MurCMurF) in the low micromolar range [68]. Moreover, an appearance of the benzene dicarboxylic moiety and benzene-based substitution of the five or six-membered heterocyclic moiety were demonstrated as prerequisites for multiple Mur ligase inhibition [68]. O

O

O

HO

HO

S

NH N O HO

N R

N

X

O

HO

O

R1

N R

O (27)

(28)

Chart (9) .

4.2. Peptide Deformylase (PDF) A metalloenzyme peptide deformylase (PDF) catalyzes the reaction of water and formyl-L-methionyl by

removing the N-terminal formyl groups. The products of this reaction are methionyl peptide and formate, which are important for bacteria survive. Therefore PDF is considered in antimicrobial chemotherapy as a potential target [69,70]. PDF is an α/β type protein where a central helix is wrapped by a five-stranded-, as well as two-stranded- anti-parallel β-sheets and by an anti-parallel β-sheet (Fig. 4) [71]. Differently to other metalloproteases, the available crystal structures of PDF show that this enzyme adopts a fold, as well as has the non-prime side [72]. On the other hand, the metal-binding site resembles the thermolysin. These two proteins interact with the bound metal by means of two histidine residues, from a conserved HEXXH motif [72]. Furthermore, as identified by the site-directed mutagenesis, a conserved glutamate and glutamine residues in the active site are essential factors for the catalytic activity [72].

Fig. (4). Escherichia coli crystal structure PDF in ribbon representation containing Ni2+ (magenta sphere) and hydroxy(3-phenylpropyl)amino]methanol (carbon atoms in cyan), PDB ID: 2AI8 [72].

Cui et al. 73 evaluated a collection of phenolic compounds (29, 30) (Chart 10) from the propolis. Researchers studied these compounds for their enzyme inhibition against H. pylori PDF. A one of the propolis components, caffeic acid phenethyl ester (CAPE), a derivative of (29) (Chart 10) is a competitive inhibitor against HpPDF. Its activity was very promising, IC50 value was equal to 4.02 µM [73]. Importantly, majority of PDF inhibitors are pseudopeptides, while the CAPE structure is different [73]. The authors also postulated that CAPE binds in the active site region without interaction with the catalytic Co2+. It may decrease CAPE interactions with metalloproteins, which are present in a human body. As a consequence, it should decrease the amount and intensity of side effects [73].



O R1

R2

HO

O

R1 R2

HO OH O (30)

(29)

O

N

N

O

H HO (31)

O

R1 N R2

O R 1

H HO

N

N O

O

R2 N R3

(32)

Chart (10).

Shi and coworkers [74] synthesized 2,5dihydropyrrole formyl hydroxyamino derivatives (31) (Chart 10) and analyzed their antibacterial activity. The in vitro antibacterial activity of several compounds from that group was better than that of existing antibiotics, such as penicillin, vancomycin, linezolid and ciprofloxacin. It was demonstrated that replacing the pyrrolidine moiety of LBM415 with a 2,5dihydropyrrole ring led to new derivatives with good activity against drug-resistant bacteria [74]. Moreover, it was also found that derivatives of aliphatic amines had weak antimicrobial activity. On the other hand, derivatives of aromatic amine and heterocyclic amine were much better antibacterial agents. Shi et al. synthesized new methylenepyrrolidine formyl hydroxyamino derivatives (32) (Chart 10). They tested activity of that compounds against many drugresistant bacteria, among them were also clinical isolates [75]. Investigated compounds best’s activity was 0.0625-0.5 µg/mL against Staphylococcus aureus, MSSA, MRSA and Staphylococcus epidermidis [75]. Lee et al. [76] reported the synthesis of novel compounds based on a natural PDF inhibitor actinonin and structure-activity relationship studies of retro-amide inhibitors (33-34) (Chart 11). The derivatives were tested by P. aeruginosa Ni-PDF enzyme assay. Moreover, authors tested new compounds in vitro activity against S. pneumoniae, M.catarrhalis and H. influenzae [76]. The structure-activity relationship was done for searching compounds with potent enzyme inhibition, as well as antibacterial activity against S. pneumoniae, M. catarrhalis and H. influenzae. The most promising derivative of (33) bore cyclobutylmethyl substituent and displayed IC50 of 13 nM. The equally potent derivative of (34) bore valine moiety. As the continuation of the efforts described above Lee et al. [77] reported the synthesis of non-peptide

15

PDF inhibitors (35-36) (Chart 11) and respective structure-activity relationship. A Pseudomonas aeruginosa Ni-PDF enzyme assay, together with in vitro antibacterial activity were tested against Moraxella catarrhalis, Streptococcus pneumoniae, Haemophilus influenzae as well as Staphylococcus aureus. It was determined that the size of the amino acid side chain at the modified position significantly affects the antimicrobial activity against Staphylococcus aureus, as well as the enzyme inhibition activity. Interestingly, the best activity was obtained for a derivative with valine moiety. Furthermore, it was found that a side chain bigger than isopropyl group (e.g. leucine) cannot be accommodated by the enzyme binding pocket. Urea derivatives also exhibited PDF inhibition, as well as antimicrobial activity towards tested strains. What is important, either alkyl, or aromatic urea’s modification were well tolerated [77]. Kwon et al. isolated two 1,3-dihydroisobenzofuran compounds from Aspergillus flavipes as PDF inhibitors. Some derivatives of compounds (37) (Chart 11) inhibited S. aureus PDF with very good IC50 values. They were equal respectively 3.6 and 2.5 µM. Moreover, MIC values of these compounds were equal to 25 µg/ml [78]. By the next studies Kwon et al. isolated from cultures of Aspergillus flavipes novel compounds to act as inhibitors of PDF. Among them, two were the most promising: flavimycins A and B, a 1,3dihydroisobenzofurans (38) and (39) (Chart 11). Some derivatives of compound (38) inhibited Staphylococcus aureus PDF dose-dependently with IC50 (35.8 and 100.1 µM). Moreover, (38) stopped cell growth of S. aureus, MRSA, as well as QRSA. The MIC values ranged from 32 to 64 µg/mL. On the other hand, the compound (39) derivatives had much weaker activity. Their MIC values were equal to 64-128 µg/mL [79]. 4.3. DNA Gyrase and Type IV Topoisomerase (TPIV) DNA topoisomerases catalyze the changes in the topology of the deoxyribonucleic acid [80]. In particular, they are capable of interconversion of relaxed and supercoiled forms. Moreover, they can introduce and remove catenanes and knots [80]. As an effect of their key nature and due to their mechanisms of action, topoisomerases are important targets for chemotherapy, as well as antimicrobial therapy [80]. There are two types of DNA topoisomerases (type I and II). This classification takes into consideration if they catalyze breakage of one (topoisomerase I) or two (topoi-


O HO

R N

N H

Kaczor et al.

O N H

O

O N H

HO O

O N

N H

( 33) O H

H N

N OH

O

R O

H

O N OH

( 35 )

O

H N

O

[AA] O (36) R1

HO

HO

HO R2

OH

H

H

R2

O

O OH

N H

(34)

O

R1

[AA] N H O

H

HO

OH O

OH OH

HO OH

O

H

H

OH O H

O

OH ( 37)

(38)

R3

(39)

R

O H

Chart (11).

somerase II) DNA strands [80]. Importantly, all topoisomerases are able to relax supercoiled DNA. However, only DNA gyrase may introduce negative supercoils in a reaction requiring the hydrolysis of ATP [80]. Prokaryotic enzymes, like gyrase and topoisomerase IV (TPIV), consist of two subunits: A and B. Fig. (5) shows gyrase forming an A2B2 complex in the active enzyme (Fig. 5). Generally, the A subunit is responsible for interactions with DNA. Moreover, it contains responsible for DNA cleavage, the active-site tyrosine. On the other hand, it is the B subunit, where the ATPase active site is located [80]. The applicability of gyrase as an antibacterial target results from its mechanism of supercoiling [80]. Unfortunately, this mechanism is not fully known yet. However, a theoretical model, widely known as a “two-gate mechanism” is extensively supported by experimental data [80]. The gyrase supercoiling cycle constitutes wide possibilities for disruption by various inhibitors. It may interfere with: ATP hydrolysis, DNA binding, DNA cleavage, as well as strand passage [80]. Currently a crystal structure of Staphylococcus aureus DNA gyrase (Fig. 5) in complex with AM8191 inhibitor, as well as many crystals containing A or B subunit are available [81]. What is important, AM8191 is bactericidal. It has the ability to selective inhibition of DNA synthesis and S. aureus gyrase with IC50 of 1.02 µM and topoisomerase IV with IC50 of 10.4 µM [81]. A crystal structure of AM8191 bound to Staphylococcus aureus DNA-gyrase. Moreover, it exhibited interactions very similar to GSK299423, more precisely, it displays a key contact of Asp83 and the basic amine was displayed at position-7 of the linker [81].

Fig. (5). Crystal structure of Staphylococcus aureus DNA gyrase ((DNA gyrase subunit B (UNP P0A0K8 residues 410-543 and 580-644), DNA gyrase subunit A (UNP P20831 residues 2-491)) in ribbon representation in complex with inhibitor (AM8191) shown with cyan carbon atoms. DNA shown in orange, PDB ID: 4PLB [81].

TPIV is the second bacterial type II enzyme, which acts as a heterotetramer. It is built from two ParC and two ParE subunits similarly to gyrase. What is interesting, topoisomerase IV cannot generate negative supercoiling in contrast to gyrase [82]. Gyrase, as well as TPIV are able to remove negative or positive supercoiling. However, TPIV rather more binds and as a result relaxes positive supercoils [82]. These two enzymes, although sharing high peptide sequence identity (about 40% sequence identity, as well



as a much higher sequence similarity), but vary considerably in catalyzing abilities. The main difference is seen in their ability to catalyze inter- versus intramolecular strand passage reactions [82]. TPIV favors intermolecular (decatenation) reactions, while gyrase has higher specificity for intramolecular (supercoiling) reactions [82].

Sherer et al. [85] synthesized substituted pyrrolamide analogs (42) (Chart 12). The improved enzyme activity of one of derivatives of (42) is connected with the presence of two lipophilic electron withdrawing groups (R1 and R2) on the pyrrole moiety. Presence of these two groups increases hydrophobic interactions in the adenine pocket. On the other hand, decreased activity of the derivative without such substituents confirms the observations that increasing potency is connected with an electron withdrawing group [85]. Furthermore, all the modifications of heterocyclic substituents R3 maintained satisfactory enzyme level, with the exception of the compound with the pyrrolidyne substituent. As a consequence, it can be concluded that an aromatic nature of these heterocycles is crucial for optimization of pi-stacking with Arg84. Improvement of the spectrum of antimicrobial activity against gram-negative bacteria, like Moraxella catarrhalis and Haemophilus influenzae can be connected with the enhanced permeability of the cells thanks to the presence of a heterocyclic carboxylate substituent [85]. Such compounds maximize interactions of stacking and hydrogen bonding outside of the ATP pocket and additively optimize pyrrole hydrogen bond to Asp81, and are hydrophobic in the ATP pocket [85].

Abdullah et al. [83] synthesized quinoline based chalcones (40) (Chart 12). A fourteen new compounds were obtained by condensation of 2,7-dichloro-8methyl-3-formyl quinoline with acetylthiophenes and acetophenone. It was found that the position of substituent has an impact on the activity. Additionally, a greater hydrophobic effect of the bromo substituent was observed at 4-position of thiopene and benzene, whereas this effect decreased at the second position. The docking experiment, as well as bioassay, confirmed that chemical compounds with 4-chlorophenyl and 4-bromophenyl were the most active DNA gyrase inhibitors [83]. Foss et al. [84] characterized new DNA gyrase inhibitors. They analyzed N-benzyl-3-sulfonamidopyrrolidines (gyramides) (41) (Chart 12). It was earlier demonstrated that gyramide A displays antibacterial activity. It is believed to inhibit bacterial cell division. It was found that gyramide A primary target is a DNA gyrase. Importantly, the gyramide A resistancedetermining fragment in DNA gyrase is adjacently located to the DNA cleavage gate. It can be used as a potential site for new inhibitors design [84]. The authors analyzed antibacterial activity of gyramides AC and Gram-negative efflux pump inhibitor MC207,110 (60 µM) combination [84]. Value of IC50 against E. coli DNA gyrase of gyramides A-C was equal to 0.7-3.3 µM. In conclusion, the N-benzyl-3sulfonamidopyrrolidines can be regarded as a good point of reference for furtherer progress of compounds affecting a novel site of a DNA gyrase [84]. O R Cl

N

Cl

N

Werner et al. [86] performed computer-aided identification of novel 3,5-substituted rhodanine derivatives (43) (Chart 12) with activity against Staphylococcus aureus DNA gyrase [86]. The IC50 of all the compounds was in a micromolar range, with the most active compound bearing furane moiety and two carboxylic groups which displayed IC50 of 30µM. Brvar et al. [87] carried out identification and characterization of a novel class of 4′-methyl-N2-phenyl[4,5′-bithiazole]-2,2′-diamine (44) (Chart 12). They were characterized as bacterial gyrase inhibitors. They have micromolar activity in a lower range (the most promising derivative displayed IC50 of 1.1± 0.2 µM). NH O S O

R1

R

R2 N S

(41)

O R1

NH R1

N

O

Chart (12).

R3

N S

(43)

NH O

N

R3

(42)

S

S

R2

N H

F (40)

(44)

17

NH

R2

O NH

NH

NH O O (45)

N


Authors implemented a structure-based design approach consisting of two steps. Furthermore, application of a set of in silico, structural and biophysical methods, beginning from the accessible structural data and the pharmacophore model description of the basic ligands interactions (consisting of overall 12 characteristic pharmacophore portions: an aromatic ring, two hydrogen bond acceptors, two hydrogen bond donors and seven hydrophobic features), yielded six active compounds of the 4,5-bithiazole structural class [87]. Hung et al. [88] worked for a workflow with many methods used in de novo design for identification of DNA gyrase inhibitors. In this research, authors built 2D-QSAR model, which employed molecular fingerprints. It involved extraction of molecular fingerprints, characterised by a new mechanism and structure from a set of DNA gyrase inhibitors data. In the next step, the fingerprints were divided into the molecular fragments and recombined thus generating a compound library, which was screened applying LigandFit, and Gold software for docking. Next, the obtained results were analyzed by binding mode analysis and pharmacophore validation analysis. Research identified a potential novel DNA gyrase inhibitor (45) (Chart 12) [88]. Angehrn et al. [89] optimized bicyclic lactones (46, 47) (Chart 13), which were a derivatives of the naturally occurring cyclothialidine. The optimization was done according to the hypothesis: only an accurate value of hydrophilicity can enable a good antibacterial activity into in vivo potency [89]. In accordance with the above statement, efficacious compounds can only be identified in a narrow lipophilicity range [89]. Continuous efforts to elaborate better hydrophilic substitution patterns for the 14-membered monolactams yielded compounds of similar efficacies as those previously reported. However, a chloro- or bromosubstituents used for the 1-methyl group of monolactams turned out to be beneficial for antimicrobial potency for in vitro, as well as in vivo [89]. Introducing of an additional amide group on lactone ring resulting in an increase of polarity of a new scaffold rendered to be profitable for antibacterial activity. In comparison to the monolactams, dilactams had a little worse antimicrobial activity. It can be connected with their enhanced hydrophilic character. On the other hand they had more favorable physicochemical properties [89]. Interestingly, the dilactam inhibitors displayed similar antimicrobial spectrum comparing to monolactams, but were particularly more active against multiresistant strains of E. faecium, E.faecalis and S. pneumonia [89].

Kaczor et al.

Manchester et al. [90] synthesized the azaindole series (48) (Chart 13). They began with a virtual screening hit, and optimized its activity by an optimization of structure-based design combination with physical properties. About ten compounds synthesis was essential for achieving inhibition of the growth of methicillin-resistant Staphyloccus aureus (MRSA) at the concentrations of 1.56 µM. The obtained derivatives inhibited DNA gyrase and TPV at almost the same nanomolar concentrations. It was determined that the best biochemical and antibacterial activity was observed in ethylaminocarboxamide substitution at the 2-position. [90]. In contrast, introduction of a methyl substituent into trifluoromethylpyrazole at the 4-position of azaindole led to more than a 10-fold decrease in activity [90]. When the nicotinic acid was converted to nicotinamide at the azaindole 5-position, its biochemical potency was maintained. Moreover, a 30-fold increase in antimicrobial activity against Streptococcus pneumoniae was observed. Alkylation reaction of acetamide resulted in a two-fold drop in MIC value against Streptococcus pneumoniae. Moreover, it resulted in 6.25 µM activity against methicillin-sensitive and 12.5 µM activity towards multidrug-resistant strains of Staphylococcus aureus. Dougherty et al. [91] worked on a new bacterial type II topoisomerase inhibitors. (49) (Chart 13) is a compound with an increased activity towards gramnegative bacteria, especially P. aeruginosa. Contrary to fluoroquinolones, it was observed that received derivative failed to create a double-strand DNA cleavable complex with DNA and E. coli DNA gyrase. However, it was a strong inhibitor of DNA gyrase, as well as E. coli TPIV [91]. Moreover, against Pseudomonas aeruginosa, (49) was bactericidal. Animal infection studies displayed that (49) was effective in ascending urinary tract infections, thigh and lung in mouse models [91]. Perola et al. [92] optimized the in vivo efficacy in a series of benzimidazole and benzimidazole urea derivatives (50) (Chart 13). They were both inhibitors of bacterial gyrase B, as well as TPIV, earlier designed and investigated by Charifson et al. [93]. The compounds were optimized to mid-to-low nanomolar potency against a variety of bacteria. Tari et al. [94] designed and synthesized pyrrolopyrimidine DNA gyrase B and TPIV inhibitors (51-54) (Chart 14). Authors based on the binding modes analysis of several scaffolds to E. faecalis GyrB selected pyrrolopyrimidine scaffold for optimization. They built substituents into three different places of the binding pocket: (i) the lipophilic active-site interior,

New Antibacterial Substances and their Drug Targets O OH


N N

S HN

OH

R1

HN O

OCH3 R3

O

N

S

x

O

OCH3

N

O

R4

R1

N

O

H R2

R2

x

H N

R3

H N

NH2

N

H N

N

R1 R2

Y

R3

( 47)

(46)

19

(48)

O

R2

N HN

O

N

O

O

HN O

N (49)

N H

R1

(50)

Chart (13).

along the R1 group and a neighbouring substituent which was optimized to ethyl group, (ii) the salt-bridge pocket, along the R3 vector, and (iii) the residues and ordered solvent network located at the mouth of the lipophilic pocket, along the R2 vector [94]. An ethyl substituent and small group at R1 resulted in inhibitors with 2-3 log increase, comparing with the lead compound. This class has high ligand efficiency. Additionally, it tolerates considerable chemical variety at two analogous vectors without disturbing enzyme spectrum and potency [94]. Moreover, R2 and R3 positions substituents have solvent facing synthetic vectors allowing small groups to be incorporated. It can change charge distribution and physicochemical properties of the series [94]. As a continuation of the efforts described above Tari et al. [95] synthesized tricyclic inhibitors (55) (Chart 14) of DNA gyrase and topoisomerase IV. They used structure-based drug design to create a new dualtargeting pyrimidoindole inhibitors with similar activity towards DNA gyrase, as well as type IV topoisomerase from a broad range of microbes [95]. That compounds exhibited potent, broad-spectrum activity against Gram-positive and Gram-negative bacteria, including pathogens such as fluoroquinolone resistant or multidrug resistant strains [95]. Trzos et al. [96] applied structure-based inhibitor design and improved activity of a new pyrrolopyrimidine inhibitors (56) (Chart 14). They possess potent, dual targeting activity towards DNA gyrase and type IV topoisomerase. They found derivatives having broad antibacterial spectrum, active towards Escherichia Coli, Acinetobacter baumannii and Pseudomonas aeruginosa. The compounds possessing 1-

aminopropan-2-ol R2 substituent owe their potency to a hydrogen bond involving the conserved Asn. It should be stressed, however, that thanks to the three rotatable bonds, there is involved a pronounced entropic penalty by the 1-aminopropan-2-ol to form the hydrogen bond. Interestingly, the presence of amino- and hydroxyazetidine moieties with a hydrogen-bond donor attached to a more rigid molecular skeleton, resulted in significant activity increase. Moreover, the hydroxyl moiety replacement in the azetidin-3-ol moiety with a basic amine resulted in antimicrobial activity against wild-type E. coli. Curiously enough, a properly placed basic amine at the R2 position increased enzyme activity and Gram-negative antimicrobial potency, when it was applied to synthesize series of R2 analogs with similar basic amine moieties [96]. Surivet et al. [97] synthesized new dual DNA gyrase and type IV topoisomerase inhibitors. They possess a tetrahydropyran moiety (57) (Chart 15).The studies enabled identification of an ethylenediol moiety as a proper factor, which connects a main tetrahydropyran nucleus with aromatic left-hand side moiety. This study was aimed to improve pharmacological and antimicrobial activity and diminish block of hERG K+ channel. Analyzed compounds had good activity against clinically important bacteria including Streptococci, Staphylococci and Enterococci. Moreover, the authors depicted that dual inhibition of gyrase, as well as topoisomerase is necessary in order to decrease bacteria resistance development [97]. Alt et al. [98] analyzed the antimicrobial, as well as inhibitory activity of natural aminocoumarin antibiotics together with their six structural analogues (novclobiocins) (58) (Chart 15). They were tested against DNA


N H N

Cl

N

N Cl

NH

NH

NH R1

Kaczor et al.

N R2

S (51)

N

N

S

N

X

H N

N

N R2

N

S

R3

(53) R4

R3

N N

H2N

(52)

N

Cl

Cl

N

H N R1

N

R3

(55)

R1 N

R3

R2

(54)

N

R4 R2

(56)

Chart (14). OH

OH

H N

O

N

Y

O

R

O R2

(57)

H2N

O O O O

OH

O

Cl

(58 ) Cl

O O H N O

R4

R3

O

R1 O

OH

O

O

Z

H N

OH

HN

O

HO

H

O H H O

NH O

OH

(59 )

Chart (15).

gyrase and TPIV from Escherichia. coli and Staphylococcus aureus. Moreover, they analyzed the effect of potassium and sodium glutamate on these enzymes. Among four aminocoumarin antibiotics occurring in nature, clorobiocin and coumermycin A1 displayed the best inhibitory potency against all tested enzymes. Clorobiocin exhibited the lowest IC50 against E. coli and Staphylococcus aureus topoisomerase IV. It was found that optimization of the substituents at position 3′′-OH of noviose revealed that methylpyrrolcarbonyl is necessary for efficient inhibition activity of gyrase and type IV topoisomerase. Moreover, removing at position 8′′ the methyl group of noviose yielded novclobiocin 105, which was totally not active against all tested enzymes. This confirms interactions between the methyl group and hydrophobic parts in enzyme binding pocket had a crucial role. Interestingly, the study made it possible to find very potent clorobiocin and novobiocin derivatives with better antibacterial activity comparing to natural compounds [98]. Accordingly, optimization of the ring A, linked to the amino group of the coumarin moiety, resulted in better activity towards gyrase, especially against Escherichia. coli

enzyme. However, there were observed the decrease of activity against type IV topoisomerase from both bacteria. It was proposed that compounds with the best activity against Staphylococcus aureus and E. coli topoisomerases II need to have an 8′′-CH3 group, a methylpyrrolcarbonyl moiety at position 3′′, and an 8′-CH3 motif. It suggests that gyrase should be the key target for all new aminocoumarin antibiotics investigated in in vitro research [98]. Phillips and coworkers [99] studied a Staphylococcus aureus fitness test strategy for screening of natural products. They discovered kibdelomycin (59) (Chart 15), a new group of antibiotics produced by a novel element of the genus Kibdelosporangium. Importantly, kibdelomycin displays broad spectrum of action, including Gram-positive antimicrobial activity, as well as it is a strong DNA synthesis inhibitor. By means of chemical genetic fitness test profiling, as well as biochemical enzyme assays, kibdelomycin was shown to be a structurally novel group of bacterial type II topoisomerase inhibitor which inhibits the ATPase activity of DNA gyrase and type IV topoisomerase [99]. It should be stressed that kibdelomycin is a first of a


novel type of a bacterial topoisomerase II inhibitor that possesses potent antibacterial activity. Moreover, it is based on natural product sources and was discovered in more than six decades [99]. 4.4. Fatty Acid Biosynthesis (FAB) Fatty acids biosynthesis (FAB) is a metabolic process, necessary for both eukaryotic, as well as prokaryotic cells. However, there are significant differences in this process in bacteria in comparison to human cells. These differences make (an) enzyme involved in bacterial FAB excellent targets for antibiotics. The human fatty acid synthesis requires only one enzyme, whereas bacteria’s pathway involves different enzymes, each of them can be an antibacterial target [100]. The βketoacyl-acyl carrier protein synthase III (FabH) is an enzyme present in the bacterial FAB pathway, which is involved in initiating of the FAB cycle. FabH catalyzes the first step of condensation reaction of malonyl-ACP with acetyl-CoA. It plays an important role for the bacterial FAB cycle and is considered to be essential for the survival of the microorganisms [100]. The FabH monomer exhibits structure internal pseudo-symmetry which can suggest a gene duplication event during its evolution [101]. As a consequence, the structure consists of two domains: N-terminal and Cterminal (Fig. 6). Interestingly, the sequence similarity within these two domains is very low.

Fig. (6). E. coli FabH crystal structure in ribbon representation, PDB ID: 3IL9 [101].

Enoyl-acyl-carrier-protein reductase (ENR), named as well FabI, is another important FAB bacterial enzyme [102]. FabI works in the fatty acid chain elongation cycle working as a catalyst for the stereospecific reduction of double bonds joining the C2 and C3 positions in an increasing fatty-acid chain [102]. The most clinically important inhibitors of FabI are isoniazid and


21

triclosan. The FabI consists of 7 β-strands and 11 helices with a number of loops (Fig. 7). Wang et al. [103] synthesized novel vanillic acylhydrazone derivatives (60) (Chart 16) and tested them against E. coli, S. aureus, B. subtilis and P. aeruginosa. Among 30 vanillic acylhydrazone derivatives, one compound with 2-hydroxy-3,5-dichlorophenyl moiety exhibited best MIC equal to 0.39-1.56 µg/mL against bacteria strains mentioned above. Moreover it displayed the IC50 of 2.5 µM [103]. Molecular modeling study, as well as structure-activity relationships supplied important information about interactions of enzymes and ligands. In particular, it was demonstrated that compound without any substituent group on benzene ring did not have good antimicrobial activity. Their MIC was equal to 12.5µg/mL against P. aeruginosa and 25 g/mL against B. subtilis, S. aureus and E. coli [103]. Subsequently, addition of -OH moieties on the benzene ring, significantly increased activity against tested bacteria. As an example, addition of a -OH moiety at the ortho-position of benzene ring, increased MIC values of compound to 3.13-6.25 µg/mL. On the other hand, the compound with -OH moiety at the para-position of benzene ring had MIC value equal to 12.5-50 µg/mL [103]. Furthermore, a derivative with -OH moiety at the ortho-position, as well as at paraposition of benzene ring had MIC equal to 1.56-3.13 µg/mL. While the compound containing -OH moiety at the para-position, as well as in meta-position of benzene ring had worse MIC value equal to 12.5-25 µg/mL [103]. Above outcomes demonstrated the sequence of inhibitory activity of substances with -OH group(s) on the benzene ring, which displayed the following order: ortho > 2,4-disubstituted > 3,4disubstituted > para [103]. Finally, compounds obtained in a condensation of substituted salicylaldehyde and 4-hydroxy-3-methoxybenzohydrazide had favorable activity against above mentioned bacteria with MIC of 0.39-25 µg/mL, in particular for a compound with two chlorine atoms at the positions 3 and 5 of salicylaldehyde. The MIC values for that compound were 0.39-1.56 µg/mL [103]. Lee et al. [104] tried to identify novel and potent antimicrobial FabH inhibitors. To do that, they used two pharmacophore maps from receptor-oriented pharmacophore-based in silico screening of the X-ray structure of FabH. After conducting of biological assays the authors identified compound (61) (Chart 16) as a new and strong antimicrobial inhibitor of FabH. The antimicrobial activity of (61) against Staphylococcus aureus and MRSA had MIC value equal to 2-4 µg/mL.


Kaczor et al.

It was significantly stronger than against other bacteria. This data stressed the significant function of the benzene-1,3-diol motif on binding to FabH. The key function of the hydrazone motif on antibacterial action was also demonstrated. Furthermore, analogs of (61), i.e. (62, 63) were also synthesized and tested. Whole series of nine analogs exhibited a reasonable affinity to all tested FabH enzymes with micromolar to nanomolar dissociation constants [104].

teractions of this compound with the E. coli FabH active pocket [105]. Li et al. [106] synthesized 24 vinylogous carbamates (65) (Chart 17) and tested them against E. coli FabH. Some derivatives displayed anti-Gram-negative bacteria activities. It includes good activity for two Gram-negative bacteria: P. aeruginosa and E. coli. In particular the compound with R1=R2=R4=H and R3=Br and the compound with R1=R2=R4=H and R3=Me, showed the strongest E. coli FabH inhibitory activity with IC50 equal to 3.3 and 2.6 µM, respectively. Molecular docking was carried out to determine the orientation of the active compounds in the FabH binding pocket [106]. O

R1 R2

R1

OC2H5

NH

R3

R2

N N

O R4

O

(65)

(66)

R3

O S

S

Fig. (7). Staphylococcus aureus FabI crystal structure in ribbon representation in complex with nicotinamide-adeninedinucleotide phosphate (NADP, cyan carbon atoms) and triclosan (magenta carbon atoms), PDB ID: 3GR6 [102].

N N

N

OH

O

N NH

R1

R1 R2

R2

(67) O 2N

N

NH

N

F (60)

N

F

HO

HO

O

OH

R

(61)

F

R3

HO

HO

NH R2

R2

OH N

N

O 2N

N N

R

(70)

Chart (17).

N R1

(63)

R1 (69)

OH N

R3

(68)

(62)

R4

R1

X

X

O O

N

N N

R5

R2

R3 OH

(64)

Chart (16).

Li et al. [105] designed and synthesized new haloid deoxybenzoin derivatives. Next, they tested them as antibacterials against E. coli FabH. Promising activity against anti-Gram-negative bacteria were observed in Schiff bases from deoxybenzoins. Some derivatives exhibited excellent activities against Gram-negative P. aeruginosa and E. coli. Importantly, derivative with R1=OH, R2 =R3 =H and R4=R5=Cl exhibited the best E. coli FabH inhibitory activity (IC50 of 1.87 µM). Molecular docking was carried out to find the binding in-

Li et al. [107] synthesized 56 1-acetyl-3,5-diphenyl4,5-dihydro-(1H)-pyrazole derivatives (66) (Chart 17) as potent inhibitors of FabH. Assay and docking simulation of E. coli FabH inhibitors showed that the 1-(5(4-fluorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro1H-pyrazol-1-yl)ethanone and 1-(5-(4-chlorophenyl)-3(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone are potent inhibitors of E. coli FabH with IC50 of 4.2 µM and 7.6 µM, respectively. Moreover, molecular docking was performed to determine the mode of interactions of the most promising compound with the E. coli FabH active site [107]. Yang et al. [108] synthesized new 27 sulfurcontaining heterocyclic pyrazoline derivatives (67, 68) (Chart 17) and screened them for FabH inhibitory activity. The derivative of (67) with R1=Me, R2 =R3 =H and X=O displayed the best activity against Staphylo-



coccus aureus, Pseudomonas aeruginosa, E. coli and Bacillus subtilis with MIC values equal to 1.56-3.13 µg/mL. The results were akin to the positive control, whereas the derivative of (68) with R1 =F, R2 =H and X=O performed the best in the thiazolidinone series with MIC values in the range of 3.13-6.25 µg/mL. Above mentioned compounds also showed the best E. coli FabH inhibitory activity with IC50 of 4.6 and 8.4 µM, respectively. Molecular docking was carried out to find the binding mode of these compounds with the E. coli FabH binding pocket [108]. Zhang et al. [100] obtained a group of cinnamic acid secnidazole ester derivatives (69) (Chart 17) and tested them for the antibacterial activities against B. subtilis , P. aeruginosa, E. coli and S. aureus. (E)-1-(2methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl-3-(biphenyl-4-yl)acrylate exhibited very promising antibacterial activity with MIC in the range of 1.56-6.25 µg/mL against the tested bacteria, as well as displayed the strongest E. coli FabH inhibitory activity with IC50 of 2.5 µM, which was comparable to kanamycin. Moreover, the molecular docking was carried out in order to identify the binding system of this compound with the E. coli FabH active pocket. It was found that the compound forms hydrogen bonding interactions with Asn247 [100]. Duan et al. [109] prepared a series of novel 2-styryl 5-nitroimidazole derivatives (70) (Chart 17) by straightforward chemistry and tested them for their inhibitory activities against Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, as well as Bacillus thuringiensis. Most of them displayed potent antibacterial and E. coli FabH inhibitory activities. Compounds with 4-nitrophenyl and 4-benzyloxyphenyl substituents exhibited the most potent FabH inhibition activities (IC50=2.1, 3.1 µM, respectively) and antimicrobial acN

NO2 N

tivities (IC50=33.0, 34.3 µg/mL for E. coli, IC50 =15.5, 14.5 µg/mL for Pseudomonas aeruginosa, IC50= 13.9, 9.8 µg/mL for Bacillus thuringiensis and IC50 = 9.0, 6.3 µg/mL for Bacillus subtilis) [109]. Li et al. [110] synthesized novel secnidazole analogs, which were based on oxadiazole scaffold (71) (Chart 18). The substances were checked for their activity against P. aeruginosa, E. coli, S. aureus and B. subtilis. Importantly, these new nitroimidazole-based compounds exhibited promising antibacterial activities. E. coli FabH inhibitory assay and molecular docking demonstrated that the compounds 2-(2-methoxyphenyl)-5-((2-methyl-5-nitro-1H-imidazol-1-yl)methyl)-1,3,4-oxadiazole with MIC equal to 1.56-3.13 µg/mL against the tested bacterial strains and 2-((2-methyl-5nitro-1H-imidazol-1-yl)methyl)-5-(2-methylbenzyl)1,3,4-oxadiazole with MIC equal to 1.56-6.25 µg/mL were the strongest inhibitors of E. coli FabH with IC50 of 4.3 and 5.1 µM, respectively [110]. As the continuation of these efforts, Li et al. [111] designed novel nitroimidazole derivatives (72) (Chart 18) and tested the inhibitory activity against S. aureus, E. coli, B. subtilis and P. aeruginosa. Many of them showed strong antimicrobial and E.coli FabH inhibitory activity. In particular, (E)-2-(2-methyl-5-nitro-1Himidazol-1-yl)-N‘-(3,4,5-trimethylbenzylidene) acetohydrazide with MIC equal to 3.13-6.25 µg/mL was proved to be the most promising compound. Compounds with para-substituted methyl group displayed better E. coli FabH inhibitory activity than compounds with para-substituted fluorine group on benzaldehyde component. Compounds with para-substituted methoxy group displayed better E. coli FabH inhibitory activity than compounds with ortho-substituted methoxy group on benzaldehyde moiety [111].

N

R

O

R

NO2

N

NH N

N N

R

N N N

(72)

N

Chart (18).

S (73)

OH R1

N

R2

R5 R4

(74)

R1

NH

OH

SH

N

O N

O (71)

R3

HO

O

O O OH O (75)

23

OH O

HO

OH (76)


Li et al. [112] prepared new thiazole derivatives with an amide skeleton (73) (Chart 18) and evaluated their activity against B. subtilis, P. aeruginosa, E. coli, as well as S. aureus. Certain investigated substances exhibited moderate antibacterial activities which were generally consistent with their potencies as FabH inhibitors. In particular, compound with R=Br and R1=3BrPh was the strongest with MIC equal to 1.56-6.25 µg/mL. It also exhibited the best E. coli FabH inhibitory activity with IC50 of 5.8 µM. The compounds bearing a substituent on the benzene ring located at the C4 position of the thiazole ring showed better E. coli FabH inhibitory activity than compounds which had no substituent on this ring. It was also shown that compounds with m-substituted Cl or Br at the benzene ring attached to the amide displayed better inhibitory activity than those compounds with p-substituents [112]. Zhang et al. [113] obtained a number of Schiff bases containing pyrazine and triazole moiety (74) (Chart 18) and checked their activity against Staphylococcus aureus, Pseudomonas aeruginosa, E. coli and Bacillus amyloliquefaciens. Some investigated derivatives displayed potent antibacterial and E. coli FabH inhibitory activities. The compound bearing R1 =OH and R2=R3=R4=R5=H was the most potent with MIC values equal to 0.39-1.56 µg/mL and revealed the best inhibitory activity for E. coli FabH with IC50 of 5.2 µM. The binding model of this compound inferred that the introduction of pyrazine group enabled the ligand to combine with FabH, compared with the reported compounds [113]. Applying high throughput docking followed by in vitro verification, luteolin (75) and curcumin (76) (Chart 18), two Chinese medicine monomers, were reported as uncompetitive inhibitors of enoyl-ACP reductase from E. coli (EcFabI) with Ki of 7.1 µM and 15.0 µM, respectively. Moreover, curcumin displayed a significant antibacterial activity against E. coli with MIC90 of 73.7 µg/mL. It is worth stressing that FabIoverexpressing E. coli had decreased susceptibility to the inhibitor in comparison to the wild-type strains, displaying that the mechanism of antibacterial activity is governed by the inhibition of EcFabI [114]. 4.5. FtsZ FtsZ is a bacterial protein with high degree of conservation, which is implicated in cytokinesis and is potential target for antibiotics [115,116]. As the first protein reported to be located at the division site, it polymerizes into the Z ring which is a considerably dynamic form [117]. The dynamics of polymerization of FtsZ

Kaczor et al.

depends on its GTPase properties. Binding with GTP results in longitudinal formation of protofilaments from monomers, and hydrolysis results in protofilament dissociation [117]. Latest studies indicate that, apart its role in formation of septum, FtsZ might be important for cell wall elongation [117]. The Bacillus subtilis FtsZ crystal structure is presented in Fig. (8). So far there are known FtsZ inhibitors such as viriditoxin [118], dichamantin, 2-hydroxy-5-benzylisouvarinol-B and 2,4,6-trihydroxy-3,5-di(2′-hydroxybenzyl)-acetophenone [119], which inhibit Grampositive bacteria, while sanguinarine [120] and zantrins [121] show broad spectrum of action. Domadia et al. reported cinnamaldehyde as FtsZ inhibitor [122].

Fig. (8). Bacillus subtilis FtsZ crystal structure in ribbon representation, PDB ID: 2VAM [117].

Ma et al. [123] designed and synthesized novel 3elongated arylalkoxybenzamide derivatives (77) (Chart 19) and evaluated them as antibacterials. The 3alkyloxybenzamides displayed significantly better activity against both Bacillus subtilis and three phenotypes of Staphylococcus aureus compared with 3methoxybenzamide. Contrary, 3-phenoxyalkyloxybenzamides, 3-heteroarylalkyloxybenzamides and 3heteroarylthioalkyloxybenzamides exhibited a considerably better antibacterial activity against Bacillus subtilis only. 4-chlorophenyl derivative presented the best antibacterial activity against Bacillus subtilis and 3butoxypentyloxy derivative exhibited the most promising antibacterial activity against resistant Staphylococcus aureus. Furthermore, 3-chlorobutoxy derivative, 3bromobutoxy derivative, and 3-butoxypentyloxy derivative showed the most considerably uprated antibacterial activity against Staphylococcus aureus. Importantly, the addition of small terminal groups to 3alkoxy chains increased antibacterial activity against



Bacillus subtilis and Staphylococcus aureus while the change of the small terminal groups of the 3-elongated alkoxy chains into large heteroaryl moieties only increases antibacterial activity against Bacillus subtilis [123].

Zhang et al. [126] synthesized a number of substituted 1,6-diphenylnaphthalenes (80) (Chart 19) and tested them for antibacterial activity against Enterococcus faecalis and Staphylococcus aureus. The existence of a basic functional group or a quaternary ammonium moiety on the 6-phenylnaphthalene appeared to be fundamental for considerable antibacterial activity. Diphenylnaphthalene derivatives had a pronounced effect on bacterial FtsZ polymerization and do not seem to cross-react with mammalian tubulin [126].

It is well-known that the benzo[c]phenanthridinium sanguinarine and the dibenzo[a,g]quinolizin-7-ium berberine are two plant alkaloids which are structurally similar and change FtsZ functioning. Importantly, the hydrophobic group at the 1-position of 5methylbenzo[c]phenanthridinium derivatives or the 2position of dibenzo[a,g]quinolizin-7-ium derivatives is linked with considerably increased antibacterial activity. Kelley et al. [124] synthesized a number of 3phenylisoquinolines and 3-phenylisoquinolinium derivatives (78) (Chart 19) and checked their antibacterial activity against Enterococcus faecalis and Staphylococcus aureus [124].

Ruiz-Avila et al. [127] used virtual screening and employed a fluorescence anisotropy primary assay against in-house synthetic library to search for small non-nucleotide synthetic inhibitors of bacterial division. They identified compound (81) (Chart 19) [127]. Parhiet et al. [128] determined the antibacterial activity of a number of phenyl substituted quinoxalines, quinazolines and 1,5-naphthyridines (82-85) (Chart 20) against methicillin-sensitive and methicillin-resistant Staphylococcus aureus and vancomycin-sensitive and vancomycin-resistant Enterococcus faecalis. The antibacterial activities of the more potent quinoxaline, quinazoline, and 1,5-naphthyridine derivatives where checked for their bactericidal or bacteriostatic nature. The non-quaternary quinoxaline, quinazolin, and 1,5naphthyridine compounds were all connected with an MBC/MIC ratio of 1-2, indicative of a bactericidal mode of action against MSSA, MRSA, and VRE [128].

As the continuation of the efforts described above Kelley et al. [125] determined the activities against Staphylococcus aureus of a number of 4- and 5substituted 1-phenylnaphthalenes (79) (Chart 19). In particular, the antibacterial activities against methicillin-resistant Staphylococcus aureus ranged between 2.0 and 4.0 µg/mL. A comparable activity against methicillin-resistant Staphylococcus aureus was found for sanguinarine or chelerythrine. The ADMET properties of 1-phenylnaphthalenes are beneficial for in vivo absorption and distribution due to constitutive cationic charge of these alkaloids. Moreover, the relative MICs for 1phenylnaphthalenes against vancomycin-resistant Enterococcus faecalis are lower than the registered antibiotics and the investigated alkaloids [125].

Sun et al. [129] identified 9-phenoxyalkyl berberine derivatives (86) (Chart 20) as potent FtsZ inhibitors, using in silico structure-based design and in vitro biological assays. In comparison to the parent compound berberine, the synthesized compounds exhibited a con-

O

R2

O

H2N

O ( )n R1

R2

R3

R1

N

O

R4

R1

(77)

(78) R1 R2

(79)

OH

HO

O

O O

R5

Chart (19).

O

R3 R4

(80)

25

(81)

OH OH


Kaczor et al. R1

N

R1

N +

N

N

R1 I

OH

O

N R2

(83)

OH

N

-

R2 (82)

R1 N

(84)

N

+ N Cl

O

R2

-

O

(85)

(86)

R2

O

OH N

HN N Cl

Cl

Cl

N Cl

(87)

(88)

Chart (20).

siderable improvement of antibacterial activity against clinically relevant bacteria, and a better potency against the GTPase activity and polymerization of FtsZ [129]. The best compound was a potent inhibitor of the proliferation of Gram-positive bacteria, along with methicillin-resistant Staphylococcus aureus and vancomycinresistant Enterococcus faecium, with MIC values in the range 2 - 4 mg/mL, and was potent against the Gramnegative E. coli and Klebsiella pneumoniae, with MIC values equal to 32 and 64 µg/mL, respectively [129]. Keffer et al. [130] elaborated a model system applying a permeable E. coli strain and an inducible FtsZyellow fluorescent protein construct to demonstrate using fluorescence microscopy that chrysophaentin A and hemi-chrysophaentin disrupt living bacteria Zrings. They further investigated the E. coli system by reproducing the phenotypes recognized for zantrins (87) and (88) (Chart 20) and showed that berberine, a known FtsZ inhibitor, exhibits auto-fluorescence, which makes it incompatible with systems that use GFP or YFP tagged FtsZ. Importantly, the reports constitute an interesting example of non-nucleotide, competitive FtsZ inhibitors that disrupt FtsZ in vivo, accompanied by a model system that should be usable for in vivo testing of FtsZ inhibitor leads identified through in vitro screens but are not capable of penetrating the Gram-negative outer membrane [130]. Plaza et al. [131] investigated 8 novel chrysophaentins A-H (89) (Chart 21) which are antimicrobial natural products isolated from the marine chrysophyte alga Chrysophaeum taylori. It was found that the most potent chrysophaentin A was active against clinically relevant Gram-positive bacteria including methicillinresistant Staphylococcus aureus (MIC50 1.5-0.7 µg/mL), multidrug-resistant Staphylococcus aureus

(1.3-0.4 µg/mL), and vancomycin-resistant Enterococcus faecium (MIC50 2.9-0.8 µg/mL). It was proved using in vitro enzyme assays and transmission electron microscopy that chrysophaentin A inhibits the GTPase activity of FtsZ with an IC50 value of 6.7-1.7 µg/mL, as well as GTP-induced formation of FtsZ protofilaments [131]. Stokes et al. [132] reported the design, synthesis and SAR of a number of oxazole-benzamide inhibitors (90) (Chart 21) of the indispensable bacterial cell division protein FtsZ. The investigated derivatives displayed promising anti-staphylococcal activity and inhibited the cytokinesis of the clinically-significant bacterial pathogen Staphylococcus aureus. Interestingly, some compounds bearing a 5-halo oxazole also inhibited a strain of Staphylococcus aureus harbouring the glycine-to-alanine amino acid substitution at residue 196 of FtsZ which conferred resistance to inhibitors previously reported in the series. Moreover, substitutions to the pseudo-benzylic carbon of the scaffold enhanced the pharmacokinetic properties by improving metabolic stability and provided a mechanism for creating pro-drugs. Finally, combining multiple substitutions based on the findings reported in this study has provided small-molecule inhibitors of FtsZ with enhanced in vitro and in vivo antibacterial efficacy [132]. Chan et al. [133] used structure-based virtual screening, substructure searches, and in vitro assays to identify pyrimidine-substituted quinuclidines (91) (Chart 21) as a novel chemotype for FtsZ inhibitors. The majority of the compounds in a group of 2-methyl6-(thiophen-2-yl)pyrimidin-4-yl-quinuclidines had better antimicrobial activity than the initial hit [133]. Interestingly, most active molecules share a trifluoromethyl moiety which increases lipophilicity and facili-



tates bacterial cell permeation. An increased bacterial permeation may be also contributed by absence of the negatively charged carboxylic acid (R1-substituent) [133]. OR1

OR1

Cl R2 OR1

O

O

R1O

R3

Cl

R1

OR1

R1O (89)

O

NH2

F

F

R3

R1 O

N

R1

R3

O

R2

(90)

N

N N R2 (91)

Chart (21).

4.6. Ribosomes The bacterial ribosome constitutes a cytoplasmic nucleoprotein particle. The major function of ribosome, composed of two subunits labeled as 30S (small subunit) and 50S (large), is acting as the site of mRNA translation and protein synthesis. In the process of protein synthesis a ribosome is shifted alongside an mRNA chain, reads the codon and adds the appropriate amino acid, belonging to the corresponding aminoacyl

27

tRNA, to the augmenting protein. After accomplishing a stop codon, translation is discontinued, and the mRNA and protein can be released. Synthesis of a single protein is performed in the peptidyl transferase center (PTC), which is situated in the large (50S) subunit of the ribosome [134]. Voorhees et al. [134] presented structures of the 70S ribosome, at 3.6-A and 3.5-A resolution respectively, in the form of complex with A- and P-site tRNAs that are able to mimic pre- and post-peptidyl-transfer states (Fig. 9). Before this report none of the high-resolution structures of the intact ribosome involving a complete active site with both A- and P-site tRNAs was available. Moreover, despite the fact that previous structures of the 50S subunit identified no ordered proteins at the PTC, biochemical proof proposes that specific proteins are able to interact with the 3' ends of tRNA ligands [134]. Oxazolidinones are antibacterial agents which inhibit protein synthesis through binding with bacterial ribosomes [135]. Bacterial protein synthesis is a sequence of reactions that are necessary in the process of the bacterial protein translation [135]. Abovementioned reactions are initiation, elongation, and termination. Linezolid targets the step of initiation, which involves forming of a ternary complex containing Nformylmethionyl-transfer RNA (tRNA), messenger RNA, and the ribosome (Fig. 9). The interactions of oxazolidinone with the ribosomal peptidyltransferase centre disrupts the binding/processing of the N-

Fig. (9). Structure of the Thermus thermophilus 70S ribosome in complex with mRNA, paromomycin, acylated A- and P-site tRNAs, and e-site tRNA. A - 30S subunit in surface representation, PDB ID: 2WDK [134]; B - 50S subunit in surface representation, PDB ID: 2WDL [134].


Kaczor et al.

formylmethionyl-tRNA by the ribosome. As a consequence, oxazolidinone does not allow the formation of a functional 70S initiation complex, which is the necessary constituent of the bacterial translation process [136]. This leads to the conclusions that oxazolidinones have the same binding sites on the 50S ribosome as chloramphenicol, only the manner of operation of the oxazolidinones is not the same as in case of the chloramphenicol [136]. Oxazolidinones inhibit bacterial protein synthesis, but they do not influence the peptidyl transferase in contrast to the chloramphenicol or lincomycin [136]. Colca et al. demonstrated that oxazolidinones are bound specifically to 23S rRNA, tRNA [137].

Simultaneously De Rosa et al. [139] published the study involving the preparation of a new series of 5substituted oxazolidinones (93) (Chart 22) derived from linezolid, having urea and thiourea moieties at the C-5 side chain of the oxazolidinone ring, and evaluated their in vitro antibacterial activity. Minimal inhibitory concentrations (MICs) were calculated for all linezolid analogues against methicillin-susceptible Staphylococcus aureus. Some of the investigated compounds had a MIC higher than 100 µg/mL, however one of them was able to inhibit the Staphylococcus aureus at a concentration of 10 µg/mL. The compound with X=S and R=NO2 was the most active having a MIC of 2 µg/mL comparable to the activity of linezolid. These finding are in accordance with the results of the molecular modelling analysis of linezolid derivatives which suggests that urea and thiourea derivatives should bind to the ribosome with very different orientations [139].

Recent achievements in the area of ribosomal antibiotics were described by Fortuna et al. [138] who reported synthesis and in vitro antibacterial activity of novel linezolid-like oxadiazoles (92) (Chart 22). They demonstrated that changing of the linezolid morpholine C-ring into 1,2,4-oxadiazole leads to an antibacterial activity against Staphylococcus aureus both methicillin-susceptible and methicillin-resistant similar or better than that of linezolid. It was proved as well that acetamidomethyl or thioacetoamidomethyl groups in the C(5) sidechain are necessary, fluorination of the phenyl B ring has a fine effect on an antibacterial activity but its existence reduces the compounds cytotoxicity. Molecular modeling demonstrated that the novel compounds have a binding pose similar to the crystallographic pose of linezolid. In particular, the 1,2,4oxadiazole group mimics excellently the activity of the morpholinic ring, because of the retention of the Hbond - U2585 interaction [138].

Phillips et al. [140] synthesized a number of 1H1,2,3-triazolyl piperazinooxazolidinone derivatives (94) (Chart 22) with different glycinyl moieties and evaluated their antibacterial activity against Grampositive and selected Gram-negative bacteria. The studies confirmed that the N-aroyl- and N-heteroaroylglycinyl (MIC: 0.06-4 µg/mL) derivatives were more potent than the N-acylglycinyl (2-8 µg/mL) derivatives against all Gram-positive bacteria tested. Nitro group on aryl and heteroaryl rings considerably increased activity against Gram-positive bacteria, as it was found for the 3,5-dinitrobenzoyl and 5-nitro-2-furoyl derivatives with MIC ranges of 0.25-0.5 and 0.06-0.5 µg/ml, respectively. The nitro derivatives exhibited as well activity against Moraxella catarrhalis, with MICs O

O

O

N

O

N

N O

N

O R1

NH R (93)

O O N

NH R

N F

R1

O N

N

O

N

NH

HO (95)

(96)

NH2 ()n

HO

H2N O NH2 OH

R NH

HO

OH

HO

Chart (22).

H2N O

HO

(94) R

X

F

R2

(92)

N

NH

N

O HO

R NH OH (97)

OH


ranges of 0.25-1 µg/mL, compared to linezolid (MIC: 8 µg/mL). Thus, the introduction of the N-aroyl and/or N-heteroaroyl glycinyl moieties as spacer group could considerably improve the antibacterial activities of 1H1,2,3-triazolyl oxazolidinones [140]. Stathakis et al. [141] reported 4 new 6,7-spiro bicyclic aminoglycoside mimics (95) (Chart 22). The presence of a lipophilic side chain considerably regains a part of the biological activity. The presence of hydrophilic chains leads to great improvement in the binding potential. The interactions of 6,7-spiro bicyclic aminoglycoside mimics with the target RNA were also discussed. Importantly, the general action of these compounds in the inhibition process of bacterial protein translation does not pursue the same goal [141]. Mavridis et al. [142] synthesized and evaluated a series of diversely functionalized 5,6-, 6,6- and 7,6spiroethers (96, 97) (Chart 22). These derivatives successfully mimic natural aminoglycosides regarding their binding to the decoding center of the bacterial ribosome. Their potential to inhibit prokaryotic protein production in vitro along with their antibacterial potencies have also been examined. Realizing the serious socio-economic challenge of emerging antibiotic resistance and the associated urgent need for novel potent antibiotics, the identified scaffolds and their proposed RNA target were suggested as important tools for studying the biological processes involved and the development of effective pharmaceutical agents [142]. SUMMARY AND PERSPECTIVE Searching for novel safe and effective antibacterial drugs is one of the greatest challenges of contemporary medicinal chemistry. After golden era of antibiotics in the 50th and 60th of the past century not many original antibacterial drugs have been introduced into market. Unfortunately, bacterial pathogens managed to develop resistance to most of the currently used antibiotics. Thus, elaboration of new antibacterials for wellestablished targets as well as identification of new targets are two complementary strategies leading to modern antibiotics. It should be kept in mind, however, that frequently lead compounds identified in HTS campaigns or in virtual screening experiments exhibit activity against bacterial proteins but not against microorganisms as they are not able to permeate inwards bacterial cells. The compound properties needed for prokaryotic cell penetration need to be urgently elaborated as they essentially differ from Lipinski rule filters for eukaryotic targets.


29

Although the market for antibacterial drugs is considerably smaller than this for drugs used in chronic diseases, it is attractive enough to promote development of novel antibacterials in not only academia but also by pharmaceutical companies. However, the development of resistant pathogens remains unchallenged while there is no effective platform for antibiotic discovery. Novel platforms involve studies concerning unused before resources of natural products, like uncultured bacteria, progress in the area of synthetic antibiotics by establishing rules of compound penetration, designing species-specific antibiotics and identification of prodrugs able to eliminate latent persisters, frequently liable for infections difficult to cure [26]. LIST OF ABBREVIATIONS AMP-PCP

= Adenylyl 50-(β,γmethylene)diphosphonate

ATP

= Adenosine triphosphate

CADD

= Computer-Assisted Drug Discovery

CAPE

= Caffeic acid phenethyl ester

FAB

= Fatty acid biosynthesis

FABH

= β-ketoacyl-acyl carrier protein synthase III

FabI

= Enoyl-acyl-carrier-protein reductase (ENR), also known as FabI

FAD

= Flavin adenine dinucleotide

GTP

= Guanosine triphospate

HTS

= High-Throughput Screening

LBDS

= Ligand-based drug screening

MDR

= Multidrug-resistant bacteria

MES

= Multi-drug efflux systems

MetAP

= Methionine aminopeptidase

MIC

= Minimal inhibitory concentration

MMP

= Matched molecular pairs

MRSA

= Methicillin-resistant Staphylococcus aureus

NADP

= Nicotinamide-adenine-dinucleotide phosphate

PDF

= Peptide deformylase

QSAR

= Quantitative structure-activity relationship

SBDS

= Structure-based drug screening

TPIV

= Topoisomerase IV


Kaczor et al.

UDP-MurNAc = Uridine 5′-(trihydrogen diphosphate), P′-[2-(acetylamino)-3-O-(1R-carboxyethyl)-2-deoxy-R-Dglucopyranosyl] ester

[10]

UMA

[12]

= UDP-MurNAc-L-Ala

CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest.

[11]

[13] [14]

ACKNOWLEDGEMENTS The paper was developed using the equipment purchased within the project ‘‘The equipment of innovative laboratories doing research on new medicines used in the therapy of civilization and neoplastic diseases’’ within the Operational Program Development of Eastern Poland 2007-2013, Priority Axis I modern Economy, operations I.3 Innovation promotion. The research was partially performed during the postdoctoral fellowship of Agnieszka A. Kaczor at University of Eastern Finland, Kuopio, Finland under Marie Curie fellowship.

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