Fighting bacterial resistance: approaches, challenges, and opportunities in the search for new antibiotics. Part 1. Antibiotics used in clinical practice: mechanisms of action and the development of bacterial resistance Текст научной статьи по специальности «Биологические науки»

MIRJournal

MicrobioLogy Independent Research JournaL

REVIEW

Fighting bacterial resistance: approaches, challenges, and opportunities in the search for new antibiotics.

Part 1. Antibiotics used in clinical practice: mechanisms of action and the development of bacterial resistance

Alexander Zhivich#

Framingham State University, Massachusetts, USA

# Corresponding author: Alexander Zhivich, e-mail: [email protected]

Keywords: bacteria, antibiotic, bacterial resistance, multidrug-resistant pathogens DOI: 10.18527/2500-2236-2017-4-1-31-51

Received September 24, 2017 Accepted December 20, 2017 Published December 25, 2017

ABSTRACT

Hundreds of thousands of people are dying every year in the world from infections caused by drug resistant bacteria. Antibiotic resistance is a rapidly increasing problem mostly as a result of the worldwide overuse and misuse of antibiotics for conditions that do not require them. The rapid spread of antibiotic resistance in bacteria makes it necessary to intensify the development of new antibiotics and new methods to combat drug resistant bacteria. The goal of this publication is to review the approaches to finding new antibiotics that are active against drug resistant bacteria. The first part of this review is focused on an analysis of the mechanisms of action of antibiotics that are used in clinical practice as well as the mechanisms of bacterial resistance. The molecular structure and modes of action of these antibiotics are reviewed with examples of detailed mechanisms of drugs interaction with the targets in bacteria. General and specific mechanisms of bacterial resistance to these antibiotics are described. Examples of new antibiotics development active against the drug resistant bacteria are presented.

INTRODUCTION

Antibiotic resistance is the natural tendency for bacteria to change under the process of natural selection in such a way that they survive when exposed to the action of previously potent antibiotics. Antibiotic resistance is a rapidly increasing problem, largely as a result of the worldwide misuse and overuse of antibiotics for conditions that do not require them.

Hundreds of thousands of people are dying every year in the world from infections caused by antibiotic-resistant bacteria [1]. In February 2017, the WHO published [2] a list of the most dangerous antibiotic-resistant bacteria - 'priority pathogens' that includes 12 families (Table 1). The purpose of this publication was to raise awareness in the world's scientific community in order to organize and guide the research and development efforts in academia and pharmaceutical industry focused on the search for new antimicrobial agents.

Microorganisms acquire antibiotic resistance through chromosomal DNA mutations and horizontal gene transfer that eventually lead to a change of bacterial proteins. There are several mechanisms of horizontal gene transfer in bacteria - transformation, transduction, and conjugation [3]. Multi-resistant bacteria or 'superbugs' have emerged recently due to the horizontal transfer of genetic material that has spread antibiotic resistance.

Table 1. The most dangerous antibiotic-resistant bacteria

Priority Families

Critical 1. Acinetobacter baumannii, carbapenem-resistant 2. Pseudomonas aeruginosa, carbapenem-resistant 3. Enterobacteriaceae, carbapenem-resistant, ß-lactamase producing

High 1. Enterococcus faecium, vancomycin-resistant 2. Staphylococcus aureus, methicillin-resistant, vancomycin-intermediate and resistant 3. Helicobacter pylori, clarithromycin-resistant 4. Campylobacter spp., fluoroquinolone-resistant 5. Salmonellae, fluoroquinolone-resistant 6. Neisseria gonorrhoeae, cephalosporin-resistant, fluoroquinolone-resistant

Medium 1. Streptococcus pneumoniae, penicillin-non-susceptible 2. Haemophilus influenzae, ampicillin-resistant 3. Shigella spp., fluoroquinolone-resistant

Recently, scientists from Harvard Medical School (USA) and Technion - Israel Institute of Technology - have designed an experiment visualizing how bacteria acquire resistance to antibiotics [4]. The most dangerous group of multidrug resistant bacteria include E. coli, Klebsiella, Acinetobacter, Serratia, Pseudomonas, Proteus and some

other bacteria that cause severe diseases such as bloodstream bacteremia and pneumonia. These infections are easily spread in hospitals and nursing homes and often lead to a lethal outcome.

The goal of this publication is to introduce the challenging and exciting field of the design and discovery of new antibiotics to the wide community of medicinal chemists and scientists interested in drug discovery and development.

The structure of bacteria and types of bacterial resistance to antibiotics

Bacteria being simple organisms contain a well-developed cell structure (Fig. 1). The cell wall provides the structural integrity to bacterial cell and, therefore, it is one of the most important components of the cell envelope. Both Gram-positive and Gram-negative bacteria contain an inner membrane in the form of a phospho-lipid bilayer. Gram-negative bacteria also have the outer membrane. Gram-positive bacteria do not contain the outer membrane but the cell wall in this type of bacteria is much thicker than that in Gram-negative bacteria. Both Gram-positive and Gram-negative bacteria have DNA, ribosomes and the corresponding enzymes for DNA replication, transcription and translation in order to produce the necessary proteins for the bacterial cell. All the main elements of the bacterial cell envelope as well as organelles and DNA, mRNA, and tRNA molecules and their complexes that are formed in the course of the protein synthesis can be the targets for antibiotics (Fig. 2). The main targets for antibiotics in bacteria are: a) intermediates in the cell wall biosynthesis, b) cell membrane, c) biomolecules and organelles involved in the processes of protein biosynthesis, d) bacterial DNA and RNA, and e) enzymes involved in the biosynthesis of tetrahydrofolate.

Fig. 1. Structure of Gram-positive bacteria (reprinted from [5]).

Bacteria use several mechanisms for protection from antibiotics that function as mechanisms of resistance. The most common mechanisms of resistance among bacteria are:

a) biosynthesis of enzymes in the bacterial cell that deactivate antibiotics [7, 8].

b) active efflux of antibiotics from the bacterial cell that is accomplished by integral membrane transporters also known as efflux pumps or drug pumps [9, 10].

c) change in the structure of bacterial organelles or enzymes that are the antibiotic targets in order to prevent or diminish the action of antibiotics [11, 12].

d) change in the structure of the intermediates involved in the process of bacterial cell wall synthesis preventing the disruption of this process by antibiotics [13].

e) change in the bacterial membrane leading to prevention of antibiotic - membrane interaction [14, 15].

Fig. 2. The main antibiotic targets in bacteria and the main mechanisms of bacterial resistance (reprinted from [6]).

Some scientists consider the formation of bacterial biofilms as one of the mechanisms of bacterial resistance, but bacterial biofilms will not be discussed in this review.

Since the efflux of antibiotics that is used by both Gram-positive and Gram-negative bacteria is a general mechanism of resistance to antibiotics that belong to different classes and have quite different molecular structures, it will be briefly discussed in this section. Active efflux is a method that bacteria use for transporting unwanted toxic compounds and antibiotics out of the cell. Efflux pumps are single proteins (Gram-positive bacteria) or a combination of several proteins (Gram-negative bacteria) that are embedded in the bacterial membrane and function like transporters for chemical compounds. There are five distinct types of efflux pumps known to date: MFS (major facilitator class), RND (resistance-nodulation-division), SMR (small multidrug resistance class), MATE (multidrug and toxin exclusion), and ABC (ATP-binding cassette). Since the pumping of antibiotics is accomplished often against the concentration gradient, this process requires energy. Most of the efflux pumps -MFS, RND, SMR, and MATE - are powered by the flow of the protons that decreases the proton gradient across the bacterial membrane. The ABC pumps use ATP (adenosine triphosphate) hydrolysis as the energy source.

Mechanisms of action of known antibiotics and the modes of bacterial resistance to them

Natural, semi-synthetic and synthetic antibiotics are successfully used today in clinical practice to fight bacterial infection. Following the generally accepted classification based mostly on molecular structure [16] all marketed

antibiotics can be divided into several major classes: p-lactams, glycopeptides and lipoglycopeptides, sulfonamides, aminoglycosides, tetracyclines, macrolides, rifa-mycines, fluoroquinolones, streptogramines, oxazolidi-nones, and lipopeptides. An overview of the history of search for antibiotics is given in a paper by Mohr [17]. The mechanisms of action of drugs within each of these antibiotic classes are similar. On the contrary, the mechanisms of action of drugs from different antibiotic classes are significantly different.

The antibiotics structure, mode of action, and mode of bacterial resistance to these antibiotics are presented below. The molecular mechanisms for these processes are discussed.

a. p-Lactams

One of the most widely used classes of antibiotics is p-Lactams (e.g. Fig. 3).

The first antibiotic penicillin was isolated from Peni-allium fungi by Alexander Fleming in 1928 [18].

Penicillin belongs to the p-lactam family of antibiotics that have the distinctive structural fragment - four membered p-lactam ring. Cephalosporins (e.g. cefaclor, Fig. 3) also contain p-lactam cycle. The first member of this class of antibiotics - cephalosporin C - was isolated from Cephalosporium acremonium.

A

Fig. 3. Penicillin G (A) and cefaclor (B).

B

There are a number of semisynthetic antibiotics among p-lactams.

Later attempts to find new p-lactam antibiotics led to the development of carbapenems (Fig. 4).

A B

Fig. 4. Meropenem (A) and doripenem (B).

p-Lactam antibiotics act by the inactivation of the transpeptidases that catalyze the last step in peptidogly-can cross-linking. The main component of the cell wall that provides its structural integrity in both Gram-positive and Gram-negative bacteria is peptidoglycan - the cross-linked peptide-sugar copolymer. The cross-linking of the glycan polymer chains that makes the cell wall strong and rigid is accomplished by the formation of peptide strands that connect the glycan chains. The linking of two pep-tide strands attached to different glycan chains is the last

step in the bacterial cell wall synthesis. It is catalyzed by transpeptidases - enzymes that have serine at their active sites. This enzyme attacks the D-alanyl-D-alanyl site of the first peptide strand and forms active acyl-enzyme intermediate with the simultaneous displacement of the terminal alanine (Fig. 5). Then, in Gram-negative bacteria, the amino group of diaminopimelic acid of the second peptide strand reacts with the acyl-enzyme intermediate and the crosslinking reaction is finished by the formation of the peptide bond between the two peptide strands. In Gram-positive bacteria, where diaminopimelic acid in the second peptide strand is replaced by lysine, the connection with the first peptide strand is accomplished through the peptide bridge that can contain several amino acids. For example, in Staphylococcus aureus this peptide bridge contains five glycine amino acids. In this case, the amino group of the last glycine reacts with the acyl-enzyme intermediate to finish the cross-linking process. Penicillins as well as cephalosporins inhibit the enzyme transpepti-dase by binding to the active center of enzyme that leads to the formation of the stable acyl enzyme intermediate. As a result of this process, the enzyme cannot catalyze the cross-linking of glycan chains and the process of bacterial wall formation is disrupted as shown in Figs. 6 and 7 [19].

Bacteria developed resistance to penicillin by the production of enzymes p-lactamases [20, 21] and later to cephalosporin drugs by production of cephalosporinases (e. g. class C p-lactamases) [21] and other p-lactamase enzymes known as extended-spectrum p-lactamases (ESBL) [22, 23]. These enzymes cleave the peptide bond in the p-lactam cycle of the corresponding penicillin molecule that leads to the loss of drug activity. (Fig. 8).

Fig. 5. Cross-linking step in the synthesis of peptidoglycan.

A

B

Fig. 6. D-alanyl-D-alanyl terminus of the peptide strand (A) and penicillin (B).

Fig. 7. Different products of transpeptidase reactions with D-ala-nyl-D-alanyl fragment of the peptide strand and with p-lactam antibiotic.

(Fig. 11) are the only group of p-lactam antibiotics that can effectively inhibit those enzymes [32, 33]. Thus, the results published by Marshall et al. [34] suggest the combination of ceftazidime-avibactam and aztreonam could be used to overcome resistance of bacteria producing metallo-p-lactamases.

In addition, known p-lactamase enzyme inhibitors are not active enough against the extended-spectrum cephalosporinases [35, 36]. Today, the search for the potent inhibitors of this class of enzymes is an area of active exploration [26, 37]. On the other hand, the search for the new types of p-lactamases inhibitors continues. Thus, Venugopal et al. [38] published interesting results on using peptidyl boronate analogues as broad-spectrum p-lactamase inhibitors.

Another approach to combatting p-lactamase resistance is to block one of the key steps in the biosynthesis

Fig. 8. Deactivation of p-lactam antibiotics by enzyme p-lactamase.

The search for methods to overcome bacterial resistance to penicillin antibiotics led to the discovery of a number of p-lactamase enzymes inhibitors like clavu-lanic acid, sulbactam and tazobactam (Fig. 9) [24, 25]. Since that time, a number of penicillin antibiotics (e.g. ampicillin) were marketed by pharmaceutical companies as mixtures with the corresponding p-lactamase inhibitors. Kuzin et al. [25] presented the X-ray crystallography data for the SHV-1 class A p-lactamase-tazobactam complex and discussed the mechanism of this enzyme inhibition by tazobactam. Mechanism of action of avibactam (Fig. 9), the effective inhibitor of class A, C and some class D p-lactamases and the first non-p-lactam p-lactamase inhibitor, is discussed in recent publications [26, 27].

The combination of tazobactam with the new antibiotic ceftolozane (Fig. 10) that belongs to the 5th generation of cephalosporins is marketed as Zerbaxa. This antibiotic shows high potency against several drug resistant strains [28, 29].

The strategies to discover new p-lactam antibiotics and the development of bacterial resistance to them are reviewed by Llarrull et al. [30]. Unfortunately, the known p-lactamase enzymes inhibitors that are active against class A, C, and D p-lactamases cannot deactivate class B metallo-p-lactamases. These enzymes, containing Zn in their active site, utilize a different mechanism to cleave the p-lactam cycle of antibiotics. Monobactams [31]

C

D

Fig. 9. p-Lactamase enzymes inhibitors. Clavulanic acid (A), sulbactam (B), tazobactam (C), avibactam (D).

Fig. 10. Ceftolozane.

A

Fig. 11. Aztreonam (A) and sulfazecin (B).

B

of these enzymes that was demonstrated by Bouquet et al. for AmpC p-lactamase [39]. Recently discovered 2-aminobenzimidazoles were found to significantly reduce the bacterial resistance to a number of p-lactam antibiotics [40]. It is believed that these compounds have a different mechanism of action from the p-lactamase inhibitors.

Expression by the bacteria of the mutant enzyme transpeptidase (mutant penicillin-binding protein -PBP2 enzyme) that has a reaction center practically inaccessible to the p-lactam antibiotics but, on the other hand, perfectly capable of catalyzing the crosslinking reaction in the course of peptidoglycan synthesis, serves as another way of bacterial resistance to p-lactam antibiotics [41]. It was discovered that in order to catalyze the crosslinking of peptidoglycan the active center of the enzyme should undergo conformational change. This conformational change is probably triggered by the interaction of the corresponding peptidoglycan fragments with the allosteric site of this enzyme [41]. Then, it was shown that some p-lactams like ceftaroline are able to interact with the allosteric site of the enzyme leading to the conformational change - opening of its active site - that enables the second molecule of cef-taroline to react with the enzyme active site, thereby finishing the enzyme inactivation [41, 42]. Fishovitz et al. reported that the triggering of the allosteric cite of PBP2 enzyme by one p-lactam antibiotic enables another p-lactam drug to interact with the active cite of that protein. Therefore, they report that there is synergy in the action of ceftaroline and some other p-lactams like oxacillin, imipenem, and meropenem against the MRSA bacterial strains [43].

Other mechanisms of antibiotic resistance in Gramnegative bacteria involve structural and/or functional changes in porin superfamily transporters that are imbedded in the bacterial outer membrane. These changes strongly affect the rate of antibiotics permeation though the bacterial outer membrane. It was shown that mutations of porins or switching to the expression of the other type porins with a different pore size or different electrostatic interactions in the porin eyelet region lead to a decrease in the permeability of p-lactams in Gram-negative bacteria that serves as the reason for bacterial resistance to these antibiotics [44, 45].

b. Glycopeptides and Lipogycopeptides Another class of antibiotics that act by preventing the biosynthesis of the bacterial cell wall is glycopeptides (e. g. Fig. 12) and their semisynthetic derivatives - lipo-glycopeptides (e. g. Fig. 17).

Both vancomycin and teicoplanin are natural antibiotics produced by microorganisms. Vancomycin was isolated from the soil bacteria Amycolatopsis orientalis. Tei-coplanin is a mixture of natural antibiotics isolated from bacteria Actinoplanes teichomyceticus. Glycopeptides act as inhibitors of the peptidoglycan synthesis in bacterial cell wall. Vancomycin binds to the D-alanyl-D-alanyl end fragment in the peptide strands of the peptidoglycan intermediate and prevents the cross-linking of the peptide polymer chains (Fig. 13).

A

B

Teicoplanin core

R (side chain)

Fig. 12. Vancomycin (A) and teicoplanin (B). 1 - Teicoplanin A2-1; 2 - teicoplanin A2-2; 3 - teicoplanin A2-3; 4 - teicoplanin A2-4; 5 - teicoplanin A2-5.

The bacterial strains resistant to vancomycin emerged as a response to the action of this antibiotic. These new bacterial strains have cell walls with a different structure of peptide chains terminus - D-alanyl-D-lactate - instead of D-alanyl-D-alanyl. Since vancomycin forms 5 hydrogen bonds with a D-alanyl-D-alanyl fragment and only 4 hydrogen bonds with D-alanyl-D-lactate fragment (Fig. 14) its activity decreases 1,000-fold against

these vancomycin-resistant bacteria. Xie et al. [46] reported the synthesis of vancomycin aglycon (Fig. 15) specially designed to interact with both D-alanyl-D-alanyl and modified D-alanyl-D-lactate fragments in peptide strands. This compound showed high potency against vancomycin-resistant bacteria.

Silverman et al. [47] described the synthesis of van-comycin dimers and showed that these compounds have

Fig. 13. Interaction of vancomycin with the D-alanyl-D-alanyl end fragment in peptide strand in the course of peptidoglycan synthesis.

increased potency against methicillin-resistant Staphylococcus aureus and some vancomycin resistant strains.

An interesting approach to improve vancomycin activity against resistant bacteria was used by Okano et al. [48]. They changed one functional group and introduced 2 new functional groups in the molecule of vancomycin (Fig. 16). These new functional groups enable the new drug to act as an antibacterial agent according to two different mechanisms. As a result, the new vancomycin drug molecule has three possible different mechanisms of action and showed high activity against vancomycin-resistant bacteria.

Kang et al. [49] showed that combinations of vancomycin with non-p-lactam antibiotics like ciprofloxacin and gentamicin give a synergetic effect against bacteria with reduced susceptibility to vancomycin. Yarlagadda et al. [50] showed that lipophilic vancomycin aglycon di-mer is 300 times more active compared to vancomycin against some vancomycin resistant bacteria. They also reported that vancomycin-sugar derivatives, especially lipophilic vancomycin-sugar analog, exhibit significantly higher activity against the vancomycin resistant strains than vancomycin itself [51].

In attempt to improve the activity of vancomy-cin a number of its semisynthetic derivatives has been

Fig. 15. Interaction of vancomycin aglycon with D-alanyl-D-alanyl (X = NH) or D-alanyl-D-lactate (X = O).

Fig. 14. Interaction of vancomycin with D-alanyl-D-alanyl fragment (X = NH, hydrogen bond is formed with vancomycin amide oxygen) or D-alanyl-D-lactate (X = O, no hydrogen bond) fragment of peptide strain.

Fig. 16. Structure of improved vancomycin [48].

isolated and studied [52]. As a result of these research efforts, three new drugs - dalbavancin, telavancin and oritavancin, that belong to the subclass of lipoglyco-peptides (Fig. 17) - were introduced to the market. They have certain advantages as compared with vancomycin. Dalbavancin requires fewer frequent dosing because it has much longer half-life. The lipophilic fragments in the structures of both telavancin and oritavancin enable these drugs to have a second mechanism of action - to induce the membrane depolarization in bacterial cell.

A

Due to this dual-action mechanism, both antibiotics show excellent antibacterial activity. Telavancin also has significantly improved ADME (adsorption, distribution, metabolism and excretion) profile [52, 53].

c. Sulfonamides

Sulfonamides represent another important class of antibiotics that inhibit the synthesis of the folic acid metabolite - tetrahydrofolate. This compound is an important intermediate in different biosynthetic reactions and also plays an important part in the cell division process. Sul-fanilamide and sulfamethoxazole are the main examples of sulfonamides (Fig. 18). Sulfanilamide is a synthetic antibiotic the structure of which is close to that of p-Ami-nobenzoic acid (PABA) (Fig. 19).

A B

Fig. 18. Sulfanilamide (A) and sulfamethoxazole (B).

-S—NH2

A

B

Fig. 19. Sulfanilamide (A) and p-aminobenzoic acid (PABA) (B).

Sulfonamide antibiotics mimic p-aminobenzoic acid in the process of tetrahydrofolate synthesis (Fig. 20) and are potent against both Gram-negative and Gram-positive bacteria. By interacting with enzyme Dihydroptero-ate Synthase (DHPS) this class of antibiotics prevents the formation of intermediate I in the synthesis of tetra-hydrofolate (Fig. 20) [54, 55].

Fig. 17. Lipoglycopeptide antibiotics: A - dalbavancin, B - telavancin, C - oritavancin.

Tetrahydrofolate

Fig. 20. Synthesis of tetrahydrofolate.

B

C

Resistance to sulfonamide antibiotics developed because the small fraction of bacteria can use very small concentrations of folic acid provided by the host organism. Since bacteria reproduce fast, even a small number of surviving bacterial cells results in the spread of infectious disease in humans. Another mechanism of bacterial resistance to sulfonamides [56] involves certain mutations in enzyme DHPS that make it practically insensitive to these drugs. At the same time, the mutant enzyme preserves the ability to bind the PABA that is structurally very similar to sulfonamides.

Search for the inhibitors of the enzyme dihydrofolate reductase (DHFR) that catalyzes the conversion of dihydro-folic acid into tetrahydrofolate, led to the discovery of di-aminopyrimidines - the second class of synthetic drugs that disrupt the tetrahydrofolate synthesis in bacteria. These compounds (e.g. trimethoprim, Fig. 21) prevent the reduction step in the synthesis of tetrahydrofolate.

Fig. 21. Trimethoprim.

Trimethoprim inhibits the reduction of dihydrofolic acid to tetrahydrofolic acid by binding to the enzyme di-hydrofolate reductase (DHFR). Therefore, the combination of sulfonamide antibiotics with diaminopyrimidines was successfully used in fighting both Gram-positive and Gram-negative bacteria. Resistance to trimethoprim in some pathogenic bacteria developed as a result of the single amino acid Ile100Leu substitution in dihydrofo-late reductase (DHFR) [57]. Recently, Nepka et al. [58] showed that the combination of sulfamethoxazole-trimethoprim with colistin could be effectively used for fighting carbapenem-resistant bacterial strains. Bavadi et al. describe the synthesis of the series of new dihydropyr-rol-2-one compounds containing sulfonamide groups. Most of these compounds showed antibacterial activity against P. aeruginosa and S. epidermidis and were more active against the P. aeruginosa than the combination of trimethoprim-sulfamethoxazole [59].

d. Aminoglycosides

Another group of antibiotics (streptogramins, macro-lides, oxazolidinones, tetracyclines and aminoglyco-sides) kills bacteria by disrupting its protein synthesis. The targets for these drugs are both large 50S and small 30S ribosomal subunits. One of the antibiotic classes that target the bacterial ribosome is aminoglycosides (e.g. streptomycin and kanamycin Fig. 22).

Aminoglycosides are natural antibiotics. Streptomycin - the first member of this class of antibiotics - was isolated from the Gram-positive bacteria Streptomyces griseus. Streptomycin inhibits the protein synthesis in bacteria by binding to the 30S subunit of the bacterial ri-bosome. That leads to codon misreading over the course of the translation process, resulting in the synthesis of

proteins with wrong structures and eventually to bacterial death. Interaction of neomycin with 50S ribosomal subunit is shown below (Fig. 23) [60].

Streptomycin-resistant bacterial strains (both Grampositive and Gram-negative) emerged after the introduction of this antibiotic and its analogues. Action of several bacterial enzymes such as acetylase, adenylase, and phosphorylase leads to the deactivation of aminoglycoside antibiotics [7, 8] (Fig. 24). Enzymatic acetyla-tion, phosphorylation, and adenylylation produce modified drug molecules that have lost their antibacterial activity.

In order to overcome bacterial resistance, new semi-synthetic aminoglycoside antibiotic amikacin (Fig. 25) was designed. This compound is stable to enzymatic deactivation. The amikacin binds to the A-site of 16S rRNA that disrupts the protein synthesis.

In this molecule, the L-hydroxyaminobutyramide moiety attached to the central ring fragment protects the drug from attacks by certain bacterial antibiotic inactivating enzymes. Another semisynthetic drug candidate that is stable to enzymatic acylation, phosphorylation, and adenylylation - plazomicin (Fig. 26) - is now in the development stage.

Fig. 22. Streptomycin (A) and kanamycin A (B).

I Uracil ] oh

Fig. 23. Interaction of neomycin with 50S ribosomal subunit.

Adenosine monophosphate (AMP)

Fig. 24. Enzymatic deactivation of aminoglycoside antibiotics. I -acetylation, II - phosphorylation, III - adenylylation.

Fig. 25. Amikacin.

bacterial cell (an example: tigecycline, Fig. 28) [62] and disrupt the translation (synthesis of proteins) process by preventing aminoacyl-tRNA from binding to the corresponding site of mRNA - ribosome complex.

Since this class of antibiotics was used extensively, the bacterial resistance emerged without delay. Three major mechanisms of resistance to tetracyclines have been recognized to date: efflux of the drug molecules from the bacterial cell by the efflux pumps, enzymatic modification of tetracycline molecule leading to its inactivation and ri-bosomal protection [63]. The latter involve the synthesis of ribosomal protection proteins (RPPs) (e.g. Tet(O) and Tet(M)) that significantly reduce the binding of tetracy-cline antibiotics to the ribosome subunit or displace them from the binding site. Ribosomal protection is considered to be one of the two major mechanisms of bacterial resistance against tetracyclines [64]. This approach is used by both Gram-positive and Gram-negative bacteria.

One of the efficient ways to fight the drug resistant strains of bacteria is the chemical modification of existing antibiotics. By using this approach, new representatives of this class of drugs efficient against the resistant strains were discovered [65, 66] (Fig. 29).

Another major mechanism of tetracyclines resistance in bacteria is drug efflux from bacterial cell. It was proven that membrane-associated proteins (e.g. Tet(A), Tet(B), Tet(C)) are responsible for the efflux of tetracyclines from bacteria [63]. Most of them belong to the major

A

Fig. 27. Chlorotetracycline (A) and Oxytetracycline (B).

Fig. 26. Plazomicin. e. Tetracyclines

Another class of broad-spectrum antibiotics that target the 30S ribosomal subunit is tetracyclines (Fig. 27).

Tetracyclines are natural products. The first member of this class of antibiotics - chlorotetracycline - was isolated from the bacteria Streptomyces aureofaciens. There are also a number of semisynthetic tetracycline drugs. In the review by Liu et al. practical methods of the synthesis of tetracyclines are summarized including an example of the successful synthesis of a new potent antibiotic erava-cycline that is currently in clinical trials [61].

Tetracyclines are broad-spectrum antibiotics active against both Gram-negative and Gram-positive bacteria. Tetracyclines bind to the 30S subunit of the ribosome in

Fig. 28. Mg-tigecycline complex interactions with residues of 16S rRNA (30S ribosome subunit).

B

facilitator superfamily (MFS) of efflux transporters [9, 10, 67] though some of them (e.g. EfrAB [9, 68]) belong to the ATP-binding cassette (ABC) family of efflux pumps. Thus, Tet proteins transport tetracycline molecules coordinated with the divalent cations (Ca2+ or Mg2+) through the bacterial membrane in exchange for the protons [67, 10]. Nelson et al. [69] demonstrated that cyclopentylthio-tetracycline (13-CPTC) is an efficient inhibitor of Tet(B) protein. The uptake of tetracycline into the resistant bacteria was significantly increased in the presence of 13-CPTC (Fig. 30) as a result of bacterial efflux pumps blockage. Therefore, the simultaneous administration of 13-CPTC with tetracycline can restore the activity of this antibiotic against the resistant bacteria.

A new antibiotic fluorocycline TP-271 (Fig. 31), described recently by Grossman et al. [70], demonstrates high in vitro potency against multidrug-resistant bacteria. These results once more show that changing the antibiotic's structure is a promising approach to combat bacterial resistance.

The third - less prevalent - mechanism of resistance is the chemical modification of tetracyclines studied by Speer et al. [71]. They found that tetracycline is chemically modified in bacteria that carry the *Tcr gene in aerobic conditions. The structures of modified tetracycline products were not determined. The impact of tetracyclines coordination with metals on antimicrobial activity was reviewed by Guerra et al. [72].

A

Fig. 29. Tigecycline (A) and pentacycline (B).

Fig. 30. Cyclopentylthiotetracycline (13-CPTC).

f. Streptogramins

One of the antibiotic classes that target the large 50S ribosomal subunit is streptogramins (e.g. dalfopristin (streptogramin A) and quinupristin (streptogramin B), Fig. 32).

A

Fig. 31. Fluorocycline TP-271.

Fig. 32. Dalfopristin(A) and quinupristin(B).

Both dalfopristin and quinupristin are semisynthetic modifications of the natural streptogramin antibiotics produced by various Streptomyces bacteria. Strep-togramins inhibit the bacterial protein synthesis by binding to the 50S ribosomal subunit. Group A streptogramins (dalfopristin) bind in the peptidyltransferase center (PTC) and interfere with binding of aminoacyl-tRNAs, which prevents the formation of a peptide bond during the elongation step. Streptogramin B components (quinupristin) bind to the proximal end of the exit tunnel resulting in the release of the small incomplete oligopeptide chains [73, 74]. Each compound shows moderate activity, whereas the combination of both drugs demonstrates a synergetic effect. The binding of dalfopristin results in a specific conformational change of the 50S ri-bosomal subunit that significantly facilitates the consequent binding of quinupristin. As a result, administration of the dalfopristin and quinupristin mixture (marketed as Synercid) produces much higher antibacterial activity.

Bacteria have developed several mechanisms of resistance to these antibiotics including active efflux from the bacterial cell, 23S rRNA methylation, and enzymatic deactivation. The acetylation of the OH group in dalfopristin and its analogues catalyzed by O-acetyltransferases (e.g. virginiamycin acetyltransferase) results in the formation of O-acetyl products that do not have the binding affinity to 50S ribosome subunit [73]. Enzymatic de-activation of quinupristin and its structural homologues proceeds by lactone opening catalyzed by virginiamycin B lyase (Vgb) resulting in the formation of linear products that have lost their antibacterial activity [75].

B

B

This development accelerated the search for new strep-togramins active against the resistant strains. The main efforts to modify the structure of type A streptogramins were concentrated on the introduction of different sub-stituents R in position 16 of 16-desoxopristinamycin II (Fig. 33). On the other hand, the substitution of the 4-oxo-2-piperidinecarboxylic acid fragment in pristinamycin I (Fig. 33) by other heterocyclic amino acids led to the discovery of several new active compounds [73]. An interesting approach to the modification of the streptogramin structure was demonstrated by Mukhtar et al. [76]. Using synthetic and enzymatic methods, they prepared a series of chimeric compounds containing structural elements of streptogramins B and antibiotic tyrocidine. These compounds showed a broad range of activity against Grampositive bacteria. It is believed that their mechanism of action is different from that of the parent antibiotics.

Fig. 33. 16-Desoxopristinamycin II (A) and pristinamycin I (B).

In a recent publication by Li et al., a new practical scalable approach to the synthesis of streptogramin antibiotics is described. This method can lead to the discovery of new potent drugs that will overcome the limitations of known streptogramins [77].

g. Macrolides

Another class of antibiotics that target the large ri-bosomal subunit is macrolides (e.g. azithromycin and erythromycin, Fig. 34).

Erythromycin is a natural compound isolated from the bacteria Saccharopolyspora erythraea. Azithromycin is one of semisynthetic analogues of erythromycin. Erythromy-cin binds close to the peptidyl transferase center (PTC) in the 50S ribosomal subunit at the end of the polypeptide exit tunnel. It affects the polypeptide chain elongation step and consequently disrupts the whole process of protein biosynthesis [78]. The mechanism of action of these antibiotics is discussed by Gaynor et al. [79].

During last two decades, we witnessed the emergence of bacterial resistance to macrolides and particularly to erythromycin [80]. There are three known mechanisms of resistance. The primary mechanism involves post-transcriptional methylation of the 23S ribosomal RNA that prevents the antibiotic from binding to bacterial ribosome [79]. The other mechanism of resistance is based on the action of efflux pumps that exert antibiotics from the bacterial cell [81]. One more possible mechanism involves the ribosomal synthesis of small peptides (4-6 amino acids) that interact with the drug molecule bound to the 50S subunit of the ribosome and promote its release from the active site [79]. In order to fight bacterial resistance, the second (clarithromycin) and then the third (telithromycin) generation of macrolides were developed (Fig. 35). The latter has a significantly improved affinity to both the native and mutant 50S ribo-some subunits, although the new mutations that lead to this antibiotic resistance have already been reported [81].

A

A B

Fig. 34. Azithromycin (A) and erythromycin (B).

Fig. 35. Clarithromycin (A) and telithromycin (B).

Summary of practical methods of synthesis of new macrolide antibiotics is presented by Seiple et al. [82].

h. Oxazolidinones

Oxazolidinones (e.g. linezolid and tedizolid, Fig. 36) are the only completely synthetic class of antibiotics that target bacterial ribosome.

The distinctive structural fragment of this class of antibiotics is the five-membered oxazolidinone ring. This class of compounds was first investigated at DuPont and

B

B

then at Pharmacia [73]. Linezolid and other oxazolidi-nones bind to the 50S ribosomal subunit and inhibit the process of protein synthesis. The possible mechanism of action of oxazolidinones is binding at the P site of the 50S ribosomal subunit and inhibition of the initiation step of translation [73]. Resistance to oxazolidinones occurs by mutations in the binding site of these antibiotics at the 50S subunit. The second generation of oxazolidi-nones - antibiotic tedizolid, which was approved for use in humans in 2014, - has improved potency and broader antimicrobial activity. A series of new oxazolidinones containing fused heterocyclic rings were synthesized and showed higher activity than linezolid against some Gram-positive and Gram-negative bacteria [83].

Fujisaki et al. report the synthesis of novel oxazolidi-none dimers that showed significant activity against both Gram-negative and Gram-positive bacteria [84].

Interesting approach in search for new antibiotics was demonstrated by Gordeev et al. [85] and Hubschwerlen et al. [86, 87]. They synthesized new antibiotics by linking ciprofloxacin (fluoroquinolone) and oxazolidinone fragments together. The best representative of this class of compounds (Fig. 37) showed broad antibacterial activity including activity against fluoroquinolone- and linezolid-resistant bacteria.

A

A

Fig. 36. Linezolid (A) and tedizolid (B).

Fig. 37. New antibiotic obtained by linking ciprofloxacin and oxa-zolidinone fragments together.

i. Flouroquinolones

Another important group of antibiotics selectively targets certain steps of bacterial DNA replication and transcription. Synthetic antibiotics that belong to the flouro-quinolone class inhibit DNA replication while the natural and semisynthetic rifamycins disrupt the transcription process.

Fluoroquinolones (e.g. ciprofloxacin and norfloxacin, Fig. 38) are considered to be the most successful class of synthetic antibiotics.

Both ciprofloxacin and norfloxacin are synthetic antibiotics containing the central quinolone fused ring system.

Fruoroquinolones are broad-spectrum antibiotics. They interact with enzymes DNA gyrase and topoi-somerase IV that leads to the distraction of the DNA

Fig. 38. Ciprofloxacin (A) and norfloxacin (B).

replication process in the bacterial cell and also prevents the bacterial DNA repair and disrupts the cell division process. In the course of the DNA replication process, the double stranded DNA is unwound by the DNA helicase enzyme. That leads to an under wound DNA sector (negative supercoils) on one side of the replication fork and super wound DNA sector (positive supercoils) on the other side. The main role of DNA gyrase is to relieve the DNA supercoiling strand. This is achieved by DNA double strand cleavage that leads to the relief of both negative and positive supercoiling, and then the successful resealing of the DNA double strand break. Flouroquinolones interact with the double-strand cleaved DNA-enzyme complexes, stabilize them, and prevent DNA double-strand resealing [88]. The role of enzyme topoisomerase IV is to release the two daughter chromosomal circles after the replication step that is essential in the process of cell division. This is achieved by cutting the DNA double -strand of one of the chromosomes, moving the cut site out of the second chromosome and the consequent resealing of the cut. Fluoroquinolones interact with the complex formed by enzyme topoisomerase IV with DNA double-strand cut and stabilize it (Fig. 39) [88]. That, in turn, prevents the sealing of the double-strand DNA cuts and leads to DNA breakage and eventually to bacterial cell death. In spite of the wide use of fluoroquinolones in medical practice, the detailed molecular mechanism of action of these drugs was discovered quite recently.

Bacterial resistance to this class of antibiotics emerged as a result of their widespread use for the treatment of infections in humans. In many cases, these antibiotics were misused for the treatment of infections that could be treated with other narrow spectrum antimicrobials and even for viral infections.

There are several known mechanisms of resistance to quinolones in Staphylococcus aureus. One of them - plas-mid-mediated - is an active efflux of these antibiotics from the bacterial cell by drug pumps. It has been shown [89-92] that the NorA efflux pumps are responsible for the decreasing of the concentration of some (hydro-philic) fluoroquinolone drugs in the bacterial cell and consequently for the loss of activity of these antibiotics in Staphylococcus aureus. According to the recent publication by Tintino et al. [93], the tannic acid is an efficient inhibitor of the NorA efflux pumps in this bacterial strain. Thus, they showed that the administration of norfloxacin

B

B

together with tannic acid (Fig. 40C) leads to a significant decrease in the minimum inhibitory concentration (MIC) for this antibiotic. It was also shown that reserpine is an efficient inhibitor of the NorA efflux pumps [94]. Other possible inhibitors for these efflux pumps include pro-chlorperazine, paroxetine, verapamil, and omeprazole (Fig. 40 A, B, D, E, correspondingly) [9], although in order

to achieve significant inhibition a relatively high concentration of these compounds is necessary.

Since active efflux is one of the major mechanisms of resistance [95], the search for the new efflux pumps inhibitors continues. Coelho et al. [96] showed that ne-rol (Fig. 41) acting as a drug pump inhibitor can significantly increase the activity of norfloxacin against the

Fig. 39. Drug-stabilized topoisomerase IV - DNA cleavage complex.

D

Fig. 40. Prochlorperazine, paroxetine, tannic acid, verapamil, and omeprazole (A, B, C, D, E, correspondingly).

Staphylococcus aureus strain. According to Felicetti et al. [97] 2-(3,4-dimethoxyphenyl)quinoline (Fig. 41) is an active NorA pumps inhibitor and can be successfully used in combination with ciprofloxacin.

A B

Fig. 41. Nerol (A) and 2-(3,4-dimethoxyphenyl)quinoline (B).

Other plasmid-mediated resistance mechanisms include the bacterial production of pentapeptide repeat proteins that prevent the drugs from binding to DNA gyrase and action of bacterial enzyme aminoglycoside acetyltransferase AAC (6')-Ib-cr leading to the drug in-activation [98, 99]. Two codon changes in aac (6')-Ib gene directing two substitutions Trp102Arg and Asp179Tyr lead to structural changes in aminoglycoside acetyl-transferase that enable this enzyme to acetylate the ciprofloxacin. After the acylation of NH-group of the piper-azinyl fragment (Fig. 42), the drug shows practically no antibacterial activity.

A

of Helicobacter pylori. Some of these compounds have shown synergetic effects when combined with metronidazole. Compounds from other chemical classes are also considered as topoisomerase IV inhibitors. A new gyrase inhibitor spiropyrimidinetrione AZD0914 (Fig. 44) was developed by AstraZeneca [101]. Another new lead compound for gyrase inhibition (Fig. 44, compound 2) was reported by Chan et al. [102].

A

Fig. 42. Acylation of the piperazinyl fragment of ciprofloxacin.

Obviously, this mechanism of resistance affects only those fluoroquinolones that have a piperazinyl ring like ciprofloxacin, norfloxacin, etc. Other representatives of this class of antibiotics like rufloxacin, perfloxacin, and fleroxacin that have an N-methylpiperazinyl fragment (Fig. 43) are unaffected by bacterial aminoglycoside acetyltransferase AAC(6')-Ib-cr [99].

Fig. 43. Rufloxacin (A), perfloxacin (B), and fleroxacin (C).

In order to overcome the bacterial resistance, a search for the new fluoroquinolone antibiotics is ongoing. According to Abu-Qatouseh et al. [100], a number of 8-nitrofluoroquinolones are active against some strains

Fig. 44. AZD0914 (A) and compound 2 (B). j. Rifamycins

Rifamycins represent the natural and semisynthetic class of antibiotics that target RNA polymerase. Natural rifamycins are produced by Amycolatopsis rifamycinica (previously known as Amycolatopsis mediterranei) although they can also be synthesized in the laboratory. Rifampi-cin, rifapentine, and rifabutin are examples of semisynthetic rifamycins (Fig. 45).

Semisynthetic drugs of this class (e.g. rifampicin) have rather broad-spectrum antibiotic activity. Rifamy-cins bind to the RNA polymerase in the bacterial cell and distract the transcription process - DNA dependent synthesis of mRNA [103]. Interaction of the drug molecule with the enzyme leads to the formation of a complex that prevents the elongation of the mRNA oligonucleotide chain. These drugs are active against the Gram-positive and some strains of Gram-negative bacteria. Mutations in the bacterial RNA polymerase, which can develop very quickly, lead to the strong bacterial resistance to these antibiotics. Rifamycin producer Amycolatopsis rifamycinica uses this approach for self-protection. The effect of different mutations in this enzyme on antibiotic activity of rifamycins was discussed recently [104, 105]. General overview of this class of antibiotics with a focus on zwit-terionic compounds is given in [106]. Since these antibiotics are used to treat tuberculosis, there is an ongoing search for new active compounds. Thus, Bujnowski et al.

[107] reviewed the development of new methods for the synthesis of novel rifamycins, whereas Czerwonka et al.

[108] described a new series of these antibiotics containing L-amino acid esters and showed that some of them have antibacterial activity comparable to rifampicin.

k. Lipopeptides

Lipopeptides are the unique class of antibiotics that target the bacterial membrane. Bacterial membranes have

B

B

C

Fig. 45. Rifampicin (A), rifapentine (B), and rifabutin (C).

significantly higher content of anionic phospholipids than membranes of eukaryotic cells. That allows for the selective targeting of bacterial membranes by antibiotics. There are several known active lipopeptide antimicrobials and at least three of them -polymyxin B, polymyxin E (colistin) (Fig. 46) [109], and daptomycin (Fig. 47) [109] -are or have been in clinical practice.

Polymyxins are natural antibiotics produced by different strains of Paenibacillus polymyxa. Daptomycin is also a natural compound isolated from bacteria Strepto-myces roseosporus, although recently a synthetic method for this compound was developed. Daptomycin as well as polymyxins is synthesized in bacteria by complexes of nonribosomal peptide synthetase enzymes. Polymyxins are a mixture of several compounds; the major components are shown in Fig. 46.

The action of these antibiotics leads to a drastic change in bacterial membrane permeability and eventually causes bacterial cell death. Thus, it is believed that daptomycin initially interacts with the negatively charged bacterial membrane by means of coordination through the Ca2+ ions, then its lipophilic 'tail' is inserted in the membrane bilayer and the consequent interaction with several other daptomycin molecules leads to oligomerization, and finally ion channel formation (Fig. 48). The formation of an ion channel leads to potassium ions efflux from the bacterial cell, membrane depolarization, and eventually bacterial cell death [111]. Resistance to daptomycin in several bacterial strains has

A

Fig. 46. Major components of polymyxin E (colistin) (A) and polymyxin B (B) [110].

Fig. 47. Daptomycin.

Fig. 48. The proposed mechanism of action of daptomycin. Step I - binding and insertion, step II - oligomerization, step III -channel formation and ion efflux.

been reported. The mechanism of resistance is related to the changes in bacterial membrane - from significant decrease to the absence of anionic phospholipids in the membrane structure [14]. These changes are connected with the loss of cdsA activity that encodes the cdsA enzyme involved in synthesis of membrane anionic lipids

B

B

[15]. In the absence of a negative charge on the bacterial membrane daptomycin cannot interact with it through the Ca2+ ions and thereby bacteria become resistant to this antibiotic.

The recent developments in search for new macrocy-clic polypeptide antibiotics including glycopeptides and lipopeptides was recently reviewed by Luther et al. [112]. In general, antimicrobial peptides (AMPs) are getting more and more attention as novel promising therapeutic agents against antibiotic resistant bacteria [113-115].

CONCLUSION

In conclusion, we can summarize that the general methods to overcome bacterial resistance are:

1. Discovery of new antibiotics that are active against drug resistant bacteria due to:

a. New or improved mechanism of action;

b. New physicochemical properties that makes it im-

possible for bacterial drug efflux pumps to recognize them and to remove them from bacterial cell;

c. Ability to withstand the action of bacterial deactivating enzymes;

d. Ability to bind to the modified target in bacteria [41-43, 116-119].

2. Synthesis of new antibiotics with dual or triple mechanism of action by the introduction of new functional groups in the molecule of the antibiotic [48, 120, 121] in order to restore the activity of the drug against the resistant bacteria.

3. Synthesis of new dual action antibiotics by linking two different antibiotic molecules [85-87, 122]. That makes it much more difficult for bacteria to develop resistance to these drugs.

4. Administration of antibiotics together with adjuvants that help to overcome bacterial resistance [28, 29, 123, 124]

a. By blocking the action of bacterial drug efflux pumps;

b. By blocking the action of bacterial enzymes that deactivate the antibiotic;

5. Simultaneous administration of two antibiotics with different mechanisms of action [49, 58].

6. Simultaneous administration of two antibiotics with a synergetic effect.

REFERENCES

1. Antimicrobial resistance: global report on surveillance. World Health Organization. 2014. ISBN 978 92 4 156474 8. Available: http://www.who. int/drugresistance/documents/surveillancereport/ en/ (Accessed on December 22, 2017).

2. WHO publishes list of bacteria for which new antibiotics are urgently needed. News release. World Health Organization 2017. Available: http://www. who.int/mediacentre/news/releases/2017/bacteria-antibiotics-needed/en/. (Accessed on December 22, 2017).

New antibiotics that are in the late stages of clinical development were recently reviewed by Fernandes et al. [125].

It should be mentioned that there are a number of new approaches in the development of antibiotics active against the drug resistant bacteria e.g. development of pleuromutilins, quorum-sensing inhibitors, FabI-inhib-itors, peptidomimetics, inhibitors of bacterial virulence etc.

These are definitely very important and promising areas of research, although no approved antibiotics for use in humans (except retapamulin approved for topical use) have been discovered so far.

The rapid spread of antibiotic resistance in bacteria makes it necessary to intensify the development of new antibiotics and expand the search for new methods to fight bacterial resistance. Over the course of these research efforts, scientists are gaining a better understanding of the mechanisms of antibiotic action, the mechanisms of bacterial resistance, and the key biochemical processes of the bacterial life cycle. On the other hand, new targets for antibiotics in bacteria are being discovered [126].

CONFLICT OF INTEREST

The author does not pursue commercial or financial interests.

CITATION

A. Zhivich. Fighting bacterial resistance: approaches, challenges, and opportunities in the search for new antibiotics. Part 1. Antibiotics used in clinical practice: mechanisms of action and the development of bacterial resistance. MIR J, 2017; 4(1), 31-51, doi: 10.18527/25002236-2017-4-1-31-51

COPYRIGHT

© 2017 Zhivich. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International Public License (CC-BY-NC-SA), which permits unrestricted use, distribution, and reproduction in any medium, as long as the material is not used for commercial purposes, provided the original author and source are credited.

3. Juhas M. Horizontal gene transfer in human pathogens. Crit Rev Microbiol. 2015; 41(1), 101-8. doi: 10.3109/1040841X.2013.804031.

4. Baym M, Lieberman TD, Kelsic ED, Chait R, Gross R, Yelin I, Kishony R. Spatiotemporal microbial evolution on antibiotic landscapes. Science. 2016; 353(6304), 1147-51; doi: 10.1126/science.aag0822.

5. Biology. Available: https://cnx.org/contents/GFy_ [email protected]:pOpVdIwp@11/Prokaryotic-Cells. (Accessed on December 22, 2017).

6. Wright GD. Q&A: Antibiotic resistance: where does it come from and what can we do about it? BMC Biol. 2010; 8(123). doi: 10.1186/1741-7007-8-123.

7. Shaw KJ, Rather PN, Hare RS, Miller GH. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modi-fying enzymes. Microbiol Rev. 1993; 57(1), 138-63. PubMed PMID: 8385262.

8. Wright GD. Bacterial resistance to antibiotics: enzymatic degradation and modification. Adv Drug Deliv Rev. 2005; 57(10), 1451-70. doi: 10.1016/j. addr.2005.04.002.

9. Schindler BD, Kaatz GW. Multidrug efflux pumps of Gram-positive bacteria. Drug Resist Updat. 2016; 27, 1-13. doi: 10.1016/j.drup.2016.04.003.

10. Levy SB. Active efflux mechanisms for antimicrobial resistance. Antimicrob Agents Chemother. 1992; 36(4), 695-703. PubMed PMID: 1503431.

11. Alekshun MN, Levy SB. Molecular mechanisms of antibacterial multidrug resistance. Cell. 2007; 128(6), 1037-50. doi: 10.1016/j.cell.2007.03.004.

12. Wilson DN. Ribosome-targeting antibiotics and mechanisms of bacterial resistance. Nat Rev Microbiol. 2014; 12(1), 35-48. doi: 10.1038/nrmicro3155.

13. Walsh C. Molecular mechanisms that confer antibacterial drug resistance. Nature. 2000; 406(6797), 77581. doi: 10.1038/35021219.

14. Mishra NN, Tran TT, Seepersaud R, Garcia-de-la-Maria C, Faull K, Yoon A, et al. Perturbations of Phosphatidate Cytidylyltransferase (CdsA) Mediate Daptomycin Resistance in Streptococcus mitis/ora-lis by a Novel Mechanism. Antimicrob Agents Chemother. 2017; 61(4). doi: 10.1128/AAC.02435-16.

15. Adams HM, Joyce LR, Guan Z, Akins RL, Palmer KL.

Streptococcus mitis and S. oralis Lack a Requirement for CdsA, the Enzyme Required for Synthesis of Major Membrane Phospholipids in Bacteria. Antimicrob. Agents Chemother. 2017; 61(5). doi: 10.1128/ AAC.02552-16.

16. Coates ARM, Halls G, Hu Y. Novel classes of antibiotics or more of the same? Br J Pharmacol. 2011; 163, 184-194. doi: 10.1111/j.1476-5381.2011.01250.x

17. Mohr KI. History of Antibiotics Research. In: Current Topics in Microbiology and Immunology. 2016; 398 (How to Overcome the Antibiotic Crisis), 237-72. doi: 10.1007/82_2016_499.

18. Fleming A. Classics in infectious diseases: on the antibacterial action of cultures of a penicillium, with special reference to their use in the isolation of B. influenzae. Br J Exp Pathol. 1929; 10(3), 226-236. PMCID: PMC2048009.

19. Lee W, McDonough MA, Kotra L, Li ZH, Silvag-gi NR, Takeda Y, et al. A 1.2-A snapshot of the final step of bacterial cell wall biosynthesis. Proc Natl Acad Sci USA. 2001; 98(4), 1427-31. doi: 10.1073/ pnas.98.4.1427.

20. Bachmann BO, Li R, Townsend CA. ß-Lactam synthetase: a new biosynthetic enzyme. Proc Natl

Acad Sci USA. 1998; 95(16), 9082-6. doi: 10.1073/ pnas.95.16.9082.

21. Bush K, Jacoby GA, Medeiros AA. A functional classification scheme for ß-lactamases and its correlation with molecular structure. Antimicrob Agents Chemother. 1995; 39(6), 1211-33. doi: 10.1128/ AAC.39.6.1211.

22. Pfeifer Y, Cullik A, Witte W. Resistance to cephalosporins and carbapenems in Gram-negative bacterial pathogens. Int J Med Microbiol. 2010; 300(6), 371-9. doi: 10.1016/j.ijmm.2010.04.005.

23. Delmas J, Chen Y, Prati F, Robin F, Shoichet BK, Bonnet R. Structure and dynamics of CTX-M enzymes reveal insights into substrate accommodation by extended-spectrum ß-lactamases. J Mol Biol. 2008; 375(1), 192-201. doi: 10.1016/j.jmb.2007.10.026.

24. Trehan I, Morandi F, Blaszczak LC, Shoichet BK. Us-

ing steric hindrance to design new inhibitors of class C ß-lactamases. Chem Biol. 2002; 9(9), 971-80. doi: 10.1016/S1074-5521(02)00211-9.

25. Kuzin AP, Nukaga M, Nukaga Y, Hujer A, Bonomo RA, Knox JR. Inhibition of the SHV-1 ß-lactamase by sul-fones: crystallographic observation of two reaction intermediates with tazobactam. Biochemistry. 2001; 40(6), 1861-6. doi: 10.1021/bi0022745.

26. Choi H, Paton RS, Park H, Schofield CJ. Investigations on recyclisation and hydrolysis in avibactam mediated serine ß-lactamase inhibition. Org Biomol Chem. 2016; 14(17), 4116-28. doi: 10.1039/ c6ob00353b.

27. Krishnan NP, Nguyen NO, Papp-Wallace KM, Bono-mo RA, van den Akker F. Inhibition of Klebsiella ß-Lactamases (SHV-1 and KPC-2) by Avibactam: A Structural Study. PLoS One. 2015; 10(9), e0136813. doi: 10.1371/journal.pone.0136813.

28. Sader HS, Rhomberg PR, Farrell DJ, Jones RN. Antimicrobial activity of CXA-101, a novel cephalospo-rin tested in combination with tazobactam against Enterobacteriaceae, Pseudomonas aeruginosa, and Bacteroides fragilis strains having various resistance phenotypes. Antimicrob Agents Chemother. 2011; 55(5), 2390-4. doi: 10.1128/AAC.01737-10.

29. Taneja N, Kaur H. Insights into Newer Antimicrobial Agents Against Gram-negative Bacteria. Microbiol Insights. 2016; 9, 9-19. doi: 10.4137/MBI.S29459.

30. Llarrull LI, Testero SA, Fisher JF, Mobashery S. The future of the ß-lactams. Curr Opin Microbiol. 2010; 13(5), 551-7. doi: 10.1016/j.mib.2010.09.008.

31. Asai M, Haibara K, Muroi M, Kintaka K, Kishi T. Sulfazecin, a novel ß-lactam antibiotic of bacterial origin. Isolation and chemical characterization. J Antibiot (Tokyo). 1981; 34(6), 621-7. doi: 10.7164/ antibiotics.34.621.

32. Braga D, Lackner G. One Ring to Fight Them All: The Sulfazecin Story. Cell Chem Biol. 2017; 24(1), 1-2. doi: 10.1016/j.chembiol.2017.01.001.

33. Li R, Oliver RA, Townsend CA. Identification and Characterization of the Sulfazecin Monobactam

Biosynthetic Gene Cluster. Cell Chem Biol. 2017; 24(1), 24-34. doi: 10.1016/j.chembiol.2016.11.010.

34. Marshall S, Hujer AM, Rojas LJ, Papp-Wallace KM, Humphries RM, Spellberg B, et al. Can Ceftazidime-Avibactam and Aztreonam Overcome ß-Lactam Resistance Conferred by Metallo-ß-Lactamases in En-terobacteriaceae? Antimicrob Agents Chemother. 2017; 61(4). doi: 10.1128/AAC.02243-16.

35. Skalweit MJ, Li M, Taracila MA. Effect of asparagine substitutions in the YXN loop of a class C ß-lactamase of Acinetobacter baumannii on substrate and inhibitor kinetics. Antimicrob Agents Chemother. 2015; 59(3), 1472-7. doi: 10.1128/AAC.03537-14.

36. Powers RA. Structural and Functional Aspects of Extended-Spectrum AmpC Cephalosporinases. Curr Drug Targets. 2016; 17(9), 1051-60. doi: 10.2174/157 3399811666150615144707.

37. Powers RA, Swanson HC, Taracila MA, Florek NW, Romagnoli C, Caselli E, et al. Biochemical and structural analysis of inhibitors targeting the ADC-7 cephalosporinase of Acinetobacter baumannii. Biochemistry. 2014; 53(48), 7670-9. doi: 10.1021/ bi500887n.

38. Venugopal DVR, Karteek RA, Uma Devi P, Sastry YN, Aruna Lakshmi K, Tarak Ramji M, and Shiralgi Y. Design, synthesis and characterization of peptidylbo-ronate analogues as effective antimicrobial agent. Res Chem Intermed. 2017; 43(10), 5755-5778. doi: 10.1007/s11164-017-2961-0.

39. Bouquet J, King DT, Vadlamani G, Benzie GR, Iorga B, Ide D, et al. Selective trihydroxylatedazepane inhibitors of NagZ, a glycosidase involved in Pseudomonas aeruginosa resistance to ß-lactam antibiotics. Org Biomol Chem. 2017; 15(21), 4609-19. doi: 10.1039/ c7ob00838d.

40. Nguyen TV, Blackledge MS, Lindsey EA, Min-rovic BM, Ackart DF, Jeon AB, et al. The Discovery of 2-Aminobenzimidazoles That Sensitize Mycobacterium smegmatis and M. tuberculosis to ß-Lactam Antibiotics in a Pattern Distinct from ß-Lactamase Inhibitors. Angew Chem Int Ed Engl. 2017; 56(14), 3940-4. doi: 10.1002/anie.201612006.

41. Fishovitz J, Hermoso JA, Chang M, Mobashery S. Penicillin-binding protein 2a of methicillin-resis-tant Staphylococcus aureus. IUBMB Life, 2014; 66(8), 572-7. doi: 10.1002/iub.1289.

42. Fishovitz J, Rojas-Altuve A, Otero LH, Dawley M, Carrasco-Lopez C, Chang M, Hermoso JA, Mobash-ery S. Disruption of allosteric response as an unprecedented mechanism of resistance to antibiotics. J Am Chem Soc. 2014; 136(28), 9814-7, doi: 10.1021/ ja5030657.

43. Fishovitz J, Taghizadeh N, Fisher JF, Chang M, Mobashery S. The Tipper-Strominger Hypothesis and Triggering of Allostery in Penicillin-Binding Protein 2a of Methicillin-Resistant Staphylococcus aureus (MRSA). J Am Chem Soc. 2015; 137(20), 6500-5, doi: 10.1021/jacs.5b01374.

44. Pages JM, James CE, Winterhalter M. The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria. Nat Rev Microbiol. 2008; 6(12), 893-903, doi: 10.1038/nrmicro1994.

45. Lou H, Chen M, Black SS, Bushell SR, Ceccarelli M, Mach T, Beis K, Low AS, Bamford VA, Booth IR, Bayley H, Naismith JH. Altered antibiotic transport in OmpC mutants isolated from a series of clinical strains of multi-drug resistant E. coli. PLoS ONE. 2011; 6(10), e25825, doi: 10.1371/journal. pone.0025825.

46. Xie J, Pierce JG, James RC, Okano A, Boger DL. A redesigned vancomycin engineered for dual D-Ala-D-ala and D-Ala-D-Lac binding exhibits potent antimicrobial activity against vancomycin-resistant bacteria. J Am Chem Soc. 2011; 133(35), 13946-9. doi: 10.1021/ ja207142h.

47. Silverman SM, Moses JE, Sharpless KB. Reengineering Antibiotics to Combat Bacterial Resistance: Click Chemistry [1,2,3]-Triazole Vancomycin Dimers with Potent Activity against MRSA and VRE. Chemistry. 2017; 23(1), 79-83. doi: 10.1002/chem.201604765.

48. Okano A, Isley NA, Boger DL. Peripheral modifications of [¥[CH2NH]Tpg4]vancomycin with added synergistic mechanisms of action provide durable and potent antibiotics. Proc Natl Acad Sci USA. 2017; 114(26), E5052-E61. doi: 10.1073/pnas.1704125114.

49. Kang YR, Chung DR, Kim J, Baek JY, Kim SH, Ha YE, et al. In vitro synergistic effects of various combinations of vancomycin and non-ß-lactams against Staphylococcus aureus with reduced susceptibility to vancomycin. Diagn Microbiol Infect Dis. 2016; 86(3), 293-9. doi: 10.1016/j.diagmicrobio.2016.08.009.

50. Yarlagadda V, Sarkar P, Manjunath GB, Haldar J. Lipo-philic vancomycin aglycon dimer with high activity against vancomycin-resistant bacteria. Bioorg Med Chem Lett. 2015; 25(23), 5477-80. doi: 10.1016/j. bmcl.2015.10.083.

51. Yarlagadda V, Konai MM, Manjunath GB, Ghosh C, Haldar J. Tackling vancomycin-resistant bacteria with 'lipophilic-vancomycin-carbohydrate conjugates'. J Antibiot (Tokyo). 2015; 68(5), 302-12. doi: 10.1038/ja.2014.144.

52. Ashford P-A, Bew SP. Recent advances in the synthesis of new glycopeptide antibiotics. Chem. Soc. Rev. 2012; 41, 957-978. doi: 10.1039/C1CS15125H.

53. James RC, Pierce JG, Okano A, Xie J, Boger DL. Redesign of Glycopeptide Antibiotics - Back to the Future. ACS Chem Biol. 2012; 7(5), 797-804. doi: 10.1021/cb300007j.

54. Boothe DM. Sulfonamides and Sulfonamide Combinations. Sulfonamides Mechanism of Action. Merck Veterinary Manual. Available: http://www.merckvet-manual.com/pharmacology/antibacterial-agents/ sulfonamides-and-sulfonamide-combinations (Accessed on December 22, 2017).

55. Capasso C, Supuran CT. Sulfa and trimethoprim-like drugs - antimetabolites acting as carbonic anhy-drase, dihydropteroate synthase and dihydrofolate

reductase inhibitors. J Enzyme Inhib Med Chem. 2014; 29(3): 379-387. doi: 10.3109/14756366.2013.787422.

56. Sköld O. Sulfonamide resistance: mechanisms and trends. Drug Resist Updat. 2000; 3(3), 155-60. doi: 10.1054/drup.2000.0146.

57. Rudolph KM, Parkinson AJ, Roberts MC. Mechanisms of erythromycin and trimethoprim resistance in the Alaskan Streptococcus pneumoniae serotype 6B clone. J Antimicrob Chemother. 2001; 48(2), 3179. doi: 10.1093/jac/48.2.317.

58. Nepka M, Perivolioti E, Kraniotaki E, Politi L, Tsakris A, Pournaras S. In Vitro Bactericidal Activity of Trimethoprim-Sulfamethoxazole Alone and in Combination with Colistin against Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates. Antimicrob Agents Chemother. 2016; 60(11), 69036. doi: 10.1128/AAC.01082-16.

59. Bavadi M, Niknam K, Gharibi M. Synthesis of new dihydropyrrol-2-one derivatives bearing sulfon-amide groups and studies their antibacterial activity Monatsh Chem. 2017; 148(6), 1025-34; doi: 10.1007/ s00706-016-1847-y.

60. Wang L, Pulk A, Wasserman MR, Feldman MB, Alt-man RB, Cate JH, et al. Allosteric control of the ribo-some by small-molecule antibiotics. Nat Struct Mol Biol. 2012; 19(9), 957-63. doi: 10.1038/nsmb.2360.

61. Liu F, Myers AG. Practical synthesis of new tetracycline antibiotics. Curr Opin Chem Biol. 2016; 32, 4857; doi: 10.1016/j.cbpa.2016.03.011.

62. Jenner L, Starosta AL, Terry DS, Mikolajka A, Filo-nava L, Yusupov M, et al. Structural basis for potent inhibitory activity of the antibiotic tigecycline during protein synthesis. Proc Natl Acad Sci USA. 2013; 110(10), 3812-6. doi: 10.1073/pnas.1216691110.

63. Chopra I, Roberts M. Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbi-ol Mol Biol Rev. 2001; 65(2), 232-60. doi: 10.1128/ MMBR.65.2.232-260.2001.

64. Connell SR, Tracz DM, Nierhaus KH, Taylor DE. Ribosomal protection proteins and their mechanism of tetracycline resistance. Antimicrob Agents Chemother. 2003; 47(12), 3675-81. doi: 10.1128/ AAC.47.12.3675-3681.2003.

65. Rose WE, Rybak MJ. Tigecycline: first of a new class of antimicrobial agents. Pharmacotherapy. 2006; 26(8), 1099-110. doi: 10.1592/phco.26.8.1099.

66. Sun C, Hunt DK, Clark RB, Lofland D, O'Brien WJ, Plamondon L, et al. Synthesis and antibacterial activity of pentacyclines: a novel class of tetracycline analogs. J Med Chem. 2011; 54(11), 3704-31. doi: 10.1021/jm1015395.

67. Levy SB. Active efflux, a common mechanism for biocide and antibiotic resistance. J Appl Microbiol. 2002; 92 Suppl: 65S-71S. PubMed PMID: 12000614.

68. Chopra I. New developments in tetracycline antibiotics: glycylcyclines and tetracycline efflux pump inhibitors. Drug Resist Updat. 2002; 5(3-4), 119-25. doi: 10.1016/S1368-7646(02)00051-1.

69. Nelson ML, Levy SB. Reversal of tetracycline resistance mediated by different bacterial tetracycline resistance determinants by an inhibitor of the Tet(B) antiport protein. Antimicrob Agents Chemother. 1999; 43(7), 1719-24. PubMed PMID: 10390229.

70. Grossman TH, Fyfe C, O'Brien W, Hackel M, Minyard MB, Waites KB, et al. Fluorocycline TP-271 Is Potent against Complicated Community-Acquired Bacterial Pneumonia Pathogens. mSphere. 2017; 2(1), e00004-17. doi: 10.1128/mSphere.00004-17.

71. Speer BS, Salyers AA. Novel aerobic tetracycline resistance gene that chemically modifies tetracycline. J Bacteriol. 1989; 171(1), 148-53. PubMed PMID: 2644186.

72. Guerra W, Silva-Caldeira PP, Terenzi H, Pereira-Maia EC. Impact of metal coordination on the antibiotic and non-antibiotic activities of tetracycline-based drugs. Coord. Chem. Rev. 2016; 327-328, 188-199. doi: 10.1016/j.ccr.2016.04.009.

73. Mukhtar TA, Wright GD. Streptogramins, oxazolidi-nones, and other inhibitors of bacterial protein synthesis. Chem Rev. 2005; 105(2), 529-42. doi: 10.1021/ cr030110z.

74. Werner G, Klare I, Witte W. Molecular analysis of streptogramin resistance in enterococci. Int J Med Microbiol. 2002; 292(2), 81-94. doi: 10.1078/14384221-00194.

75. Korczynska M, Mukhtar TA, Wright GD, Berghuis AM. Structural basis for streptogramin B resistance in Staphylococcus aureus by virginiamycin B lyase. Proc Natl Acad Sci USA. 2007; 104(25), 10388-93. doi: 10.1073/pnas.0701809104.

76. Mukhtar TA, Koteva KP, Wright GD. Chimeric strep-togramin-tyrocidine antibiotics that overcome streptogramin resistance. Chem Biol. 2005; 12(2), 229-35. doi: 10.1016/j.chembiol.2004.12.009.

77. Li О, Seiple IB. Modular, Scalable Synthesis of Group A Streptogramin Antibiotics. J Am Chem Soc. 2017; 139(38), 13304-7. doi: 10.1021/jacs.7b08577.

78. Tenson T, Lovmar M, Ehrenberg M. The mechanism of action of macrolides, lincosamides and strepto-gramin B reveals the nascent peptide exit path in the ribosome. J Mol Biol. 2003; 330(5), 1005-14. doi: 10.1016/S0022-2836(03)00662-4.

79. Gaynor M, Mankin AS. Macrolide antibiotics: binding site, mechanism of action, resistance. Curr Top Med Chem. 2003; 3(9), 949-61. doi: 10.2174/1568026033452159.

80. Varaldo PE, Montanari MP, Giovanetti E. Genetic elements responsible for erythromycin resistance in streptococci. Antimicrob Agents Chemother. 2009; 53(2), 343-53. doi: 10.1128/AAC.00781-08.

81. Leclercq R, Courvalin P. Resistance to macrolides and related antibiotics in Streptococcus pneumoniae. Antimicrob Agents Chemother. 2002; 46(9), 2727-34. PubMed PMID: 12183222.

82. Seiple IB, Zhang Z, Jakubec P, Langlois-Merci-er A, Wright PM, Hog DT, et al.; A platform for the

synthesis of new macrolide antibiotics. Nature. 2016; 533(7603), 338-45; doi: 10.1038/nature17967.

83. Deshmukh MS, Jain N. Design, Synthesis, and Antibacterial Evaluation of Oxazolidinones with Fused Heterocyclic C-Ring Substructure. ACS Med Chem. Lett. Article ASAP. 2017; 79. doi: 10.1021/ acsmedchemlett.7b00263.

84. Fujisaki F, Hiromatsu S, Matsumura Y, Fukami A, Kashige N, Miake F, Sumoto K. Synthesis and Antibacterial Activity of 3-(Substituted arylmethyl)-4-acylaminomethyloxazolidin-2-ones and De-rivatization to Symmetrical Twin-Drug Type Molecules. J Heterocyclic Chem. 2013; 50(2), 417-24. doi: 10.1002/jhet.1522.

85. Gordeev MF, Hackbarth C, Barbachyn MR, Ban-itt LS, Gage JR, Luehr GW, et al. Novel oxazolidi-none-quinolone hybrid antimicrobials. Bioorg Med Chem Lett. 2003; 13(23), 4213-6. doi: 10.1016/j. bmcl.2003.07.021.

86. Hubschwerlen C, Specklin JL, Sigwalt C, Schroeder S, Locher HH. Design, synthesis and biological evaluation of oxazolidinone-quinolone hybrids. Bioorg Med Chem. 2003; 11(10), 2313-9. doi: 10.1016/ S0968-0896(03)00083-X.

87. Hubschwerlen C, Specklin JL, Baeschlin DK, Borer Y, Haefeli S, Sigwalt C, et al. Structure-activity relationship in the oxazolidinone-quinolone hybrid series: influence of the central spacer on the antibacterial activity and the mode of action. Bioorg Med Chem Lett. 2003; 13(23), 4229-33. doi: 10.1016/j. bmcl.2003.07.028.

88. Laponogov I, Pan X-S, Veselkov DA, McAuley KE, Fisher LM, Sanderson MR. Structural Basis of GateDNA Breakage and Resealing by Type II Topoisom-erases. PLoS ONE. 2010; 5(6): e11338. doi: 10.1371/ journal.pone.0011338

89. Yoshida H, Bogaki M, Nakamura S, Ubukata K, Konno M. Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones. J Bacteriol. 1990; 172(12), 6942-9. doi: 10.1128/jb.172.12.6942-6949.1990.

90. Kaatz GW, Seo SM. Mechanisms of fluoroquinolone resistance in genetically related strains of Staphylococcus aureus. Antimicrob Agents Chemother. 1997; 41(12), 2733-7. PubMed PMID: 9420048

91. Kaatz GW, Seo SM, Ruble CA. Efflux-mediated fluo-roquinolone resistance in Staphylococcus aureus. Antimicrob Agents Chemother. 1993; 37(5), 108694. doi: 10.1128/AAC.37.5.1086.

92. Ng EY, Trucksis M, Hooper DC. Ouinolone resistance mediated by norA: physiologic characterization and relationship to flqB, a quinolone resistance locus on the Staphylococcus aureus chromosome. Antimicrob Agents Chemother. 1994; 38(6), 1345-55. doi: 10.1128/AAC.38.6.1345.

93. Tintino SR, Oliveira-Tintino CD, Campina FF, Silva RL, Costa M, Menezes IR, et al. Evaluation of the tannic acid inhibitory effect against the NorA efflux

pump of Staphylococcus aureus. Microb Pathog. 2016; 97, 9-13. doi: 10.1016/j.micpath.2016.04.003.

94. Neyfakh AA, Borsch CM, Kaatz GW. Fluoroquinolone resistance protein NorA of Staphylococcus au-reus is a multidrug efflux transporter. Antimicrob Agents Chemother. 1993; 37(1), 128-9. doi: 10.1128/ AAC.37.1.128.

95. Machuca J, Briales A, Diaz-de-Alba P, Martinez-Martinez L, Pascual A, Rodriguez-Martinez JM. Effect of the efflux pump OepA2 combined with chromosomally mediated mechanisms on quinolone resistance and bacterial fitness in Escherichia coli. J Antimicrob Chemother. 2015; 70(9), 2524-7. doi: 10.1093/jac/dkv144.

96. Coelho ML, Ferreira JH, de Siqueira Junior JP, Kaatz GW, Barreto HM, de Carvalho Melo Caval-cante AA. Inhibition of the NorA multi-drug transporter by oxygenated monoterpenes. Microb Pathog. 2016; 99(1), 173-7. doi: 10.1016/j.mic-path.2016.08.026.

97. Felicetti T, Cannalire R, Burali MS, Massari S, Man-froni G, Barreca ML, et al. Searching for Novel Inhibitors of the S. aureus NorA Efflux Pump: Synthesis and Biological Evaluation of the 3-Phenyl-1,4-ben-zothiazine Analogues. ChemMedChem. 2017; 12(16), 1293-302. doi: 10.1002/cmdc.201700286.

98. Rodriguez-Martinez JM, Cano ME, Velasco C, Martinez-Martinez L, Pascual A. Plasmid-mediated quino-lone resistance: an update. J Infect Chemother. 2011; 17(2), 149-82. doi: 10.1007/s10156-010-0120-2.

99. Strahilevitz J, Jacoby GA, Hooper DC, Robicsek A. Plasmid-mediated quinolone resistance: a multifac-eted threat. Clin Microbiol Rev. 2009; 22(4), 664-89. doi: 10.1128/CMR.00016-09.

100. Abu-Oatouseh L, Abu-Sini M, Mayyas A, Al-Hiari Y, Darwish R, Aburjai T. Synthesis of New Nitrofluo-roquinolone Derivatives with Novel Anti-Microbial Properties against Metronidazole Resistant H. pylori. Molecules. 2017; 22(1), 71. doi: 10.3390/mol-ecules22010071.

101. Biedenbach DJ, Huband MD, Hackel M, de Jonge BL, Sahm DF, Bradford PA. In Vitro Activity of AZD0914, a Novel Bacterial DNA Gyrase/Topoisomerase IV Inhibitor, against Clinically Relevant Gram-Positive and Fastidious Gram-Negative Pathogens. Antimicrob Agents Chemother. 2015; 59(10), 6053-63. doi: 10.1128/AAC.01016-15.

102. Chan PF, Germe T, Bax BD, Huang J, Thalji RK, Bacque E, et al. Thiophene antibacterials that al-losterically stabilize DNA-cleavage complexes with DNA gyrase. Proc Natl Acad Sci USA. 2017; 114(22), E4492-E4500. doi: 10.1073/pnas.1700721114.

103. Floss HG, Yu TW. Rifamycin-mode of action, resistance, and biosynthesis. Chem Rev. 2005; 105(2), 621-32. doi: 10.1021/cr030112j.

104. Islam MM, Hameed HM, Mugweru J, Chhotaray C, Wang C, Tan Y, et al. Drug resistance mechanisms and novel drug targets for tuberculosis therapy. J Genet Genomics. 2017; 44(1), 21-37. doi: 10.1016/j. jgg.2016.10.002.

105. Goldstein BP. Resistance to rifampicin: a review. J Antibiot (Tokyo). 2014; 67(9), 625-30. doi: 10.1038/ ja.2014.107.

106. Bujnowski K, Synoradzki L, Darlak RC, Zevaco TA, Dinjus E. Semi-synthetic zwitterionic rifamycins: a promising class of antibiotics; survey of their chemistry and biological activities. RSC Advances. 2016; 6(115), 114758-72. doi: 10.1039/C6RA22880A.

107. Bujnowski K, Synoradzki L, Zevaco TA, Dinjus E, Augustynowicz-Kopec E, Napiorkowska A. Rifa-mycin antibiotics - new compounds and synthetic methods. Part 4: Study of the reaction of 3-formyl-rifamycin SV with secondary amines and ketones. Tetrahedron. 2015; 71(1), 158-69. doi: 10.1016/j. tet.2014.10.079.

108. Czerwonka D, Domagalska J, Pyta K, Kubicka MM, Pecyna P, Gajecka M, et al. Structure-activity relationship studies of new rifamycins containing l-ami-no acid esters as inhibitors of bacterial RNA poly-merases. Eur J Med Chem. 2016; 116, 216-21. doi: 10.1016/j.ejmech.2016.03.061.

109. Patel S, Ahmed S, Eswari JS. Therapeutic cyclic lipo-peptides mining from microbes: latest strides and hurdles. World J Microbiol Biotechnol. 2015; 31, 1177-93. doi: 10.1007/s11274-015-1880-8.

110. Gallardo-Godoy A, Muldoon C, Becker B, Elliott AG, Lash LH, Huang JX, Butler MS, Pelingon R, Kava-nagh AM, Ramu S, Phetsang W, Blaskovich MAT, Cooper MA. Activity and Predicted Nephrotoxic-ity of Synthetic Antibiotics Based on Polymyxin B. J Med Chem. 2016; 59, 1068-1077. doi: 10.1021/acs. jmedchem.5b01593

111. Taylor SD, Palmer M. The action mechanism of dap-tomycin. Bioorg Med Chem. 2016; 24(24), 6253-68. doi: 10.1016/j.bmc.2016.05.052.

112. Luther A, Bisang C, Obrecht D. Advances in macro-cyclic peptide-based antibiotics. Bioorg Med Chem. 2017. Ahead of print. doi: 10.1016/j.bmc.2017.08.006.

113. Mondal NR, Jagdale DM. Antimicrobial peptides: a review on its types, mechanism of action, synthesis and therapeutic applications. International Journal for Pharmaceutical Research Scholars. 2016; 5(2), 72-81.

114. Kosikowska P, Lesner A. Antimicrobial peptides (AMPs) as drug candidates: a patent review (20032015). Expert Opin Ther Pat. 2016; 26(6), 689-702. doi: 10.1080/13543776.2016.1176149.

115. Bala P, Kumar J. Antimicrobial peptides: a review. International Journal of Life Sciences Biotechnology and Pharma Research. 2014; 3(1), 62-71.

116. Surivet JP, Zumbrunn C, Bruyere T, Bur D, Kohl C, Locher HH, et al. Synthesis and Characterization of Tetrahydropyran-Based Bacterial Topoisomerase Inhibitors with Antibacterial Activity against GramNegative Bacteria. J Med Chem. 2017; 60(9), 377694. doi: 10.1021/acs.jmedchem.6b01831.

117. Lahiri SD, Kutschke A, McCormack K, Alm RA. Insights into the mechanism of inhibition of novel bacterial topoisomerase inhibitors from characterization of resistant mutants of Staphylococcus aureus. Antimicrob Agents Chemother. 2015; 59(9), 5278-87. doi: 10.1128/AAC.00571-15.

118. Zhang J, Yang O, Cross JB, Romero JA, Poutsiaka KM, Epie F, et al. Discovery of Azaindole Ureas as a Novel Class of Bacterial Gyrase B Inhibitors. J Med Chem. 2015; 58(21), 8503-12. doi: 10.1021/acs. jmedchem.5b00961.

119. Klahn P, Bronstrup M. New Structural Templates for Clinically Validated and Novel Targets in Antimicrobial Drug Research and Development. Curr Top Microbiol Immunol. 2016; 398, 365-417. doi: 10.1007/82_2016_501.

120. Schmidt NW, Wong GCL, Kasko A, Deshayes S, Xian W. Multifunctional membrane-active ami-noglycoside-peptide conjugates. Patent No. WO 2016025627 A1, USA, 2016.

121. Klahn P, Bronstrup M. Bifunctional antimicrobial conjugates and hybrid antimicrobials. Nat Prod Rep. 2017; 34(7), 832-85. doi: 10.1039/c7np00006e.

122. Shavit M, Pokrovskaya V, Belakhov V, Baasov T. Cova-lently linked kanamycin - Ciprofloxacin hybrid antibiotics as a tool to fight bacterial resistance. Bioorg Med Chem. 2017; 25(11), 2917-25. doi: 10.1016/j. bmc.2017.02.068.

123. Wright GD. Antibiotic Adjuvants: Rescuing Antibiotics from Resistance. Trends Microbiol. 2016; 24(11), 862-71. doi: 10.1016/j.tim.2016.06.009.

124. Vishwakarma R, Kumar A, Khan IA, Bharate SB, Joshi P, Singh S, Satti N. A pharmaceutical composition for the treatment of multi-drug resistant infections comprising boeravinone B in combination with an antimicrobial agent. Patent No. WO 2016067309 A1, India, 2016.

125. Fernandes P, Martens E. Antibiotics in late clinical development. Biochem Pharmacol. 2017; 133, 15263. doi:10.1016/j.bcp.2016.09.025.

126. Chaudhary AS, Chen W, Jin J, Tai PC, Wang B. SecA: a potential antimicrobial target. Future Med Chem. 2015; 7(8), 989-1007. doi: 10.4155/FMC.15.42.