Pharmaceutical Approaches to Target Antibiotic Resistance

Perspective Cite This: J. Med. Chem. 2017, 60, 8268-8297

pubs.acs.org/jmc

Pharmaceutical Approaches to Target Antibiotic Resistance Mechanisms Domenico Schillaci, Virginia Spanò, Barbara Parrino, Anna Carbone, Alessandra Montalbano, Paola Barraja, Patrizia Diana, Girolamo Cirrincione, and Stella Cascioferro* Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, Sezione di Chimica e Tecnologie Farmaceutiche, Università degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy

ABSTRACT: There is urgent need for new therapeutic strategies to fight the global threat of antibiotic resistance. The focus of this Perspective is on chemical agents that target the most common mechanisms of antibiotic resistance such as enzymatic inactivation of antibiotics, changes in cell permeability, and induction/activation of efflux pumps. Here we assess the current landscape and challenges in the treatment of antibiotic resistance mechanisms at both bacterial cell and community levels. We also discuss the potential clinical application of chemical inhibitors of antibiotic resistance mechanisms as add-on treatments for serious drug-resistant infections. Enzymatic inhibitors, such as the derivatives of the β-lactamase inhibitor avibactam, are closer to the clinic than other molecules. For example, MK-7655, in combination with imipenem, is in clinical development for the treatment of infections caused by carbapenem-resistant Enterobacteriaceae and Pseudomonas aeruginosa, which are difficult to treat. In addition, other molecules targeting multidrug-resistance mechanisms, such as efflux pumps, are under development and hold promise for the treatment of multidrug resistant infections.

1. INTRODUCTION The development of antibiotics has contributed greatly to the increase in life expectancy and improvement of the quality of life during the last 75 years. Unfortunately, multidrug resistance developed by common pathogens has seriously limited the efficacy of antibiotic therapy.1 Antibiotic resistance is an issue of great concern that has attracted the attention of health agencies, media, and global leaders. It has a high social and economic burden, and it has been estimated that failure to treat drug-resistant infections, in 2009 had a cost of $20 billion in the United States.2 The excessive use and misuse of antibiotics in livestock and humans and the continual evolution and adaptation of microorganisms are the main causes of multidrug resistance. A group of common pathogens including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., acronymically known as “the ESKAPE pathogens”, are the cause of severe and difficult-to-treat nosocomial infections due to their antibiotic resistance.3 Nosocomial infections are often caused by pathogens that produce biofilms on the surfaces of host tissues and medical devices. Formation of biofilms is © 2017 American Chemical Society

associated with multidrug resistance and complicates the treatment of infections in patients with vascular, valvular, and orthopedic prosthesis.4 There is an urgent medical need for new classes of antibiotics that are refractory to most common mechanisms of bacterial resistance or that may restrain or inhibit the mechanisms of resistance. These new antibiotics should harbor the following features: (i) a low selectivity pressure to avoid the rise of antibiotic-resistance strains,5,6 (ii) the ability to kill pathogens without affecting the microbiota, (iii) the ability to counteract natural forms of resistance such as the biofilms, and (iv) the ability to eliminate “dormant” bacteria that are metabolically inert and naturally resistant to current antibiotics. Antivirulence agents (agents that specifically target the molecular determinants of virulence) meet these requirements7 but are still at their infancy. The focus of this Perspective is on agents that target the most common mechanisms of antibiotic resistance. Other important strategies to tackle antibiotic resistance, such as Received: February 8, 2017 Published: June 8, 2017 8268

DOI: 10.1021/acs.jmedchem.7b00215 J. Med. Chem. 2017, 60, 8268−8297

Journal of Medicinal Chemistry

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Figure 1. Resistance mechanisms at cellular level (A) and at community level (B).

Figure 2. Mechanisms of action of β-lactamase and β-lactamase inhibitors (e.g., clavulanic acid).

limit the penetrations of antibiotics in resident bacterial cells. In addition, the deepest layers of biofilms contain bacterial subpopulations that are endowed with low metabolic activity and proliferation rate and are therefore intrinsically resistant to conventional antibiotics. In addition, the biofilm represents an ideal ecological niche for the horizontal transfer of antibiotic resistance genes among bacteria.

antibiotic alternatives (e.g., antivirulence agents, inhibitors of the transpeptidase sortase A, quorum sensing inhibitors, phage therapy) careful antibiotic stewardship, and campaigns aimed at reducing antibiotic prescriptions, are beyond the scope of this review.

2. MECHANISMS OF ANTIBIOTIC RESISTANCE Antibiotic resistance may develop at cellular and community levels.8 In the former case, it is caused by chromosomal mutation or by horizontal transfer mediated by mechanisms of transformation, transduction, or conjugation. In the latter case, it reflects a mechanism of adaptive resistance to environmental challenges, which involves the switch from a free-living form of life (planktonic) to a sessile multistratified community (biofilm).9,10 At the cellular level, bacteria can become resistant to antibiotics by three major mechanisms: (i) enzymatic resistance, causing inactivation of the antibiotic, (ii) chemical modification of the antibiotic target or expression of an alternative target, and (iii) changes in cell permeability or expression of efflux pumps (Figure 1). These mechanisms may still operate at single cell level after biofilm formation. Biofilms consist of a matrix that includes exopolysaccharides or other extracellular polymeric molecules (extracellular DNA, amyloid fibers, etc.), as well as molecules originating from the host, such as mucus and DNA. Biofilms

3. TARGETING MECHANISMS OF ANTIBIOTIC RESISTANCE The main biochemical mechanisms of antibiotic resistance, in particular, enzymatic resistance and efflux pumps, represent good candidate drug targets for add-on treatment of bacterial infections. The combination of the penicillin analogue amoxicillin, and the β-lactamase inhibitor, clavulanic acid, provides a remarkable example of a combination therapy that permits extension of the pharmacodynamic spectrum of amoxicillin to bacteria that produce conventional or extended-spectrum β-lactamase (Figure 2). Penicillin belongs to the broad class of β-lactam (four-membered cyclic amide) antibiotics, which act by interfering with the biosynthesis of the cell wall in bacterial pathogens. β-lactamases, produced by bacteria, are able to hydrolyze the β-lactam ring, thereby inactivating it. 8269



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Avibactam, sodium [(2S,5R)-2-carbamoyl-7-oxo-1,6diazabicyclo[3,2,1]octan-6-yl]sulfate, 4 (Figure 4), known as AVE1330A and NXL104, is a reversible, covalent serine βlactamase inhibitor bearing the non β-lactam diazabicyclooctane (DBO) moiety, which shows greater efficacy with respect to clavulanic acid 1, sulbactam 2, and tazobactam 3 in inhibiting classes A (TEM-1 and KPC-2) C (AmpC, CTX-M-15, P99) and some D β-lactamase and KPC.17 Avibactam in combination with ceftazidime, a thirdgeneration cephalosporin, was recently approved by the FDA for the treatment of patients with complicated intra-abdominal and urinary tract infections caused by resistant Gram-negative bacteria, like carbapenem-resistant Enterobactericeae (CRE), for which there are few or no alternative therapeutic options.18,19 Combinations of avibactam with aztreonam,20 or ceftaroline fosamil,21 have completed phase 1 clinical trials. Aztreonam MIC values against enteric bacteria producing ESBLs or KPC decreased from >16 μg/mL to 1000 μM). Compound 16a in combination with meropenem was also studied, and it was found to have an excellent in vitro activity against Escherichia coli, K. pneumoniae, and Enterobacter

possibility to introduce a polar group, e.g., a carboxylate, in compound 12 to obtain broader-spectrum inhibitors able to form an additional hydrogen-bond interaction with the C3(4′)carboxylate binding pocket present in all serine-active βlactamases. Compound 12 binds Tyr221 and Asn152 of the AmpC binding site through quadrupole−quadrupole and quadrupole− dipole interactions, respectively. The 3-carboxy derivative, 3-(2carboxyvinyl)benzo(b)thiophene-2-boronic acid 11, was 1250fold more active against classes A and C β-lactamase than the lead compound 12, showing a low nanomolar potency in inhibiting class A β-lactamase as well as AmpC (Ki = 40 nM vs TEM-1 and CTX-M-9 and 90 nM vs AmpC).35 The improvement in activity against CTX-M-9 might be due to an additional hydrogen bond between the 3-(2-carboxy-vinyl) group and Arg276 as observed in the X-ray complex of compound 11 with CTX-M-9 (Figure 7). As compared to the glycylboronic derivative 10, compound 11 was less active against AmpC but more potent against CTXM-9 (Table 1). Further studies on the SAR of compound 11 revealed that replacing the carboxy group with a keto group, activity against AmpC was reduced, but still present, whereas activity against class A β-lactamases was completely lost. Many α-aminoboronic acid derivatives were described as broad spectrum inhibitors of β-lactamases with IC50 < 10 nM against class A carbapenemases such as SHV, CTX-M-15, and KPC-2, and class C β-lactamase, like AmpC. These are exemplified by analogues 13, 14, and by the heterocyclic compound 15 (Figure 8).39 The presence of a hydroxyl group on the aromatic ring in derivative 13 was found to increase the inhibitory activity against TEM-1. Hecker et al. investigated the utility of cyclic boronate ester formation to constrain compound 13 into a conformation that is expected to have 8272



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Figure 8. α-Aminoboronic acid derivatives.

Figure 9. Sulfonamidic boronic acids.

compound 21b, showing an improvement of drug-like properties and potent activity against AmpC (Ki of 26 nM).45 A fragment-based lead discovery (FBLD) approach was employed by Eidam et al. to optimize the boronic acid derivatives to obtain better in vivo antimicrobial activity. Compounds 22 and 23 (Figure 10) showed selective affinity for

spp., including multidrug-resistant KPC-producing Enterobacteriaceae strains, but to be ineffective against A. baumannii and P. aeruginosa.42 Potent E. coli AmpC inhibitors with Ki values as low as 25 nM were synthesized by replacing the carboxamide group, which is common to all penicillins and cephalosporins with a sulfonamide moiety. These derivatives were several times more potent than their carboxamide analogues. 4,7-Dichloro-1benzothien-2-yl sulfonylaminomethyl boronic acid 17 (DSABA) (Figure 9) was the first boronic acid derivative showing activity against class D β-lactamases, with an IC50 of 5.6 μM against OXA-40. It also showed IC50 values in the micromolar range (0.57−5.6 μM) against class A (TEM-1, SHV-5) and C (AmpC, P99) β-lactamases.43 The advantage of replacing the carboxamide group with a sulfonamide group has been highlighted by Evidam et al., who investigated a series of sulfonamide boronic acids.44 The authors, comparing the methanesulfonamide boronic acid 18 with the carboxamide analogue 19 (Figure 9) in inhibiting AmpC, found a 23-fold increase in potency of (Ki of 789 nM vs 18.5 μM). The benzylic sulfonamide derivative 20 (Figure 9) was the most active of this series, with a AmpC Ki of 25 nM. By performing a comparative analysis of five X-ray crystallographic complexes of AmpC with boronic acids inhibitors, including compound 21a (PDB codes: GA9, 1KDS, 1KDW, 1KE0, and 1KE3), Tondi et al. identified

Figure 10. Chemical structures of boronic acid derivatives 22 and 23.

AmpC, with Ki values of 0.8 and 0.05 nM, respectively. The pyridine derivative 22 in combination with cefotaxime showed in vivo efficacy in the treatment of mice infected with hospitalderived strain of E. coli, overproducing AmpC and highly resistant to cefotaxime.46 Compound 22 reduced the MIC values of cefotaxime for E. coli from 8−128 to 0.5−1 μg/mL. Rojas et al. synthesized a series of boronic acid transition state inhibitors to obtain more potent compounds against the two class A β-lactamases of K. pneumonia: SHV-1 and KPC-2 (Figure 11).47 Three regions were selectively modified: (i) R1, 8273



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activity. Compound 25 displayed a lower potency than the reference compound, tazobactam 3, in inhibiting KCP-2 activity (IC50 values of 200 and 99 μM, respectively). 4.1.4. Dicarboxylic Acids. A variety of dicarboxylic acid derivatives have been described as metallo-β-lactamase inhibitors, with derivatives of succinic acid,51 phthalic acid,52 and N-heterocyclic dicarboxylic acid53 being recently described as inhibitors of IMP-1. IMP enzyme family belongs to subclass B1 that is widespread in clinical isolates.54 (2S,3S)-Disubstituted succinic acid 27 (Figure 13) has been reported by Toney et al. as a potent inhibitor of the IMP-1 with an IC50 value of 9 nM.51 Two key structural features have been identified for the activity of these compounds as β-lactamase inhibitors: the stereochemistry and the presence of two hydrophobic substituents on the succinic acid scaffold. The importance of the stereochemistry was investigated by comparing the activity of the three isomers of the 2,3dibenzylsuccinic acid against IMP-1. The (S,S)-isomer showed the highest activity with an IC50 of 2.7 nM, followed, in order of decreasing potency, by the (R,R)-isomer (IC50 = 0.21 μM) and the (R,S)-isomer (IC50 = 200 μM). Hiraiwa et al. identified the phtalic acid derivative 28 (Figure 13) as a lead for inhibitory activity against IMP-1 (IC50 = 16 μM).52 The most active compound was the derivative substituted at position 3 with a phenyl group, 29 (Figure 13), which showed an IC50 against IMP-1v of 0.968 μM. The phthalic derivative 29 was also effective in reducing the MIC value of biapenem against P. aeruginosa KG5002/pMS363 (ΔMexAB) and P. aeruginosa PAO1/pMS363. The presence of a bulky group at position 3 was found to increase the inhibitory activity against IMP-1; the introduction of a p-hydroxyl group on the phenyl in position 3 produced compound 30, which displayed the best inhibitory activity when associated with biapenem. New 3-alkyloxy and 3-amino phthalic acid derivatives were synthesized and evaluated for their activity against IMP-1.55 Among the 3-alkyloxy derivatives, long carbon chain compounds, such as 3-buthoxyphthalic acid, (3-cyclohexylpropoxy)phthalic acid, and (3-phenylpropoxy)phthalic acid, showed the highest inhibitory activity with IC50 values against IMP-1 ranging from 1.7 to 5.10 μM. In the series of the 3aminophthalic derivatives, piperidine compounds were the most active. In particular, 4′-hydroxypiperidine 31 showed an IC50 value of 2.7 μM against IMP-1 and was highly active in combination with biapenem against the P. aeruginosa strains,

Figure 11. Chemical structure of compound 24.

with side chains typical of β-lactam, like cephalotin and benzylpenicillin, (ii) R2, with groups that resemble the thiazolidine and dihydrothiazine rings of penicillins and cephalosporins, and (iii) the amide group, with bioisosteric groups like urea, thiourea, and sulfonamide (Figure 8). A thiophene ring in R1 and a triazole with a carboxyl group in R2 were found to be the best substituents to increase the activity against KPC and SHV. The most active compound, 24 (S02030), showed IC50 values of 84 and 130 nM against KPC and SHV, respectively. It could also considerably reduce the MIC values of cefepime, ceftazidime, and ertapenem against K. pneumoniae and E. coli. The crystal structures of 24 bound to the active site of SHV1 and KPC-2 were recently reported.48 Compound 24 assumes similar binding modes in forming a complex with the two enzymes. In particular, in the interaction between derivative 24 and KPC-2, the boron atom binds to catalytic Ser 70, the amide moiety interacts with the residues Asn 132 and Thr 237, and the N2 of the triazole ring forms a hydrogen bond with Ser 130. A comparison with the binding mode of the DBO avibactam49 highlighted some common structural features: (i) an oxygen atom that occupies the oxyanion hole of the active site, (ii) the oxygen atom of the amide group, which interacts with Asn 132, an, (iii) a group with a negative charge (the carboxyl group of 24 and the sulfate of avibactam) that interacts with the carboxyl recognition pocket of class A β-lactamases. 4.1.3. Sulfonamides. Two potential inhibitors of the carbapenemase, KPC-2 25 (ZINC01807204) and 26 (ZINC02318494), were identified by a structure-based virtual screening of a ZINC database (Figure 12).50 These two molecules were tested in combination with carbapenems against a carbapenem-resistant clinical strain of K. pneumoniae (NP6). Although 25 and 26 reduced the MIC value of meropenem and ertapenem by 8-fold and the MIC value of imipenem by 4fold, they were not highly effective in inhibiting KPC-2 enzyme

Figure 12. Chemical structures of the carbapenemase KPC-2 inhibitors 25 and 26. 8274



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Figure 13. Examples of dicarboxylic acids inhibitors of metallo-β-lactamases.

Figure 14. Heterocyclic derivatives with MBLs inhibitory activity.

Figure 15. Sulfur-containing inhibitors of β-lactamases.

5-(4-pyridyl)-2-mercapto-1,3,4-triazole 38 (Figure 14) were synthesized and tested for their inhibitory activity against the metallo-β-lactamases, CcrA, ImiS, and L1.53 Derivatives 32 and 34 acted as competitive inhibitors for CcrA and L1 with IC50 values of 0.69−1.9 μM but did not show significant activity against ImiS, which contains a single zinc ion. Pyridylmercaptothiadiazoles 35 and 36 were noncompetitive inhibitors for CcrA (Ki < 7 μM) and ImiS (Ki values of 3.5 and 6.8 μM, respectively) but failed to inhibit L1. In contrast, pyridylmercaptotriazole analogues 37 and 38 were inactive against all three enzymes at concentration up to 50 μM. Derivative 33 was the only compound showing activity against CcrA, ImiS, and L1 with IC50 values ranging from 0.64 to 7.1 μM. Recently, Zhang et al. described 18 new diaryl-substituted azolylthioacetamides as broad spectrum inhibitors of metallo-βlactamases. Docking studies highlighted that the most active compounds coordinated the Zn ion through the triazole

KG5002/pMS363 (ΔMexAB) and PAO1/pMS363 (MIC values: ≤0.25 and 1 μg/mL, respectively). The crystal structure of compound 31 complexed with IMP1 provided insights into the binding mode and suggested the possibility to introduce an additional substituent on position 6 to reinforce the hydrophobic interaction with Trp64 and His263.56 Hiraiwa et al. synthesized various 3,6-disubstituted phthalic acid derivatives and the 3,6-bis(4-hydroxy piperidine-1yl) derivative was found 10-fold more potent than compound 31 in inhibiting IMP-1 activity (IC50 = 0.27 μM) and was highly active in combination with biapenem. 4.1.5. Heterocyclic Compounds. N-Heterocyclic dicarboxylic acid derivatives were identified as broad-spectrum inhibitors of metallo-β-lactamases. 2,4-Oxazolidinedicarboxylic acid 32, 2,4-thiazolidinedicarboxylic acid 33, 2,5-pyrrolidinedicarboxylic acid 34, and thiol compounds 5-(2-pyridyl)-2mercapto-1,3,4-thiadiazole 35, 5-(4-pyridyl)-2-mercapto-1,3,4thiadiazole 36, 5-(2-pyridyl)-2-mercapto-1, 3,4-triazole 37, and 8275



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moiety.57 Subsequently, Christopeit et al. reported the crystal structure of one of these triazolylthioacetamide derivatives with Verona integron-encoded metallo-β-lactamase 2 (VIM-2).58 4.1.6. Other Sulfur-Containing Inhibitors of β-Lactamases. Recently several thiol containing molecules including derivatives of L-amino acids,59 captopril analogues,60,61 tetrahydropyrimidine-2-thiones,62 and N-sulfonyloxy β-lactams were described as β-lactamase inhibitors. Among L-amino acid derivatives, (S)-3-(4-(benzyloxy)phenyl)-2-(2-mercaptoacetamido) propanoic acid 39 (Figure 15) was observed to be a potent competitive inhibitor with a Ki of 86 nM. It also reduced by 3.9−5.2-fold the MIC of imipenem for bacteria expressing IMP-1 or IMP-4 (E. coli BL21 and Enterobacter cloacae ECL1).59 Several marketed drugs which have no antibiotic properties and employed for different clinical purposes, were recently described as antibiotic resistance breakers.63 For example, the antihypertensive drug L-captopril 40 was shown to inhibit subclasses B1−B3 of MBLs,64,65 with a Ki of 12.5 μM against IMP-1 enzyme. SAR studies carried out on a series of captopril analogues showed that the inhibitory activity: (i) could be increased by shortening the mercaptoalkanoyl side chain, (ii) is not affected by the methyl group, and (iii) depends on the presence of the thiol group that binds to the zinc ions of the active site. In addition, the replacement of the proline residue with pipecolic acid is efficiently resisted. Efforts to optimize the captopril structure resulted in compounds L-41 and D-41, which showed Ki values against IMP-1 of 2.2 and 2.3 μM, respectively.60 Vella et al. performed a fragment-based screening of a library of 500 compounds that identified the 4-methyl-5-(trifluoromethyl)-4H-1,2,4-triazole-3-thiol 42 (IMP-1 Ki = 0.97 mM) as lead compound for the development of new IMP-1 inhibitors.66 A series of 1,2,4-triazole-3-thiols were prepared and tested against IMP-1. Data suggested that the N-methyl group and the trifluoromethyl are not necessary for the activity, as opposed to the thiol group, which is required for IMP-1 inhibition. Furthermore, a significant increase of the inhibitory activity was observed for the 5-(4-benzoylphenyl) derivative. The highest effect in inhibiting the enzyme was found for a thiosemicarbazide, 43 (Figure 15), a synthetic precursor of triazole-thiols, which showed a comparable potency with respect to L-captopril (40, Ki = 11 μM).67 Carosso et al. investigated the β-lactamases inhibitory activity of multielectrophilic N-sulfonyloxy β-lactams. The mechanism of action of this class of compounds seems to be related to the sulfonyloxy group, which acts as a good leaving group. Compounds 44, 45, and 46 (Figure 16) were found to be selectively active against the class A β-lactamase, SHV-12, with Ki values ranging from 0.190 to 0.779 μM.68 Papp-Wallace et al. described the inhibition of classes A and C of β-lactamases by 6β-(hydroxymethyl) penicillanic acid

sulfone 47. This compound showed IC50 values against TEM-1 (class A) of 12 nM and against PDC-3 (class C) of 180 nM. It was also able to enhance the antibacterial activity of ampicillin against E. coli and P. aeruginosa.69 4.2. Inhibition of Aminoglycoside Modifying Enzymes. Enzymatic resistance is also the most common resistance mechanism toward aminoglycosides (AGs) like amikacin, tobramycin, and gentamycin. AG-acetyltransferases (AACs) transfer the acetyl group of acetyl coenzyme A (AcCoA) to the amine moieties of AG. AACs are the most representative class of aminoglycoside-modifying enzymes (AMEs). Other AMEs include AG O-phosphotransferases (APHs) and the AG O-nucleotidyltransferases (ANTs). Over 50 AACs have been described, and they are responsible for the acetylation at the 3, 3″, 2′, or 6′ positions of various AGs (Figure 17). In Mycobacterium tuberculosis, the resistance toward AGs is due to a particular AAC, called the enhanced intracellular survival protein (Eis), which, as opposed to other AACs that act on a single amino group, are able to inactivate a broad set of AGs by acetylating several positions.70 Among AMEs, bifunctional enzymes were also described such as AAC(6′)-Ie/APH(2″)-Ia from S. aureus, ANT(3″)-Ii/ AAC(6′)-IId from Serratia marcescens, and AAC(3)-Ib/ AAC(6′)-Ib′ and AAC(6′)-30/AAC(6′)-Ib from P. aeruginosa.71 Attempts to overcome AG bacterial resistance led to the development of AME inhibitors but, unfortunately, are not used in the clinic. On the basis of the ability of Zn2+ to inhibit aminoglycoside 6′-N-acetyltransferase type Ib (AAC(6′)-Ib) (IC50 = 15 μM) and to reduce as a complex with the ionophore pyrithione (ZnPT) the resistance toward amikacin in Acinetobacter baumannii and E. coli,72 Li et al. investigated the inhibitory effects of numerous metals (Zn2+, Mg2+, Cr3+, Cr6+, Mn2+, Co2+, Ni2+, Cu2+, Cd2+, and Au3+) against various AACs [AAC(2′)-Ic, AAC(3)-Ia, AAC(3)-Ib, AAC(3)-IV, AAC(6′)-Ib′, AAC(6′)-Ie, AAC(6′)-IId, and Eis].73 Cu2+ was the most potent in inhibiting AACs in UV−vis assays, followed by Zn2+ and Cd2+. Although less active than Cu2+ salts, Zn2+salts displayed a better safety profile in rats with a LD50 values of 794 and 1.710 mg/kg bw for oral for Zn(OAc)2 and ZnSO4, respectively. Owing to its limited membrane permeability, ZnCl2 did not affect the MIC values of amikacin and tobramycin as opposed to what observed for the membrane-permeant zinc complex, zinc pyrithione (ZnPT). Moving from the evidence that type Ii AG 6′-Nacetyltransferase (AAC(6′)-Ii) encoded by Enterococcus faecium forms a thioester with acetylcoenzyme A that breaks down to acetylate the 6′-NH2 group of aminoglycosides. Gao et al. designed a series of bisubstrates as novel AAC(6′)-Ii inhibitors.74 Among them, derivative 48 (Figure 18) showed the best inhibitory activity with a Ki of 43 nM. The strategy proved effective in identifying new AAC inhibitors is the virtual screening of libraries of compounds. Micromolar inhibitors of AAC(6′)-Ib were identified by an in silico screening approach of a ChemBridge library using the Xray crystal structure of the enzyme complexed with the AG kanamycin C and coenzyme A (PDB 1V0C).75,76 In particular, the derivative 2-{5-[(4,6-dioxo-1,3-diphenyl-2-thioxotetrahydro-5(2H)-pyrimidinylidene)methyl]-2-furyl}benzoic acid 49 (ChemBridge library compound 5646906) (Figure 18), showed the lowest Ki values when tested for its inhibitory activity

Figure 16. Chemical structures of N-sulfonyloxy β-lactams. 8276



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Figure 17. Representative modifications generated by AAC, ANT, and APH enzymes.

Figure 18. Significant examples of AMEs inhibitors.

against kanamycin A and acetyl CoA (Ki of 12 and 35 μM, respectively). The same strategy was followed by Green et al.77 who screened 23000 compounds from three libraries (ChemDiv, BioFocus NCC, and MicroSource MS2000 spectrum) and found 25 compounds with IC50 values against Eis in the lowmicromolar range. The most potent Eis inhibitor, compound 50 (Figure 18), showed an IC50 of 188 nM. Compound 50 was also tested against other negatively charged AAC enzymes, showing weak inhibitory activity against AAC(2′)-Ic from M. tuberculosis and no activity against AAC(3)-IV from E. coli and AAC(6)/APH(2″) from S. aureus. Similarly, the 1-[3-(2-aminoethyl)benzyl]-3-(piperidin-1ylmethyl)pyrrolidin-3-ol, 51, was able to inhibit acetylation of kanamycin A and amikacin with IC50 values of 39.7 and 34.9 μM, respectively. This compound was identified through a molecular docking of a collection of 280000 compounds into the AAC(6′)-Ib active site (1V0C).78 Addition of 50 μM of compound 51 to amikacin (8 μg/mL) caused a complete inhibition of growth of A. baumannii A155 but had no effect on growth of K. pneumoniae.

Gram-negative pathogens by which microorganisms actively remove antibiotics from the cell. Moreover, the resulting sublethal drug concentration may lead to the acquisition of another resistance mechanism associated with mutation accumulation. Efflux pumps are classified into five families: (i) the resistance-nodulation-division family or RND, (ii) the major facilitator superfamily or MFS, (iii) the ATP -binding cassette superfamily or ABC, (iv) the small multidrug resistance family or SMR, and (v) the multidrug and toxic compound extrusion family or MATE. All families of efflux pumps are found in Gram-positive and Gram-negative bacteria, with the exception of RND, which is exclusively expressed by Gram-negative bacteria.79 An association between efflux pump inhibitors (EPIs) and antibiotics represents a valid antimicrobic strategy aimed at increasing the antibacterial activity and widening the antimicrobic spectrum of antibiotics at the same time.80 Interestingly, EPIs were also shown to interfere with antibiotic resistance of biofilms.81 In spite of these potential benefits, no EPIs are still used in the clinic because of their high toxicity and, in some cases, low in vivo efficacy.82 There are excellent reviews on EPIs.83−85 Here, we will focus on the recent development (2010−2016) of natural and

5. EFFLUX PUMP INHIBITORS Efflux pumps represent one of the most important mechanism of resistance against antibiotics in both Gram-positive and 8277



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addition to EPI activity, compound 56 also exhibited intrinsic antibacterial activity against several S. aureus strains including MSSA (methicillin-sensitive), MRSA (methicillin-resistant), and GISA (glycopeptide-intermediate), with MIC values ranging from 17.6 to 35.1 μg/mL. To confirm the inhibitory activity of 56 on NorA, its effect on S. aureus 1199B and on the norA knockout strain K1712 was evaluated. Compound 56 was equipotent against norA knockout strain K1712 and the mother strain 8325.4, suggesting that the lack of NorA has no effect on the intrinsic bactericidal activity of the compound. Moreover, 56 displayed a synergistic activity with CPX against strains 1199, 1199B, and 8325.4, with an improved effect on 1199B, whereas it proved inactive in enabling CPX activity against K1712, highlighting the important role of NorA in this activity. This hypothesis has been further corroborated by studying the ability of 56 in inhibiting EtBr efflux in S. aureus 1199B. Compound 56 showed an EPI activity comparable to that of reserpine (IC50 = 18 μM vs reserpine IC50 = 14 μM). In vitro (HME-1 cells) and in vivo (BALB/cByJ mice) toxicity studies indicated that derivative 56 was not toxic at the tested concentrations. Sabatini et al., in an attempt to obtain new potent NorA inhibitors, described the synthesis of 2-phenyl-4-(1H)-quinolone and 2-phenyl-4-hydroxyquinoline derivatives structurally related to the flavone and flavolignane EPIs,91 which are characterized by the presence of the 2-phenyl-4H-chromen-4one moiety.92 2-Phenylquinolone nucleus differs from the 2phenyl-4H-chromen-4-one only for a nitrogen atom replacing the endocyclic oxygen and share two important featureswith the NorA inhibitors: a hydrophobic area and two sites, the N-1 and the carbonyl group, available for hydrogen bonding with the NorA active site. The two new series of compounds were initially tested at a concentration of 50 μM to evaluate EtBr efflux inhibition and dose−response curves were calculated for the most active derivatives, 57−60 (Figure 20), which showed an inhibition

synthetic EPIs acting on efflux pumps of Gram-positive and Gram-negative bacteria. Another approach to increase the intracellular antibiotic concentration is to permeabilize the outer membrane of Gramnegative bacteria using polymyxin analogues, which are cationic cyclic lipodecapeptides particularly active against important pathogens such as E. coli, K. pneumoniae, P aeruginosa, and A. baumanii. For this class of compounds, we suggest recent reviews by Vaara86 and Brown et al.87 5.1. Synthetic Gram-Positive Efflux Pump Inhibitors. NorA, a member of the MFS family of efflux pumps, is the predominant protein efflux pump in S. aureus. NorA is a drug/ proton antiporter, which extrudes a broad spectrum of antimicrobic compounds such as phenothiazines, fluoroquinolones, quaternary ammonium compounds, rhodamine, acridines, and ethidium bromide (EtBr) inter alia.88 5.1.1. Quinolines and Quinolones. In an effort to overcome fluoroquinolone resistance associated with overexpression of MFS and MATE efflux pumps, Pieroni et al. synthesized three new series of 6-fluoro-, 6-amino, and 6-amino-8-methylquinolones bearing a heterobicycle thiopyranopyridine group at C-7 position.89 Oximes 52−55 (Figure 19) caused the

Figure 19. Quinolones with EPI activity.

strongest inhibition of EtBr efflux of SA-1199B at the screening concentrations of 50 μM (74.5−91.2%). At concentrations of 10 μM derivatives 53, 54, and 55 demonstrated 3−4-fold more potent than reserpine used as a reference compound. The synergistic activity of compounds 50−53 with ciprofloxacin (CPX) was evaluated against five strains of S. aureus: ATCC 25923, SA-K1902, SA-1199, and NorA overexpressing SAK1904 and SA-1199B. Quinolones 53 and 54 were able to reduce the MIC value of CPX against SA-K1904 by 16- and 32fold, respectively. The best synergistic effect was observed for compound 55, which was found to lower the CPX MIC values to 6.25 μg/mL against SA-K1904 and 50 μg/mL against SA1199B, respectively. Further studies were carried out in order to evaluate the inhibitory activity of this class of compounds on MATE efflux pumps, which also mediate fluoroquinolone resistance. Their synergistic effects on EtBr efflux were estimated in the S. aureus strain, SA-K2886, in which MepA is overexpressed. All tested compounds caused a decrease of the EtBr MIC. In particular, oximes 54 and 55 proved very effective in restoring the antibacterial activity of EtBr against this resistant strain reducing its MIC value by 32-fold. Through a screening on different subclasses of aryl quinolones, Dolèans-Jordheim et al. identified 5-hydroxy-7methoxy-4-methyl-3-phenylquinolin-2-one 56 (Figure 19), which proved to be able to restore the antibacterial activity of ciprofloxacin on S. aureus ATCC 29213, causing a 4-fold reduction of the MIC at a concentration of 31.25 μM.90 In

Figure 20. Chemical structures of derivatives 57−60.

percentage higher than 65%. The most potent NorA inhibitors, 2-{[2-(4-propoxyphenyl)-4-quinolinyl]oxy}ethanamine derivatives 58 and 60, showed IC50 in the range 7−10 μM. Compounds 57−60 did not show intrinsic antibacterial activity against S. aureus strains ATCC 25923, SA-K1902, SA-K2378, SA-1199, and SA-1199B. Isobolograms suggested no synergistic activity for the compounds when used in combination with CPX against the control wild-type ATCC 25923, SA-K1902, which is norA knockout and the norA wild-type SA-1199, while derivatives 58 and 60 showed a potent synergistic activity with CPX against norA overexpressing strains SA-K2378 and SA1199B, which is higher than paroxetine and reserpine used as 8278



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Figure 21. Chemical structures of 2-phenylquinoline derivatives 61a−z.

[1,2]benzothiazines 5,5 dioxide,95 and 5,6,7,8-tetrahydroquinolines96 was used to develop the pharmacophore model. Sixtyfive compounds were chosen in order to obtain high homogeneity in the biological data; in particular, derivatives with a well-defined chirality for which NorA inhibitory activity was evaluated at the screening concentration of 50 μM on the NorA overexpressing S. aureus strain SA-1199B. EtBr efflux inhibitory activity of the obtained quinolines was determined in SA-1199B and compounds 61e, 61g, 61i, 61l, 61q, and 61r proved effective in inhibiting EtBr efflux by more the 90%. For all compounds that showed an inhibition percentage higher than 80%, dose−response studies were performed and derivatives 61e, 61f, 61h, 61i, 61m, 61q, 61s, and 61x showed IC50 values lower than 9 μM. These

reference drugs, with a 16-fold reduction of CPX MICs, to 3.13 and 6.25 μg/mL respectively The SAR of these compounds was further investigated by synthesizing a series of 2-phenylquinoline derivatives 61a−z (Figure 21) introducing an O-alkyl or O-alkylamino chains at the C-4 position of the 2-(4′-propoxyphenyl)quinoline scaffold.93 The design of these compounds was developed on the basis of a NorA inhibitors pharmacophore model developed by employing Phase software. A library of seven quinolines, 61a−c, 61j, 61t, 61w, and 61y, together with other known EPIs such as 3-phenyl-1,4benzothiazines,94 6-amino-7-thiopyranopyridinyl quinolone esters,89 2-(4′-propoxyphenyl)quinolines,92 pyrazolo[4,3-c]8279



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Figure 22. 3-(3,4-Dihydronapth-2-yl)-propenoic acid isobutyl amide 62 and 3-(3,4-dihydronaphth-2-yl)-propenoic acid N,N-diisopropylamide 63.

μM, respectively. Moreover, chalcone 64 proved more potent than reserpine in reducing CPX MIC against the two S. aureus strains SA-1199B and K1758 (8-fold reduction to 6.25 μg/mL and 16-fold to 12.5 μg/mL). Although compound 64 showed intrinsic antibacterial activity (MIC = 25 μg/mL), it exhibited a synergistic activity at much lower concentrations than the MIC. Surprisingly 65, in contrast with its potent EtBr efflux inhibitory activity, did not show any potentiating activity when combined with CPX. This could be due to its structural similarity to pentamidine, which is a good substrate not only for NorA but also for QacA and MepA. 5.1.4. Boronic Acid Derivatives. Starting from screening a library of 150 heterocyclic boronic compounds to test their synergistic effect with CPX against the NorA-overexpressing strain SA-1199B, Fontaine et al. identified two new promising NorA inhibitors, 6-benzyloxypyridine-3-boronic acid 66 and 4benzyloxybenzene boronic acid 67 (Figure 24), able to restore the activity of CPX with MMC4 (minimum modulatory concentration of inhibitor to achieve a 4-fold CPX MIC reduction) values of 1 and 0.5 μg/mL, respectively.100 Among the tested compounds, pyridine-3-boronic derivatives and benzene boronic compounds showed the highest synergistic activity. Pyridine-3-boronic acids were more active than the corresponding esters and the 4-boronic derivatives, with MMC4 ranging from 0.5 to 8 μg/mL. In an attempt to obtain more potent efflux pump inhibitors, a series of 6-(aryl)alkoxypyridine-3-boronic acids were synthesized and evaluated in combination with CPX against S. aureus 1199B.101 Although NorA structure has not been determined so far, in silico investigations suggested that it presents 12 transmembrane helices and a large hydrophobic binding site. To explore the size of this pocket, the effect of the replacement of the 6-benzyloxy group of 66 with various alkoxy chains was investigated. 6-(3-Phenylpropoxy)pyridine-3-boronic acid 68 and 6-(4phenylbutoxy)pyridine-3-boronic acid 67 were found to be 4fold more potent than the parent compound 66 in potentiating CPX activity not showing intrinsic antibacterial activity. Unfortunately, they were less effective than reserpine in inhibiting EtBr efflux. An improvement of the synergistic effect of these compounds with the increase of the chain length was observed. 5.1.5. Indole Derivatives. As part of a program concerning the development of new indole derivatives with antibacterial properties a series of 3-substituted indoles were obtained, through a nucleophilic addition of indoles to nitrones, and tested against a panel of 28 reference bacterial strains of common human Gram-positive and Gram-negative pathogens.102 Several types of indole derivatives were screened: primary and secondary amines, N-hydroxyamines, aldonitrones, formamides, amides, and N-hydroxycarbamides. One of the tested sublibraries of indole compounds, the aldonitrones, proved completely inactive against all used strains, with MIC values higher than 128 μg/mL. However, they showed a good

compounds did not have any intrinsic effect on bacterial viability, but they were able to reduce CPX MIC of 4−32-fold (2.5−0.08 μg/mL) against SA-K2378 (norA++). The best NorA inhibitors, 61h, 61i, 61m, 61q, and 61s, were also tested for their inhibitory activity against the MepA (MATE) by evaluating their synergistic activity with EtBr, which is a better MepA substrate than CPX, against the two S. aureus strains SAK2885 (mepA−) and SA-K2886 (mepA++). All the compounds showed synergistic activity with EtBr, in particular, quinolines 61m and 61q reduced EtBr MIC 16-fold to 0.78 and 1.56 μg/ mL, respectively. All derivatives bearing the 2-ethylamino alkyl chain proved to be potent efflux pump inhibitors. Compounds with the nitrogen atom included in an aliphatic ring retained the activity, whereas when the nitrogen atom was included in an aromatic ring the EPI activity was completely lost. 5.1.2. Naphthene Compounds. Thota et al. described a series of 3-(substituted-3,4-dihydronaphthyl)-2-propenoic acid amides as NorA inhibitors capable to reduce the MIC of CPX 2 from 4 to 0.5 μg/mL against NorA overexpressing S. aureus 1199B. The most active compounds, 3-(3,4-dihydronaphth-2yl)-propenoic acid isobutyl amide 62 and 3-(3,4-dihydronaphth-2-yl)-propenoic acid N,N-diisopropylamide 63 (Figure 22) when combined with CPX decreased its MIC against S. aureus 1199B from 8 to 0.5 μg/mL. It was observed that the introduction of methoxy or alkoxy substituents at the 6,7-position of 3,4-dihydronaphtalene moiety, as well as the saturation of the double bond, resulted in a reduction of EPI activity. To confirm that these compounds acted as inhibitors of NorA protein, they were tested against norA knock out strain S. aureus SA-K1758. No change was observed in the MIC of CPX in the presence of 62 and 63. Further, their effect on the efflux of EtBr was evaluated and they proved to be able to prevent ethidium bromide extrusion.97 5.1.3. Chalcones. In the light of the discovery of the potent NorA inhibitory activity of a kaempferol rhamnoside,98 which will be discussed in section 5.2, a series of 117 chalcones were screened for their EPI activity.99 Derivatives 4-phenoxy-4′-dimethylaminoethoxychalcone 64 and 4-dimethylamino-4′-dimethylaminoethoxychalcone 65 (Figure 23) displayed comparable activity to the positive control reserpine in inhibiting EtBr efflux, with IC50s of 9 and 7

Figure 23. 4-Phenoxy-4′-dimethylaminoethoxychalcone 64 and 4dimethylamino-4′-dimethylaminoethoxychalcone 65. 8280



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Figure 24. Chemical structures of the 6-aryl-alkoxypyridine-3-boronic acids 66−69 (Bn = benzyl group).

Figure 25. Chemical structures of the aldonitrones 70−72 and bis-indolyl derivatives 73a−c.

Figure 26. Chemical structures of NorA heterocyclic inhibitors 74−77.

synergistic concentration-dependent effect with CPX against SA-1199B. In particular, 5-haloaldonitrones 70−72 (Figure 25), showed MIC values in the range 0.5−2 μg/mL in combination with subinhibitory (MIC/4) concentrations of CPX. To confirm that the synergistic effect of these compounds was due to the inhibition of NorA and not to the mutation in the DNA topoisomerase gene grlA, second mechanism of resistance of SA-1199B, the compounds were tested also against the S. aureus strain K2378, which overexpresses NorA but does not present any mutation on DNA gyrase encoding genes.94 In the presence of MIC/4 concentration of CPX, compounds 70−72 proved to be active, with MIC values ranging from 2 to 4 μg/mL, corroborating the hypothesis that they act as NorA inhibitors. In addition, aldonitrones proved to be 2−5-fold more active than reserpine in inhibiting EtBr efflux from SA-1199B.

To deepen the SAR of this class of compounds, 24 bisindolyl derivatives were prepared and tested against reference and clinical strains of S. aureus and CoNS (coagulase negative staphylococci) species.103 The compounds that did not show antibacterial activity (MIC > 32 μg/mL), 73a−c, were tested for NorA efflux pump inhibition. Bis-indole 73b reduced CPX MIC for SA-1199B from 16 to 4 μg/mL at a concentration of 32 μg/mL, whereas in the case of derivative 73a, the same result was observed at a concentration of 0.25 μg/mL. Compound 73c, bearing an α-keto-amide linker, potentiated CPX activity against SA-1199B, showed a decrease in CPX MIC from 4 to 2 μg/mL at concentrations of 0.5 and 2 μg/mL, respectively. Contrary to what was observed for the monoindoles 70−72, for the bis-indoles with EPI activity, halogenation of the indole core did not have any effect on the activity. 8281



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the increased activity. In the fluorescent EtBr efflux assay, compound 80 (Table 2) showed the best activity at a

5.1.6. Other NorA Heterocyclic Inhibitors. Most of the synthetic EPIs described in literature have a heterocyclic structure and include, in addition to quinolines, quinolones and indoles described above, phenylpiperidines,104 benzothiophenes,105 phenothiazines, and thioxantenes.94,106 Here we focus on the most recently described heterocyclic inhibitors. Brincat et al. using the FLAP procedure (fingerprints for ligands and proteins) described by Baroni et al.107 for the virtual screening of about 300000 compounds, identified four novel NorA inhibitors, 4-methyl-N-[2-(1-methyl-1H-pyrrol-2yl)-1H-benzimidazol-5-yl]benzensulfonamide 74, 2-{[3(benzyloxy)benzyl]-amino}-1-phenylpropan-1-ol 75, 4-({[3cyano-6-ethyl-4-(trifluoromethyl)-5,6,7,8-tetrahydroquinolin-2yl]thio}methyl)-benzoic acid 76, and 3-{5-[(Z)-(3-sec-butyl2,4-dioxo-1,3-thiazolidin-5-ylidene)methyl]2-furyl}-4-chlorobenzoic acid 77 (Figure 26).96 Compounds 76−77 evaluated for their EtBr efflux inhibitory activity caused an inhibition rate of more than 70% at 50 μM. Two derivatives, 76 and 77, exhibited variable intrinsic antibacterial activity, whereas compounds 74 and 75 did not show any intrinsic antibacterial activity (MICs > 100 μg/mL) and proved to be more potent than reserpine in inhibiting EtBr efflux at concentrations greater than 30 μM. All four compounds displayed synergistic activity with CPX, higher than reserpine, against NorA overexpressing bacterial strains at concentrations greater than 6.25 μg/mL. The sulfonamide 74 was effective in potentiating CPX activity against S. aureus ATCC25923 and SA-1199. This effect could be due to the inhibition of other pumps, different from NorA, responsible of the extrusion of CPX. Preliminary SAR studies highlighted that modifications to the sulfone and amide group of 74, as well as the removal of the methyl group of the 2-(1phenyl-1-propanol) moiety of 75, are sufficiently tolerated, whereas changes in the substituents of the imidazole nucleus of 74 drastically decreased the EPI activity. A similar approach was subsequently employed on a library of 150 celecoxib analogues characterized by the presence of 1,4dihydropyrazolo[4,3-c]-benzothiazine 5,5-dioxide nucleus.95 Celecoxib, a known cyclooxygenase-2 inhibitor, was recently described as being able of increasing the sensitivity of multidrug resistant bacteria to the antibiotics ampicillin, kanamycin, chloramphenicol, and ciprofloxacin by gathering the drugs inside the cell.108 Prediction of the activity obtained using FLAP pharmacophoric model led to the identification of three potential NorA inhibitors, 78 and 79 (Figure 27). The in silico results suggested that the presence of a NH instead of NMe in the benzothiazine scaffold and a potent electron-withdrawing group, such as a para-nitro group or a meta- or para-fluorine in the phenyl ring linked at the pyrazole, were advantageous for

Table 2. EtBr Efflux Inhibition (%) at 50 μM against SA1199B compd

R

EtBr efflux inhibition (at 50 μM, %)

78 79 80 celecoxib reserpine paroxetine

3-F 4-F 4-NO2

33.9 15.5 76.9 64.5 84.8 89.7

concentration of 50 μM (76.9%), and although the dose− response curves of derivative 77 and celecoxib were different, they displayed the same IC50 values (10 μM). Checkerboard assays were performed to confirm the NorA inhibitory activity of compound 80 (Figure 27) to estimate its synergistic activity with CPX. It proved to be able to reduce of 16-fold CPX MIC at concentrations greater than 12.5 μg/mL against SA-1199B. Bharate et al. discovered a polysubstituted pyrrole acting as a dual inhibitor of human P-glycoprotein (P-gp), a pump responsible of MDR in cancer cells, and NorA efflux pump.109 Although there is no evidence in the literature concerning a structural similarity between these two proteins, there are several examples of P-gp inhibitors which are active against efflux pumps of bacteria, like piperine and capsaicin.110,111 4-Acetyl-3-(4-fluorophenyl)-1-(p-tolyl)-5-methylpyrrole showed a MEC4 value of 6.25 μM. 5.2. Natural Based Gram-Positive Efflux Pump Inhibitors and Their Synthetic Analogues. Significative advances in the discovery of new compounds with efflux pump inhibitory activity have been done by phytochemists. Several natural compounds were identified as potent NorA inhibitors. The most common one, the alkaloid reserpine, is often used as a reference drug in pump inhibitory assays. Considering the activity showed by the extracts of Mirabilis jalapa Linn. (Nyctaginaceae) in reversing fluoroquinolone resistance in SA-1199B strain, Michalet et al. isolated, from the methanolic extracts of leaves and stems, the N-trans-feruloyl 4′O-methyldopamine, which is able to give an 8-fold reduction of norfloxacin MIC at a concentration of 100 μg/mL (292 μM). To improve the activity of this natural compound, a series of analogues were prepared and the N-trans-3,4-O-dimethylcaffeoyl tryptamine proved to be more potent than reserpine in restoring norfloxacin antibacterial activity against MDR S. aureus. It causes a 4-fold norfloxacin MIC reduction at a concentration of 29 μM.112 Following the discovery of piperine (Figure 28) as a potentiator of CPX against methicillin-resistant S. aureus strains, many studies to develop more potent EPIs were carried out. Piperine, or 5-(3,4-methylenedioxyphenyl)-2E,4E-pentadienoic acid piperidine amide, is an alkaloid mainly found in black pepper (Piper nigrum) and long pepper (Piper longum). Initially, it was described as having inhibitory activity against several cytochrome P450-mediated pathways and against the human P-glycoprotein.113 Recently, the application of this alkaloid as NorA inhibitor was reported.114 Piperine did not have any intrinsic antibacterial activity but, when tested in combination with CPX, it was able to restore the fluoroquinolone antibacterial activity, causing a 2-fold and 4fold reduction in its MIC against ATCC29213 at concen-

Figure 27. 1,4-Dihydropyrazolo[4,3-c]-benzothiazine 5,5-dioxides 78− 80. 8282



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Figure 28. Piperine analogues with CPX potentiating activity.

trations of 12.5 and 50 μg/mL, respectively. Several attempts to synthesize more potent analogues of piperine and QSAR studies115 to identify a reliable model for the prediction of NorA inhibition were carried out. The introduction of an alkyl chain at the C-4 position and the replacement of a piperidine ring with an aromatic amine led to a marked improvement of the CPX potentiation activity. Other important structural features for the synergistic activity of piperine seem to be the unsaturation and the amide carbonyl group. Recent examples of piperine analogues, which are more potent than the parent molecule, are the derivatives 81−84 (Figure 28), whose activities are summarized in Table 3.116,117 Table 3. Effect of Piperine Analogues on the Activity of CPX against S. aureus 1199B and S. aureus 1199, Fluoroquinolone-Resistant and -Susceptible Isolates, Respectively S. aureus 1199B MIC of CPX (μg/mL)

S. aureus 1199 MIC of CPX (μg/mL)

compd

without EPI

with EPI

fold reduction

without EPI

with EPI

fold reduction

81 82 83 84 piperine reserpine

8 8 8 8 8 8

1 1 2 2 4 2

8 8 4 4 2 4

0.25 0.25 0.25 0.25 0.25 0.25

0.06 0.06 0.12 0.12 0.12 0.12

4 4 2 2 2 2

Figure 29. Chemical structure of kaempferol-3-O-α-L-(2,4-bis-E-pcoumaroyl)rhamnoside 85.

reserpine (IC50 = 9 μM).98 The NorA inhibitory activity was also tested in enriched everted membrane vesicles for excluding intracellular interfering pathways. Compound 85 at 2.76 μM was able to give 40% inhibition of the efflux of Hoechst 33342, proving to be significantly more potent than verapamil, used as a reference drug. Synergistic studies highlighted that the kaempferol rhamnoside 85 increased by 8-fold the antibacterial activity of CPX against SA-1199B at 1.56 μg/mL. Recently, Tintino et al. described the EPI activity of tannic acid.119 The evaluation of the effect of MIC/8 tannic acid in combination with EtBr or Norfloxacin against SA-1199B resulted in a 3-fold reduction in the MICs of both compounds. 5.3. Synthetic Gram-Negative Efflux Pump Inhibitors. Multidrug resistance in Gram-negative pathogens constitutes an important threat for the effectiveness of the treatment of infections caused by pathogens such as Enterobacteriaceae and P. aeruginosa. An important role in the development of MDR phenotype in Gram-negative bacteria is played by the resistance-nodulationdivision (RND)-type efflux pumps (Figure 30), which extrude, outside of the cell, a wide range of antibiotics including βlactams and β-lactamase inhibitors, tetracyclines, fluoroquinolones, and oxizolidines. RND pumps are characterized by a tripartite structure: (i) an inner membrane (IM) efflux transporter, (ii) an outer membrane channel, and (iii) a periplasmic adapter protein.120 The main difficulty in designing EPI compounds for Gramnegative pathogens is the need to obtain derivatives able to cross the outer membrane and bind the RND. In fact, the penetration of the outer membrane depends on channel-

Capsaicin, 8-methyl-N-vanillyl-6-nonenamide, the main component of hot chili (Caspicum), was, similarly to piperine, initially reported as an P-glycoprotein inhibitor118 and because of its interesting biological properties was tested in order to evaluate its EPI activity against S. aureus.110 Capsaicin not only caused by 2−4-fold reduction of the MIC value at concentrations of 12.5 and 25 μg/mL against the NorA overexpressing SA-1199B but also increased the postantibiotic effect of CPX at MIC concentration. Moreover, capsaicin proved to be able to reduce the invasiveness in J774 macrophage cell lines of SA-1199B by 2 log10 magnitude orders at a concentration of 25 μg/mL. This is further evidence of its antivirulence properties. Through a bioassay-guided fractionation of the ethanolic extract of Persea lingue, Holler et al. isolated the kaempferol-3O-α-L-(2,4-bis-E-p-coumaroyl)rhamnoside 85 (Figure 29), which showed an EtBr efflux inhibitory activity with an IC50 value of 2 μM, making it more than 4-fold more potent than 8283



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causing an increased influx of the antibiotics into the cell, improving the antibiotic effect in an efflux-independent manner.120 5.3.2. Pyranopyridines. Opperman et al., using a cell-based reporter assay for identifying new compounds with synergistic effect with CPX, screened a library of 183400 compounds and discovered a novel potent inhibitor of AcrAB efflux pump of E. coli, the pyranopyridine 86 (MBX2319) (Figure 32).127 RND

Figure 30. RND efflux pump: integral membrane pump protein (AcrB), an outer membrane channel (TolC), and a protein adapter (AcrA).

Figure 32. Chemical structure of the pyranopyridine 86, inhibitor of AcrAB-TolC efflux pump of E. coli.

forming proteins called porins and is easier for zwitterionic or hydrophilic molecules. In contrast, the binding with RND pumps requires a hydrophobic structure. Therefore, the identification of new EPIs for Gram-negative bacteria requires the use of rational drug design to obtain compounds with both structural features.120 This approach was made possible by the discovery of the three-dimensional structures of pumps belonging to RND family such as AcrB and MexB.121−123 5.3.1. Peptidomimetics EPIs. The EPI activity of the dipeptide phenylalanine-arginine β-naphthylamide (PAβN) (Figure 31) against Gram-negative pathogens was described

pumps in E. coli consist, as already mentioned, of an integral membrane pump protein (AcrB), an outer membrane channel (TolC) and a protein adapter (AcrA). The potentiating activity of 86 was tested for two fluoroquinolones, CPX and LVX, and for the β-lactam, piperacillin (PIP), against E. coli AB1157. The compound at a concentration of 12.5 μM proved to be able to decrease the MIC values of CPX, LVX, and PIP by 2-, 4-, and 8fold, respectively. At 3.13 μM, 86 in combination with 0.016 μg/mL of CPX (CPX MIC) decreased the E. coli AB1157 viability by 10000-fold after 4 h of exposure. The authors proved that the activity of 86 was due to the inhibition of the AcrAB-TolC efflux pump of E. coli. They tested the ability of 86 in potentiating the antibacterial activity of CPX, LVX, and PIP against efflux-defective mutant strains of E. coli such as ΔtolC and ΔacrB mutants, and it proved inactive. Docking studies and molecular dynamics simulations were carried out in order to deepen the understanding of the binding mode of 86 with the active site of AcrB.128 The results showed that the pyranopyridine derivative binds with high affinity the lower part of the distal pocket in the B protomer and interacts with the phenylalanine residues inside the hydrophobic area of AcrB. SAR studies performed on 60 synthetic analogues of 86 have identified the structural features required for a good synergistic activity and a low toxicity.129 In particular it was observed that: (i) the gem-dimethyl group, the nitrile, and the dimethylenesulfide were crucial for the activity, in fact, their replacement with hydrogen atoms produced compounds devoid of activity, (ii) the introduction of nonacidic substituents on the phenyl ring improved the potency and the PK properties, and (iii) the replacement of the morpholinyl group with other amino groups, such as dimethylamino, diethylamino, pyrrolidinyl, Nmethylpiperazine, N-(2-methoxyethyl)piperazinyl, thiomorpholinyl, 2,6-dimethylmorpholinyl, homopiperazinyl, N-methylhomopiperazinyl, and homomorpholinyl, produced compounds with higher synergistic activity with PIP (MPC4 ≤ 25 μg/mL) except in the case of the pyrrolidine analogue. 5.3.3. Pyridopyrimidines. A series of 4-oxo-4H-pyrido[1,2a]pyrimidine derivatives, decorated at the 2-position with

Figure 31. Chemical structure of phenylalanine-arginine β-naphthylamide.

for the first time in 1999.124 The synergistic effect of this compound resulted in being heavily dependent on the antibiotic considered. In particular, PaβN, when tested against 115 P. aeruginosa strains at a concentration of 50 μg/mL in combination with CPX and levofloxacin (LVX), proved to be able to reduce the MIC mean value from 16 to 2 μg/mL, showing the best potentiating effect in association with LVX against a strain overexpressing MexAB-Oprm (RND family),125 whereas, in Acinetobacter baumannii, the synergistic effect proved more potent for clindamycin than for trimethoprim and chloramphenicol.126 This biological effect also seemed to be due to another mechanism of action: it was shown that PaβN was able to increase outer membrane permeability, 8284



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Figure 33. Chemical structures of the most representative pyridopyrimidine derivatives acting as inhibitors of Gram-negative efflux pumps.

carbon-linked substituents,130 aromatic rings,131 and piperidines bearing quaternary ammonium salt side chains,132 were synthesized and tested for their ability to potentiate the antibacterial activity of CPX, LVX, and aztreonam (AZT) against the laboratory strain of P. aeruginosa, MexAB-OprM overexpressing in which MexCD-OprJ and MexEF-OprN pumps are disrupted. In analogy to the 2-nitrogen-linked derivatives, previously described,133,134 the 2-carbon-linked analogues, obtained through palladium-mediated cross-coupling reactions, caused a potent synergistic activity, particularly in the case of the compounds bearing cyclic substituents, as for compound 87 (Figure 33), which showed an MPC8 value (minimum concentration of inhibitor required to decrease 8fold the antibacterial MIC) for LVX and AZT of 1 μg/mL. Unfortunately, these compounds, because of their high lipophilicity, had low solubility in an aqueous medium and, consequently, less active in the presence of 0.125% human serum albumin (HSA) (87 MPC8 = 4 μg/mL). Introduction of hydrophilic substituents on the aryl moiety led to the morpholine derivative 88 (Figure 33) with improved solubility and in vivo potentiating activity. The in vivo synergistic activity was tested against P. aeruginosa PAM 1020 in a pneumonia model and at a dose of 10 mg/kg in combination with 1000 mg/kg of AZT caused a moderate survival in comparison with the treatment with AZT alone.131 A higher solubility and an improved safety profile were observed when a piperidine bearing a quaternary ammonium salt side chain was attached at the 2-position as for compound 89 (D13-9001), which exhibited a MPC8 of 2 μg/mL for both LVX and AZT against PAM 1723. Synergistic activities and the solubility data of the derivatives 87−89 are reported in Table 4. Nakashima et al. reported that derivative 89 did not have any effect on the inner or outer membranes and that it was not a substrate of AcrB.123 5.3.4. Piperazine Analogues. Through a high-throughput screening (HTS) of a library of N-heterocyclic organic compounds, 1-(1-naphthylmethyl)-piperazine 90 (NMP)

Table 4. Potentiation Activity and Aqueous Solubility of Pyridopyrimidines 87−89 MPC8 (LVX) (μg/mL) compd

without HSA

with 0.125% HSA

MPC8 (AZT) (μg/mL)

Sol (pH 6.8) (μg/mL)

87 88 89

1 4 2

1 16 4

1 2 2

nta 140 747

a

nt = not tested.

(Figure 34) has been identified as a potentiator of LVX against multidrug resistant E. coli strains, overexpressing RND efflux

Figure 34. 1-(1-Naphthylmethyl)-piperazine.

pumps.135 The synergistic effect of NMP was tested in combination with several antibiotics against the efflux pumps overexpressing strains 2-DC14PS and 3-AG100MKX and against the efflux pump-deficient control strains 1-DC14PS and HS276. Compound 90 proved to be able to reduce the MIC of LVX by 8−16-fold in acrAB or acrEF overexpressing strains, with a MCR4 against 3-AG100MKX of 50 μg/mL. A similar, or slightly smaller, value was observed for 100 μg/ mL 90 in combination with oxacillin, chloramphenicol, rifampin, and clarithromycin (8−16-fold MIC reduction).136 Further studies showed the ability of 90 to reverse antibacterial resistance in MDR E. coli strains isolated from animals. At a concentration of 100 μg/mL, it induced at least a 4-fold decrease of the MIC values of florfenicol, CPX, and 8285



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past decade, many attempts were done to obtain new antibacterial agents suitable for the treatment biofilm-associated infections.141 Antibiofilm agents act through three main mechanisms of action: (i) inhibiting the adhesion of bacteria to surfaces at the first step of bacterial pathogenesis, (ii) disrupting the biofilm architecture during the maturation process,142 and (iii) interfering with the quorum sensing system.143 6.1. Imidazole Derivatives. The 2-aminoimidazole scaffold has been widely used for the development of antibiofilm agents. Reyes et al. synthesized a series of 2-aminoimidazole triazoles (2-AITs), which were found to possess biofilm inhibition activity at low micromolar concentrations when assayed against multidrug-resistant Acinetobacter baumannii (MDRAB) and methicillin-resistant S. aureus (MRSA).144 Compounds 97a−e (Figure 36) were tested as inhibitors of MRSA biofilm formation at 100 μM. Compounds able to

tetracycline against the overexpressing strain AG112 and it showed no potentiating effect against the pump-deficient strain AG100A.137 Evaluation as efflux pump inhibitor was also carried out in other 167 clinical isolates of Enterobacteriaceae, in addition to E. coli. 90 significantly improved the antibacterial activity of linezolid, in Citrobacter freundii, Enterobacter aerogenes, and K. pneumoniae clinical isolates, and of levofloxacin, tetracycline, and chloramphenicol in E. aerogenes, whereas no significant effect was observed in Serratia marcescens.138 5.4. Natural Gram-Negative Efflux Pump Inhibitors. Dwivedi et al. described the EPI activity of the natural compound 4-hydroxy-α-tetralone, 91, extracted from Ammania spp., and of its five acyl and aryl semisynthetic derivatives 92− 96 (Figure 35). No compounds showed intrinsic antibacterial

Figure 35. Chemical structures of the natural compound 91 and its semisynthetic derivatives 92−96.

activity against five E. coli strains: the MDR clinical isolates MDREC-KG4 and MDREC-KG1, the MDR mutants MDRECEM5 and MDREC-EM7, and the multidrug-sensitive MDSEC (MICs in the range 250−1000 μg/mL). Compounds 91 and 96 exhibited the highest potentiating activity of tetracycline (TET) generating a 4- and 8-fold MIC reduction, respectively, at a concentration of 25 μg/mL. The EPI effect of these derivatives was confirmed by a fluorescence based EtBr efflux assay using MDREC-KG4. Additionally, 91 and 96 also proved to be able to inhibit ATPase at 25 μg/mL, suggesting the capability of these compounds to inhibit ATP dependent efflux pumps.139 To identify RND efflux pump inhibitors, 26 natural and synthetic small heterocyclic compounds were screened for their synergistic effect with CPX, TET, chloramphenicol, erythromycin, and EtBr against a wide range of Enterobacteriaceae and P. aeruginosa.140 The compounds were able to increase the antibacterial activity of CPX toward Salmonella typhimurium AcrAB-TolC overexpressed strains and inactive against mutant strains devoid of AcrA, AcrB, or TolC. Their efflux inhibitory activity was confirmed by measuring the accumulation of the fluorescent dye Hoechst 33342. Among the tested compounds, the three natural phenylethylamines epinephrine, norepinephrine, and cathinone showed the greatest synergy. Additionally, epinephrine significantly increased (2.5-fold) the accumulation of Hoechst 33342 by S. typhimurium, highlighting a mechanism involving the inhibition of efflux pumps.

Figure 36. 2-Aminoimidazole based biofilm inhibitors 97a−e and 98a−l.

produce >94% biofilm inhibition were subjected to a dose− response study. The 2-AITs 97a, 97c, and 97d exhibited IC50 values of 9.86, 8.55, and 4.50 μM, respectively. Additionally, derivative 97c showed the highest activity against dispersing preformed A. baumannii biofilms, with an IC50 of 44.70 μM. Subsequently, Yeagley et al. prepared a series of analogues, 98a−l (Figure 36), with the purpose of obtaining more potent biofilm inhibitors against three strong biofilm forming strains of MRSA: BAA-1770, BAA-1765, and 43300.145 Results suggested the importance of the length of the alkyl side chain. In fact, analogues bearing alkyl chains longer than four carbons (98j) showed the best antibiofilm activities (IC50 in the range 16−18 μM). In contrast, the presence of short aliphatic chains (98c and 98d) resulted in being advantageous for the synergistic activity with oxacillin against MRSA. Derivatives 98c and 98d

6. INHIBITORS OF BACTERIAL BIOFILM FORMATION The biofilm is a bacterial community characterized by a high level of multifactorial resistance. The majority of antibiotics effective on planktonic cells are inactive against biofilms. In the 8286



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caused an 8-fold MIC reduction of oxacillin at concentrations of 16 and 8 μ/mL, respectively. Several 1,4- and 4,5-disubstituted 2-aminoimidazole analogues were prepared and screened for their antibiofilm activity against MRSA and MDRAB.146,147 Generally, the 4,5-disubstituted derivatives proved to be more potent than the other series. In particular, compounds 99a−c (Figure 37) exhibited the highest potency in inhibiting

Figure 38. Phenylhydrazinylidene derivatives as inhibitors of S. aureus biofilm formation.

vivo model (the wax moth Galleria mellonella), and the compound was demonstrated as being not toxic at a concentration of 1 mg/mL. With the aim of investigating the mechanism of action of this class of derivatives, all compounds were tested for their activity against sortase A, a transpeptidase responsible for catalyzing the covalent anchoring of surface proteins to the Gram-positive bacterial cell wall involved in the bacterial adhesion to host tissues,5 but no correlation was found between the biofilm inhibition and the activity on the enzyme. 6.3. Carbazole and Indole Derivatives. Liebens et al. identified the N-alkylated 3,6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol 103 (Figure 39) with antipseudomonal activity (MIC = 18.5 μg/mL) through a medium-throughput screening of a library of 23909 compounds.152 Derivative 103 was able to totally inhibit P.aeruginosa biofilm formation at 3.7 μg/mL. Additionally, it showed activity against the mature biofilm at a concentration of 4.63 μg/mL. Moreover, compound 103 caused bacteriostatic and antibiofilm activity on other clinically tested Gram-negative bacteria, E. coli and Porfiromonas gingivalis, with a MBIC (minimal biofilm inhibitory concentration) of 2.22 and 5.11 μg/mL, respectively, and on the two Gram-positive strains S. aureus SH1000 and Staphylococcus epidermidis RP62A with a MBIC of 9.25 μg/mL. A class of indole-based analogues of the marine alkaloid oroidin were synthesized and screened for their antibiofilm activity against MRSA, Streptococcus mutans, and P. aeruginosa.153 No compound was effective in inhibiting P. aeruginosa biofilm formation. The most active compound of the series was derivative 104 (Figure 39), which exhibited MBIC50 against MRSA and S. mutans of 20 μM. 6.4. Pyrazole Derivatives. A class of 4-diazopyrazole derivatives were prepared and tested in vitro for their antimicrobial and antibiofilm activity on staphylococcal strains.154 Compounds 105a−c (Figure 40) were found to be active against S. aureus strains ATCC 29213 and ATCC 43866 and S. epidermidis RP62A, with MIC values ranging from 1.5 to 12.5 μg/mL. Diazopyrazoles 105a and 105c showed remarkable biofilm inhibition at 25 μg/mL (69−96%). In particular, derivative 105c exhibited 86.5% inhibition of S. aureus ATCC 29213 at 6.2 μg/mL. SAR studies directed to obtain more potent antibiofilm agents led to the identification of compound 106, which showed at 3.1 μg/mL an inhibition of 45.7, 38, and 25% against S. aureus strains ATCC 29213, ATCC 25923, and 708.155 Nagender et al. reported a series of pyrazolo[3,4-b]pyridines 107a,b (Figure 41) which exhibited good activity in inhibiting biofilm formation of Micrococcus luteus MTCC 2470, S. aureus MTCC 96, S. aureus MLS-16 MTCC 2940, P. aeruginosa

Figure 37. 4,5-Disubstituted 2-aminoindazole derivatives 99a−f and 1,2-disubstituted 2-aminoimidazole derivative 100 with biofilm inhibitory activity.

MRSA biofilm formation, showing IC50 values of 3.7, 7.2, and 4.2 μM, while 99d−f (Figure 37) were the most active compounds against MDRAB, with IC50 values ranging from 25.8 to 69.4 μM; in both cases, compounds were able to inhibit the biofilm not interfering with microbial viability. Among the 1,4-disubstituted 2-aminoimidazoles, derivative 100 displayed an IC50 against MRSA and MDRAB biofilms of 4.14 and 31.4 μM, respectively. This class of compounds was also endowed with inhibitory activity against the biofilm formation of P. aeruginosa. Biofilm growth by P. aeruginosa has drawn particular attention because of its involvement in the fatal chronic lung infections in most of cystic fibrosis patients. 5,6-Dimethyl-2-aminoimidazole is one of the most potent P. aeruginosa biofilm inhibitors (IC50 = 4.0 μM) so far.148 6.2. 2-Phenylhydrazinylidene Derivatives. Recently, two new classes of 2-phenylhydrazinylidene compounds were synthesized and tested for their antibiofilm activity against the three S. aureus reference strains ATCC 25923, ATCC 29213, and ATCC 6538.149−151 Ethyl 2-[2-(3,4-dichlorophenyl)hydrazinylidene]-3-(1-ethyl3-methyl-1H-pyrazol-5-yl)-3-oxopropanoate 101 and 2-[2(3,4-dichlorophenyl)hydrazinylidene]-3-(5-methyl-1,2-oxazol3-yl)-3-oxopropanoic acid 102 (Figure 38) showed the best activity against S. aureus ATCC 29213, with IC50 values of 1.7 and 0.8 μM, respectively. For derivative 101, which proved to be able also to interfere with the biofilm formation of the other two tested strains with IC50 of 42 μM against ATCC 25923 and of 13 μM against ATCC 6538, the toxicity was studied in an in 8287



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Figure 39. Chemical structures of the carbazole and indole derivatives 103 and 104 with antibiofilm properties.

7. CHEMICAL INHIBITORS THAT INTERFERE WITH THE PROPAGATION OF ANTIBIOTIC-RESISTANCE GENES Horizontal gene transfer (HGT) between a donor cell and a recipient is the most efficient mechanism in propagating antibiotic resistance genes among bacterial species and in the evolution of antibiotic resistant pathogens. HGT is achieved using the processes of transformation, transduction, and conjugation, with the latter process being more common in clinical setting. The appearance and propagation of CRE in clinical isolates is an example of the importance of the conjugation process.159 The complex protein machinery (type IV secretion system) and the bacterial enzymes involved in the DNA transport and recombination during the conjugation, for example in the conjugative relaxase, could be interesting targets for inhibitors that interfere with the propagation of antibiotic-resistance genes. The conjugative relaxase is a transesterase enzyme responsible for nicking DNA in the transferred strand (Tstrand) at the origin of transfer (oriT), forming a covalent 5phosphotyrosine intermediate that virtually combines the nicked DNA strand and the enzyme into one molecule. The nicked T-strand moves from the donor cell (plasmid+) to the recipient cell (plasmid−) via a conjugation bridge mediated by a type IV secretion system. The relaxase completes the plasmid transfer by reversing the covalent phosphotyrosine linkage and releasing the T-strand. Small molecules as bisphosphonates (BSRCs), clodronate and ethidronate (Figure 43), can inhibit F plasmid conjugative relaxase activity in E. coli, at nanomolar concentrations, as they interfere with two phosphotyrosine intermediates within their divalent metal-containing active sites.160 Some authors later investigated the impact of BSRCs on conjugative plasmids and bacterial cell survival and confirmed that the effects of BSRCs are variable and are dependent on plasmid type and experimental conditions, while their potential use to curb

Figure 40. 4-Diazopyrazoles inhibitors of staphylococcal biofilms.

MTCC 2453, and Klebsiella planticola MTCC 530 bacterial strains, with IC50 values ranging from 3.9 to 7.8 μg/mL.156 Another class of pyrazole derivatives described as antibiofilm agents were 1,3,5-triazine-pyrazoles, reported by Singh et al., which, however, were only moderately active against S. aureus NCIM-2079, with IC50 ranging from 15.62 to 125 μg/mL.157 6.5. Pyrrolomycins. Pyrrolomycins, polyhalogenated metabolites of the Actinosporangium and Streptomyces species, were described, for the first time, as potent antibiofilm agents by Schillaci et al. in 2010.10 Pyrrolomycins D, F1, F2a, F2b, and F3 showed a biofilm inhibition percentage higher than 60% at 1.5 μg/mL against S. aureus (ATCC 29213, ATCC 25923, 657, and 702) and S. epidermidis (DSM 3269 and RP62A). The most potent derivative was the pyrrolomycin F3 (Figure 42), which exhibited an inhibition higher than 50% against all tested strains at the concentration of 0.045 μg/mL and, interestingly, a selectivity index (ratio between IC50 on human cell and antibiofilm concentration) of 1333. Among the synthetic derivatives, the bromo-analogues 108 and 109 (Figure 42) were particularly interesting because they showed, at 1.5 μg/ mL, inhibition ranges of 68.5−100% and 67.9−86.7%, respectively, against the above-mentioned strains.158 The ability to inhibit biofilm formation of this class of compounds proved closely related to their halogen content: compounds with the highest degree of halogenation displayed the best potency.

Figure 41. Pyrazolo[3,4-b]pyridines with antibiofilm activity. 8288



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Figure 42. Pyrrolomycins potent antibiofilm agents.

Figure 43. Examples of chemical inhibitors that interfere with the propagation of antibiotic-resistance genes.

obtained from natural sources such as tropical fruits, was found to be the most active, among a number of 2-AFAs, in inhibiting the conjugation process.163 Starting from the chemical structure of DHCA, several derivative were easily synthesized and tested for the ability to interfere with the spread of resistance genes. In particular, the 2-hexadecynoic acid (2-HDA) (Figure 43), a 2-AFA with a chain length of 16 carbon atoms including a triple bond at C-2, can tackle the dissemination of antibiotic resistance genes of many different Gram negative pathogens, for example, Salmonella enterica, A. baumanni, and Vibrio cholerae. In plate conjugation inhibition assay, 2-HDA inhibited plasmid R388 conjugation with an inhibitory effect of 98% at 0.3 mM. Moreover, it has been shown that donor cells and not the recipients are the target of 2-HDA.164 These inhibitors might be advantageous for decelerating antibiotic resistance spread among bacterial cells and species, and the investigation of their effects in specific environments (e.g., farm, fish factory, or even clinical settings) is desirable.

antibiotic-resistance genes propagation in clinical setting is not promising.161 The relaxase enzyme for conjugative transfer of antibioticresistance determinants to a range of antibiotics, including vancomycin, amynoglycosides, and mupirocin, is known in S. aureus as nicking enzyme (NES). Some authors explained the molecular basis for resistance transmission mediated by NES, the DNA complex crystal structure and interactions NES− DNA (that is the interaction between the enzyme and the nicked DNA strand and into an unique complex), essential in vitro for conjugation. This useful structural information was the basis for the design of a polyamide molecule (Figure 43) aimed to target the GC-rich DNA minor groove of the NES substrate, inhibiting NES at low micromolar concentration.162 Unsaturated fatty acids (uFAs) can interfere with horizontal spread of resistance determinants, as it has been shown that uFAs inhibit conjugation of resistance plasmids IncW and IncF, without affecting cell growth. In particular, dehydrocrepenynic acid (DHCA) (Figure 43), a 2-alkynoic fatty acid (2-AFA), 8289


β-lactams oxyimino-cephalosporins

enzymatic resistance: CRE

8290

β-lactams, aminoglycosides, macrolides, tetracyclines, fluoroquinolones, etc.


efflux pumps (RND family)): Gram negative

HGT


aminoglycosides

β-lactams

enzymatic resistance: metallo-β-lactamases (MBL) (class B)

enzymatic resistance: aminoglycoside modifying enzymes efflux pumps (NorA etc.): Gram positive

β-lactams

antibiotics

enzymatic resistance: serine β-lactamases (SBL) (classes A, C, D)

mechanism of resistance

bisphosphonates polyamide dehydrocrepenyc acid and synthetic derivatives

pyranopyridines pyridopyrimidine piperazine analogues

quinolones and quinolines (compound 53) naphthenic compounds chalcones boronic acid derivatives indole derivatives other heterocyclic inhibitors celecoxib analogues reserpine analogues alkaloids (piperine analogues) peptidomimetics

avibactam derivatives FPI-1465 FPI-1523 FPI-1602 MK7655 1-[3-(2-aminoethyl)benzyl]-3-(piperidin-1-ylmethyl)pyrrolidin-3-ol

avibactam

FPI-1465 FPI-1523 FPI-1602 boronic acid derivatives dicarboxylic acids sulfur-containing inhibitors

clavulanic acid tazobactam sulbactam avibactam boronic acid derivatives sulfur-containing inhibitors avibactam avibactam derivatives

AMI

Table 5. Antibiotic-Resistance Mechanism Inhibitors (AMI) and Their Stage of Development as Therapeutics development stages

in vivo studies early preclinical (with ciprofloxacin, tetracycline, linezolid) in vitro studies

early preclinical (with ciprofloxacin and aztreonam)

early preclinical (with ciprofloxacin or norfloxacin); in vitro studies

preclinical (with imipenem) early preclinical (with amikacin)

in vitro studies, early preclinical (with imipenem) in vitro studies clinical (with ceftazidime) phase1 (with aztreonam)

early preclinical (with biapenem)

clinical clinical170 clinical171 clinical172 early preclinical173 early preclinical68 phase1 (with aztreonam) early preclinical (with aztreonam), early preclinical (with imipenem)

169

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Additionally, the conjugative F pilus could be a target for the prevention of the propagation of antibiotic resistance genes, but, as far as we know, there are not any studies of chemical molecules that have as objective conjugative pili.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +39 091 23891920. E-mail: stellamaria.cascioferro@ unipa.it. ORCID

8. CONCLUSIONS AND FUTURE DIRECTIONS It is time for action in order to address antibiotic resistance, which is a threat to global health care and security. While there is a lack of new antibiotics, the microorganisms have become increasingly resistant across many species and genera and against all classes of current antibiotics. Human beings face a serious risk of not having more weapons available to effectively control pathogens. In this review, we have presented chemical inhibitors of principal mechanisms of resistance in pathogens, both at the bacterial cell level and at the community level.165 In the past decade, significant attempts have been done to develop a new potent EPI able to restore the antibacterial activity of those antibiotics decreased by efflux pumps but, unfortunately, to date, no EPI is used for clinical purposes, generally because of their high toxicity. However, a topical use of such inhibitors seems to be closer. Although important progress has been done in this field, in particular on the structure, mechanism, regulation, and functions of the most common bacterial efflux pumps, new approaches to anticipate the overexpression of efflux genes and to obtain new more potent, and less toxic EPI candidates are needed. A valid approach to overcome the toxicity of these compounds should be directed to the discovery of derivatives able to inhibit only efflux pumps operating in prokaryotes and not in human cells.166 One main obstacle is that the mechanisms of action of most EPIs remain unknown, even if the first inhibitor-bound structures of AcrB and MexB advanced our understanding.123 Efforts should be focused on the development of biological assays, including fitness and in vivo studies that can facilitate the application of EPIs in clinics. To develop ideal candidates for clinical drugs, it is important to consider, in addition to the planktonic form of life, the biofilm phenotype. Despite the growing interest in antibiofilm drugs and the large number of new potent compounds that are able to interfere with the biofilm formation, the treatment of biofilm-associated infections still remains a crucial issue because the most promising candidates are still in early stages of drug development and no antibiofilm compound has reached the stage of clinical use. In particular, the PK/PD profile needs to be optimized for the targeting of bacteria in biofilms. It would be particularly useful for the treatment of chronic infections. Moreover, new compounds able to destroy biofilm architecture and simultaneously to kill dispersed bacterial cells could be helpful but are still rare.141,167 In Table 5, we summarized the main molecules targeting enzymatic resistance, efflux pumps, and HGT and their stage of development as therapeutics. Inhibitors of enzymatic resistance, such as derivatives of the β-lactamase inhibitor avibactam, are closer to the clinics than other molecules. For example, a piperidine diazabicyclooctane, in association with imipenem, is under clinical development for the treatment of infections caused by CRE and P. aeruginosa, which are very hard to treat because they show high levels of resistance to last resort antibiotics. It is possible that in the not to distant future many more inhibitors of antibiotic-resistance mechanisms, at present in the preclinical stage, will reach clinical trials.168

Stella Cascioferro: 0000-0002-4725-1881 Notes

The authors declare no competing financial interest. Biographies Domenico Schillaci is a microbiologist and is the head of the Laboratory of Microbiology and Biological Assays of the Department of Biological, Chemical and Pharmaceutical Science and Technology (University of Palermo, Italy). He is the head of, or collaborates with, research projects regarding the discovery of new anti-infective and antibiofilm agents, in particular against staphylococcal biofilms. He concentrates on both synthetic organic compounds and natural products as antimicrobial peptides. He is the author of 74 publications in peer reviewed journals. From 2001 to date, he has been teaching General Microbiology to the students of the School of Pharmacy of the University of Palermo. He has served as a reviewer for several journals in the field of applied microbiology. Virginia Spanò graduated with a degree in Pharmaceutical Chemistry and Technology (magna cum laude) from the University of Palermo (Italy) in 2005. She received her Ph.D. in Medicinal Chemistry in 2009 under the supervision of Professor Paola Barraja. During her Ph.D., she was a research fellow at the laboratory of Prof. Christopher Moody, School of Chemistry, University of Nottingham (U.K.). In 2009, she received a postdoctoral fellowship at the Faculty of Pharmacy of Palermo. At present she is Researcher at the Faculty of Pharmacy of Palermo. Her current research interests include the synthesis, identification, and biological evaluation of nitrogen heterocyclic compounds as antitumor agents. She is the coauthor of 24 scientific papers. Barbara Parrino graduated with a degree in Medicinal Chemistry and Technology at the University of Palermo with full honors in 2007. From January to April 2010, she was a Ph.D. visiting student at Semmelweiss University. She was a Ph.D. visiting student at the University of Nottingham from May to September 2011. In March 2012, she received the “Doctor Europaeus” Ph.D. in Pharmaceutical Sciences. On September 2012, she was awarded the P. Ehrlich MedChem Euro-PhD Network Certif icate. Since November 2012, she is a Postdoc Research fellow at the University of Palermo. She is the author of 24 papers and one Italian patent. Anna Carbone obtained in 2000 her degree (cum laude) in Medicinal Chemistry and Technology and in 2004 her Ph.D. in Medicinal Chemistry at the University of Messina. She went on to work from 2004 to 2010 as a Post Doctoral Fellow at the University of Palermo. She spent two years (2010-2012) as a Marie Curie Fellow at the University of Nottingham. She is currently a Senior Scientist in the Department of STEBICEF, University of Palermo. Her research interests include the design, synthesis, and biological evaluation of heterocyclic compounds with potential biological properties. She is the author of 46 papers and one Italian patent. Alessandra Montalbano graduated with a degree in Pharmaceutical Chemistry and Technology (magna cum laude) from the University of Palermo (Italy) in 1996. She received her Ph.D. in Medicinal Chemistry in 2000, and in 2002 she joined to Faculty of Pharmacy of Palermo as a Researcher; at present, she is an Associate Professor of Drug Analysis. Her current research interests include the synthesis, identification, and biological evaluation of nitrogen heterocyclic 8291



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multidrug and toxic compound extrusion family; MBIC, minimal biofilm inhibitory concentration; MBL, metallo-βlactamase; MDRAB, multidrug resistant A. baumannii; MFS, major facilitator superfamily; MDR, multidrug resistance; NDM, New Dehli metallo β-lactamase; NES, nicking enzyme; NMP, 1-(1-naphthylmethyl)-piperazine; OXA, oxacillinase; PaβN, dipeptide phenylalanine−arginine β-naphthylamide; PBP, penicillin binding protein; PIP, piperacillin; RND, resistance-nodulation-division family; SBL, serine β-lactamase; SMR, small multidrug resistance family; TET, tetracycline; uFAs, unsaturated fatty acids; VIM-2, Verona integron-encoded metallo-β-lactamase 2

compounds as antitumor agents. She is the coauthor of 55 scientific papers. Paola Barraja graduated with a degree in Pharmacy (University of Palermo) in 1992 with full honors. From October 1992 to September 1994, she was a CNR fellow and she was a guest fellow at the Cancer Research Campaign (Nottingham, UK), working under the supervision of Prof. M. F. G. Stevens. From April 1996 to December 2002, she was a researcher (SSD CHIM/08) at the University of Palermo and she was a winner of the Marie Curie fellowship from February 1998 to February 1999 at the Department of Chemistry of the University of Exeter under the supervision of Prof. C. J. Moody. She became an associate professor (SSD CHIM/08), from December 2002 to present. She is the author of 91 papers, two international and four Italian patents, and serves as a reviewer for several journals in the area of medicinal chemistry.

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REFERENCES

(1) Ventola, C. L. The Antibiotic Resistance Crisis. Pharm. Ther. 2015, 40 (4), 277−283. (2) Roberts, R. R.; Hota, B.; Ahmad, I.; Scott, R. D., II; Foster, S. D.; Abbasi, F.; Schabowski, S.; Kampe, L. M.; Ciavarella, G. G.; Supino, M.; Naples, J.; Cordell, R.; Levy, S. B.; Weinstein, R. A. Hospital and Societal Costs of Antimicrobial-Resistant Infections in a Chicago Teaching Hospital: Implications for Antibiotic Stewardship. Clin. Infect. Dis. 2009, 49 (8), 1175−1184. (3) Pendleton, J. N.; Gorman, S. P.; Gilmore, B. F. Clinical Relevance of the ESKAPE Pathogens. Expert Rev. Anti-Infect. Ther. 2013, 11 (3), 297−308. (4) Høiby, N.; Bjarnsholt, T.; Givskov, M.; Molin, S.; Ciofu, O. Antibiotic Resistance of Bacterial Biofilms. Int. J. Antimicrob. Agents 2010, 35 (4), 322−332. (5) Cascioferro, S.; Totsika, M.; Schillaci, D. Sortase A: An Ideal Target for Anti-Virulence Drug Development. Microb. Pathog. 2014, 77, 105−112. (6) Cascioferro, S.; Raffa, D.; Maggio, B.; Raimondi, M. V.; Schillaci, D.; Daidone, G. Sortase A Inhibitors: Recent Advances and Future Perspectives. J. Med. Chem. 2015, 58 (23), 9108−9123. (7) Cascioferro, S.; Cusimano, M. G.; Schillaci, D. Antiadhesion Agents against Gram-Positive Pathogens. Future Microbiol. 2014, 9, 1209−1220. (8) Penesyan, A.; Gillings, M.; Paulsen, I. T. Antibiotic Discovery: Combatting Bacterial Resistance in Cells and in Biofilm Communities. Molecules 2015, 20 (4), 5286−5298. (9) Sánchez-Romero, M. A.; Casadesús, J. Contribution of Phenotypic Heterogeneity to Adaptive Antibiotic Resistance. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (1), 355−360. (10) Schillaci, D.; Petruso, S.; Raimondi, M. V.; Cusimano, M. G.; Cascioferro, S.; Scalisi, M.; La Giglia, M. A.; Vitale, M. Pyrrolomycins as Potential Anti-Staphylococcal Biofilms Agents. Biofouling 2010, 26 (4), 433−438. (11) Ambler, R. P.; Coulson, A. F.; Frère, J. M.; Ghuysen, J. M.; Joris, B.; Forsman, M.; Levesque, R. C.; Tiraby, G.; Waley, S. G. A Standard Numbering Scheme for the Class A Beta-Lactamases. Biochem. J. 1991, 276, 269−270. (12) Bush, K.; Jacoby, G. A. Updated Functional Classification of βLactamases. Antimicrob. Agents Chemother. 2010, 54 (3), 969−976. (13) Yigit, H.; Queenan, A. M.; Rasheed, J. K.; Biddle, J. W.; Domenech-Sanchez, A.; Alberti, S.; Bush, K.; Tenover, F. C. Carbapenem-Resistant Strain of Klebsiella Oxytoca Harboring Carbapenem-Hydrolyzingβ -Lactamase KPC-2. Antimicrob. Agents Chemother. 2003, 47 (12), 3881−3889. (14) Bonomo, R. A. New Delhi Metallo-β-Lactamase and Multidrug Resistance: A Global SOS? Clin. Infect. Dis. 2011, 52 (4), 485−487. (15) Khan, A.; Faheem, M.; Danishuddin, M.; Khan, A. U. Evaluation of Inhibitory Action of Novel Non β-Lactam Inhibitor against Klebsiella Pneumoniae Carbapenemase (KPC-2). PLoS One 2014, 9 (9), e108246. (16) Bratu, S.; Mooty, M.; Nichani, S.; Landman, D.; Gullans, C.; Pettinato, B.; Karumudi, U.; Tolaney, P.; Quale, J. Emergence of KPCPossessing Klebsiella Pneumoniae in Brooklyn, New York: Epidemi-

Patrizia Diana is full Professor of Medicinal Chemistry at the University of Palermo. She is currently coordinator of the Medicinal and Biological Section of the Department of Science and Technology: Chemical, Biological and Pharmaceutical (STEBICEF), and is a member of the Italian Chemical Society and International Society of Heterocyclic Chemistry. Girolamo Cirrincione is full Professor of Medicinal Chemistry at the University of Palermo. He is a member of the Drug Discovery Commettee of the Pharmacology and Molecular Mechanisms (PAMM) Group of the European Organization for Research and Treatment of Cancer; the Italian Chemical Society, where he has been President of the Medicinal Chemistry Division, and the International Society of Heterocyclic Chemistry, where he served as vice president for 2004−2005. He is currently Research Pro-rector of the University of Palermo. Stella Cascioferro graduated with a degree in Pharmacy with honors in 1999, and she got her Ph.D. in Medicinal Chemistry in 2004 at the University of Palermo. In 2004, she joined the Physical and Theoretical Chemistry Laboratory at the University of Oxford as part of the group led by Professor Graham Richards. Her research interests include the design, synthesis, and biological evaluation of heterocyclic compounds as antitumoral, antinfective, and antiinflammatory agents. Currently, she is working in the Department of Biological, Chemical, and Pharmaceutical Science and Technology (University of Palermo, Italy). She is the author of 33 scientific papers published in peer reviewed international journals and a reviewer for several journals in the field of medicinal chemistry.

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ABBREVIATIONS USED AAC, aminoglycoside N-acetyltransferase; ABC, ATP-binding cassette superfamily; AFA, alkynoic fatty acid; AG, aminoglycoside; 2-AITs, 2-aminoimidazole triazoles; AME, aminoglycoside-modifying enzymes; AMI, antibiotic-resistance inhibitors; ANT, aminoglycoside O-nucleotidyltransferases; APH, aminoglycoside O-phosphotransferase; AZT, aztreonam; BIPM, biapenem; BLI, β-lactamase inhibitor; BSRCs, bisphosphonates; CoNS, coagulase negative staphylococci; CPX, ciprofloxacin; CRE, carbapenem-resistant Enterobacteriaceae; DBO, diazabicyclooctane; DHCA, dehydrocrepenynic acid; Eis, enhanced intracellular survival protein; EPI, efflux pump inhibitors; ESBL, extended spectrum β-lactamase; EtBr, ethidium bromide; FBLD, fragment-based lead discovery; FLAP, fingerprints for ligands and proteins; 2-HAD, 2hexadecynoic acid; HGT, horizontal gene transfer; HSA, human serum albumin; HTS, high-througput screening; KPC, K. pneumoniae carbapenemase; LVX, levofloxacin; MATE, the 8292



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