Review Derivatives as Antibacterial and Anticancer Agents: Historic Problems and Current Solutions

George P. Dinos 1, Constantinos M. Athanassopoulos 2,*, Dionissia A. Missiri 2, Panagiota C. Giannopoulou 1, Ioannis A. Vlachogiannis 1, Georgios E. Papadopoulos 3, Dionissios Papaioannou 2 and Dimitrios L. Kalpaxis 1,*

1 Department of Biochemistry, School of Medicine, University of Patras, GR-26504 Patras, Greece; [email protected] (G.P.D.); [email protected] (P.C.G.); [email protected] (I.A.V.) 2 Laboratory of Synthetic Organic Chemistry, Department of Chemistry, University of Patras, GR-26504 Patras, Greece; [email protected] (D.A.M.); [email protected] (D.P.) 3 Department of Biochemistry and Biotechnology, University of Thessaly, Ploutonos 26, GR-41221 Larissa, Greece; [email protected] * Correspondence: [email protected] (C.M.A.); [email protected] (D.L.K.); Tel.: +30-2610997909 (C.M.A.); +30-2610996124 (D.L.K.)

Academic Editor: Claudio O. Gualerzi Received: 24 February 2016; Accepted: 24 May 2016; Published: 3 June 2016

Abstract: Chloramphenicol (CAM) is the D-threo isomer of a small molecule, consisting of a p-nitrobenzene ring connected to a dichloroacetyl tail through a 2-amino-1,3-propanediol moiety. CAM displays a broad-spectrum bacteriostatic activity by specifically inhibiting the bacterial protein synthesis. In certain but important cases, it also exhibits bactericidal activity, namely against the three most common causes of , Haemophilus influenzae, Streptococcus pneumoniae and Neisseria meningitidis. Resistance to CAM has been frequently reported and ascribed to a variety of mechanisms. However, the most important concerns that limit its clinical utility relate to side effects such as neurotoxicity and hematologic disorders. In this review, we present previous and current efforts to synthesize CAM derivatives with improved pharmacological properties. In addition, we highlight potentially broader roles of these derivatives in investigating the plasticity of the ribosomal catalytic center, the main target of CAM.

Keywords: chloramphenicol; antibiotics; resistance; side effects; anticancer agents; chemical synthesis; ; ; ; reaction

1. Introduction Sixty decades of biochemical studies and more recent crystallographic studies, have revealed the molecular basis by which chloramphenicol (CAM) specifically inhibits bacterial protein synthesis. CAM is an old broad-spectrum antibiotic but its medical and veterinary applications as antibacterial are full of pros and cons. Besides resistance to CAM frequently reported, the major disinclined concerns for the clinical use of CAM relate to the side effects in long antibiotic courses causing neurotoxicity and hematologic disorders. This has prompted many investigators to attempt the synthesis of new CAM derivatives with improved pharmaceutical properties. In this review, we discuss how the information gained from these efforts has advanced our knowledge on the mode of action of CAM and how these CAM derivatives have contributed to compacting resistant and addressing side effects of the drug.

Antibiotics 2016, 5, 20; doi:10.3390/antibiotics5020020 www.mdpi.com/journal/antibiotics Antibiotics 2016, 5, 20 2 of 21

2. Mode of Action of CAM Antibiotics 2016, 5, 20 2 of 21 Upon CAM discovery [1], it was recognized that the D-threo isomer (Figure1) inhibits bacterial Upon CAM discovery [1], it was recognized that the D-threo isomer (Figure 1) inhibits bacterial protein synthesis. The other stereoisomers of CAM, not occurring in nature, were found inactive [2]. protein synthesis. The other stereoisomers of CAM, not occurring in nature, were found inactive [2]. Antibiotics 2016, 5, 20 Cl Cl 2 of 21 Upon CAM discovery [1], it was recognized that the D-threo isomer (Figure 1) inhibits bacterial protein synthesis. The other stereoisomers of CAM, not occurring in nature, wereO foundNH inactive [2]. NO 2 Cl ClH OH

NH OH Cl HOO H C H NO2 H 2 R N H OH HO H R Cl OH Cl HOH C H H NH COCHCl H O 2 2 O2N R NOH HO H R Cl CH2OH NO2 H NH COCHCl O 2 O2N OH CH OH NO A 2 B 2 C A B C Figure 1. AlternativeAlternative structures structures for CAM. CAM. ( (ΑA)) Fischer Fischer projection projection (D- (D-threothreo isomer);isomer); ( (ΒB)) Skeletal Skeletal formula formula showingFigure the configurationconfiguration1. Alternative structures of thethe two for CAM. stereogenic (Α) Fischer centers; projection (C)) (D- NewmanNewmanthreo isomer); projection.projection. (Β) Skeletal formula showing the configuration of the two stereogenic centers; (C) Newman projection. Two bindingbinding sites sites in in ribosomes were were identified identified by earlyby early equilibrium equilibrium studies studies [3], CAM1 [3], CAM1 and CAM2, and Two binding sites in ribosomes were identified by early equilibrium studies [3], CAM1 and CAM2,with affinityCAM2, with constantsaffinitywith affinity constants equal constants to equal 2 equalµM to andto 2 2 μΜ 200μΜ andµM, 200200 respectively. μΜ μΜ, respectively., respectively. Recently, Recently, Recently, cross-linking cross-linking cross-linking of of CAM of to CAMribosomes toCAM ribosomes isolated to ribosomes from isolated theisolated bacterium from from the theEscherichia bacterium bacterium coli EscherichiaEscherichiaand the archaealcoli coli and and Halobacteriumthe archaealthe archaeal Halobacterium halobium Halobacteriumlocalized halobiumCAM2halobium at localized the entrance localized CAM2 toCAM2 theat the ribosomalat the entrance entrance peptide to to thethe ribosomal exit tunnel peptide peptide [4]. Aexit similar exit tunnel tunnel binding[4]. A [4]. similar siteA similar binding was observed binding siteby crystallography wassite observed was observed inby the crystallographybyHaloarcula crystallography marismortui inin thethe ribosome,Haloarcula marismortui overlappingmarismortui ribosome theribosome , overlapping, overlapping binding the site the [5] macrolide(Figure2macrolideA). binding Nevertheless, binding site [5]site high(Figure [5] (Figure concentrations 2A). 2A). Nevertheless, Nevertheless, of CAM high high were concentrations concentrations used by both of CAM studiesof CAM were to usedwere cross-link by used both orby bindboth studies to cross-link or bind the drug at the exit-tunnel, a fact that limited the functional significance studiesthe drug to at cross-link the exit-tunnel, or bind a the fact drug that at limited the exit-tunnel, the functional a fact significance that limited of the CAM2 functional binding significance site. of CAM2 binding site. of CAM2 binding site.

Figure 2. Binding positions of CAM (in green) in the (A) Haloarcula marismortui and (B) Deinococcus Figure 2. Binding positions of CAM (in green) in the (A) Haloarcula marismortui and radiodurans ribosome, as detected by crystallography (PDB ID code 1NJI and 1K01, respectively). (B) Deinococcus radiodurans ribosome, as detected by crystallography (PDB ID code 1NJI and 1K01, respectively).Quite recently we revealed a second kinetically cryptic binding site for CAM at the nucleotide FigureG2611 2. ofBinding the 50S positions ribosomal of subunit CAM of(in E. green) coli, using in the much (A) Haloarculalower concentrations marismortui (<60 and μΜ (B) )of Deinococcus a CAM radioduranshomodimer, ribosome, than those as detectedutilized previously by crystallography [6]. A plausible (PDB explanationID code 1NJI for and this 1K01, raised respectively). affinity is that Quite recently we revealed a second kinetically cryptic binding site for CAM at the nucleotide binding of the dimer to the high affinity site (CAM1) facilitates targeting of a cryptic, low affinity site G2611 of the 50S ribosomal subunit of E. coli, using much lower concentrations (<60 µM) of a CAM Quite(CAM2) recently via the we second revealed edge ofa thesecond homodimer. kinetically Consistently, cryptic ,binding site afor macrolide CAM atthat the binds nucleotide homodimer, than those utilized previously [6]. A plausible explanation for this raised affinity is that G2611 ofat the entrance50S ribosomal of the exit subunit tunnel, of in E.turn coli inhibits, using binding much lowerof CAM concentrations [7], while CAM (<60enhances μΜ) theof a CAM bindingpremature of the dimer release to theof highshort affinityoligopeptidyl-tRNAs, site (CAM1) facilitatesreminiscent targeting of macrolide of a cryptic, antibiotics low affinity[8]. site homodimer, than those utilized previously [6]. A plausible explanation for this raised affinity is that (CAM2)Nevertheless, via the second due to edge its low of affinity the homodimer. for CAM, the Consistently, CAM2 binding erythromycin, site seems unlikely a macrolide to play a central that binds at binding of the dimer to the high affinity site (CAM1) facilitates targeting of a cryptic, low affinity site the entrance of the exit tunnel, in turn inhibits binding of CAM [7], while CAM enhances the premature (CAM2) via the second edge of the homodimer. Consistently, erythromycin, a macrolide that binds at the entrance of the exit tunnel, in turn inhibits binding of CAM [7], while CAM enhances the premature release of short oligopeptidyl-tRNAs, reminiscent of macrolide antibiotics [8]. Nevertheless, due to its low affinity for CAM, the CAM2 binding site seems unlikely to play a central Antibiotics 2016, 5, 20 3 of 21 release of short oligopeptidyl-tRNAs, reminiscent of macrolide antibiotics [8]. Nevertheless, due to its low affinity for CAM, the CAM2 binding site seems unlikely to play a central role in inhibition of protein synthesis, an hypothesis also strengthened by the fact that most of the resistance mutations or modificationsAntibiotics cluster 2016, 5, 20 around the CAM1 binding site [9–12]. 3 of 21 In contrastrole in inhibition to the of CAM2 protein bindingsynthesis, site,an hypothesis CAM1 also seems strength toened be strongly by the fact relevant that most forof the inhibition. Numerousresistance biochemical mutations and or modifications structural studies,cluster around like the competition CAM1 binding studies site [9–12]. between CAM and small In contrast to the CAM2 binding site, CAM1 seems to be strongly relevant for inhibition. tRNA fragmentsNumerous forbiochemical the A-site and structural of the peptidyl studies, like transferase competition (PTase) studies between center [CAM13], and inhibition small of the puromycintRNA reaction fragments by CAMfor the [14A-site–17 ],of andthe peptidyl crystallographic transferase analyses(PTase) center of CAM [13], inhibition bound to of 50S the subunits from bacteriapuromycin [18– reaction20] demonstrated by CAM [14–17], that and the crystallog CAM1raphic binding analyses site of liesCAM within bound to the 50S ribosomal subunits A-site (Figure2B).from bacteria [18–20] demonstrated that the CAM1 binding site lies within the ribosomal A-site (Figure 2B). CAM has been considered as iso-structural to both puromycin and the 31-end of aminoacyl-tRNA CAM has been considered as iso-structural to both puromycin and the 3′-end of aminoacyl- (Figure3)[tRNA21]. (Figure The initial 3) [21]. observation The initial inobservation favor of thisin favor consideration of this consideration was undertaken was undertaken in a computational in a study thatcomputational related CAM study tothat the related peptide CAM to moiety the peptide of peptidyl-tRNAmoiety of peptidyl-tRNA bound bound to theto the P-site P-site and the correspondingand thetransition corresponding state transition of peptide state of bond peptide formation bond formation with aminoacyl-tRNAwith aminoacyl-tRNA [22 [22].].

Figure 3.FigureStructures 3. Structures of CAM, of CAM, puromycin puromycin and and Phe-tRNAPhe-tRNAPhePhe in anin iso-structural an iso-structural orientation. orientation.

Nevertheless, this proposal ignored that CAM is essentially an A-site inhibitor. A few years later, Nevertheless,Bhuta et al. [23] this oriented proposal the ignoredp-nitrophenyl that group CAM ofis CAM essentially and the methylated-tyrosyl an A-site inhibitor. moiety A of few years later, Bhutapuromycinet al. [23 to] the oriented hydrophobic the p crevic-nitrophenyle of the A-site, group so that of CAMthe CO and-NH thesequence methylated-tyrosyl of the amido bonds moiety of puromycinin puromycin to the hydrophobic and CAM were crevice in the of opposite the A-site, direction. so that Thus, the CAM CO-NH and puromycin sequence were of theregarded amido bonds in puromycinas retro-inverso and CAM analogs, were which in the is at opposite variance with direction. a crystallographic Thus, CAM model and of CAM puromycin in complex were with regarded Deinococcus radiodurans 50S subunit (Figure 2B) [18] but in line with current crystallographic evidence as retro-inversofrom CAM-50Sanalogs, ribosomal which subunit is at variance (Figure 4) with complexes a crystallographic from Thermus thermophilus model of and CAM E. coli in [19,20]. complex with DeinococcusThe radiodurans concept of retro-inverso50S subunit orientation (Figure of 2CAMB) [ 18in relation] but in to line the substrate with current of the A-site crystallographic is particularly evidence from CAM-50Simportant ribosomal if we take subunitinto consideration (Figure 4that) complexes retro-inverso from analogsThermus of peptides thermophilus exhibit high andbiologicalE. coli [19,20]. The conceptactivity of retro-inverso [24]. orientation of CAM in relation to the substrate of the A-site is particularly In the course of kinetic studies, early evidence suggested that CAM behaves as a simple important if we take into consideration that retro-inverso analogs of peptides exhibit high biological competitive inhibitor of peptide bond formation, a view compatible with the structural similarity of activity [24CAM]. with puromycin, a pseudo-substrate of A-site of PTase and the 3′-end of aminoacyl-tRNA In the(Figure course 3) [25,26]. of kinetic However, studies, besides competitive early evidence kinetics, suggested there is also thatevidence CAM for noncompetitive behaves as a simple competitive[15] or inhibitor mixed-noncompetitive of peptide bondtype of formation,inhibition mechanism a view [14,16,27], compatible depending with theon the structural buffer ionic similarity of CAM withconditions puromycin, used, the a inhibitor pseudo-substrate concentration, of th A-sitee pre-incubation of PTase andeffect the and 31 -endthe nature of aminoacyl-tRNA of the substrates. Evidence for the dependence of CAM inhibition kinetics on mono- and divalent cations (Figure3)[was25 provided,26]. However, by high-resolution besides competitive structures of CAM-ribosome kinetics, there complexes, is also evidence indicating for the noncompetitiveinvolvement [15] or mixed-noncompetitiveof cations mediating typecoordination of inhibition bonding between mechanism the drug [14 ,and16, 27ribosomal], depending residues on[18,20]. the The buffer ionic conditions used, the inhibitor concentration, the pre-incubation effect and the nature of the substrates. Evidence for the dependence of CAM inhibition kinetics on mono- and divalent cations was provided by high-resolution structures of CAM-ribosome complexes, indicating the involvement of cations mediating coordination bonding between the drug and ribosomal residues [18,20]. The inhibitor Antibiotics 2016, 5, 20 4 of 21 concentration effect and the significance of ribosome pre-incubation with the drug before adding the substrate were understood when it was realized that CAM behaves as a slow-binding competitive Antibiotics 2016, 5, 20 4 of 21 inhibitor of peptide bond formation, acting via a two-sequential reaction; CAM (I) reacts rapidly inhibitor concentration effect and the significance of ribosome pre-incubation with the drug before with an initiatoradding the ribosomal substrate were complex understood (C) towhen form it was the realized encounter that CAM complex behaves CI as that a slow-binding is then isomerized slowly tocompetitive a more tight inhibitor complex of peptide C*I [bond17]. Consistently,formation, acting as via observed a two-sequential in some reaction; studies, CAM noncompetitive (I) reacts or mixed-noncompetitiverapidly with an initiator modes ribosomal of action complex could be(C) explained to form the by encounter the slow-onset complex CI inhibition that is then theory [28] accordingisomerized to which slowly the inhibition to a more patterntight complex under C*I “pre-incubation” [17]. Consistently, conditions as observedcan in some be misinterpreted studies, as noncompetitive or mixed-noncompetitive modes of action could be explained by the slow-onset indicating mixed, noncompetitive inhibition. Regarding the effects of the nature of the substrate, it inhibition theory [28] according to which the inhibition pattern under “pre-incubation” conditions Phe was observedcan be thatmisinterpreted tRNAs bearing as indicati bulkyng mixed, amino noncompetitive acids, like inhibition. Phe-tRNA Regardingare lessthe effects prone of tothe inhibition by CAMnature than tRNAsof the substrate, carrying it was smaller observed or chargedthat tRNAs amino bearing acids bulky [amino8,29]. acids, The naturelike Phe-tRNA of thePhe P-site are bound substrateless can prone also to influence inhibition CAMby CAM inhibitory than tRNAs activity, carrying ansmaller effect or consistentcharged amino with acids the [8,29]. crystallographic The observationnature that of the the P-sitep-nitro bound moiety substrate of CAMcan also is influe in closence CAM proximity inhibitory to activity, the aminoacyl an effect consistent moiety of P-site with the crystallographic observation that the p-nitro moiety of CAM is in close proximity to the bound tRNAaminoacyl [19,20 moiety]. of P-site bound tRNA [19,20].

Figure 4. Binding positions of CAM (in green) in the (A) Thermus thermophilus and (B) Escherichia coli Figure 4.ribosome,Binding as positions detected by of crystallography CAM (in green) (PDB in ID the code (A 4V7W) Thermus and 3OFC, thermophilus respectively).and (B) Escherichia coli ribosome, as detected by crystallography (PDB ID code 4V7W and 3OFC, respectively). Beyond the inhibition of peptide bond formation, CAM can perturb additional ribosomal functions, such as termination [30], translational accuracy [31] and biogenesis of 50S ribosomal Beyond the inhibition of peptide bond formation, CAM can perturb additional ribosomal subunits [32]. With regards to the latter study, more recent observations suggested that ribosomal functions,assembly such asdefects termination are due to a [ 30specific], translational inhibition of the accuracy biosynthesis [31] of and ribosomal biogenesis proteins of by 50S CAM ribosomal subunits[33]. [32 ]. With regards to the latter study, more recent observations suggested that ribosomal assembly defects are due to a specific inhibition of the biosynthesis of ribosomal proteins by CAM [33]. 3. Bacterial Survival Strategies to Combat CAM Activity 3. Bacterial SurvivalThe misuse Strategiesof antibiotics to in Combatclinical practice CAM and Activity the widespread use in agriculture has seriously contributed to the rise in global resistance to antibiotics. Nevertheless, the microbial world has had Theper misuse se the molecular of antibiotics tools to in drive clinical resistance; practice the anti andbiotic the resistome widespread pre-dates use inthe agriculturemodern antibiotic has seriously contributedera by to millions the rise of in years. global Therefore, resistance development to antibiotics. of resistance Nevertheless, cannot be completely the microbial eradicated world and has had per se theis molecular only a matter tools of when. to drive The anti resistance;biotic-resistance the antibiotic crisis is further resistome aggravated pre-dates by the thepaucity modern of new antibiotic era by millionsantibiotics of in years. the pipeline Therefore, [34]. development of resistance cannot be completely eradicated and Resistance or decreased sensitivity to CAM has been frequently observed and is mediated by is only anumerous matter of mechanisms. when. The Target antibiotic-resistance mutations or alterations crisis are is a usual further mechanism aggravated of bacterial by the resistance paucity of new antibioticsto CAM. in the At pipeline least eight [34 mutations]. in the V domain of 23S rRNA and one methyl-modification of A2503 Resistanceconfer high or CAM decreased resistance sensitivity in bacteria to [9–12], CAM especially has been when frequently occurring in observed most of rRNA and operons is mediated by numerous(7 mechanisms.in E. coli). More Targetcrucial are mutations mutations or and/or alterations deletions are of aribosomal usual mechanism proteins, which of bacterialare usually resistance to CAM.encoded At least by eight single mutations copies. in However, the V domain mutation ofs 23Sin ribosomal rRNA and proteins one methyl-modificationcausing CAM-resistance of A2503 have been identified only in a few cases so far [35,36]. Because interactions of CAM with ribosomal confer highproteins CAM have resistance not been detected in bacteria by crystallographic [9–12], especially analyses when [18,20], occurring such resistance in most is suspected of rRNA to operons (7 in E. coli). More crucial are mutations and/or deletions of ribosomal proteins, which are usually encoded by single gene copies. However, mutations in ribosomal proteins causing CAM-resistance have been identified only in a few cases so far [35,36]. Because interactions of CAM with ribosomal proteins have not been detected by crystallographic analyses [18,20], such resistance is suspected to arise indirectly, probably by induced conformational changes in rRNA. It has been found that combination of both ribosomal protein and rRNA mutations lead to increased resistance; e.g. G-to-A Antibiotics 2016, 5, 20 5 of 21 nucleotide change at position 2073 (2058, E. coli numbering) in all three copies of the 23S rRNA gene in combination with G14D modification in ribosomal protein L4 confers CAM resistance in Campylobacter jejuni [36]. Nevertheless, such mutations are often accompanied by fitness cost and consequently are not extensively spread. The most frequently encountered mechanism of bacterial resistance to CAM is the enzymatic inactivation of the drug warhead by different types of , extensively reviewed by Schwarz et al. [37]. Although O-phosporylation [38–40], hydrolytic degradation to p-nitrophenylserinol [41,42], and nitroreductation [43] have been reported previously, the major enzymatic mechanism that inactivates CAM is acetylation via several types of CAM acetyltransferases (CATs). All CAM acetyltransferases share similar molecular strategies in acetylating CAM. The O-acetyl derivatives of CAM fail to act as antibiotics, because they do not bind to bacterial ribosomes. Beyond the above mechanisms, there are also publications on other mechanisms of CAM resistance, such as permeability barriers and efflux systems. CAM gains access to the periplasm through pore-forming porins [44]. Permeation of the outer membrane by antibiotics through porins depends on the molecular dimensions of the drugs [45]. Therefore, perturbations of the outer membrane or derivatization of CAM may reduce its capacity for internalization into the cell. On the other hand, bacteria defend themselves against antibiotics by pumping out the drugs. Export of CAM from the bacterial cell can be mediated either by specific and/or multi-drug efflux pumps, the former mediating higher levels of resistance compared to those of nonspecifically driving out the drug [37]. associated with CAM specific efflux pumps have been identified and characterized in a wide variety of bacteria. Eight different groups of specific exporters, reviewed by Schwarz et al. in 2004 [37], are constantly enriched by new data [46,47]. CAM is a substrate of most multi-drug efflux pumps, namely the major facilator superfamily (MPS) [48], the small multi- (SMR) family [49], the resistance nodulation and cell division (RND) family [50], the ATP-binding cassette (ABC) family [51] and the multidrug and toxin extrusion (MATE) family [52]. Worth noting, CAM may induce expression of efflux pumps, such as MexXY in that belongs to RND family [53] or enhance the efflux of other ligands by AcrB pump in E. coli [54]. Such effects, not beginning nor ending with these special examples, complicate the use of CAM for the treatment of bacterial .

4. Side Effects of CAM Soon after its discovery and subsequent synthesis, CAM became very popular due to its antimicrobial effectiveness and low cost. However, in the following years when CAM was issued for general clinical use, it was realized that CAM can cause hematological disorders like bone marrow depression and [55]. While an irreversible and fatal aplastic anemia appeared to be a rare complication, probably caused by nitrobenzene metabolites of CAM that act on DNA [56–58], mild and reversible was found to occur quite frequently and was ascribed to a mitochondrial protein synthesis inhibition, particularly in cases in which underlying mitochondrial mutations favor CAM binding to mitochondrial ribosome (mitoribosome) [59,60]. Despite mitochondria may have originated from bacteria (endosymbiosis hypothesis), the mitochondrial ribosomal machine is substantially different from that found in bacterial and human cells [61]. Nevertheless, the catalytic core of PTase and decoding center architecture is conserved in mitoribosome and bacterial ribosome. This explains why CAM is capable of inhibiting mitochondrial protein synthesis. Further studies in mice indicated that CAM inhibits protein synthesis in mitoribosomes of the marrow progenitor cells at the differentiation rather than the replication stage [62]. Due to its lipoidal nature (MLogP = 1.23), low number of OH and NH H-bond donors (=3) and low number of N and OH-bond acceptors (=7), CAM penetrates well in all tissues, including many privileged sites such as the bone marrow and the brain [63]. It remains relatively unbound to proteins and its small molecular size (MW = 323.14) facilitates crossing of the blood-brain barrier (BBB) [64]. Penetration of BBB was further improved when optimized palm kernel oil nanoemulsion-loaded with chloramphenicol were used to cross BBM [65]. These properties made the CAM-loaded Antibiotics 2016, 5, 20 6 of 21 nanoemulsions a first-choice therapeutic for meningitis treatment. Although less available in brain and cerebrospinal fluid (CSF) relative to plasma, CAM can achieve high levels in brain and CSF after prolonged use and high cumulative dose that may cause drug-related mitochondrial neuropathies [66]. CAM treatment over a long period may also cause irreversible panmyelopathy which is lethal and may be associated with genetic defects. Dermatological studies disclosed lymphocyte sensitization by chloramphenicol and azidamphenicol in allergic contact dermatitis [67], while experiments in mice splenocytes revealed immunosuppressive activity of a CAM analog, florfenicol, on the immune response to stimulators [68]. In neonates and infants lacking glucuronidation reactions (phase II detoxification) and sufficient renal capacity for excreting unconjugated drugs, accumulated toxic CAM metabolites can cause Gray syndrome, a disease manifested by , cyanosis, hypothermia, cardiovascular collapse, ashen gray color of the skin and vomiting [69]. There is also evidence that CAM causes mitochondrial stress that in turn promotes matrix metalloproteinase-13-associated cancer cell invasion [70]. Due to the above harmful effects, the clinical use of CAM is currently limited in developed countries to infections not responding to other antibiotics. Nevertheless, a recent study suggests that the inhibitory effects of antibiotics on the host mitochondrial protein synthesis and the subsequent negative consequences on mitochondrial biogenesis would allow us to eradicate cancer stem cells, across multiple tumor types, by repurposing antibiotics for anti-cancer therapy [71]. This suggestion was based on the hypothesis that cancer stem cells are strongly anabolic and they may require robust mitochondrial biogenesis and activity for survival and proliferative expansion. Indeed, treatment of MCF7 cells with CAM caused inhibition of tumor-sphere formation with an IC50 of approximately 200 µM. Consistently, previous in vitro studies have indicated that CAM alone, or in combination with other anticancer drugs can cause inhibition of growth in several cancer lines including leukemic cell lines [6,72,73]. Taking into account the critical role of CAM in induced leukemogenesis [74], it is clear that additional cellular studies using different solid tumor cell lines and graded doses of CAM are needed to explore the potentially beneficial role of CAM in cancer therapy. To define which regions of the parent molecule are essential for antibacterial activity or strongly associated with toxicity, numerous CAM analogs have been synthesized and biologically evaluated. For the sake of brevity, we present below only the most important of them, particularly those investigated as drugs.

5. Modifications of CAM to Obtain Antibacterials with Improved Properties

5.1. Modifications of the p-Nitrophenyl Moiety Soon after recognizing that the specific metabolism of the nitrobenzene ring system causes the appearance of partial or total reduction products in the human body, like nitroso-compounds and hydroxylamine-derivatives, capable of causing DNA damage and irreversible aplastic anemia, the interests of the scientific community orientated to the development of CAM derivatives with changes in the p-nitrobenzene moiety, by substituting the nitro group with a number of other electron withdrawing groups (Figure5; compounds 2–6) or by replacement of the whole aromatic system (Figure5; compounds 7–10). Most of them exhibited gentler toxicity, without severe loss of pharmaceutical potency [2,57]. A very potent member in this class of derivatives was the perchloryl analog of CAM (compound 2), with a twofold larger activity than that of CAM. However, this derivative was never introduced in therapy, due to its explosive properties [75]. The only member that received clinical applications was (compound 3). This compound has never been associated with aplastic anaemia, despite its extensive use in human [76]. Nevertheless, its weaker antibacterial activity, compared to CAM, and reversible toxicity on bone marrow and kidneys have currently shifted its use primarily for veterinary. Tevenel (compound 4), although stronger inhibitor of peptide bond formation in vitro than thiamphenicol [16], was also found to be a potent inhibitor of mitochondrial protein synthesis and was never used on humans. Antibiotics 2016, 5, 20 7 of 21 Antibiotics 2016, 5, 20 7 of 21

OH Cl H N Cl O X OH Me Cl OH H N N 1. CAM: X = NO2 Y Cl 2. Perchloryl analog of CAM: X = ClO 3 O 3. Thiamphenicol: X = SO CH 2 3 X OH 4. Tevenel: X = SO2NH2 8. 4-nitropyrrole analog of CAM: X = NO2, Y = H 5. Adamantylmethyl analog of CAM: X= CH2 9. 5-nitropyrrole analog of CAM: X = H, Y = NO2 6. Cetophenicol: X = COCH3

OH Cl OH Cl H H O N O N S N Cl 2 Cl Me S N O O OH O OH 7. Thiophene analog of CAM 10. 1-methylsulfonylpyrrole analog of CAM

FigureFigure 5. 5. AnalogsAnalogs of of CAM CAM derived derived through through modi modificationfication or or replacement replacement of the p-nitrobenzene moiety.moiety.

AnAn effort by Mullen and Georgief to to replace the nitro group of CAM with a bulky nonpolar substituent,substituent, namely namely the the adamantylmethyl adamantylmethyl group group (compound (compound 55),), was was detrimental for for antibacterial activityactivity [[77].77]. ThisThis replacement replacement decreases decreases the hydrophilicitythe hydrophilicity of the of molecule, the molecule, but increases but increases the molecular the molecularsize that negatively size that negatively correlates withcorrelates bacterium with bacterium penetration penetration [78]. Today, [78]. it is Today, easy to it explain is easy thisto explain failure thisfor anfailure additional for an additional reason; recent reason; crystallographic recent crystallographic studies revealedstudies revealed that a bulky that a substituentbulky substituent of the ofbenzene the benzene ring at ring the pat-site the could p-site hampercould hamper stacking stacking of the aromatic of the aromatic ring on C2452ring on of C2452 23S rRNA of 23S [19 rRNA,20]. [19,20].Synthesis of pyrrole or thiophene analogs of CAM has been also reported [57,79]. Some of them are illustratedSynthesis of in pyrrole Figure5 or (compounds thiophene analogs8–10). Compoundof CAM has 10been, in also which reported the p-nitrophenyl [57,79]. Some moiety of them of areCAM illustrated was replaced in Figure by 1-methylsulfonylpyrrole 5 (compounds 8–10). Compound was found 10 to, in exhibit whichin the vitro p-nitrophenylactivity close moiety to that of CAMthiamphenicol, was replaced suggesting by 1-methylsulf that the onylpyrrolep-nitrobenzene was moiety found ofto CAMexhibit is in susceptible vitro activity of replacementclose to that byof thiamphenicol,alternative aromatic suggesting systems. that Althoughthe p-nitrobenzene compound moiety10 was of lessCAM toxic is susceptible than CAM, of it replacement has never had by alternative aromatic systems. Although compound 10 was less toxic than CAM, it has never had therapeutic applications. therapeutic applications. 5.2. Modifications of the 2-Amino-1,3-propanediol Moiety 5.2. Modifications of the 2-Amino-1,3-propanediol Moiety Early efforts to improve the pharmacokinetic properties of CAM indicated that substitutions in theEarly 2-amino-1, efforts to 3-propanediol improve the pharmacokinetic moiety are associated properties with of severeCAM indicated loss of biological that substitutions activity [in2]. theExceptionally, 2-amino-1, three 3-propanediol examples were moiety deviated are associated from this rule. with First, severe various loss hydrolysableof biological esters activity of CAM [2]. Exceptionally,were prepared three (Figure examples6), useful were for deviated . from this rule. They First, were various found hydrolysable to be easily esters hydrolyzed of CAM wereinto freeprepared CAM in(Figure the gastrointestinal 6), useful for oral tract administration. (CAM-palmitate, They compound were found11) orto inbe plasma, easily hydrolyzed , lungs intoand free kidneys CAM (CAM-hemisuccinate, in the gastrointestinal compound tract (CAM-palmitate,12)[80]. Second, compound transformation 11) or in of plasma, the secondary liver, lungs OH andgroup kidneys into a keto(CAM-hemisuccinate, group with consequent compound elimination 12) [80]. of stereoisomerism,Second, transformation led to the of synthesisthe secondary of nicetin OH group(compound into a13 keto), an efficaciousgroup with drug consequent for the treatment eliminat ofion fungous of stereoisomerism, infections of the led skin to [the81]. synthesis Two relative of nicetincompounds, (compound additionally 13), an modifiedefficacious at drug the OHfor the group treatment in position of fungous 3, that infections is compounds of the14 skinand [81].15, Twowere relative found tocompounds, be many times additional morely active modified than CAMat the inOH combating group in position resistant 3, strains that is overexpressingcompounds 14 andacetyltransferase 15, were found activity to be [82 many,83]. The times high more efficacy active of compoundsthan CAM 14in andcombating15 may resistant be attributed strains to overexpressingthe modifications acetyltransferase at positions 1 andactivity 3 of [82,83]. CAM that The preventhigh efficacy enzymatic of compounds inactivation 14 and of the 15 drugmay be by attributedacetyl- and to phospho-transferases. the modifications at positions 1 and 3 of CAM that prevent enzymatic inactivation of the drugBulky by substitutions acetyl- and phospho-tr at positionsansferases. 1 and 3 lead to an opposite effect. For instance, we recently prepared a CAM-polyamineBulky substitutions conjugate at positions by introducing 1 and a3 lead to an molecule opposite into effect. position For 3 instance, of the 1,3-propanediol we recently preparedbackbone a ofCAM-polyamine CAM, following conjugate oxidation by introducing of the primary a spermine hydroxyl molecule group into (compound position 316 of)[ the73 1,3-,84]. propanediolWe anticipated backbone that the of conjugatedCAM, following polyamine oxidat portionion of the could primary enhance hydroxyl the binding group (compound of CAM to 16 the) [73,84].ribosome, We byanticipated mimicking that the the function conjugated of an polyamine analogous portion cationic could center enhance found the by binding crystallography of CAM to to thecoordinate ribosome, the by interaction mimicking between the function the OHof an group analogous at position cationic 3 center of CAM found and by nucleotide crystallography U2506 to in coordinate the interaction between the OH group at position 3 of CAM and nucleotide U2506 in D. radiodurans [18]. Unfortunately, the activity of compound 16 was found lower than expected. Our failure can be explained by a more recent crystallographic study conducted in E. coli ribosome [19]; Antibiotics 2016, 5, 20 8 of 21

D. radiodurans [18]. Unfortunately, the activity of compound 16 was found lower than expected. OurAntibiotics failure 2016can, 5 be, 20 explained by a more recent crystallographic study conducted in E. coli ribosome8 of 21 [19 ]; ˝ thethe new new orientation orientation of CAMof CAM is rotated is rotated by 180by 180°,, thus thus directing directing the the OH OH group group in positionin position 3 of 3 CAMof CAM away fromaway U2506 from (Figure U25064B). (Figure Noteworthy, 4B). Noteworthy, CAM efficacy CAM is decreased efficacy byis decreased mono-acetylation by mono-acetylation (compound 17) and(compound nearly disappeared 17) and nearly with disappeared di-acetylation with (compound di-acetylation18)[ (compound85]. 18) [85].

Cl OH H O Cl N H N Cl Cl O O N OR O 2 O2N

11. CAM-palmitate: R = CO(CH2)14CH3 15. α-dichloroacetamido-α-methylene-p-nitroacetophenone 12. CAM-hemisuccinate: R= CO(CH2)2COOH

Cl OH Cl O H H N N Cl Cl O H O N O2N X O2N O N N NH2 H H 13. Nicetin: X = OH 14. Bromo-nicetin: X = Br 16. CAM-spermine

1 Cl OR H N Cl O O2N OR2

1 2 17. 3-acetyl-CAM: R = H, R = COCH3 1 2 18. 1,3-diacetyl-CAM: R = R = COCH3

FigureFigure 6. 6.Derivatives Derivatives of of CAM CAM modifiedmodified at the 2-amino-1,3-propanediol 2-amino-1,3-propanediol moiety. moiety.

5.3. Modifications at Both the p-Nitrophenyl and the 2-Amino-1,3-propanediol Moieties 5.3. Modifications at Both the p-Nitrophenyl and the 2-Amino-1,3-propanediol Moieties The necessity for avoiding both toxic effects and resistance to CAM led to the synthesis of The necessity for avoiding both toxic effects and resistance to CAM led to the synthesis of (Figure 7, compound 19). Florfenicol was prepared from thiamphenicol by replacing the florfenicolOH group (Figure in position7, compound 3 by a fluorine19). atom. Florfenicol Florfenicol was was prepared found fromto be a thiamphenicol broad spectrum by antibiotic replacing thein OH vitro group, with activity in position similar 3 byor stronger a fluorine than atom. those of Florfenicol CAM and thiamphenicol was found to [83,86–88]. be a broad However, spectrum antibioticthe mostin interesting vitro, with pharmaceutical activity similar features or stronger of flor thanfenicol those were of first, CAM its andability thiamphenicol to combat CAM- [83, 86and–88 ]. However,thiamphenicol-resistant the most interesting strains pharmaceutical bearing features that possess of florfenicol cat gene wereand second, first, its a more ability favorable to combat CAM-toxicity and profile thiamphenicol-resistant than CAM, due to the strains lack of bearing an aromatic plasmids nitro that group possess [83,88].cat Ingene fact, florfenicol and second, cannota more favorabledeal with toxicity strains profilebearing thanresistance CAM, genes, due like to the floR lack, encoding of an protein aromatic components nitro group of drug [83, 88exporters]. In fact, florfenicol[46,47,89–92], cannot or deal mutations/modifications with strains bearing of resistance the target genes, [93]. Due like tofloR its, encodingfavorable proteinpharmacokinetics, components of drugflorfenicol exporters like CAM [46,47 penetrates,89–92], or well mutations/modifications into CSF of calves, appearing of the an target availability [93]. Dueof 46% to itsrelative favorable to ,plasma. Therefore florfenicol it has been like introduced CAM penetrates to treat meni wellngitis into CSFin cattle of calves, and pigs appearing [94]. It is an expected availability that of 46%florfenicol relative to use plasma. will extend Therefore to huma it hasn medicine been introduced in the near to future. treat meningitis in cattle and pigs [94]. It is expected that florfenicol use will extend to human medicine in the near future. 5.4. Modifications at the Dichloroacetyl Moiety 5.4. ModificationsThe dichloroacetyl at the Dichloroacetyl moiety of CAM Moiety is essential for antibacterial potency [2]. However, numerous studiesThe dichloroacetyl have been published moiety in of which CAM CAM is essential is modified for antibacterial by substituting potency this portion [2]. However, of the molecule. numerous studiesEarly have studies been investigated published inthe which incidence CAM of is replacing modified the by substitutingdichloroacetyl this moiety portion by of a themono- molecule. or polysubstituted with F, Br, or I (Figure 7, compounds 20–23) [2,86,87,95–97]. All of them Early studies investigated the incidence of replacing the dichloroacetyl moiety by a mono- or were found less active than CAM. Interestingly, compounds 21 and 22 with a haloacyl tail are polysubstituted with F, Br, or I acetyl group (Figure7, compounds 20–23)[2,86,87,95–97]. All of chemically reactive and have been employed as affinity probes of the CAM binding site in ribosomes them were found less active than CAM. Interestingly, compounds 21 and 22 with a haloacyl tail are [2]. Compound 22 was found to be an irreversible inhibitor of CAM acetyltranferase and used as chemicallyaffinity probe reactive of andthe havecatalytic been site employed of this asenzyme affinity [97]. probes Compound of the CAM 23 is binding a fluorinated site in ribosomes analog of [2]. Compoundflorfenicol22 withwas less found activity to be than an irreversibleCAM or florfenicol inhibitor [86,87]. of CAM acetyltranferase and used as affinity probe of the catalytic site of this [97]. Compound 23 is a fluorinated analog of florfenicol with less activity than CAM or florfenicol [86,87]. Antibiotics 2016, 5, 20 9 of 21 Antibiotics 2016, 5, 20 9 of 21

FigureFigure 7. 7. FlorfenicolFlorfenicol and and derivatives derivatives of of CAM CAM modified modified at at the the dichloroacetyl moiety.

Prompted byby thethe structural structural similarity similarity of of CAM CAM to theto the 31-terminus 3′-terminus of aminoacyl- of aminoacyl- or peptidyl-tRNA, or peptidyl- tRNA,manyinvestigators many investigators synthesized synthesized various (amino)acyl- various (amino)acyl- and peptidyl-analogs and peptidyl-analogs of CAM and of evaluated CAM and the evaluatedbiological activitythe biological of the conjugatesactivity of the [23, 98conjugates–106]. Harza [23,98–106].et al. synthesized Harza et aal. series synthesized of amides a series derived of amidesfrom CAM derived base from and cholicCAM acidbase orand deoxycholic cholic acid acidor deoxycholic [98]; one of acid them, [98]; compound one of them,24, iscompound illustrated 24 in, isFigure illustrated7. It was in expected Figure 7. that It was these expected derivatives that of these CAM derivatives could easily of penetrate CAM could the bacterialeasily penetrate membranes the bacterialand inhibit membranes cell growth. and Although inhibit cell active growth. against Although various active Gram-positive against various bacteria, Gram-positive their potency bacteria, was theirmuch potency less than was that much of CAM. less than that of CAM. The synthesis of aminoacyl and peptidyl conjugates of CAM was originally started in order to enrich the construct with additional binding sites, through the introduction of amino and carboxyl functions. However, However, none of them them was was found found to to have have superior superior activity activity than than CAM, CAM, particularly particularly inin vivo vivo.. Some of the most active conjugates are illustrated in Figure8 8.. Correlation of the inin vitro and in vivo activities led to the conclusion that steric properties of the acyl moiety significantly influence the antibacterial potency of the constructs [[2].2]. Nevertheless, Nevertheless, these these efforts had a great contribution in understandin understandingg the mode of action of CAM at a time when information from from crystallographic crystallographic works works was was missing. missing. More More specifically, specifically, the the group group of of Vince Vince using using a seta set of ofanalogs analogs of ofpuromycin puromycin and and CAM CAM proposed proposed that that the theamino-acylated amino-acylated tail tailof CAM of CAM simulates simulates the aminoacyl-tailthe aminoacyl-tail of tRNA of tRNA placed placed at the at A-site the A-site in a inparallel a parallel orientation, orientation, a hy apothesis hypothesis extended extended to the to suggestionthe suggestion that thatthe dichloroacetyl the dichloroacetyl tail of tail CAM of CAM points points away away from fromthe exit the tunnel, exit tunnel, towards towards the CCA the endCCA of end an incoming of an incoming aminoacyl-tRNA aminoacyl-tRNA [100,101]. [100 Based,101]. Basedon this on proposal this proposal and the and D. radiodurans the D. radiodurans model [18],model Johansson [18], Johansson et al. designedet al. designed a set of anucleotide set of nucleotide CAM conjug CAMates conjugates that met little that success met little (Figure success 8, compounds(Figure8, compounds 27 and 28) [104].27 and Another28)[104 CAM]. Another analog CAM synthesized analog synthesizedin the framework in the of framework this study, CAM- of this pyrenestudy, CAM-pyrene conjugate (compound conjugate 29 (compound), was found29 ),to was protect found U2506 to protect from CMCT U2506 modification. from CMCT modification. As U2506 is knownAs U2506 to be is knowna footprint to be signal a footprint of CAM signal bind ofing, CAM the binding,authors supposed the authors that supposed compound that 29 compound may bind to29 themay drug bind site. to theHowever, drug site. other However, expected other interactions expected between interactions compound between 29 and compound the ribosome29 and were the notribosome clearly were demonstrated, not clearly nor demonstrated, the effect on nor the thegrow effectth of on bacterial the growth cells ofwas bacterial further cells investigated. was further In contrast,investigated. Bhuta In et contrast, al. using Bhuta anotheret al. setusing of aminoacyl-CAM another set of aminoacyl-CAM analogs proposed analogs that proposed CAM binds that to CAM the ribosomebinds to the in a ribosome retro-inverso in a orientationretro-inverso toorientation the aminoacyl-tRNA to the aminoacyl-tRNA [23]. This hypothesis [23]. This that hypothesis is compatible that withis compatible recent crystallographic with recent crystallographic studies [19,20] studies was adopted [19,20] wasby other adopted groups by otherto explain groups in toa molecular explain in basis,a molecular the activity basis, of the aminoacyl activity of and aminoacyl peptidyl and constructs peptidyl of constructs CAM [103,105,106]. of CAM [103 ,105,106]. Antibiotics 2016, 5, 20 10 of 21 Antibiotics 2016, 5, 20 10 of 21

FigureFigure 8. 8.Conjugates Conjugates of ofCAM CAM withwith aminoamino acids, peptides, peptides, nucleotides nucleotides and and pyrene. pyrene.

Some of the pentapeptidyl conjugates of CAM synthesized by Mamos et al. showed strong Some of the pentapeptidyl conjugates of CAM synthesized by Mamos et al. showed strong affinity for the binding site of CAM (e.g. compound 32). More interestingly, compound 33 was found affinity for the binding site of CAM (e.g. compound 32). More interestingly, compound 33 was found to orientate its peptidyl-chain into the ribosomal tunnel and establish interactions with the region to orientate its peptidyl-chain into the ribosomal tunnel and establish interactions with the region surrounding nucleotide A752 in domain II of 23S rRNA [106]. Similar interactions of a series of surroundingstalling peptides nucleotide with this A752 region in domainof the exit II tunnel of 23S have rRNA been [106 found]. Similar to inactivate interactions allosterically of a seriesthe A- of stallingsite of peptidesPTase [107]. with Recent this regionstudies ofhave the been exit focused tunnel haveon a special been found class of to antimicrobial inactivate allosterically peptides, the the A-siteproline-rich of PTase antimicrobial [107]. Recent peptides, studies which have beenare actively focused transported on a special inside class the of bacterial antimicrobial cell, bind peptides, into thethe -rich exit tunnel antimicrobial of the ribosome peptides, and influence which are the actively function transported of the A-site inside [108]. the Therefore, bacterial lessons cell, bind intolearned the exit from tunnel both of studies the ribosome could promote and influence the develop the functionment of new of the drugs, A-site espe [108cially]. Therefore, those targeting lessons learnedGram-negative from both bacteria. studies could promote the development of new drugs, especially those targeting Gram-negative bacteria. Antibiotics 2016, 5, 20 11 of 21 Due to the cellular origin of polyamines (PAs), numerous derivatives and analogs of PAs have been synthesized for medicinal purposes. An advantage of these compounds is that they are Due to the cellular origin of polyamines (PAs), numerous derivatives and analogs of PAs have been internalized into the bacterial cell by destabilizing the liposaccharide layer of the outer membrane synthesized for medicinal purposes. An advantage of these compounds is that they are internalized [109] and penetrating the bacterial inner membrane through specific uptake systems [110]. The into the bacterial cell by destabilizing the liposaccharide layer of the outer membrane [109] and eukaryotic cells also have available specific polyamine transporters, however their molecular penetrating the bacterial inner membrane through specific uptake systems [110]. The eukaryotic demands for selective delivery of PAs differ. Therefore, conjunction of CAM with PAs could facilitate cells also have available specific polyamine transporters, however their molecular demands for the import of constructs into the cells. However, the role of PAs not merely serves as means of a selective delivery of PAs differ. Therefore, conjunction of CAM with PAs could facilitate the import of “Trojan Horse”; PAs could provide the constructs with additional binding sites through their amino constructs into the cells. However, the role of PAs not merely serves as means of a “Trojan Horse”; functions or other incorporated groups. The above information combined with the observation that PAs could provide the constructs with additional binding sites through their amino functions or other PAs are implicated in the binding of CAM to bacterial ribosome [17], motivated us to design and incorporated groups. The above information combined with the observation that PAs are implicated synthesize recently a series of CAM-PA conjugates [73,84]. The most potent members of this series in the binding of CAM to bacterial ribosome [17], motivated us to design and synthesize recently a are shown in Figure 9 (compounds 34 and 35). Compounds 34 and 35 were synthesized by attaching series of CAM-PA conjugates [73,84]. The most potent members of this series are shown in Figure9 the CAM base on N4 and N1 positions of the N8,N8-dibenzylspermidine (Bn2SPD), respectively, via a (compounds 34 and 35). Compounds 34 and 35 were synthesized by attaching the CAM base on N4 succinate1 (Su) linker. 8 8 and N positions of the N ,N -dibenzylspermidine (Bn2SPD), respectively, via a succinate (Su) linker.

Figure 9. CAM-polyamine conjugates.

Uptaken bybyE. E. coli colicells cells through through the SPD-preferential the SPD-preferential transport transport system, bothsystem, compounds both compounds decreased decreasedthe protein the and protein PA content and PA of content the cells of [ the84]. cells The [84]. antibacterial The antibacterial activity ofactivity compounds of compounds34 and 3534 wasand 35comparable was comparable or superior or tosuperior that of CAM, to that particularly of CAM, against particularly CAM-resistance against CAM-resistance strains. Moreover, strains. these Moreover,compounds these exploiting compounds the highly exploiting active the PA-transporters highly activein PA-transporters cancer cells [111 in] andcancer their cells capability [111] and to theirrecognize capability the ionic to surfacerecognize of mitochondria,the ionic surface were of toxic mitochondria, against HS-Sultan were toxic and against Jurkat cells HS-Sultan and human and Jurkatmesothelioma cells and cells human ZL34. mesothelioma Instead, their cells toxicity ZL34. against Instead, healthy their human toxicity cells against was lowhealthy to negligible human cells [73]. wasKinetic low and to negligible footprinting [73]. analysis Kinetic disclosed and footprinting that the CAM-scaffold analysis disclosed of the that constructs the CAM-scaffold binds to the A-siteof the constructsof PTase, while binds the to PA-tailthe A-site interacts of PTase, with while nucleotides the PA-tail U2585 interacts and U2586 with of nucleotides 23S rRNA thatU2585 interfere and U2586 with ofthe 23S rotary rRNA motion that interfere of the 31 -terminuswith the rotary of aminoacyl-tRNAs motion of the 3′-terminus (Figure 10 ).of Current aminoacyl-tRNAs efforts by our (Figure research 10). Currentteam to improveefforts by the our lead research compound team to34 improveled to a slightthe lead strengthening compound 34 of theled targetingto a slight properties, strengthening but offailed the totargeting improve theproperties, antibacterial but potencyfailed to of impr the constructove the (unpublishedantibacterial results),potency thusof the emphasizing construct (unpublishedthe need to touch results), up the thus internalization emphasizing procedure. the need to touch up the internalization procedure. Antibiotics 2016, 5, 20 12 of 21

Antibiotics 2016, 5, 20 12 of 21

FigureFigure 10. 10.Binding Binding positionspositions of compounds 3434 andand 3535 inin the the EscherichiaEscherichia coli coli ribosome,ribosome, as asdetected detected by by kinetickinetic analysis, analysis, footprinting footprinting assays, assays, and and MD MD simulations simulations [ 73[73].]. ((AA)) CompoundCompound 3434 (in(in green)green) bindsbinds toto the ribosomalthe ribosomal A-site A-site through through its CAM its scaffold,CAM scaffold, while the while dibenzyl-PA the dibenzyl-PA tail makes tailπ -stackingmakes π-stacking interactions withinteractions nucleotides with U2585 nucleotides and U2586 U2585 of and 23S U2586 Rrna; (ofB) 23S Compound Rrna; (B)35 Compound(in green) 35 exhibits (in green) a similar exhibits pattern a ofsimilar interactions pattern with of theinteractions A-site but with the the dibenzyl-PA A-site but tail the stacks dibenzyl-PA only on tail U2586, stacks thus only behaving on U2586, as a thus weaker inhibitorbehaving of as peptide-bond a weaker inhibitor formation. of peptide-bond formation.

6. CAM Hybrids and Dimers 6. CAM Hybrids and Dimers

6.1.6.1. CAM CAM Hybrids hybrids Segments from two antibiotics covalently connected into one molecule and inhibiting dissimilar Segments from two antibiotics covalently connected into one molecule and inhibiting dissimilar targets in the ribosome named hybrid antibiotics; and may show improved properties including targets in the ribosome named hybrid antibiotics; and may show improved properties including enhanced affinity for the target, reduced potential for generating drug-resistance, elevated activity enhanced affinity for the target, reduced potential for generating drug-resistance, elevated activity against drug-resistance strains and broader spectrum of activity. against drug-resistance strains and broader spectrum of activity. Sparsophenicol (Figure 11, compound 36) was the first CAM hybrid that was synthesized and Sparsophenicol (Figure 11, compound 36) was the first CAM hybrid that was synthesized and evaluated [112]. It strongly inhibited the puromycin reaction (Ki = 3 μΜ) but failed to inhibit the µ evaluatedpoly(Phe) [ 112synthesis]. It strongly and the inhibitedgrowth of thebacterial puromycin cells, including reaction E. (K colii = strains. 3 M) butIt was failed suggested to inhibit that the poly(Phe)sparsophenicol synthesis is capable and the of growth inhibiting of bacterialthe synthesis cells, of including only a singleE. coli peptidestrains. bond It was but suggestedis unable to that sparsophenicolpenetrate the bacterial is capable envelope, of inhibiting as it is the less synthesis lipophilic of than only CAM. a single Sparsophenicol peptide bond was but devoid is unable of to penetratein vitro antitumor the bacterial activity envelope, when tested as it in is inhibiting less lipophilic the growth than of CAM. murine Sparsophenicol L1210 was cells devoid [112]. of inWhether vitro antitumor the latter activity finding when is related tested to inthe inhibiting low lipophilic the growth character of murineof sparsophenicol leukemia L1210or inability cells [of112 ]. Whetherthis agent the to latter target finding the eukaryotic is related tocell the wa lows never lipophilic explored. character Another of sparsophenicol CAM hybrid orcombining inability of thisstructural agent to features target the of CAM eukaryotic and linc cellomycin, was never named explored. lincophenicol Another (compound CAM hybrid 37), was combining found to structural share featuressimilar ofactivity CAM profiles and , with CAM namedin inhibiting lincophenicol the puromycin (compound reaction,37 poly(Phe)), was found synthesis, to share and similar the activitygrowth profiles of bacterial with cells CAM [113]. in inhibiting Both compounds the puromycin 36 and 37 reaction, were not poly(Phe) further investigated synthesis, andas drugs the growth but ofrather bacterial used cells as tools [113 for]. Both the verification compounds of36 theand retro-inverso37 were nothypothesis. further investigated as drugs but rather used as tools for the verification of the retro-inverso hypothesis. 6.2. CAM Heterodimers 6.2. CAMEncouraged Heterodimers by the promising properties of hybrid drugs, the group of Yu designed and synthesizedEncouraged a series by theof heterodimers promising propertiescomprised of hybrid drugs, B, an theaminoglycoside group of Yu antibiotic designed that and synthesizedbinds to the adecoding series of center heterodimers of the ribosome comprised and CAM of neomycin [114]. The B, most an aminoglycosidepotent member of antibiotic them, NC2 that binds(compound to the decoding 38), exhibited center one-order of the ribosome higher affinity and CAM to mo [114del]. 16S The rRNA most than potent that member of neomycin of them, B and NC2 (compoundgood discrimination38), exhibited factor one-order between bacterial higher affinity and human to model A-sites. 16S However, rRNA than the antimicrobial that of neomycin activity B and of NC2 against a panel of was lower than that of neomycin B, probably due to its good discrimination factor between bacterial and human A-sites. However, the antimicrobial activity lower compliance with the Lipinski’s rule of 5 [63]. of NC2 against a panel of pathogenic bacteria was lower than that of neomycin B, probably due to its lower compliance with the Lipinski’s rule of 5 [63]. Antibiotics 2016, 5, 20 13 of 21 Antibiotics 2016, 5, 20 13 of 21

OH NO2 O O OH NO O 2 HN N H N OH H O N N OH H 36. Sparsophenicol 37. Lincophenicol

NH2 O HO HO H H2N H2N O OH NH H 2 N S O OH S O O O2N OH H2N NH2 O OH HO HO O

38. CAM-neomycin heterodimer (NC2)

H2N NH2 HO NO NH2 O 2 O O O O O S S S S O N OH H OH HO NH2 HO OH

NH2 39. CAM- heterodimer

OH HO S

O OH HO O H N O S S S S O N H O N O2N OH 40. CAM- heterodimer

FigureFigure 11.11. CAMCAM hybrids and and heterodimers. heterodimers.

NewNew CAM CAM heterodimers heterodimers with with improved improved antimicrob antimicrobialial properties properties were were recently recently synthesized synthesized and andevaluated evaluated by byBerkov-Zrihen Berkov-Zrihen et al.et al.[115].[115 We]. We prefer prefer to touse use the the term term “heterodimers” “heterodimers” for for these these compounds because they are constructed by connecting two different antibiotics through a linker. compounds because they are constructed by connecting two different antibiotics through a linker. The best two of them are presented in Figure 11 (compounds 39 and 40). Compound 39 was derived The best two of them are presented in Figure 11 (compounds 39 and 40). Compound 39 was derived from tobramycin, an that binds to the decoding region of the ribosome and CAM. It from tobramycin, an aminoglycoside that binds to the decoding region of the ribosome and CAM. It exhibited high activity in inhibiting an in vitro prokaryotic translating system, better than CAM. It exhibited high activity in inhibiting an in vitro prokaryotic translating system, better than CAM. It also exhibited good antibacterial activity, comparable to that of CAM and low sensitivity to CAM alsoacetyltranferase. exhibited good Compound antibacterial 40 was activity, derived comparable from clindamycin, to that of a CAM semi-synthetic and low sensitivitylincosamide to that CAM acetyltranferase.binds to the 50S Compoundribosomal subunit40 was and derived inhibits from early clindamycin, peptide chain a semi-syntheticelongation by interfering lincosamide with that bindsboth to A- the and 50S P- ribosomal sites of the subunit catalytic and center inhibits [116] early and peptideCAM. Compared chain elongation to clindamycin, by interfering compound with 40 both A-was and equally P- sites potent of the as catalytic inhibitor center of the [in116 vitro] and translation CAM. Compared but 3-fold less to clindamycin, potent antibacterial compound compared40 was equallyto CAM. potent The antimicrobial as inhibitor of activity the in of vitro compoundtranslation 40 was but strain 3-fold dependent; less potent in antibacterial the case of Streptococcus compared to CAM.aureus The ATCC51907, antimicrobial S. aureus activity NorA of compoundand Bacillus 40anthracis,was strain it was dependent; more potent in than thecase CAM, of whereasStreptococcus in aureusBacillusATCC51907, subtilis 168,S. E. aureus coli MC1061,NorA Haemophilus and Bacillus influenzae anthracis, andit was S. pyogenes more potent it was worse than CAM, than the whereas parent in Bacillus subtilis 168, E. coli MC1061, Haemophilus influenzae and S. pyogenes it was worse than the parent Antibiotics 2016, 5, 20 14 of 21

compound.Antibiotics Compared 2016, 5, 20 to CAM, compound 40 was 40% less sensitive to CAM-acetyltranferase14 of 21 but 74% more sensitive to CAM–nitroreductase [115]. compound. Compared to CAM, compound 40 was 40% less sensitive to CAM-acetyltranferase but 6.3. CAM74% Homodimersmore sensitive to CAM–nitroreductase [115]. If6.3 ribosome CAM Homodimers possesses two binding sites for an antimicrobial agent and these sites are in close proximity,If homodimerization ribosome possesses two may binding intensify sites the for action an antimicrobial of the agent agent and and improve these sites its are potency in close against resistantproximity, strains. homodimerization This hypothesis may was intensify initially the examined action of the by agent Berkov-Zrihen and improveet its al. potency, who against synthesized homodimersresistant ofstrains. tobramycin This hypothesis and CAM was initially [115]. examined CAM homodimers by Berkov-Zrihen were et al. synthesized, who synthesized by linking bis(2-mercaptoethyl)homodimers of tobramycin ether to CAM and CAM through [115]. a CAM chloroacetyl homodimers linker. were The synthesi intermediatezed by linking CAM bis(2- monomer was thenmercaptoethyl) transformed ether to compoundto CAM through41 (Figure a chloroac 12).etyl linker. The intermediate CAM monomer was then transformed to compound 41 (Figure 12).

FigureFigure 12. 12.CAM CAM homodimers.

Using a similar strategy another CAM homodimer was prepared, possessing a longer linker Using(compound a similar 42). Both strategy compounds, another 41 CAM and 42 homodimer, demonstrated was low prepared, inhibitory possessingactivities in aan longer in vitro linker (compoundprokaryotic42). Bothtranslation compounds, assay and41 withand 42a ,few demonstrated exceptions, poor low inhibitoryantimicrobial activities activities in when an in vitro prokaryoticcompared translation to CAM. assay However, and withhomodimerization a few exceptions, improved poor the antimicrobial tolerance against activities CAM-modifying when compared to CAM.enzymes. However, Better homodimerizationhomodimers were obtained improved by our the research tolerance team in against a recent CAM-modifying study, using linkers enzymes. of varying length and flexibility [6]. The rational design of this series of agents was based on the Better homodimers were obtained by our research team in a recent study, using linkers of varying hypothesis that binding of the first CAM unit to the high affinity site (CAM1) could provide the lengthsecond and flexibility CAM unit [6with]. The high rational local concentration, design of thisthus series facilitating of agents targeting was of based the low on affinity the hypothesis site that binding(CAM2). ofPrerequisite the first for CAM a successful unit to outcome the high was affinity a correctly site (CAM1)adjusted linker, could with provide appropriate the second CAMlength unit with and flexibility. high local The concentration, optimal length thus was calc facilitatingulated from targeting crystallographic of the low models affinity (Figure site 13A) (CAM2). Prerequisite[5,19], while for a the successful role of linkeroutcome flexibility was was a correctlytested by SAR adjusted experiments. linker, Compound with appropriate 43 incorporates length and flexibility.theseThe optimal optimal characteristics;length was it calculatedcontains two from CAM crystallographic base units conjugated models through (Figure a 13p-dicarboxylA) [5,19], while the rolearomatic of linker linker flexibility of six successive was tested carbon by SAR bonds experiments. (Figure 12). Compound Compound 4343 incorporateswas found by these kinetic, optimal footprinting and cross-linking tests to bind simultaneously within both the entrance to the exit tunnel characteristics; it contains two CAM base units conjugated through a p-dicarboxyl aromatic linker (nucleotide C2610) and the ribosomal catalytic center (nucleotide C2452) (Figure 13B). Compared to of six successive carbon bonds (Figure 12). Compound 43 was found by kinetic, footprinting and cross-linking tests to bind simultaneously within both the entrance to the exit tunnel (nucleotide C2610) and the ribosomal catalytic center (nucleotide C2452) (Figure 13B). Compared to CAM, compound 43 Antibiotics 2016, 5, 20 1515 of of 21 21 CAM, compound 43 was three-fold more potent in inhibiting the AcPhe-puromycin synthesis. Moreover,was three-fold it showed more potent similar in antibacterial inhibiting the acti AcPhe-puromycinvity against wild-type synthesis. or MRSA Moreover, strains it of showed S. aureus similar and wildantibacterial type Gram-negative activity against bacteria, wild-type but orsuperior MRSA strainsactivity of againstS. aureus resistantand wild strains type of Gram-negative Pseudomonas aeruginosabacteria, but and superior E. coli. Efforts activity to against improve resistant the cell strainspenetration of Pseudomonas properties aeruginosaof homodimerand E. 43coli. are currentlyEfforts to inimprove progress. the cell penetration properties of homodimer 43 are currently in progress.

Figure 13. Rationaleof of CAM CAM dimers dimers designing designing and and binding binding model model of compound of compound43.(A )43 Two. (A CAM-binding) Two CAM- bindingsites in thesitesEscherichia in the Escherichia coli ribosome: coli ribosome: at the left at side, the CAMleft side, is docked CAM is at docked the entrance at the to entrance the ribosomal to the ribosomalexit tunnel exit (CAM2) tunnel by (CAM2) simulating by simulating its crystallographic its crystallographic position in positionHaloarcula in marismortuiHaloarcula marismortui[5]; at the right [5]; atside, the CAM right isside, docked CAM at theis docked A-site ofat the the catalytic A-site of crevice the catalytic (CAM1) crevice by simulating (CAM1) its by crystallographic simulating its crystallographicposition in Escherichia position coli in[19 Escherichia]. Both molecules coli [19]. areBoth connected molecules by are a putativeconnected linker by a andputative embedded linker and into embeddeda nucleotide into environment a nucleotide taken environment from crystallographic taken from crystallographic data derived from dataEscherichia derived from coli;( BEscherichia) Binding coliposition; (B) Binding of compound position43 (inof blue,compound yellow 43 and (in red) blue, into yellow the Escherichia and red) coli into50S the ribosomal Escherichia subunit, coli 50S as ribosomalderived by subunit, MD simulations as derived [6 ].by MD simulations [6].

Compound 43 transiently displayed 5% toxicity on neutrophils during exposure of human blood Compound 43 transiently displayed 5% toxicity on neutrophils during exposure of human blood cells to 60 μΜ of this compound, peaked at 48 h [6]. In comparison, CAM at the same concentration cells to 60 µM of this compound, peaked at 48 h [6]. In comparison, CAM at the same concentration caused 12% toxicity. Toxicity of compound 43 against human monocytes or lymphocytes was caused 12% toxicity. Toxicity of compound 43 against human monocytes or lymphocytes was negligible. negligible. However, compound 43 at 60μΜ induced 43% apoptosis to Jurkat cells, while CAM was However, compound 43 at 60µM induced 43% apoptosis to Jurkat cells, while CAM was almost inactive almost inactive under the same conditions of treatment. This confers compound 43 a significant under the same conditions of treatment. This confers compound 43 a significant advantage over CAM, advantage over CAM, since the apoptotic behavior of 43 does not allow proliferating cells to since the apoptotic behavior of 43 does not allow proliferating cells to continuously survive. continuously survive. 7. Synopsis and Future Perspectives 7. Synopsis and Future Perspectives The pharmaceutical behavior of CAM has received dithyrambic praise and severe criticism during its stormyThe pharmaceutical history. Its efficacious behavior activity of CAM against has a broadreceived spectrum dithyrambic of pathogenic praise and bacteria severe is hampered criticism duringby adverse its stormy effects causinghistory. hematologicIts efficacious disorders, activity against immunosuppression a broad spectrum and cancerof pathogenic invasion. bacteria As CAM is hamperedis an ancient by microbialadverse metabolite,effects causing genetic hematolo elementsgic conferringdisorders, resistanceimmunosuppression against this and drug cancer have invasion.been retained As CAM by and is an are ancient frequently microbial dispersed metabolite in microbial, genetic communities. elements conferring In parallel, resistance resistance against was thisexpanded drug have by the been misuse retained of CAM by and in the are medical frequently and dispersed veterinary in practice. microbial Currently, communities. CAM isIn indicated parallel, resistancein developed was countries expanded only by forthe themisuse treatment of CAM of seriousin the medical infections, and in veterinary which alternative practice. medicationCurrently, CAMis ineffective is indicated or contraindicated. in developed Forcountries these reasons, only for CAM the hastreatment been modified of serious using infections, various in synthetic which alternativeapproaches medication aiming to optimize is ineffective it pharmaceutical or contraindi profile.cated. For Despite these important reasons, progressCAM has made been tomodified address usingproblems various related synthetic to side approaches effects and aiming resistance to optimize against it CAM pharmaceutical caused by CAM-modifyingprofile. Despite important enzymes, progressthe Achilles’ made heel to address of the new problems synthesized related CAM to side derivatives effects and seems resistance to be theiragainst general CAM inabilitycaused by to CAM-modifyingpenetrate the bacterial enzymes, cell envelope,the Achilles’ coupled heel of with the theirnew synthesized susceptibility CAM to multi-drug derivatives efflux seems pumps. to be theirEngineering general nanolayeredinability to penetrate particles for the specific bacterial CAM cell delivery envelope, is acoupled challenge with for their the future. susceptibility to multi-drugIt is clear efflux from pumps. the past Engineering six decades nanolayered of efforts particles that CAM for specific derivatives, CAM with delivery ideally is a improvedchallenge forpharmaceutical the future. properties are incredibly difficult to be found and that there is an urgent need to Antibiotics 2016, 5, 20 16 of 21 change our current research ideas in developing new drugs. For example, much of the work toward elucidating the molecular basis of the CAM–ribosome interactions and the mechanisms of resistance has been conducted using model organisms. However, it has been recognized that conclusions emerged from such organisms cannot safely extrapolate to clinical pathogens of interest. Therefore, deeper understanding of the biology and the interactions with the drug of a variety of microorganisms is needed. Similarly, exploring how mitoribosomes interact with a small molecule like CAM and how this drug is metabolized in the host organism, will be important for understanding issues related to drug specificity, resistance and side effects; noteworthy, the mechanisms of the irreversible aplastic anemia following administration of CAM has not been completely established. It should be also realized that while crystallographic studies of CAM complexes with empty ribosomes can provide critical insights into the interactions of CAM with the ribosome in crystalline state, the intrinsic dynamics of the ribosome at physiological translation states could lead to additional or alternative binding modes. Determining such structures will be a challenging topic for the future. Finally, we like to mention that combining CAM and proline-rich antimicrobial peptides into a construct would be an elegant strategy to expand the stability and specificity of interactions between the drug and rRNA, thus leading to new and more effective antibiotics.

Acknowledgments: The authors would like to thank the University of Patras for financial support. Author Contributions: C.M.A. and G.P.D. wrote the initial draft of the manuscript. D.A.M., P.C.G. and I.A.V. did the literature search. G.E.P. prepared the crystallographic and MD-simulation figures. D.P. and D.L.K. discussed the initial draft and together produced the final version. Conflicts of Interest: The authors declare no conflict of interest.

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