Chloramphenicol Derivatives as Antibacterial and Anticancer Agents

21 Pages·2016·4.22 MB·English

antibiotics
Review
Chloramphenicol Derivatives as Antibacterial and
Anticancer Agents: Historic Problems and
Current Solutions
GeorgeP.Dinos1,ConstantinosM.Athanassopoulos2,*,DionissiaA.Missiri2,
PanagiotaC.Giannopoulou1,IoannisA.Vlachogiannis1,GeorgiosE.Papadopoulos3,
DionissiosPapaioannou2andDimitriosL.Kalpaxis1,*
1 DepartmentofBiochemistry,SchoolofMedicine,UniversityofPatras,GR-26504Patras,Greece;
[email protected](G.P.D.);[email protected](P.C.G.);[email protected](I.A.V.)
2 LaboratoryofSyntheticOrganicChemistry,DepartmentofChemistry,UniversityofPatras,
GR-26504Patras,Greece;[email protected](D.A.M.);[email protected](D.P.)
3 DepartmentofBiochemistryandBiotechnology,UniversityofThessaly,Ploutonos26,
GR-41221Larissa,Greece;[email protected]
* Correspondence:[email protected](C.M.A.);[email protected](D.L.K.);
Tel.:[phone](C.M.A.);[phone](D.L.K.)
AcademicEditor:ClaudioO.Gualerzi
Received:24February2016;Accepted:24May2016;Published:3June2016
Abstract: Chloramphenicol (CAM) is the D-threo isomer of a small molecule, consisting of
ap-nitrobenzeneringconnectedtoadichloroacetyltailthrougha2-amino-1,3-propanediolmoiety.
CAMdisplaysabroad-spectrumbacteriostaticactivitybyspecificallyinhibitingthebacterialprotein
synthesis. Incertainbutimportantcases, italsoexhibitsbactericidalactivity, namelyagainstthe
three most common causes of meningitis, Haemophilus influenzae, Streptococcus pneumoniae and
Neisseriameningitidis. ResistancetoCAMhasbeenfrequentlyreportedandascribedtoavarietyof
mechanisms. However,themostimportantconcernsthatlimititsclinicalutilityrelatetosideeffects
suchasneurotoxicityandhematologicdisorders. Inthisreview,wepresentpreviousandcurrent
effortstosynthesizeCAMderivativeswithimprovedpharmacologicalproperties. Inaddition,we
highlightpotentiallybroaderrolesofthesederivativesininvestigatingtheplasticityoftheribosomal
catalyticcenter,themaintargetofCAM.
Keywords:chloramphenicol;antibiotics;antibioticresistance;sideeffects;anticanceragents;chemical
synthesis;translation;ribosome;peptidyltransferase;puromycinreaction
1. Introduction
Sixtydecadesofbiochemicalstudiesandmorerecentcrystallographicstudies, haverevealed
themolecularbasisbywhichchloramphenicol(CAM)specificallyinhibitsbacterialproteinsynthesis.
CAMisanoldbroad-spectrumantibioticbutitsmedicalandveterinaryapplicationsasantibacterial
arefullofprosandcons.BesidesresistancetoCAMfrequentlyreported,themajordisinclinedconcerns
fortheclinicaluseofCAMrelatetothesideeffectsinlongantibioticcoursescausingneurotoxicityand
hematologicdisorders. ThishaspromptedmanyinvestigatorstoattemptthesynthesisofnewCAM
derivativeswithimprovedpharmaceuticalproperties. Inthisreview,wediscusshowtheinformation
gainedfromtheseeffortshasadvancedourknowledgeonthemodeofactionofCAMandhowthese
CAM derivatives have contributed to compacting resistant bacteria and addressing side effects of
thedrug.
Antibiotics2016,5,20;doi:10.3390/antibiotics5020020 www.mdpi.com/journal/antibiotics
Antibiotics2016,5,20 2of21
2. ModeofActionofCAM
Antibiotics 2016, 5, 20 2 of 21
UponCAMdiscovery[1],itwasrecognizedthattheD-threoisomer(Figure1)inhibitsbacterial
Upon CAM discovery [1], it was recognized that the D-threo isomer (Figure 1) inhibits bacterial
proteinsynthesis. TheotherstereoisomersofCAM,notoccurringinnature,werefoundinactive[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, were found inactive [2].
O NH
NO2 Cl CHl OH
NO2 OHR HN Cl HOOHH2CNH OH H
R Cl
HO H
H NH COCHCl2 O2N OHR HNOHOCl HOH2C H
CHHO2OHH R O Cl NO2
H NH COCHCl2 O2N OH
A CH2OH B NO2
C
A B C
Figure1.AlternativestructuresforCAM.(A)Fischerprojection(D-threoisomer);(B)Skeletalformula
Figure 1. Alternative structures for CAM. (Α) Fischer projection (D-threo isomer); (Β) Skeletal formula
showingtheconfigurationofthetwostereogeniccenters;(C)Newmanprojection.
showinFgi gtuhree c1o. Anfltiegrunraatitvieo snt roufc ttuhree st wforo C sAteMre. o(Αg)e Fnisicch ceern ptreorjesc; t(iCon) (ND-etwhremo iasnom perro);j e(Βct)i Sokne.l etal formula
showing the configuration of the two stereogenic centers; (C) Newman projection.
TTwwoo bbiinnddininggs istietsesin inri broibsoomsoemsews ewreeirdee nidtiefinetdifibeyde baryl yeeaqrluyi leibqruiuilmibrsituumdi esstu[3d]i,eCs A[3M],1 CanAdMC1A aMnd2,
Two binding sites in ribosomes were identified by early equilibrium studies [3], CAM1 and
CwAithMa2f, fiwniitthy caoffninstiatyn tcsoenqsutaanlttso e2quµaMl taon 2d μ2Μ00 aµnMd, 2r0e0sp μeΜcti,v reelsyp.eRceticveenlytl.y ,Rcercoesnst-llyin, kcirnogsso-lfinCkAinMg otof
CAM2, with affinity constants equal to 2 μΜ and 200 μΜ, respectively. Recently, cross-linking of
CriAboMso mtCoeA srMiisb ootolsa otremibdoefssro omimseoslt ahisteoelbdaa tecfdrte orfmriou mmt htEeh secb hbaeacrctitceehrriiiauummco l EiEsacsnhcdehreitcrhhicieah aicaroc lhic oaaleni adal nHthdae l oatbhracehc taeaerraiclu hmHaaehlaoabllao cbHtieuarimluomblao cctaelriizuemd
hCaAloMbiu2mhaa tllootbhciueamlei znloetcdraa lCinzcAeedM tCo2A tMahte2 t rhaiteb t hoeesn oetrnmatrnaalcnepc eet otpo t tithdheee rreiibxboiotssotomumnalna ple elpp[et4ipd].tei Adexeis tei mtxuinitl nateurl nb[4ni]ne. dAl i[ ns4i]gm. isAliat res bimwinaidlsianrog b bsinedrvinedg
sbiytec rwysastsiat lelo obwgsaresar pvohebydse rinvbeytdh ecbryHy sactlraoylaslrtoacglulorlaagprmahpayhr iys imnin o rtththueei rHHibaaloloosaarocrmucluael ,amo avmreiasrmrliaosprmtpuoii rntrguibiot hsroeibmmoes,a ocomrvoeelri,ld apoepvbienirngld atpihnpeg insgit et[h5e]
macrolide binding site [5] (Figure 2A). Nevertheless, high concentrations of CAM were used by both
m(Faigcurorelid2Ae )b.iNndeivnegrt shietele [s5s], (hFigighucroen 2cAen).t NraetivoenrsthoeflCesAs,M hiwgher ceounsceedntbryatbioonths sotfu CdAieMst owcerroes su-sliendk boyr bbointhd
studies to cross-link or bind the drug at the exit-tunnel, a fact that limited the functional significance
sthtueddierus gtoa ctrtohses-elxinitk-t uorn nbeinl,da tfhaec tdtrhuagt alitm thitee dextiht-etufunnncetli,o an faalcsti gthnaifit cliamnciteedo fthCeA fMun2cbtiionndainl sgigsinteif.icance
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)Deinococcusradiodurans ribosome, as detected by crystallography (PDB ID code 1NJI and

1K01,reQsupietec triveceelyn)tl.y we revealed a second kinetically cryptic binding site for CAM at the nucleotide
FigGu2r6e1 21. oBf itnhdei n50gS p roibsoitsioomnsa lo sfu CbAunMit o(ifn E g. rceoelin, )u sinin gth me u(Ach) Hloawloearr ccounlac emnatrraistmioonrst u(<i 6a0n μdΜ (B) )o fD ae iCnAocMoc cus
radhioodmuoradnims reirb, othsoanm teh, oasse duettileiczteedd p bryev ciroyusstlayl l[o6g].r Aap phlyau (sPibDlBe eIxDp lcaondaeti o1Nn fJoI ra nthdis 1 rKai0s1e,d r aefsfpineicttyi vise ltyh)a.t
QuiterecentlywerevealedasecondkineticallycrypticbindingsiteforCAMatthenucleotide
binding of the dimer to the high affinity site (CAM1) facilitates targeting of a cryptic, low affinity site
G2611ofthe50SribosomalsubunitofE.coli,usingmuchlowerconcentrations(<60µM)ofaCAM
Qu(CitAe Mre2c) evniatl tyh ew seec roenvde eadlgeed oaf tsheec ohonmdo kdiinmeetri.c Calolnys icsrteynptltyic, ebryinthdrionmgy sciinte, af omra CcrAoliMde atht atth bei nndus cleotide
homodaimt tehre, ethntarnantcheo osfe tuhet ileixziet dtupnnreevl, iionu tsulryn [i6n]h.iAbitps lbaiundsiinbgle oef xCpAlaMn a[7ti]o, nwhfoilre tChAisMr aeinsheadncaefsfi nthiet yisthat
G2611 of the 50S ribosomal subunit of E. coli, using much lower concentrations (<60 μΜ) of a CAM
bindingproefmtahteurdei mreelreatsoe thoef hsihgohrta fofilnigiotypespittiedy(Cl-tARNMA1s), farceimliitnaitsecsentta rgofe timngacorofliadec ryapnttiibci,oltoicws a[8ffi]. nitysite
homodimer, than those utilized previously [6]. A plausible explanation for this raised affinity is that
(CAM2N)veviaertthheelessesc, odnued teo ditgs eloowf atfhfienihtyo mforo CdAimMe, rt.hCe CoAnsMis2t ebnintdlyin,ge rsyitteh sreoemmsy ucninli,kaelym tao cprloalyi da ecetnhtaratl bindsat
binding of the dimer to the high affinity site (CAM1) facilitates targeting of a cryptic, low affinity site
theentranceoftheexittunnel,inturninhibitsbindingofCAM[7],whileCAMenhancesthepremature
(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
Antibiotics2016,5,20 3of21
releaseofshortoligopeptidyl-tRNAs,reminiscentofmacrolideantibiotics[8]. Nevertheless,dueto
itslowaffinityforCAM,theCAM2bindingsiteseemsunlikelytoplayacentralroleininhibitionof
proteinsynthesis,anhypothesisalsostrengthenedbythefactthatmostoftheresistancemutationsor
modificatAinotnibisoticcsl u20s1t6e, 5r, 2a0r oundtheCAM1bindingsite[9–12]. 3 of 21
In coronlet rians itnhtiobittihone oCf AprMote2in bsiynndthiensgis, saint eh,ypCoAthMesi1s aslseoe mstrsentgothebneeds tbryo tnhge lfyactr ethleavt manotst foof rthien hibition.
resistance mutations or modifications cluster around the CAM1 binding site [9–12].
Numerous biochemical and structural studies, like competition studies between CAM and small
In contrast to the CAM2 binding site, CAM1 seems to be strongly relevant for inhibition.
tRNA fragments for the A-site of the peptidyl transferase (PTase) center [13], inhibition of the
Numerous biochemical and structural studies, like competition studies between CAM and small
puromyctiRnNrAea fcrtaigomnebntys CfoAr Mthe [1A4-s–i1te7 ]o,f atnhed pcerpytsidtayll lotrganrasfperhaisce a(PnTaalsyes) ecsenotferC [A13M], ibnohiubnitidont oof5 0thSe subunits
from bacpteurrioam[y1c8in– r2e0a]ctdioenm byo nCsAtMra [t1e4d–1t7h],a atndth ceryCstaAllMog1rapbhiinc dainnaglysseist eof lCieAsMw biotuhnind ttoh 5e0Sr isbuobusonimts al A-site
(Figure2Bfr)o.m bacteria [18–20] demonstrated that the CAM1 binding site lies within the ribosomal A-site
CAM(Fihgausreb 2eBe)n. consideredasiso-structuraltobothpuromycinandthe31-endofaminoacyl-tRNA
CAM has been considered as iso-structural to both puromycin and the 3′-end of aminoacyl-
(Figure3)[21].Theinitialobservationinfavorofthisconsiderationwasundertakeninacomputational
tRNA (Figure 3) [21]. The initial observation in favor of this consideration was undertaken in a
study thacotmrpeulatatteiodnaCl sAtuMdy ttohatt hreelatpede pCtAidMe tom thoei epteyptoidfe pmeopiettiyd oyfl p-teRptNidAyl-tbRoNuAn bdoutnod ttho etheP P-s-sititee and the
corresponadndin tghet croarnressiptioonndisntga ttreanosfitpioenp sttiadtee obfo pnepdtifdoer bmonadti ofonrmwaititohna wmitihn aomaicnyola-ctyRl-NtRANA[2 [22]2.].

Figure3.FiSgturruec 3t.u SrteruscotufrCesA ofM C,ApMu, rpoumroymcyincina anndd PPhhee--ttRRNNAAPheP ihne ainn isaon-stirsuoc-tsutrraul cotruiernataltoiornie. ntation.
Nevertheless, this proposal ignored that CAM is essentially an A-site inhibitor. A few years later,
Nevertheless, this proposal ignored that CAM is essentially an A-site inhibitor. A few years
Bhuta et al. [23] oriented the p-nitrophenyl group of CAM and the methylated-tyrosyl moiety of
later,Bhuptuareotmayl.ci[n2 t3o] toher iheyndtreodphthobeicp c-rneivtircoep ohf tehney Al-gsirteo,u sop tohaftC thAe MCOa-NnHd tsheqeumeneceth oyf tlhaete adm-itdyor obosnydlsm oietyof
puromyciinn ptourtohmeyhciynd arnodp ChAoMbi cwcerree vini cteheo ofptphoesiAte- dsiirteec,tsioon.t hTahutst,h CeACMO a-nNdH pusreoqmuyecninc weeorfe trhegeaardmedid obonds
as retro-inverso analogs, which is at variance with a crystallographic model of CAM in complex with
inpuromycinandCAMwereintheoppositedirection. Thus,CAMandpuromycinwereregarded
Deinococcus radiodurans 50S subunit (Figure 2B) [18] but in line with current crystallographic evidence
asretro-inversoanalogs,whichisatvariancewithacrystallographicmodelofCAMincomplexwith
from CAM-50S ribosomal subunit (Figure 4) complexes from Thermus thermophilus and E. coli [19,20].
DeinococcTuhser caodniocedputr oafn rset5ro0-Sinvseursbou onriietn(tFatiigoun roef C2AB)M[ 1in8 r]eblautitoinn tol itnhee swubitshtractue rorfe thnet Acr-syistet aisl lpoagrtriacuplharilcy evidence
fromCAMim-p5o0rStanritb ifo wsoem taakle sinutbo uconnitsi(dFeirgatuiorne t4h)atc oremtrop-ilnevxeresso farnoamlogTs hoef rpmeputsidtehse erxmhoibpiht ihliugsh abniodloEgi.ccaol li[19,20].
Theconceapcttivoitfyr [e2t4r]o. -inversoorientationofCAMinrelationtothesubstrateoftheA-siteisparticularly
In the course of kinetic studies, early evidence suggested that CAM behaves as a simple
importantifwetakeintoconsiderationthatretro-inversoanalogsofpeptidesexhibithighbiological
competitive inhibitor of peptide bond formation, a view compatible with the structural similarity of
activity[24].
CAM with puromycin, a pseudo-substrate of A-site of PTase and the 3′-end of aminoacyl-tRNA
In th(Feigcuoreu 3r)s [e25o,2f6]k. Hinoewtiecvesrt, ubdesiiedse,s ceoamrlpyetietivvied keinnecteicss, uthgegree isst ealdso tehvaidtenCcAe fMor nboenhcoamvepsetiatisvea simple
competiti[v15e] oinr hmiibxeitdo-nroonfcopmeppettiidtivee btyopned off ionrhmibiatitoino nm,eachvanieiswm [c1o4,m16p,2a7t],i bdelepewnditinhg tohne thset rbuucffteur rioanlics imilarity
ofCAMwcointdhitpiounsr oumseydc, inth,ea ipnhsiebuitdoro -csounbcesnttrraatteiono,f tAhe- siptree-oinfcPubTaatisoen aenfdfectth aen3d1 -tehned noatfuarem oinf othaec yl-tRNA
substrates. Evidence for the dependence of CAM inhibition kinetics on mono- and divalent cations
(Figure3)[25,26]. However,besidescompetitivekinetics,thereisalsoevidencefornoncompetitive[15]
was provided by high-resolution structures of CAM-ribosome complexes, indicating the involvement
or mixedo-fn coanticoonms mpeedtiiattiivneg tcyoopredionaftiionnh bibonitdioinng bmetewceheann tihsem dr[u1g4 ,a1n6d, 2r7ib]o,sdomepale rnedsiidnuges o[n18,t2h0]e. Tbhuef fer ionic
conditionsused,theinhibitorconcentration,thepre-incubationeffectandthenatureofthesubstrates.
EvidenceforthedependenceofCAMinhibitionkineticsonmono-anddivalentcationswasprovided
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
Antibiotics2016,5,20 4of21
concentrationeffectandthesignificanceofribosomepre-incubationwiththedrugbeforeaddingthe
substratewereunderstoodwhenitwasrealizedthatCAMbehavesasaslow-bindingcompetitive
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
withaninitiatorribosomalcomplex(C)toformtheencountercomplexCIthatisthenisomerized
adding the substrate were understood when it was realized that CAM behaves as a slow-binding
slowlytocoammpoetrietivteig inhhtibciotomr pofl epxepCti*dIe [b1o7n]d. Cfoormnastiisotne,n atcltyin,ga svioa bas tewrov-esedquinenstoiaml reeascttiuodn;i eCsA,Mno (In) croeamctps etitiveor
mixed-noranpciodlmy pweitthit iavne inmitoiadtoers roibfoasocmtioaln ccoomuplledx b(eC)e xtop floarimne tdheb eyntchouenstleor wco-monplseext CinI hthibati tiiso nthetnh eory[28]
accordingisotomewrihzeicdh stlohweliyn thoi bai tmioonrep taigthtet rcnomupnldexe rC“*Ip r[1e7-]i.n Ccuonbsaisttieonntl”y,c oasn dobitsieorvnesdc ainn sboemme sitsuinditeesr, pretedas
noncompetitive or mixed-noncompetitive modes of action could be explained by the slow-onset
indicatingmixed,noncompetitiveinhibition. Regardingtheeffectsofthenatureofthesubstrate,it
inhibition theory [28] according to which the inhibition pattern under “pre-incubation” conditions
wasobservedthattRNAsbearingbulkyaminoacids,likePhe-tRNAPhe arelesspronetoinhibition
can be misinterpreted as indicating mixed, noncompetitive inhibition. Regarding the effects of the
byCAMntahtaunre toRf NtheA ssubcsatrrraytei,n itg wsams oablsleerrvoedr cthhaat rtRgNedAsa bmeainrionga bcuidlksy [a8m,2in9o]. aTcihdse, nlikaet uPrhee-otRfNthAePheP a-rsei tebound
substratelecsas npraolnseo toin iflnhuibeinticoen CbyA CMAMin thhiabni ttoRrNyAas cctairvryitiyn,g asnmaelflfeer cotr ccohnarsgiesdte anmtiwnoi tahcidths e[8c,2r9y].s Ttahlel ographic
nature of the P-site bound substrate can also influence CAM inhibitory activity, an effect consistent
observationthatthep-nitromoietyofCAMisincloseproximitytotheaminoacylmoietyofP-site
with the crystallographic observation that the p-nitro moiety of CAM is in close proximity to the
boundtRNA[19,20].
aminoacyl 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
Figure4.BindingpositionsofCAM(ingreen)inthe(A)Thermusthermophilusand(B)Escherichiacoli
ribosome, as detected by crystallography (PDB ID code 4V7W and 3OFC, respectively).
ribosome,asdetectedbycrystallography(PDBIDcode4V7Wand3OFC,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, such as termination [30], translational accuracy [31] and biogenesis of 50S ribosomal
assembly defects are due to a specific inhibition of the biosynthesis of ribosomal proteins by CAM
subunits[[333]2. ]. Withregardstothelatterstudy, morerecentobservationssuggestedthatribosomal
assemblydefectsareduetoaspecificinhibitionofthebiosynthesisofribosomalproteinsbyCAM[33].
3. Bacterial Survival Strategies to Combat CAM Activity
3. BacterialSThuer vmiivsuasleS otf raantteibgiioetiscst oin Ccloinmicabl aptraCctAiceM anAd cthtiev witiydespread use in agriculture has seriously
contributed to the rise in global resistance to antibiotics. Nevertheless, the microbial world has had
Themisuseofantibioticsinclinicalpracticeandthewidespreaduseinagriculturehasseriously
per se the molecular tools to drive resistance; the antibiotic resistome pre-dates the modern antibiotic
contributeerad btyo mthileliorniss eofi nyegarlos.b Tahlerreefsoirset,a dnecveelotopmaenntti boifo rteiscisst.anNcee vcaenrnthote blee scso,mthpleetmelyic erroabdiicaaltewd oarnldd hashad
persetheism oonllye cau mlaarttetor oofl swthoend. rTivhee arnetsibisiotatinc-crees;istthaencaen ctriibsiiso itsi fcurrethseirs taogmgraevpatreed- dbya ttehse tphaeucmityo odfe nrenwa ntibiotic
erabymailnltiiobniostiocsf iyn ethaer sp.ipTehlineer e[3fo4]r.e ,developmentofresistancecannotbecompletelyeradicatedand
Resistance or decreased sensitivity to CAM has been frequently observed and is mediated by
isonlyamatterofwhen. Theantibiotic-resistancecrisisisfurtheraggravatedbythepaucityofnew
numerous mechanisms. Target mutations or alterations are a usual mechanism of bacterial resistance
antibioticsinthepipeline[34].
to CAM. At least eight mutations in the V domain of 23S rRNA and one methyl-modification of A2503
Resicsotnafnerc ehioghr CdAecMre raessiestdansceen ins ibtiavctietyriat o[9–C12A],M espheacisalblye ewnhefnr eoqccuuernrintlgy ino bmsoesrt voef drRaNnAd oipsermonesd iatedby
numerou(s7 min eEc.h caolni)i. sMmosr.e Tcarurcgiealt amreu mtauttiaotinosnso arnadl/toerr adteiloetniosnas roef aribuossuoamlaml percohteainnsi,s wmhiocfh baarec tuesruiaalllyr esistance
encoded by single gene copies. However, mutations in ribosomal proteins causing CAM-resistance
toCAM.AtleasteightmutationsintheVdomainof23SrRNAandonemethyl-modificationofA2503
have been identified only in a few cases so far [35,36]. Because interactions of CAM with ribosomal
conferhighCAMresistanceinbacteria[9–12],especiallywhenoccurringinmostofrRNAoperons
proteins have not been detected by crystallographic analyses [18,20], such resistance is suspected to
(7inE.coli). Morecrucialaremutationsand/ordeletionsofribosomalproteins,whichareusually
encodedbysinglegenecopies. However,mutationsinribosomalproteinscausingCAM-resistance
havebeenidentifiedonlyinafewcasessofar[35,36]. BecauseinteractionsofCAMwithribosomal
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
combinationofbothribosomalproteinandrRNAmutationsleadtoincreasedresistance;e.g. G-to-A
Antibiotics2016,5,20 5of21
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
Campylobacterjejuni [36]. Nevertheless, such mutations are often accompanied by fitness cost and
consequentlyarenotextensivelyspread.
The most frequently encountered mechanism of bacterial resistance to CAM is the enzymatic
inactivationofthedrugwarheadbydifferenttypesofenzymes,extensivelyreviewedbySchwarzetal.[37].
Although O-phosporylation [38–40], hydrolytic degradation to p-nitrophenylserinol [41,42], and
nitroreductation[43]havebeenreportedpreviously,themajorenzymaticmechanismthatinactivates
CAMisacetylationviaseveraltypesofCAMacetyltransferases(CATs). AllCAMacetyltransferases
sharesimilarmolecularstrategiesinacetylatingCAM.TheO-acetylderivativesofCAMfailtoactas
antibiotics,becausetheydonotbindtobacterialribosomes.
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
throughpore-formingporins[44]. Permeationoftheoutermembranebyantibioticsthroughporins
depends on the molecular dimensions of the drugs [45]. Therefore, perturbations of the outer
membraneorderivatizationofCAMmayreduceitscapacityforinternalizationintothecell. Onthe
other hand, bacteria defend themselves against antibiotics by pumping out the drugs. Export of
CAMfromthebacterialcellcanbemediatedeitherbyspecificand/ormulti-drugeffluxpumps,the
former mediating higher levels of resistance compared to those of nonspecifically driving out the
drug[37]. GenesassociatedwithCAMspecificeffluxpumpshavebeenidentifiedandcharacterized
inawidevarietyofbacteria. Eightdifferentgroupsofspecificexporters,reviewedbySchwarzetal. in
2004[37],areconstantlyenrichedbynewdata[46,47]. CAMisasubstrateofmostmulti-drugefflux
pumps, namely the major facilator superfamily (MPS) [48], the small multi-drug resistance (SMR)
family[49],theresistancenodulationandcelldivision(RND)family[50],theATP-bindingcassette
(ABC)family[51]andthemultidrugandtoxinextrusion(MATE)family[52]. Worthnoting,CAM
mayinduceexpressionofeffluxpumps, suchasMexXYinPseudomonasaeruginosathatbelongsto
RNDfamily[53]orenhancetheeffluxofotherligandsbyAcrBpumpinE.coli[54]. Sucheffects,not
beginningnorendingwiththesespecialexamples,complicatetheuseofCAMforthetreatmentof
bacterialinfections.
4. SideEffectsofCAM
Soon after its discovery and subsequent synthesis, CAM became very popular due to its
antimicrobialeffectivenessandlowcost. However, inthefollowingyearswhenCAMwasissued
for general clinical use, it was realized that CAM can cause hematological disorders like bone
marrow depression and aplastic anemia [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 bone marrow suppression 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),themitochondrialribosomalmachineissubstantiallydifferentfromthatfoundinbacterial
andhumancells[61]. Nevertheless,thecatalyticcoreofPTaseanddecodingcenterarchitectureis
conservedinmitoribosomeandbacterialribosome. ThisexplainswhyCAMiscapableofinhibiting
mitochondrialproteinsynthesis. FurtherstudiesinmiceindicatedthatCAMinhibitsproteinsynthesis
in mitoribosomes of the marrow progenitor cells at the differentiation rather than the replication
stage[62]. Duetoitslipoidalnature(MLogP=1.23),lownumberofOHandNHH-bonddonors(=3)
andlownumberofNandOH-bondacceptors(=7),CAMpenetrateswellinalltissues,includingmany
privilegedsitessuchasthebonemarrowandthebrain[63]. Itremainsrelativelyunboundtoproteins
anditssmallmolecularsize(MW=323.14)facilitatescrossingoftheblood-brainbarrier(BBB)[64].
PenetrationofBBBwasfurtherimprovedwhenoptimizedpalmkerneloilestersnanoemulsion-loaded
with chloramphenicol were used to cross BBM [65]. These properties made the CAM-loaded
Antibiotics2016,5,20 6of21
nanoemulsionsafirst-choicetherapeuticformeningitistreatment. Althoughlessavailableinbrain
andcerebrospinalfluid(CSF)relativetoplasma,CAMcanachievehighlevelsinbrainandCSFafter
prolongeduseandhighcumulativedosethatmaycausedrug-relatedmitochondrialneuropathies[66].
CAMtreatmentoveralongperiodmayalsocauseirreversiblepanmyelopathywhichislethaland
maybeassociatedwithgeneticdefects. Dermatologicalstudiesdisclosedlymphocytesensitization
by chloramphenicol and azidamphenicol in allergic contact dermatitis [67], while experiments in
micesplenocytesrevealedimmunosuppressiveactivityofaCAManalog,florfenicol,ontheimmune
response to stimulators [68]. In neonates and infants lacking glucuronidation reactions (phase II
detoxification)andsufficientrenalcapacityforexcretingunconjugateddrugs,accumulatedtoxicCAM
metabolitescancauseGraysyndrome,adiseasemanifestedbyhypotension,cyanosis,hypothermia,
cardiovascularcollapse,ashengraycoloroftheskinandvomiting[69]. Thereisalsoevidencethat
CAMcausesmitochondrialstressthatinturnpromotesmatrixmetalloproteinase-13-associatedcancer
cellinvasion[70]. Duetotheaboveharmfuleffects,theclinicaluseofCAMiscurrentlylimitedin
developedcountriestoinfectionsnotrespondingtootherantibiotics. Nevertheless,arecentstudy
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
cancerstemcells,acrossmultipletumortypes,byrepurposingantibioticsforanti-cancertherapy[71].
Thissuggestionwasbasedonthehypothesisthatcancerstemcellsarestronglyanabolicandthey
mayrequirerobustmitochondrialbiogenesisandactivityforsurvivalandproliferativeexpansion.
Indeed,treatmentofMCF7cellswithCAMcausedinhibitionoftumor-sphereformationwithanIC
50
ofapproximately200µM.Consistently,previousinvitrostudieshaveindicatedthatCAMalone,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],itisclearthatadditionalcellularstudiesusingdifferentsolidtumorcelllinesand
gradeddosesofCAMareneededtoexplorethepotentiallybeneficialroleofCAMincancertherapy.
Todefinewhichregionsoftheparentmoleculeareessentialforantibacterialactivityorstrongly
associatedwithtoxicity,numerousCAManalogshavebeensynthesizedandbiologicallyevaluated.
For the sake of brevity, we present below only the most important of them, particularly those
investigatedasdrugs.
5. ModificationsofCAMtoObtainAntibacterialswithImprovedProperties
5.1. Modificationsofthep-NitrophenylMoiety
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
andhydroxylamine-derivatives,capableofcausingDNAdamageandirreversibleaplasticanemia,
the interests of the scientific community orientated to the development of CAM derivatives with
changesinthep-nitrobenzenemoiety,bysubstitutingthenitrogroupwithanumberofotherelectron
withdrawing groups (Figure 5; compounds 2–6) or by replacement of the whole aromatic system
(Figure5;compounds7–10).
Most of them exhibited gentler toxicity, without severe loss of pharmaceutical potency [2,57].
AverypotentmemberinthisclassofderivativeswastheperchlorylanalogofCAM(compound2),
withatwofoldlargeractivitythanthatofCAM.However,thisderivativewasneverintroducedin
therapy, due to its explosive properties [75]. The only member that received clinical applications
wasthiamphenicol(compound3). Thiscompoundhasneverbeenassociatedwithaplasticanaemia,
despiteitsextensiveuseinhuman[76]. Nevertheless,itsweakerantibacterialactivity,comparedto
CAM,andreversibletoxicityonbonemarrowandkidneyshavecurrentlyshifteditsuseprimarilyfor
veterinary. Tevenel(compound4),althoughstrongerinhibitorofpeptidebondformationinvitrothan
thiamphenicol[16],wasalsofoundtobeapotentinhibitorofmitochondrialproteinsynthesisandwas
neverusedonhumans.
Antibiotics2016,5,20 7of21
Antibiotics 2016, 5, 20 7 of 21
OH Cl
H
N
Cl
O
X OH Me OH Cl
H
N N
1. CAM: X = NO2 Y Cl
2. Perchloryl analog of CAM: X = ClO
3 O
3. Thiamphenicol: X = SO2CH3 X OH
4. Tevenel: X = SO NH
2 2 8. 4-nitropyrrole analog of CAM: X = NO , Y = H
5. Adamantylmethyl analog of CAM: X=CH 2
2 9. 5-nitropyrrole analog of CAM: X = H, Y = NO
6. Cetophenicol: X = COCH 2
3
OH Cl OH H Cl
H
S N O N
O2N Cl Me S N Cl
O
O
OH O OH
7. Thiophene analog of CAM 10. 1-methylsulfonylpyrrole analog of CAM
FFigiguurer e5.5 A.Annalaologgs soof fCCAAMM ddeerirviveedd ththrroouugghh mmooddiiffiiccaattiioonn oorr rreeppllaacceemmeenntt ooff tthhee pp--nniittrroobbeennzzeennee mmooiieettyy..
An effort by Mullen and Georgief to replace the nitro group of CAM with a bulky nonpolar
An effort by Mullen and Georgief to replace the nitro group of CAM with a bulky nonpolar
substituent, namely the adamantylmethyl group (compound 5), was detrimental for antibacterial
substituent, namely the adamantylmethyl group (compound 5), was detrimental for antibacterial
activity [77]. This replacement decreases the hydrophilicity of the molecule, but increases the
activity[77].Thisreplacementdecreasesthehydrophilicityofthemolecule,butincreasesthemolecular
molecular size that negatively correlates with bacterium penetration [78]. Today, it is easy to explain
sizethatnegativelycorrelateswithbacteriumpenetration[78]. Today,itiseasytoexplainthisfailure
this failure for an additional reason; recent crystallographic studies revealed that a bulky substituent
for an additional reason; recent crystallographic studies revealed that a bulky substituent of the
of the benzene ring at the p-site could hamper stacking of the aromatic ring on C2452 of 23S rRNA
benzeneringatthep-sitecouldhamperstackingofthearomaticringonC2452of23SrRNA[19,20].
[19,20].
SynthesisofpyrroleorthiopheneanalogsofCAMhasbeenalsoreported[57,79]. Someofthem
Synthesis of pyrrole or thiophene analogs of CAM has been also reported [57,79]. Some of them
areillustratedinFigure5(compounds8–10). Compound10,inwhichthep-nitrophenylmoietyof
are illustrated in Figure 5 (compounds 8–10). Compound 10, in which the p-nitrophenyl moiety of
CAMwasreplacedby1-methylsulfonylpyrrolewasfoundtoexhibitinvitroactivityclosetothatof
CAM was replaced by 1-methylsulfonylpyrrole was found to exhibit in vitro activity close to that of
thiamphenicol,suggestingthatthep-nitrobenzenemoietyofCAMissusceptibleofreplacementby
thiamphenicol, suggesting that the p-nitrobenzene moiety of CAM is susceptible of replacement by
alternative aromatic systems. Although compound 10 was less toxic than CAM, it has never had
alternative aromatic systems. Although compound 10 was less toxic than CAM, it has never had
therapeuticapplications.
therapeutic applications.
5.2. Modificationsofthe2-Amino-1,3-propanediolMoiety
5.2. Modifications of the 2-Amino-1,3-propanediol Moiety
Early efforts to improve the pharmacokinetic properties of CAM indicated that substitutions
in thEea2rl-ya mefifnoort-s1 ,to3 -ipmrporpoavnee dthieo lpmhaorimetaycoakreinaestisco pciraotpedertwieist hofs CevAeMre ilnodsiscaotfedb itohloatg sicuablsaticttuitviiotnys[ i2n] .
tEhxec e2p-taimoninaloly-1,,t h3r-epereoxpaamnepdleisolw meroeideteyv iaatreed afsrosomcitahtiesdr uwlei.thF irssetv,evraer iolousssh oyfd rboiloylsoagbilceael staecrtsivoiftyC A[2M].
Ewxecreepptiroenpaalrlyed, th(Freigeu erxeam6),pulesse wfuelrfeo dreovriaalteadd mfroinmis tthraist irounl.e.T Fhiresyt,w vaerrieofuosu hnyddrtoolbyesaebalsei leystherysd orfo ClyAzeMd
winetoref rpereepCaAreMd i(nFitghuergea 6s)t,r ouisnetfeuslt ifnoarl otrraacl ta(dCmAiMni-sptraaltmiointa. tTe,hceoym wpeoruen fdou1n1d)o troi nbep elaassmilya ,hlyivderro,llyuznegds
ianntdo fkrieden CeyAsM(C iAn Mth-eh gemasitsruoicnctinesattien,aclo tmrapcto u(CnAdM12-)p[a8l0m].itSaeteco, cnodm,tpraonusnfdo r1m1)a toiro nino pfltahsemsae,c loinvedra,r lyunOgHs
agnrodu kpidinnteoyas k(CetAoMgr-ohuepmwisiuthcccinonatsee,q cuoemntpeoluimndin 1at2i)o [n80o]f. sSteerceoonidso, mtraernissfmor,mleadtitoont hoef styhne tsheecsoisndofarnyic OetHin
g(croomupp oiunntod a1 3k),eaton egfrfiocuapci owuisthd rcuognfsoerqtuheenttr eealtimmeinnattoiofnfu onfg osutesrienofiescotmioenrsisomft,h leedsk itno [t8h1e] .sTywnothreeslaist ivoef
ncoicmetpino u(cnodms,paodudnidti o13n)a, lalyn mefofidcaificeiodusa tdtrhueg OfoHr tghreo utrpeaitnmpenots iotifo fnun3g,othuast inisfeccotmionpso uonf dthse1 s4kiann d[811]5.,
Twweroe rfeolautnivdet coobmepmouannydst,i madedsimtioonraellayc tmivoedtihfiaend CatA thMe iOnHco gmrobuapti ning preossiistitoann t3s, ttrhaaint iss ocvoemrepxopurnedsssi n14g
aancedt y1l5tr,a nwsefreer afsoeuancdti vtioty b[e8 2m,83a]n.yT htiemhesig hmeofrfiec aaccytivoef ctohmanp oCuAnMds 1in4 acnodm1b5atminagy rbeesisattatrnitb ustteradintos
othveermexopdriefiscsaintigo nacseattylptroasnitsifoenrass1e aanctdiv3itoyf [C82A,8M3].t hTahte phriegvhe neftfiecnazcyym ofa tciocminpaocutinvdasti o14n aonfdth 1e5 dmruayg bbey
aatctertibyul-taendd top hthoes pmhood-tirfaicnastfieornass east. positions 1 and 3 of CAM that prevent enzymatic inactivation of
the dBruuglk byys uacbesttyitlu-t aionnds pathpoospsihtioo-ntsra1nasnfedra3sleesa.d toanoppositeeffect.Forinstance,werecentlyprepared
aCABMu-lpkoyl ysaumbsintietuctoionnjusg aatte pboysiitniotrnosd u1c ainngda 3s pleearmd itnoe amno olepcpuolesiitnet oefpfeocstit. ioFnor3 ionfstthaenc1e,3, -wpreo preacneendtiloyl
pbraecpkabroende a oCfACMA-Mpo,lyfaomlloinwei ncgonojuxgidataet iboyn inotfrotdheucpinrgim aa srpyerhmyidnreo mxyollegcruoluep in(toco pmopsiotiuonnd 31 o6f) th[7e3 1,8,34-].
propanediol backbone of CAM, following oxidation of the primary hydroxyl group (compound 16)
We anticipated that the conjugated polyamine portion could enhance the binding of CAM to the
[73,84]. We anticipated that the conjugated polyamine portion could enhance the binding of CAM to
ribosome, by mimicking the function of an analogous cationic center found by crystallography to
the ribosome, by mimicking the function of an analogous cationic center found by crystallography to
coordinate the interaction between the OH group at position 3 of CAM and nucleotide U2506 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];
Antibiotics2016,5,20 8of21
D. radiodurans [18]. Unfortunately, the activity of compound 16 was found lower than expected.
OuArnftaibilioutirces 2c0a1n6, 5b,e 20e xplainedbyamorerecentcrystallographicstudyconductedinE.coliriboso8m ofe 2[11 9];
thetnheew neowri eonrtiaentitoantioonf CoAf CMAiMsr oist artoetdatbedy 1b8y0 1˝8,0th°,u tshduisr edcirtiencgtinthge thOeH OgHro gurpouinp pino spitoisointio3no f3 CoAf CMAaMw ay
fromawUay2 50fr6o(mF igUu2r5e046B )(.FNigoutreew 4oBr)t.h yN,oCtAewMoretfhfiyc, acCyAiMsd eecffriecaasceyd ibs ydmecorneaos-eadce tbyyl atmioonno(c-oacmetpyolautniodn 17)
and(cnoemaprloyudnids a1p7p) aenadre ndewariltyh ddisi-aapcpeetyarlaedti ownit(hc odmi-apcoeutynldati1o8n) ([c8o5m].pound 18) [85].
OH H Cl O Cl
N H
Cl N
Cl
O
O2N OR O2N O
11. CAM-palmitate: R = CO(CH ) CH 15. α-dichloroacetamido-α-methylene-p-nitroacetophenone
214 3
12. CAM-hemisuccinate: R= CO(CH ) COOH
22
O H Cl OH H Cl
N N
Cl Cl
O O H
O2N X O2N O HN NH N NH2
13. Nicetin: X = OH
14. Bromo-nicetin: X = Br 16. CAM-spermine
OR1 Cl
H
N
Cl
O
O2N OR2
17. 3-acetyl-CAM: R1 = H, R2 = COCH
3
18. 1,3-diacetyl-CAM: R1 = R 2 = COCH3
FigFuigruer6e. 6D. Dereirviavtaitviveesso offC CAAMM mmooddiififieedd aatt tthhee 22--aammininoo-1-1,3,3-p-prorpoapnaendeidoilo mlmoieotiye.t y.
5.3. Modifications at Both the p-Nitrophenyl and the 2-Amino-1,3-propanediol Moieties
5.3. ModificationsatBoththep-Nitrophenylandthe2-Amino-1,3-propanediolMoieties
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
florfenicol (Figure 7, compound 19). Florfenicol was prepared from thiamphenicol by replacing the
florfenicol (Figure 7, compound 19). Florfenicol was prepared from thiamphenicol by replacing
OH group in position 3 by a fluorine atom. Florfenicol was found to be a broad spectrum antibiotic
the OH group in position 3 by a fluorine atom. Florfenicol was found to be a broad spectrum
in vitro, with activity similar or stronger than those of CAM and thiamphenicol [83,86–88]. However,
antibioticinvitro,withactivitysimilarorstrongerthanthoseofCAMandthiamphenicol[83,86–88].
the most interesting pharmaceutical features of florfenicol were first, its ability to combat CAM- and
Hotwhieavmerp,htehneicmolo-sretsiinsttaenret ssttirnaginps hbaeramrinagc epulatiscmalidfes athtuart epsoossfeflsos rcfaetn giecnoel wanedr eseficrosntd,i, tas maboirleit yfatvoorcaobmleb at
CAtMox-icaintyd pthroiafimle pthhaenn iCcAolM-re, dsiuseta tnot thster alaincks obfe aanri anrgomplaatsicm niidtrsot hgraotuppo [s8s3e,s8s8c].a Itng feancte, falnodrfesneiccoonl dca,nanmoto re
favdoeraalb wleittho xstircaitinys pbreoafirilnegt rheasnistCanAcMe g,ednuese, ltioket hfloeRl,a ecnkcoodfinagn parrootmeina tciocmnpitornoegnrtso uofp d[r8u3g, 8e8x]p.orItnerfsa ct,
flor[f4e6n,4ic7o,8l9c–a9n2n],o otrd meaultawtiiothnss/tmraoidnisfibcaetairoinnsg orfe tshiset atanrcgeetg e[9n3e]s. ,Dliukee tfloo Rits, efanvcoordaibnlge pphroatreminaccookminpeotincse,n ts
ofdflrourgfenexicpool rlitkeer sC[A46M,4 7p,e8n9e–t9ra2t]e,so wrmellu itnattoi oCnSsF/ omf ocdalivfiecsa, taiopnpseaorfintgh eanta arvgaeitla[b9i3li]t.yD ouf e46t%o irteslaftaivvoe rtaob le
phaprlmasamcoak. Tinheetriecsfo,rfleo irtf henasic boelelnik ientCroAdMucpeden toet trraetaets mweenlilnignittoisC inS Fcaotftlcea alnvdes p,iagpsp [9e4a]r.i nItg isa enxapveactieladb tihliatyt of
46%florerflaentiivceolt ouspel awsimll ae.xtTehnedr etofo hreumitahna smbeedeincininet irno dthuec endeatro futrteuartem. eningitisincattleandpigs[94]. Itis
expectedthatflorfenicolusewillextendtohumanmedicineinthenearfuture.
5.4. Modifications at the Dichloroacetyl Moiety
5.4. ModificationsattheDichloroacetylMoiety
The dichloroacetyl moiety of CAM is essential for antibacterial potency [2]. However, numerous
studies have been published in which CAM is modified by substituting this portion of the molecule.
ThedichloroacetylmoietyofCAMisessentialforantibacterialpotency[2]. However,numerous
Early studies investigated the incidence of replacing the dichloroacetyl moiety by a mono- or
studieshavebeenpublishedinwhichCAMismodifiedbysubstitutingthisportionofthemolecule.
polysubstituted with F, Br, or I acetyl group (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 (Figure 7, 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
themwerefoundlessactivethanCAM.Interestingly,compounds21and22withahaloacyltailare
[2]. Compound 22 was found to be an irreversible inhibitor of CAM acetyltranferase and used as
chemicallyreactiveandhavebeenemployedasaffinityprobesoftheCAMbindingsiteinribosomes[2].
affinity probe of the catalytic site of this enzyme [97]. Compound 23 is a fluorinated analog of
Compound22wasfoundtobeanirreversibleinhibitorofCAMacetyltranferaseandusedasaffinity
florfenicol with less activity than CAM or florfenicol [86,87].
probeofthecatalyticsiteofthisenzyme[97]. Compound23isafluorinatedanalogofflorfenicolwith
lessactivitythanCAMorflorfenicol[86,87].
Antibiotics2016,5,20 9of21
Antibiotics 2016, 5, 20 9 of 21

FFiigguurree 77.. FFlloorrffeenniiccooll aanndd ddeerriivvaattiivveess ooff CCAAMM mmooddiiffiieedd aatt tthhee ddiicchhlloorrooaacceettyyll mmooiieettyy..
PPrroommpptteedd bbyy ththees tsrtuructcutruarlasl imsimilairlaitryitoyf oCfA CMAtMo tthoe t3h1-et e3r′m-teinrumsinoufas moifn aomaciynlo-aocrypl-e potri dpyelp-ttRidNyAl-,
tmRaNnAy,i nmveasntyig aintovressstiygnatthoerssi zseydnvthaerisoizuesd( avmairnioou)asc y(al-manindop)aecpytli-d yaln-adn apleopgtsidoyflC-aAnMaloagnsd oefv aCluAaMte datnhde
ebvioallougaitceadl athceti vbiitoyloogfitchael caoctnijvuigtya toefs t[h23e, 9c8o–n1ju06g]a.teHsa [r2z3a,9e8t–a1l.0s6y].n Hthaersziaz eedt aals. esryiensthoefsaizmedid eas sdereireisv eodf
afrmomideCsA dMeribvaesde faronmd cChAolMic abcaisde oarndde cohxoylcich aocliicd aocri dde[9o8x]y;cohnoeliocf atchidem [9,8c]o; monpeo oufn tdhe2m4,,i csoilmlupsotruantedd 2i4n,
iFsi gilulurest7r.aIttedw ains eFxipgeucrtee d7.t hIta twthase seexdpeercitveadt ivtheasto tfhCeAseM decoriuvladtievaessi lyofp CenAeMtra cteouthlde beaacstielyri aplemneetmrabtrea nthees
baancdteirnihali bmitecmelblrgarnoews athn.dA inlthhiobuitg chelal cgtriovweathg.a Ainltshtovuagriho uacstiGvrea amg-apinossti tvivareiobuasc tGerriaam,t-hpeoisritpivotee bnaccytewriaas,
tmheuicrh ploetsesntchya nwtahsa mtoufcCh AleMss. than that of CAM.
TThhee ssyynntthheessiiss ooff aammiinnooaaccyyll aanndd ppeeppttiiddyyll ccoonnjjuuggaatteess ooff CCAAMM wwaass oorriiggiinnaallllyy ssttaarrtteedd iinn oorrddeerr ttoo
eennrriicchh tthhee ccoonnssttrruucctt wwiitthh aaddddiittiioonnaall bbiinnddiinngg ssiitteess,, tthhrroouugghh tthhee iinnttrroodduuccttiioonn ooff aammiinnoo aanndd ccaarrbbooxxyyll
ffuunnccttiioonnss.. HHoowweevveerr,, nnoonnee ooff tthheemm wwaass ffoouunndd ttoo hhaavvee ssuuppeerriioorr aaccttiivviittyy tthhaann CCAAMM,, ppaarrttiiccuullaarrllyy iinn vviivvoo..
SSoommee ooff tthhee mmoosstt aaccttiivvee ccoonnjjuuggaatteess aarree iilllluussttrraatteedd iinn FFiigguurree 88..
CCoorrrreellaattiioonn ooff tthhee iinn vviittrroo aanndd iinn vviivvoo aaccttiivviittiieess lleedd ttoo tthhee ccoonncclluussiioonn tthhaatt sstteerriicc pprrooppeerrttiieess ooff tthhee
aaccyyll mmooiieettyy ssiiggnniiffiiccaannttllyy iinnfflluueennccee tthhee aannttiibbaacctteerriiaall ppootteennccyy ooff tthhee ccoonnssttrruuccttss [[22]].. NNeevveerrtthheelleessss,, tthheessee
eeffffoorrttss hhaadd aa ggrreeaatt ccoonnttrriibbuuttiioonn iinn uunnddeerrssttaannddiinngg tthhee mmooddee ooff aaccttiioonn ooff CCAAMM aatt aa ttiimmee wwhheenn
iinnffoorrmmaattiioonn ffrroomm ccrryyssttaallllooggrraapphhiicc wwoorrkkss wwaass mmisissisningg. .MMoroer espsepceicfiicfiaclalyll,y t,hteh egrgoruopu pofo VfiVnicnec uesuinsign ga
saest eotf oafnaanloaglos gosf opfuproumroymciync ianndan CdACMA Mpropproospeods ethdatth tahtet ahmeianmoi-nacoy-alactyelda tteadil toafi lCoAfMCA sMimusilmatuesla tthees
athmeinaomaicnyol-atcayill -otaf iltRoNftAR NplAacpedla caet dthaet Ath-esiAte- sinit ea inpaarapllaerla ollreileonrtaietniotnat, iao nh,yaphotyhpeostihs eesxitseenxdteedn dtoed thtoe
sthueggseusgtigoens ttihoant tthhaet dthicehldoircohalocertoyalc teatiyl lotfa iClAofMC pAoMintps oaiwntasya fwroamy ftrhoem exthite tuexnintetlu, ntonwela,rtdosw tahred CsCthAe
eCnCdA ofe nand ionfcaonmiinncgo amminingoaamcyiln-toRaNcyAl- t[R10N0A,10[11]0.0 B,1a0s1e]d. Bonas tehdiso pnrothpiossparl oapnods athlea nDd. rtahdeioDd.urraadniso dmuordanesl
[m18o]d, eJolh[1a8n]s,soJonh eatn asls.o dneestiganl.edde as isgente odf anuscelteootfidneu CclAeoMti dceonCjuAgMatecso tnhjautg mateets ltihttalet smuectcelsitst l(eFisguucrcee s8s,
c(Foimguproeu8n,dcso 2m7 paonudn 2d8s) [21704a]n. dA2n8o)th[1e0r4 C].AAMn oatnhaelorgC sAyMnthaensaizloegd siny nththe efsraizmedewinortkh eoff rtahmis eswtuodryk, CofAtMhis-
pstyurdeyn,eC cAonMju-pgyatree n(ceocmonpjouugnadte 2(9c)o, mwpaos ufonudn2d9 )t,ow parsotfeocut nUd2t5o06p rforotemct CUM25C0T6 mfroomdifCicMatCioTn.m Aosd Uifi2c5a0t6io ins.
kAnsoUw2n5 0to6 bise kan foowotnptroinbt esiagnfoaol topfr CinAtMsig bnianldoifngC,A thMe bauinthdoinrgs ,stuhpepaoustehdo trhsastu cpopmopseodunthda 2t9c ommapyo buinndd
t2o9 tmhea ydrbuign dsitteo. tHheowdreuvgers, ioteth.eHr oewxpeevcetre,do itnhteerraecxtpioenctse bdeitnwteereanc ctioomnspobuetnwde 2e9n acnodm tphoe urinbdos2o9maen dwtehree
nriobto csloemarelyw deermeonnosttcrlaetaerdl,y ndoerm thoen setfrfaectet do,nn tohret hgreoewfftehc toof nbatchteergiraol wcetlhlso wfabsa cftuerrtihaelrc einllvsewstaigsaftuerdt.h Ienr
cinovnetrsatisgta, tBehdu.tIan ecto anlt.r uassti,nBgh auntaotehtearl .suest ionfg aamnointhoearcysel-tCoAfaMm ainnoaalocygls- CpAroMpoasneadl otghsatp CroApMos ebdinthdast tCo AthMe
rbiibnodssomtoet hine ar irbeotrsoo-mineveirnsoa orreiteron-tiantvioerns otoo trhieen atmatiionnoatcoytlh-teRaNmAin [2o3a]c.y Tl-htiRsN hyAp[o2t3h]e.sTish tihsahty ips octohmespiasttihbalet
wisicthom repcaetnibt lcerywsittahllroegcreanpthcircy ssttualdloiegsr a[1p9h,2ic0]s twudasie asd[1o9p,t2e0d] wbya soathdeorp gterdoubpys ottoh eexrpglraoiunp isn tao mexoplleaciunlainr
baamsios,l etchuel aarctbivaistiys ,otfh aemacintiovaitcyylo afnamd pineopatcidyylla ncodnpsetrputcidtsy olfc oCnAsMtru [c1t0s3o,1f0C5A,1M06][.1 03,105,106].
Antibiotics2016,5,20 10of21
Antibiotics 2016, 5, 20 10 of 21

FiFgiugruer8e .8C. Cononjujuggaatetesso offC CAAMM wwiitthh aammiinnoo aacciiddss,, ppeeppttiiddeess, ,nnuuccleleootitdidese sanadn dpypryerneen. e.
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
affinityforthebindingsiteofCAM(e.g. compound32). Moreinterestingly,compound33wasfound
to orientate its peptidyl-chain into the ribosomal tunnel and establish interactions with the region
toorientateitspeptidyl-chainintotheribosomaltunnelandestablishinteractionswiththeregion
surrounding nucleotide A752 in domain II of 23S rRNA [106]. Similar interactions of a series of
surrounding nucleotide A752 in domain II of 23S rRNA [106]. Similar interactions of a series of
stalling peptides with this region of the exit tunnel have been found to inactivate allosterically the A-
stallingpeptideswiththisregionoftheexittunnelhavebeenfoundtoinactivateallostericallythe
site of PTase [107]. Recent studies have been focused on a special class of antimicrobial peptides, the
A-siteofPTase[107]. Recentstudieshavebeenfocusedonaspecialclassofantimicrobialpeptides,
proline-rich antimicrobial peptides, which are actively transported inside the bacterial cell, bind into
thethper oelxinite t-urincnheal notfi mthiec rroibboiaslopmeep taindde si,nwfluheicnhcea rtehea cftuivnectliyontr aonf stphoer tAe-dsiitne s[i1d0e8t]h. eThbearcetfeorriea,l lceeslslo,bnisn d
intloeatrhneeedx firtotmun bnoetlho sftuthdeiersi bcoosuoldm peraonmdotine flthuee ndceevetlhoepfmuennctt ioofn noefwt hderuAg-ss, ietesp[1ec0i8a]l.lyT thheorseef otarerg,eletisnsgo ns
leaGrnraemd-fnroegmatbivoet hbasctutedriiae.s couldpromotethedevelopmentofnewdrugs,especiallythosetargeting
Gram-negativebacteria.