Download The impact of penicillin on therapeutic outcomes

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4%Δຍຟូ‫ైسݼ࣍פ‬ऱࠌ‫ش‬Ζ
Bstatic,isabiologicalorchemicalagentthatstopsbacteriafromreproducing,while
notnecessarilyharmingthemotherwise.
AdaptedfromBrettHeintz,PharmD,BCPS
Theimpactofpenicillinontherapeuticoutcomes
• The
Thewidespreaduseofpenicillin
widespread use of penicillin savedtensofmillionsof
saved tens of millions of lives
lives
aroundtheworldsinceitsdebutinWorldWarII.
• Thedevelopmentofantibioticsandtheiravailabilityfor
p
p
g
approach
pp
widespreadtherapeuticuseledtoasignificantnew
forinfectioncontrolandhealthcare.
• Penicillinrepresentsthefirstdefinedtherapeuticproduct isolatedfrommicrobialsourcesandis
the forerunner for developmentofallantibioticstodate.
theforerunnerfor
development of all antibiotics to date
• Thetechnologiesdevelopedfortheproductionofpenicillin, includingmicrobialstrain
improvementandchemical engineeringmethodstomassproducefermentationproducts,
• haveprovidedthebasisforproductionofallclasses
have provided the basis for production of all classes ofantibioticstodate.Thedevelopmentof
of antibiotics to date The development of
penicillin representsthecombinedeffortsofseveraldiversescientific disciplines,including
chemistry,biochemistry,microbiology, andchemicalengineering.
Development and production of many new bioproducts withdiverseclinicalapplicationsincluding
with diverse clinical applications including
• Developmentandproductionofmanynewbioproducts
cancer,autoͲimmunediseases,newvaccines,etc.aredependentonthesamemicrobialstrain
selectionandlargeͲscalechemicalengineeringmethodsthatwerefirstpioneeredforpenicillin.
• ThedevelopmentofpenicillinduringWorldWarIImarkedafundamentalchangeinthe
p
p
g
g
relationshipbetweengovernment,privateindustry,anduniversitiesforcollaborationandfunding
tosupportmedicalresearch.
第1頁
Thediscoveryofpenicillin
The syntheticsulphonamides weredevelopedinthe1930s.However,bacteriaquicklydeveloped
resistance tothesulphonamides,theirspectrumofactivitywas small,andtheirsideeffectswere
severe.Forexample,the sulphonamidescouldnotbeusedtotreatvenerealdisease suchas
syphilis.
• Penicillinwasaccidentlydiscoveredin1928byDr.AlexanderFleminginLondon.Atthattime,Fleming,aphysician,
obse ed t at so e o s cu tu e d s es co ta
observedthatsomeofhisculturedishescontainingStaphylococcusaureus(ᆿရ෺ပ)werecontaminatedwitha
g Stap y ococcus au eus(ᆿရ෺ပ) e e co ta
ated t a
mold.ThispathogenicbacteriumwasdestroyedbythemoldidentifiedasPenicillium notatum.Healsonotedthat
filtratesofthemoldlysed thestaphylococci andwerenontoxicinanimals.Henamedtheagent“Penicillin.”
Penicillinwasadifficultentitytoworkwithbecauseitschemicalinstabilitymadeitdifficulttoisolateand
characterize.
h
t i
• Attemptstoisolatepenicillinweremadeintheearly1930sbyanumberofBritishchemists,buttheinstabilityof
thesubstancefrustratedtheirefforts.Thisformidablechallengewasovercomein1939whenagroupattheSir
WilliamDunnSchoolofPathologyatOxfordUniversity,composedofHowardW.Florey,ErnstB.Chain,Norman
gy
y
p
y
Heatley andEdwardAbraham,andtheircolleagues,cameuponthescene.
• Theywereabletoprepareastableformofpenicillinanddemonstrateitsremarkableantibacterialactivityalong
withalackoftoxicityinmice.TheOxfordgroupalsodeterminedpenicillin'schemicalstructurein1940.
• ThelargeͲscaleproductionofpenicillinincludeslargeͲvolumefermentation,largeͲvolumeextraction,and
The large scale production of penicillin includes large volume fermentation large volume extraction and
purificationoftheantibioticwhicharethefoundationforallmodernbioreactorsandtheproductsproducedwith
thistechnology.
• theNobelPrizeinMedicinefor1945wasawardedtoAlexanderFleming,ErnstChain,andHowardFlorey.
• theɴͲlactam groupofcompoundsisthemostsuccessfulexampleofnaturalproductapplicationand
chemotherapy.TheɴͲlactams andotherclassesofantibioticsdevelopedovertheyearshavebecomethekey
weaponstodefeatpestilence.
Genetics,fermentation,andbiosynthesisofpenicillins
• Fleming'soriginalstrainproducedonlytraces ofpenicillin.Therefore,early“bruteforce”genetic
manipulationsconcentratedheavilyontheproductionofmutantsandthestudyoftheir
properties.
• Astrainisolatedfromamoldycantaloupewascapableofproducing60mg/l.
l df
ld
l
bl f
d
/l
• NRRL1951–1325,aspontaneoussectormutantofa singleͲsporeisolations,produced150
mg/l.
• XͲraytreatmentincreasetheyieldfurtherto300mg/l.
X
t t
ti
th i ld f th t 300
/l
• AultravioletͲinducedmutants,QͲ176,produced550mg/l,becametheancestorofallof
thestrainsusedinindustry.
• Today
Today'ssindustrialstrainsproduceover70g/l.
industrial strains produce over 70 g/l
• Penicillinhadbeenoriginallyproducedinsurfaceculture,buttiterswereverylow.
• Bytheuseofsubmergedculturemethod,strainimprovementandmediummodifications,
such as the use of corn steep liquor as additive the yield of penicillin was increased by 100Ͳ
suchastheuseofcornsteepliquorasadditive,theyieldofpenicillinwasincreasedby100Ͳ
fold.
• Bythe1950s,itwasrealizedtheP.chrysogenum coulduseadditionalacyl compoundsas
sideͲchain
side
chainprecursors(otherthanphenylacetic
precursors (other than phenylacetic acidforpenicillinG)andproducenew
acid for penicillin G) and produce new
penicillins,andoneofthese,penicillinV (phenoxymethylpenicillin),achievedcommercial
success.
第2頁
ThephysiologyofP.chrysogenum in
relation to penicillin production
relationtopenicillinproduction
Incommercialproduction,the usualmediumhadbeenacomplexone,composedof glucose,corn
steepliquor,sideͲchainprecursor(phenylacetic acidforpenicillinGorphenoxyacetic acidfor
penicillinV),andmineralsalts.
Æ glucosetobeexcellentforgrowthbutpoorforpenicillinformation,whilelactoseshowedthe
oppositepattern.
Æ
(1) devisedamediumcontainingbothsugarsinwhichgrowthoccurredattheexpenseof
glucose,andwhenitwasexhausted,themassofcellsbegantoproducetheantibioticatthe
expenseoflactose.
(2) intermittent or continuous feeding of the less expensive glucose could replace batch feeding
(2)intermittentorcontinuousfeedingofthelessexpensiveglucosecouldreplacebatchfeeding
oflactose.
biosynthesisofpenicillinfromitsprecursors
Æ lysinewasapotentinhibitor
l i
t t i hibit ofpenicillinbiosynthesis
f
i illi bi
th i but
b t theinhibitioncouldbe
th i hibiti
ld b
reversedbyLͲɲͲaminoadipic acid.
Æ thetripeptide ɷͲ(ɲͲaminoadipyl)Ͳcysteinylvaline (ACV)asanintracellularcompoundinP.
chrysogenum. LͲɲͲaminoadipic
chrysogenum.L
ɲ aminoadipic acidasanimportantprecursorofallpenicillins
acid as an important precursor of all penicillins
Æ thetripeptide ɷͲ(ɲͲLͲaminoadipyl)ͲLcysteinylͲDͲvaline (LLDͲACV)wasestablishedasthe
crucialintermediateofpenicillinbiosynthesis.Thefeedbackinhibitionbylysinewaslater
proventobetheinitialstepoflysinebiosynthesisinfungi,i.e.,thehomocitrate synthase
reaction.
i
The“newer”ɴͲlactams
Duringthe1950s,thefutureofpenicillins GandVbecame doubtfulasresistantstrainsofS.aureus
emergedinhospital populations.Thepenicillins describeduptothispointweresolventsoluble,
exhibiting a high degree of activity against GramͲpositive
exhibitingahighdegreeofactivityagainstGram
positiveorganismsbutweremuchlessactiveagainst
organisms but were much less active against
Gramnegatives.
• thecompound(6Ͳaminopenicillanic acid,6ͲAPA)whichwasthe“penicillin
nucleus”
l ” wasusedtomake“semisynthetic”(chemicallymodified)
dt
k “
i th ti ” ( h i ll
difi d)
versionsofpenicillinwiththebeneficialpropertiesofresistanceto
penicillinase andacid,plusbroadͲspectrumantibacterialactivity.
• thediscoveryofa completelydifferenttypeofpenicillin,ahydrophilictype, showingequivalent
activityagainstbothclassesofmicroorganisms(i.e.,penicillinN)
In1948,GiuseppeBrotzu isolatedCephalosporium
l d
h l
acremonium,a
cephalosporinͲproducingfungus,fromsewage.Brotzu's strainproducedtwo
antibiotics,i.e.,“cephalosporinP,”activeonlyagainstGramͲpositiveorganisms
aand“cephalosporinN,”whichwasactiveagainstbothGrampositive
d cep a ospo
,
c as act e aga st bot G a pos t e aandGramͲ
dG a
negativebacteria.“CephalosporinP”wasfoundtobeofsteroidalnatureand
notaɴͲlactam atall.“CephalosporinN,”ontheotherhand,wasfoundtobea
truepenicillinpossessinganɲͲDͲaminoadipyl sidechain.Itwasrenamed
“
“penicillinN.”IncomparisontopenicillinG,itwasonly1%asactiveagainst
i illi N ” I
i
t
i illi G it
l 1%
ti
i t
GramͲpositiveformsbuthadequaltoorsomewhatgreateractivityagainst
Gramnegative bacteria.However,itneverbecameacommercialantibiotic.The
hydrophilicnatureofpenicillinNanditsroughlyequivalentactivityagainst
y p
p
g y q
y g
GramͲpositiveandGramͲnegativebacteriawasduetothecarboxylgroupin
thesidechain.
第3頁
MoreɴͲlactams
AsecondantibioticproducedbyBrotzu's strainofA.chrysogenum wasfoundtobe
relatedtopenicillinNinthatitconsistedofaɴͲlactamringattachedtoasidechain
whichwasidenticaltothatofpenicillinN,i.e.,DͲaͲaminoadipic acid.Itdiffered,
however,fromthepenicillins incontainingasixͲmembereddihydrothiazine ringin
placeofthefiveͲmemberedthiazolidine ringofthepenicillins.Itwascalled
cephalosporinC.Thus,theeraofthecephalosporins waslaunched.
CephalosporinCstronglyabsorbedultravioletlight,wasstabletoacidandtopenicillinɴͲlactamase,wasnonͲtoxic,
C
h l
i C t
l b b d lt i l t li ht
t bl t
id d t
i illi ɴ l t
t i
andhadinvivoactivityinmice.Ithadonly0.1%oftheactivityofpenicillinGagainstsensitivestaphylococci,although
itsactivityagainstGramͲnegativebacteriaequaledthatofpenicillinG.
AlthoughneitherpenicillinNnorcephalosporinCwasevercommercialized,theyledtoimportantknowledgeonthe
biosynthesisofthesecompoundsandthedevelopmentofmanypowerfulsemisyntheticcephalosporins ofgreatuse
inmedicine.ThenucleusofcephalosporinCwasnamed7Ͳ aminocephalosporanic acid(7ͲACA).Bychemicalremoval
of its D aaminoadipic acidsidechainandreplacementwithphenylacetic
ofitsDͲaaminoadipic
acid side chain and replacement with phenylacetic acid,apenicillinaseͲresistantsemiͲsynthetic
acid a penicillinase resistant semi synthetic
compoundwasobtainedwhichwas100timesasactiveascephalosporinC.
Manyothernewcephalosporins withwideantibacterialspectraweredevelopedintheensuingyears,e.g.,
cephalothin,cephaloridine,andcephaloglycin,makingthesemisyntheticcephalosporins themostimportantgroupof
antibioticsatthattime.
Thestabilityofthecephalosporins topenicillinase wasevidentlyafunctionofitsdihydrothiazine ringsince(1)theDͲ
ɲͲaminoadipic acidsidechaindidnotrenderpenicillinNimmunetoattackand(2)removaloftheacetoxy
acid side chain did not render penicillin N immune to attack and (2) removal of the acetoxy groupfrom
group from
cephalosporinCdidnotdecreaseitsstabilitytopenicillinase.CephalosporinCcompetitivelyinhibitedtheactionof
penicillinase fromBacilluscereusonpenicillinG.AlthoughitdidnothaveasimilareffectontheS.aureus enzyme,
certainofitsderivativesdid.Anotheradvantagewasthatcephalosporins couldbegiventosomepatientswhowere
allergictopenicillins
MoreɴͲlactams:toravelhowpenicillinNandcephalosporinCweremade
• Fromabiosyntheticsense,therelationshipofpenicillinNandcephalosporinCwasofgreatinterest.
• UseprotoplastlysatesofA.chrysogenum toconvertlabeledvaline intoapenicillin.
• discoveryatM.I.T.in1976oftheringexpansionreaction(Kohsaka
discovery at M.I.T. in 1976 of the ring expansion reaction (Kohsaka andDemain
and Demain 1976),catalyzedby
1976), catalyzed by
the“expandase”(deacetoxycephalosporin Csynthase,DAOCsynthase)enzyme.
Æ cephalosporins wereproducedfromapenicillin.(Formanyyears,ithadbeenthoughtthatpenicillinNand
cephalosporinCwereproductsofdifferentbiosyntheticbranchesinA.chrysogenum.)
penicillinN+2Ͳoxoglutarate+O2 ÅÆ deacetoxycephalosporin C +succinate+CO2 +H2O
• cellͲfreeactivityoftheenzymefromA.chrysogenum formedtheimportanttripeptide precursorofallpenicillins
andcephalosporins,i.e.,ɷͲ(LͲɲͲaminoadipyl)ͲLͲcysteinylͲDͲvaline
d
h l
i i ɷ (L
i
di l) L
t i l D li (LLDͲACV).TheenzymeACVsynthetase
(LLD ACV) Th
ACV
th t
was
proventobeasinglemultifunctionalenzymeactingonLͲ ɲͲaminoadipic acid,LͲcysteine,andLͲvaline toproduce
LLDACV.
3ATP
3
ATP +LLͲ2Ͳaminohexanedioate
2 aminohexanedioate+LLͲcysteine
cysteine+LLͲvaline
valine +H2OÅÆ3AMP
H2O ÅÆ3 AMP +3PPi
3 PPi +N
NͲ[LͲ5ͲaminoͲ5Ͳcarboxypentanoyl]ͲLͲ
[L 5 amino 5 carboxypentanoyl] L
cysteinylͲDͲvaline
(PPi:diphosphate)
• isolationofpureisopenicillin Nsynthetase (IPNsynthase,“cyclase”)whichconvertedtheLLDͲACVto
isopenicillin N.
• ThelongͲheldnotionthatɴͲlactamswereproducedonlybyfungiwasshatteredbyareportfromMerck&Co.
that a streptomycete producedpenicillinN.
thatastreptomycete
produced penicillin N
ÆThediscoveryofcephamycin Cledtomuchresearchonand
developmentofprokaryoticcephalosporins sincethepresenceofthe
methoxy groupontheɴ
group on the ɴͲlactam
lactamringmadethemoleculemoreactive
ring made the molecule more active
againstGramͲnegativeandanaerobicpathogensandmoreresistant
toGramͲnegativeɴͲlactamases.
第4頁
Biosyntheticpathwaytopenicillin,
cephalosporin and cephamycins
cephalosporin,andcephamycins
Overall,therearethreemainandimportantstepstothe
biosynthesisofpenicillinG(benzylpenicillin).
•Thefirststepisthecondensationofthreeaminoacids—
LͲɲͲaminoadipicacid,LͲcysteine,LͲvalineintoa
tripeptide. Beforecondensingintothetripeptide,the
aminoacidLͲvalinemustundergoepimerizationto
becomeDͲvaline. ThecondensedtripeptideisnamedɷͲ
(LͲɲͲaminoadipyl)ͲLͲcysteineͲDͲvaline(ACV).The
condensationreactionandepimerizationareboth
catalyzedbytheenzymeɷͲ(LͲɲͲaminoadipyl)ͲLͲcysteineͲ
DͲvalinesynthetase(ACVS),anonribosomalpeptide
synthetaseorNRPS.
h
NRPS
•ThesecondstepinthebiosynthesisofpenicillinGisthe
oxidativeconversionoflinearACVintothebicyclic
intermediateisopenicillinNbyisopenicillinNsynthase
(IPNS) which is encoded by the gene pcbC Isopenicillin
(IPNS),whichisencodedbythegenepcbC.Isopenicillin
Nisaveryweakintermediate,becauseitdoesnotshow
strongantibioticactivity.
•ThefinalstepisatransamidationbyisopenicillinNNͲ
acyltransferase in which the ɲͲaminoadipyl
acyltransferase,inwhichtheɲ
aminoadipylside
sideͲchain
chainof
of
isopenicillinNisremovedandexchangedfora
phenylacetylsideͲchain.Thisreactionisencodedbythe
genepenDE,whichisuniqueintheprocessofobtaining
penicillins
•LikefungalcephalosporinC,cephamycin Cwasnever
usedclinicallybutwasemployedforsemiͲsynthesisof
manymedicallyusefulcompounds.AmorepotentsemiͲ
syntheticcephamycin,cefoxitin,wasrapidly
commercializedbyMerck,tobefollowedlaterby
cefmetazole,temocillin,cefotetan,andothersemiͲ
syntheticcephalosporins.
123
More ɴͲlactams:
Moreɴ
lactams:discoveredinhibitorsofE
discovered inhibitors of EͲlactamase
lactamase
Inthe1970sand1980s,thepathwaystothepenicillins andthecephalosporins including
cephamycin Cwereworkedout.
• Lateinthe1970scamereportsontheproductionofɴͲlactamantibioticswhichwere
neitherpenicillins norcephalosporins.Themostimportantwasclavulanic acidfrom
streptomycetes which possessed only weak antibiotic activity but was an excellent inhibitor
streptomycetes,whichpossessedonlyweakantibioticactivitybutwasanexcellentinhibitor
ofɴͲlactamase.Itbecamea“blockbuster”productbybeingcoformulated withbroadͲ
spectrumsemiͲsyntheticpenicillins whichweresusceptibletoɴͲlactamase,e.g.,with
amoxicillin;thecombinationwasknownasAugmentin(tradename).
• thediscoveryatMerckofthecarbapenems
• Thefirst,calledthienamycin,wasdiscoveredbyascreening
protocol based on inhibition of peptidoglycan synthesis. The
protocolbasedoninhibitionofpeptidoglycansynthesis.The
antibioticwasproducedbyStreptomycescattleya,whichalso
madecephamycinC.
• CarbapenemsresembledthepenicillinsinhavingaɴͲlactam
ringfusedtoafiveͲmemberedring.Theydifferedinthatthe
i f d t fi
b d i Th diff d i th t th
fiveͲmemberedringwasunsaturatedandcontainedacarbon
atominsteadofsulfur.Sulfurwas,however,presentinanother
locationinallthecarbapenemsproducedbystreptomycetes.
p
p
y
p
y
• thienamycinisthemostpotent,mostbroadͲspectrum,and
nonͲtoxicnaturalantibacterialagenteverfound.Itinhibited
cellwallsynthesis,asdidthepenicillinsandcephalosporins,
and was relatively resistant to microbial ɴ lactamases
andwasrelativelyresistanttomicrobialɴͲlactamases.
第5頁
Modeofactionagainst
microbials
Penicillinbindingprotein(PBP)areresponsible
Penicillin
binding protein (PBP) are responsible
forcrossͲlinkinginthebacterialcellwall.They
makepeptidebondsbetweenlysineand
alanine.Penicillin,Cephalosporins,and
carbapenems
b
bindintothereactionsiteof
PBP’srenderingtheenzymeunabletocrossͲ
linkthebacterialwallgivingbactericidal
activity.
TheproductionsofEͲlactams
p
E
• PenicillinisasecondarymetaboliteofcertainspeciesofPenicillium andisproducedwhengrowthofthe
fungusisinhibitedbystress.Itisnotproducedduringactivegrowth.Productionisalsolimitedbyfeedbackin
the synthesis pathway of penicillin
thesynthesispathwayofpenicillin.
ɲͲketoglutarate +AcCoA їhomocitrate їLͲɲͲaminoadipic acidїLͲlysine+ɴͲlactam
ThebyͲproduct,lͲlysine,inhibitstheproductionofhomocitrate,sothepresenceofexogenouslysineshouldbe
h b
d
ll
hb
h
d
fh
h
f
l
h ld b
avoidedinpenicillinproduction.
ThePenicillium cellsaregrownusingatechniquecalledfedͲbatchculture,inwhichthecellsareconstantly
g
g
q
,
y
subjecttostress,whichisrequiredforinductionofpenicillinproduction.Theavailablecarbonsourcesarealso
important:Glucoseinhibitspenicillinproduction,whereaslactosedoesnot.ThepHandthelevelsofnitrogen,
lysine,phosphate,andoxygenofthebatchesmustalsobecarefullycontrolled.
• Thebiotechnologicalmethodofdirectedevolutionhasbeenappliedtoproducebymutationalargenumber
ofPenicillium strains.ThesetechniquesincludeerrorͲpronePCR,DNAshuffling,ITCHY,andstrandͲoverlapPCR.
• Semisyntheticpenicillins arepreparedstartingfromthepenicillinnucleus6ͲAPA and7ͲACA.
第6頁
PENICILLIN:structureandmothod ofaction
Thepenicillins arenaturalorsyntheticantibacterialagentsderived
fromfungi.Allpenicillins sharethreebasicchemicalcomponents:a
thiazoldine ring,anattachedbeta
ring an attached betaͲlactam
lactamring,andasidechain.
ring and a side chain
OtherantibioticsthatsharethecharacteristicbetaͲlactamstructure
withpenicillinincludethecepahalosporins,carbapenamines,and
monobactams. Collectively, these antibiotic classes are often referred
monobactams.Collectively,theseantibioticclassesareoftenreferred
toasbetalactams.
(betalactambackbone)
Allpenicillins arebacteriocidal toactivelygrowinganddividingorganisms.Theyinhibitcellwall
synthesisbybindingtotheenzymesthatproducetheproteincrosslinks,whichformthebacterial
cellwall.Theseenzymes,locatedjustbeneaththecellwalls,arealsoreferredtoaspenicillinͲ
bindingproteins.Theresultingstructuralweaknessiscompoundedbyactivationofautolytic
enzymes,causinglysis
i l i ofthesusceptiblebacteria.Theabilityofthecellwalltobepenetrated
f th
tibl b t i Th bilit f th
ll ll t b
t t d
andtheattractionofthecellularenzymestopenicillinwhichwhenboundtothem,inhibitstheir
functionaremajorfactorsinthesusceptibilityofbacteriatopenicillin.
Bacterial resistance to penicillin has been a problem since its introduction and has increased in
Bacterialresistancetopenicillinhasbeenaproblemsinceitsintroductionandhasincreasedin
recentyears.Resistancemaybenaturaloracquired. Certainorganisms,particularlymembersof
thestaphylococcussp.havetheabilitytosecretebetaͲlactamase,anenzymethatdestroysthe
beta lactam nucleus and thereby inactivates the penicillin molecule
betalactamnucleusandtherebyinactivatesthepenicillinmolecule
Acquiredresistancetopenicillinoccursthroughseveralmechanismsincludingspontaneous
mutationofbacterialchromosome(s),recombinationofDNA,orexchangeofgeneticmaterials
with other bacteria .Givenallmechanisms,theprimaryacquiredresistancepatterntothe
withotherbacteria
. Given all mechanisms, the primary acquired resistance pattern to the
penicillins ofmostimportclinicallyistheabilitytoproducebetaͲlactamase.
Spectrumofactivity
• Thenaturalpenicillins areprimarilyeffectiveagainstbaerobic,gramͲpositive
organismssuchasstreptococi,enterococci,andsomestaphylococcithatdo
notproducebetalactamase.
Newer synthetic penicillins suchastheaminopenicillins
such as the aminopenicillins andextended
and extended
• Newersyntheticpenicillins
spectrumpenicillins haveincreasedthisspectrumtoincludeactivityagainst
somegramͲnegativeorganismssuchasHinfluenza,Ngonorrhoeae,andE
colithathavenotdevelopedresistance.
• Theadditionofbetalactamaseinhibitorstosomeaminopenicillins further
increases the spectrum of activity making the penicillin family one of the
increasesthespectrumofactivitymakingthepenicillinfamilyoneofthe
broadestspectrumclassofantibiotics.
Despiteincreasedbacterialresistanceandthedevelopmentofnewer
antibioticclasses,penicillinremainsoneofthemostusefulantibioticsavailable
today.
第7頁
Natural Penicillin
NaturalPenicillin
(1) penicillinG(benzylpenicillin)
• forparenteraluse;havethenarrowestspectrumofactivity.
p
p
y
• easilyinactivatedbygastricsecretions.
• ineffectiveagainstbetaͲlactamaseproducingorganisms.
• availableinfoursaltforms:thepotassiumandsodiumsaltformsareinterchangable
formulationsdesignedtostabilizethepenicillinmolecule
duringstorage.Thebenzathine andprocainesaltformsaredesignedforslowerabsorptionand
thereforelongerdurationofaction.
(2)penicillinV(phenoxymethylpenicillin)
• stableingastricsecretions.
• Oraladministrationofnaturalpenicillinisdesirable.
• achievesrelativelylowpeakplasmalevelsandshouldbe usedonly
inmild,localizedinfectionscausedbysusceptibleorganisms.
• PenicillinVisstillconsideredthedrugofchoiceforstreptococcalpharyngitis
SyntheticPenicillin
PenicillinaseͲresistanceprnicillins
Thisgroupofdrugsachievestheireffectivenessbytheadditionofalargesidechaintothepenicillin
molecule,whichpreventspenicillinase producedbystaphylococcusfromenteringthepenicillin
moleculeandcleavingthebetaͲlactam ring.
Nafcillin
oxacillin
cloxacillin
dicloxacillin
• Thespectrumofactivityofthesedrugsmakesthemusefulinthetreatmentofmildinfections
oftheskinandsofttissue,especiallywhenpenicillinaseͲproducingstaphylococci arethe
presumed or known causative agents.
presumedorknowncausativeagents.
• Methicillin istheprototypeforthesedrugs,butisnolongerproducedinthiscountrybecause
thedevelopmentofmethicillinͲresistantstaphylococcusaureus (MRSA)rendereditso
y
g j
ineffectivethatitsrelativelysevereadverseeffectsarenolongerjustified.
第8頁
Aminopenicillins:broadspectrumn penicillins
ampicillin
p
amoxicillin
• Theaminopenicillins werethefirstgroupofpenicillins tohaveactivityagainst
gramͲnegativebacteria.Addinganaminogrouptothebenzylpenicillin
molecule produced ampicillin the prototype for these drugs
moleculeproducedampicillin,theprototypeforthesedrugs.
• All three drugs have virtually the same broad spectrum of activity
against gram-positive and gram-negative organisms.
bacampicillin
Aminopenicillin/betaͲlactamaseinhibitorcombinations
• Despitethebroaderactivityspectrumoftheaminopenicillins,bythemselves,theyareineffective
againstbetaͲlactamase producingorganisms.TheadditionofbetaͲlactamase inhibitorstothe
aminopenicillins wasacriticalstepinimprovingtheirspectrumsofactivity.
•Thebetalactamase inhibitorshavenointrinsicantimicrobialactivity.Rather,thesecompounds,
clavulanic acid,sulbactam,andtazobactam,performineitheroftwoways:(1)bindingtotheactive
siteofthebetaͲlactamase enzyme,therebypreventingitsattackonthebetaͲlactam ring,or(2)
enhancingtheaffinityofpenicillinͲbindingproteinsinthebacteriaforthepenicillinmolecule
therebyfacilitatingbreakdownofthebacterialcellwall.
clavulanicacid
sulbactam
tazobactam
第9頁
Extendedspectrumpenicillins
Furthermanipulationoftheampicillin moleculehasproducedadditionalcategoriesofpenicillins with
evenbroaderspectrumsofactivity.
• Thecarboxypenicillin groupincludescarbenicillin andticarcillin.
• Ureidopenicillins includemezlocillin andazlocillin.
• Piperacillin isclassifiedasapiperazine penicillin.
Theprimaryindicationforthecarboxypenicillins isforthetreatmentofPseudomonasaeruginosa
i f i
infections.
Piperacillin
i
illi andtheureidopenicillins
d h
id
i illi havethewidestspectrumsofantibacterialactivity,
h
h id
f
ib
i l i i
andhaveenhancedantiͲPseudomonasactivity.
carbenicillin
ticarcillin
mezlocillin
azlocillin
ExtendedͲspectrumpenicillins,whileusefulinthetreatment
ExtendedͲspectrum
penicillins while useful in the treatment
ofnosocomial(᠔ೃऱ)andothercomplicatedinfections,have
ahigherincidenceofadverseeffectsandshouldbereserved
for complicated situations that are outside the realm of
forcomplicatedsituationsthatareoutsidetherealmof
primarycare.
Piperacillin
Adverseeffectsofpeicillins
Thepenicillins aregenerallyconsideredamongthesafestofantibiotics.Withtheexceptionofthe
newerextendedspectrum drugs,theyareclassifiedasFDApregnancycategoryBdrugs.The
i id
incidenceofadverseresponsestopenicillinrangesfrom0.7Ͳ10percentandmaymanifestinthe
f d
i illi
f
0 10
d
if i h
immune,nervous,renal,gastrointestinal,integumentary,andhematologicsystems.
Ad
Adverseeffectsarefurthersubdividedintoadversesideeffectsandallergicreactions.
ff t
f th
bdi id d i t d
id ff t
d ll i
ti
skinrashis
ki
hi
themostcommonadverseeffectandallergicandhypersensitivityresponses,including
anaphylaxis(መඕ֘ᚨ),exfoliative dermatitisandStevensͲJohnsonsyndromearethemostserious
dermatologic manifestations Gastrointestinal adverse effects are most common in response to
dermatologicmanifestations.Gastrointestinaladverseeffectsaremostcommoninresponseto
ampicillin andincludenausea,vomiting,anddiarrhea.
第 10 頁
Penicillinbindingproteins
PenicillinͲbindingproteins(PBPs)areagroupofproteinsthatare
characterizedbytheiraffinityforandbindingofpenicillin.Theyarea
normalconstituentofmanybacteria;thenamejustreflectsthewayby
l
i
f
b
i h
j
fl
h
b
whichtheproteinwasdiscovered.AllɴͲlactam antibiotics(exceptfor
tabtoxinineͲɴͲlactam,whichinhibitsglutaminesynthetase)bindto
PBP
PBPs,whichareessentialforbacterialcellwallbiogenesis.
hi h
ti l f b t i l ll ll bi
i
BacteriaoftendevelopresistancetoɴͲlactam antibioticsby
synthesizingaɴͲlactamase,anenzymethatattackstheɴͲlactam ring.
To overcome this resistance ɴ lactam antibioticsareoftengivenwith
Toovercomethisresistance,ɴͲlactam
antibiotics are often given with
ɴͲlactamase inhibitorssuchasclavulanic acid.
DiversityofPBPs
Diversity
of PBPs
TherearealargenumberofPBPs,usuallyseveralineachorganism,andtheyarefoundasboth
membraneͲboundandcytoplasmic proteins.ThedifferentPBPsoccurindifferentnumberspercell
p
y
y
g
andhavevariedaffinitiesforpenicillin.ThePBPsareusuallybroadlyclassifiedintohighͲmolecularͲ
weight(HMW)andlowͲmolecularͲweight(LMW)categories.
FunctionofandantibioticsagainstPBPs
PBPsareallinvolvedinthefinalstagesofthesynthesisofpeptidoglycan,
whichisthemajorcomponentofbacterialcellwalls.Bacterialcellwall
synthesis is essential to growth cell division (thus reproduction) and
synthesisisessentialtogrowth,celldivision(thusreproduction)and
maintainingthecellularstructureinbacteria.InhibitionofPBPsleadsto
irregularitiesincellwallstructuresuchaselongation,lesions,lossofselective
permeability and eventual cell death and lysis
permeability,andeventualcelldeathandlysis.
PBPshavebeenshowntocatalyzeanumberofreactionsinvolvedinthe
processofsynthesizingcrossͲlinkedpeptidoglycan fromlipidintermediates NͲacetylglucosamine (GlcNAc orNAG)
andNͲacetylmuramic acid(MurNAc or
andmediatingtheremovalofDͲalanine
g
fromtheprecursorofpeptidoglycan.
p
p p
gy
NAM)
PBPsbindɴͲlactam antibioticsbecausetheyaresimilarinchemicalstructuretothemodularpieces
thatformthepeptidoglycan. Whentheybindtopenicillin,theɴͲlactam amidebondisrupturedto
formacovalentbondwiththecatalyticserineresidueatthePBPsactivesite.Thisisanirreversible
reactionandinactivatestheenzyme.
TherehasbeenagreatdealofresearchintoPBPsbecauseoftheirroleinantibioticsandresistance.
BacterialcellwallsynthesisandtheroleofPBPsinitssynthesisisaverygoodtargetfordrugsof
selectivetoxicitybecausethemetabolicpathwaysandenzymesareuniquetobacteria. Resistanceto
antibioticshascomeaboutthroughoverproductionofPBPsandformationofPBPsthathavelow
affinityforpenicillins (amongothermechanismssuchaslactamase production).ResearchonPBPshas
ledtothediscoveryofnewsemiͲsyntheticɴͲlactams,whereinalteringthesideͲchainsontheoriginal
penicillinmoleculehasincreasedtheaffinityofPBPsforpenicillin,and,thus,increasedeffectivenessin
i illi
l l h i
d th ffi it f PBP f
i illi
d th i
d ff ti
i
bacteriawithdevelopingresistance.
第 11 頁
PenicillinBindingProtein3ofPseudomonasAeruginosa (PDB3OC2)
NͲterminaldomain
“head”
head sub
subͲdomain
domain
Transpeptidase domain
TheenzymehasapenicillinͲinsensitivetransglycosylaseNͲterminaldomain(involvedinformationoflinear
glycanstrands)andapenicillinͲsensitivetranspeptidaseCͲterminaldomain(involvedincrossͲlinkingofthe
peptidesubunits)andtheserineattheactivesiteisconservedinallmembersofthePBPfamily.
tid
b it ) d th
i
t th
ti
it i
d i ll
b
f th PBP f il
PenicillinbindingmodeofPBP
Theenzyme,DͲalanylͲDͲalanine carboxypeptidase/transpeptidase createsacrosslinkbetweentwo
chainsinthepeptidoglycan net.Thestructurecontainsamoleculespeciallydesignedbyresearchers
tocaptureasnapshotoftheprocessofcrosslinking.Cephalosporin,adrugsimilartopenicillin,is
bounddirectlytotheserineintheactivesite.Thedrugisinthepositionnormallyoccupiedbyone
ofthestrandstobecrosslinked,asindicatedbythetransparentdotsthatextendtotheleft.Alittle
peptidehasbeenattachedtothisdruginthepositionnormallyoccupiedbytheotherstrandinthe
crosslink.Thisstructureshowshowthecomplexmightlookjustafterthecrosslinkismade.
PDBcode:1hvb
第 12 頁
Penicillinresistance
D-alanyl-D-alanine peptidase from
Streptomyces sp. R61 (PDB: 3pte)
class A beta-lactamase (penicillinase) of Bacillus
licheniformis 749/C (PDB: 4blm)
Bacteriahavedevelopedmanywaystothwarttheactionofpenicillin.SomechangethepenicillinͲbindingproteinsinsubtleways,so
that they still perform their function but do not bind to the drugs Some develop more effective ways to shield the sensitive enzymes
thattheystillperformtheirfunctionbutdonotbindtothedrugs.Somedevelopmoreeffectivewaystoshieldthesensitive
enzymes
fromthedrugormethodstopumpdrugsquicklyawayfromthecell.Butthemostcommonmethodistocreateaspecialenzyme,a
betaͲlactamase (alsocalledpenicillinase)thatseeksoutthedruganddestroysit.
y
usethesamemachineryasusedbythepenicillinͲbindingproteinsͲͲsosimilar,infact,thanmanyresearchers
y
y
p
gp
,
,
y
ManybetaͲlactamases
believethatthebetaͲlactamases wereactuallydevelopedbyevolutionarymodificationofpenicillinͲbindingproteins.ThepenicillinͲ
bindingproteins,liketheoneshownontheleft(PDBentry3pte),useaserineaminoacidintheirreaction,coloredpurplehere.The
serineformsacovalentbondwithapeptidoglycan chain,thenreleasesitasitformsthecrosslinkwithanotherpartofthe
peptidoglycan network.Penicillinbindstothisserinebutdoesnotreleaseit,thuspermanentlyblockingtheactivesite.BetaͲ
lactamases,liketheoneshownontheright(PDBentry4blm),haveasimilarserineintheiractivesitepocket.Penicillinalsobindsto
thisserine,butisthenreleasedinaninactivatedform.OtherbetaͲlactamases dothesamething,butuseazincioninsteadofa
serineaminoacidtoinactivatethepenicillin.
Additionalfigures
Benzathine isadiamine whichstabilises
penicillin and prolongs its sojourn(ೖఎழၴ)
penicillinandprolongsitssojourn(ೖఎழၴ)
wheninjectedintotissues.
Procaineisalocalanestheticdrugoftheamino
ester group It was used primarily to reduce the
estergroup.Itwasusedprimarilytoreducethe
painofintramuscularinjectionofpenicillin,andit
wasalsousedindentistry.
betalactam
plasmidsharingbetweenbacteria
P tid l
Peptidoglycan
unit
it
penicillincore
i illi
第 13 頁
Additionalfigures
Energyminimizedcomputationalmodelofpeptidoglycan
strandsboundtoStreptomyces R61PBP
第 14 頁