Oleksyszynr.202/A2 Combinatorialchemistryisamethodofsynthesisingabignumberofcompoundsinashortperiod oftime. 1. 2. 3. 4. 5.

findaleadstructure activity toxicity clinicaltrialsinvestigationofactivitymoleculesonhumansubject FDAApproval(FederalDrugAdministration)

Plantextractswithdifferentsolventsmethanol,chloroform.Thecomponentsareseparated withHPLC.About80%(jamame50%aletoszczeg)ofdrugsinthemarketarecomingfrom naturalsources.Sometimesthey'resyntheticsanaloguesorsyntheticmodification,butcoreofthe moleculescomefromnaturalsources.ForinstanceJapaneseexploreseaplantsextracts.Moreover manyantibioticscomefrommicroorganisms.Soilmicroorganisms,organismsfromyellowstone (hotwater)etc.produceuniquestructures. Afterextractingitisimportanttotesttheactivitiesofthesecompounds.Notanyactivities, butactivitiesrelatedtothedisease.Infactitistargetingaspecificmetabolicpathwaywhichis uniqueforapathogen.Pathogenicenzymesarespecificforthem,that'swhydrugsdon'taffectour metabolicpathways(shouldn'taffect).Mainlyinhibitorsofmolecules,actsforinfectiondiseases. However,sometimesthereareenzymesthathavepathologicalactivityandactivities importantfornormalfunctionsofourbodies.That'swhywehavetotaketoxicityofadruginto account. Whenthestructureisn'ttoxicwecanstartclinicaltrialsinvestigationofactivityonhuman subjects.Thesetrialsaresubjectsofveryrestrictedregulations. FDAapprovaltheyletyoutosellthedrugonthemarket.Ineachcountrythereare institutionstogivecertificatestoselladrug. Thewholeprocesslastsusually812yearsanditcostsalotofmoney. Patentrighttosellacertainproduct(drug)totreatcertaindiseases.Patentisasourceoffinancial returnforadrugcompany.Patentslast20years.Therearethreekindsofpatents: 1. Compositionofmatter(kompozycjamateriau)nooneelsehasrightstousethis compound.It'sanexclusiverighttosellacertainstructurenotonlyasadrug.Strongestkindof protection. WTP(worldtreatypatents)about60countries.Whenyougetpatentfromthisorganisation,you haveprotectioninallofthecountries.ThereisalsoaEuropeanorganisation(yougetprotectionin alloftheEuropeancountries). Evenwhenpatentprotectionforacompoundisonlywithinonecountry,itisdifficulttoobtaina patentinanothercountry,becausetheidea/structureisn'toriginalintheworld(evenwhennot protectedbyWTPorso). 2. Seconduseyoucanapatentforusinganexistingstructureinanotherfield(orforanother disease).Usuallyyouhavetobuyrightsforsecondusepatentfromtheoriginalinventorifpatentof compositionofthematterisnotexpired.Whennewpropertiesofthedrugareexplored.

3. Compositionmixtureof23knowndrugs.Itisaveryweakprotection.Itiseasytobreak itdown.Justchangingthecompositionofthesamedrugs(amountsofeachdrug)canleadto anothercompositionpatent.Howeveritisstillsomekindofprotection. Therearesomedifferencesbetweencountries.Forinstancetherearenocompositionpatentsin somecountries. *ParadoxaboutpatentforDNAsequencepatentthatwaswithdrawn.Curcumaasan antiinflammatorydrugitcouldn'thavebeenusedasaspice(patentalsowithdrawn).Youcant patentsubstancesisolatedfromplants. *sequencecan'tbeprotectedbypatents,onlyusingit'sinsomespecificprocess *fullprotectionisneededtoproducemedicinesbypharmaceuticalCompanies. Theexistingsystemofthepatentshaveconsequences.Bussinesisalwaysfirst,anddevelopingand sellingacertaindrugforlessthan30yearsisn'tprofitable.Thereasonsforitaremainlythecostsof thepatentprocess. Therearesomeotherwaystoprotectproducts.Productsinfoodindustry.Manydrugshavedifferent names.Theydiffersometimesindoses.Worldintellectualpropertyorganisationprotectsnames andtrademarkslikeaspirin.

Adrenal,adnephine,suparemina,epinefryna,levoranina. Sometimesdrugsarechiral.Thatmeansthattherearetwoenantiomersofadrug,howeverinmost ofthecasesonlyoneenantiomerisactive.Sometimesthesecondenantiomeristoxic Talidomid

Rinducessleep Steratogeniccausesdeformationsoffetus.

Afterdiscoverythatoneofisomershastoxicinfluenceonorganismstherewasrequirementofusing justoneenantiomerindrugindustry. Inthecaseofibuprofenbothenantiomersareactive.Evenwhenweuseoneenantiomer,thedrug undergoesfastracemisationinorganism.Sometimesproductofthedrugdegradationcanhavereal activity.

ibuprofen Toxicityweneedtoinvestigatetoxicityofeachnewcompound.Todeterminethetoxicityweuse LD50(lethaldoseatwhich50%oftreatedanimalsaredead).Theexperimentsareusuallydoneon animals.However,therearesomedifferencesbetweenreactionsofhumansandanimals.For exampletherearedrugsthataretoxicforsomeanimalsandaren'ttoxicforus.Forexample penicillinistoxicforhamsters. Chronictoxicitywelookforsomeaccumulationoftoxiccompoundsinorganism(sometimes organismdieaftersometimeofusingadrugacoupleofmonths).Inthestudiesoftoxicity, mammalsshouldbeincluded. Teratologywerelookingforteratogeniceffectinfluenceonthefetus. Carcinogenicwe'relookingforcarcinogeniceffects,testof24speciesofrodentandmammal Genotoxicitysomecompoundsinterferewithgenom,mutogenproperties Reproduction/Fertility ED50effectivedose.Dosethatgivesmaximaleffecton50%ofanimals. TI=LD50/ED50therapeuticindex.Thehighertheindexisthebetteristhedrug.Howevermany drugsforcanceraren'tsafe,becausetheTIindexisverylowthesedrugsareusedonlyin hospitals,whenthedoctorsarecontrollingthestateofthepatient. LECTURE2 OrganicChemistryofdrugdesignanddrugactionSilverman MedicinalChemistryDonaldE..Weower GenericdrugsareabigchanceforacountrylikePoland.20yearsagoitwaseasiertosellgeneric drugs.However,itiseasiertogetapermissiontoproducegenericdrugs(opacasi).Itwas predictionthatPolandwillbeabigsourceofgenericdrugs. ItisdifficulttoPharmaceuticalCompaniestodevelopwidespreadgenericdrug(availablein markets)onlyforspecificapplications. Registrationprocessesareexpensive,butitisstillverygoodbuisness. Forexample:prozac,viagra Asiaproductslotoffgenericdrugs

processofproductionisverytightlycontrolled PharmaceuticalCompaniescollectlibrariesofcompounds Combinatorialchemistryisaroboticmethodofproducingalargenumberofdrugs. Thingsbecomecomplicatedwhenapathogenicproteinisourownprotein.Itisusuallyconnected tomutations.Anenzymestartstoattackourbody.Whenweindentifythispathologicalproteinwe needabigamountofthisoteinweneedtocheckmanycompoundsthatcaninhibititsactivity. Weidentifyavirusenzyme,whenweinhibittheenzymewestopthevirusfromspreading.Virusis verysmall(assayisaboutpg=1012g)anditisdifficulttoobtainenoughbigamountoftheenzyme. Geneticengineeringhelpsustoproducetheseenzymesinbiggeramounts. PCRpolymerasechainreaction(1983KaryMullis).Itisatechniqueofmolecularbiologyto obtainmorecopiesofDNA(acertainfragment).ThenwecanaddthisDNAfragmenttoabacteria whichwillproduceourprotein.Firstlywegetasmallamountofprotein,thatwegetit'ssequence. Usually10aminoacidsfromnterminalisenough.ThanwetranslateitintothesequenceofDNA. Itisthebeginningforthesequenceofgeneinourprotein.UsingthisprobeinPCRtechniqueyou cangetthegeneofthewholeproteinandtheproduceitinalargeamount. WehaveDNAoftheproteinandwehavetomodifyit,toenablethesequencetogetinsidea bacteria.Mammaliancells(proteins)areglicosilatedinadifferentwaythanbacteriacells.Also calledrecombinanttechnique.
1 Proteinasinhibitor(1PI)belongstotheserinproteaseinhibitorfamily.Itisresponsiblefor

trypsininhibitorycapacityofhumanplasma.Itinhibitsneutrophilelastasethatdegradeelastine, collagenandproteoglycanandcancauselungdamage. Inthisway1p1 was obtained from e. coli. It was not glicosilated (glicosilation occur in Golgi Aparatus) like those from human blood. Lifetime in blood of this protein was very short (about 10 minutes). It couldn't have been used as a drug. This 1pi when not glicosilated in a right way can still be used in cases of acute diseases. We could get proteins with 99,75% of purity. However, the impurities can be toxic (toxic protein). Inpurities from each batch were different (batch szara, kada partia produkowanego biaka). Milky animals are considered as a kind of living factories for human proteins, that may be used as drugs in future. Proteins are released to milk. Cows are mammals and the proteins are produced in a very similar way (like in human body). Factor 8 isolated from human blood; it participates in blood coagulation. TPA enzyme (tissue plasminogen activator) induced proteolytic dissolvation of clots(skrzepw) The concentration of the enzyme in our body is very small. It was difficult to isolate it. To isolate it an agreement with woman monastery was made. Scientists collected urine and from the urine TPA was obtained. The process was very complicated. Research on anti-AIDS drug. They identified virus enzyme which was responsible for maturation of (code)? (biaka kodujce?-chyba nie ma czego takiego...) protein. It was a small protein over 100 aminoacids. They synthesised an enzyme from 2*92 aminoacids-as homodimer-->it is difficult to synthesize it. Mature virus never will be produced because the maturation was stopped. If our target molecule ( we would like to inhibit its activity) is an enzyme, it is quite easy to establish an assay (a technique). The problem becomes more complicated, when it isn't an enzyme.

Chymotrypsin is a proteolytic enzyme. The activity is quite specific. How to find proper inhibitors. (Hydrolysis of bond in the place where phenylalanine is situated.) We produce a derivative with p-nitroaniline. It reacts with chymotripsine and we obtain dark yellow colour. Absorption at 410 nm is equal to the concentration of p-nitroanline When we add an inhibitor the reaction is slower, and the colour is changing slower. When we put larger amount of inhibitors, the inhibition is stronger. Testing millions of compounds in laboratory (in this way) is very difficult and it requires a lot of work. That's why we use 96 plates (8x12). On the screen of the computer we obtain 96 individual plots at one shot. The spectrophotometer has a small finger with light diode and it moves across the 96 plate and measuring the absorption. This system can work 24 hour without presence of an experimantator. Weneedtobesurethatourproteinisreallyresponsibleforthepathologicalprocess.Ifweare wrong,wemayloseallthemoneywe'veinvestedintheprocess. Nakedmiceareoftenusedasexperimentalanimals.StrongUVradiationeasilycausesskincancer. Itcauseschronicinflammationthatleadstocarcinogenesis.Youcanknockoutgenesresponsible forthisprocessandthemicewillbestillalive.Sometimesmanygenescanbeeliminatedandthe animalsarefine.Theresearchshowedthattheresearchersfoundtherightenzyme,andthemice weren'tattackedbyskincancer.Elastaseinhibitor(forinstancecreamwithelastine)preventsskin cancer.Verystrongscientificproofforthepathologicalfunctionsofenzymes/proteinsaresuch researchresults.Micewithoutcertainenzymesthataren'tattackedbycertaindiseasesareoftenused toprovepathologicalfunctions. Neutrophilelastaseserineproteinaseresponseforcancerogenesiswithoutthisenzymeshould notbecancerogenesis GLPGoodLaboratoryPracticehowtotreatanimals GMPGoodManufacturePractice SOPStandardOperatingProcedurehowwhenwhykillinganimals,howhealanimals Clinicaltrials Testingtherapeuticsonsickhumans. FDAUSA(FoodDrugAdministration) PIofDPoland(PolishInstituteOfDrug) Allthesetrialsmustbedoneinapharmaceuticalindustryinagoodmanufacturepractice. Forexample: chloroformisforbiddentouseintheprocessofproducingdrugs.Halogenatedhydrocarbonsmay causecancer. Aflaskcanbeusedonlyforonepurpose.Anyotherreactioncannotberuninthesameflask. Sterileconditionsarerequired. heavymetalsarealsoexludedinclinicaltrials GLPgoodlaboratorypractice Testingcompoundsontheanimals.Thewayhowanimalsaretreatedetc.Evenwhenyouwanttodo someresearchonanimals,someimportantrulesmustbeobserved SOPstandardoperatingprocedure.You'reobligedtofollowtherules(everythingisdescribed

evenkillinganimals,treating).Itmakesheprocessmoreexpensive. LECTURE3 PIDPolishInstituteofDrug Itisnoteasytogetpermissiontotestingdrugonpeople Studiesonanimals:mainly25speciesandatleastoneofthemcan'tbearodent,becauserodent havedifferentmetabolismthanmammals monkeysaregoodanimalstotestingdrugson Toxicityfertility,teratology,chronictoxicity(smalldosesinalongerperiodoftime), carcinogenic,genotoxicity,peri/postnatal,LD50. Theplanfortrialsmustbeveryprecisewhichhospital,howmanydoctors,whatkindof experiments,patient(neverchildrenandwomemareincludedonlyincaseswhentheitwoman disease,aftervolunteers,womanareincluded) Aquicktourthroughtheapprovalprocess: Alltheexperimentsaredoubleblinded.Placeboeffectischecked(glucoseandcalciumcarbonate). Itisimportanttheplaceboeffectfromthetotaltherapeuticeffectofthedrug.Placebostudiesmust berunparalleltothenormalstudiesofthedrug.Withoutthatcompanycouldclaimaboutany activityofadrug. Placibohas3040%terapeuticeffectsoweneddtoremoveallplaciboeffectfromtrack Statisticsisanotherpartitinvolveshugesumsofmoney.Thenumberofpatientsisverycarefully calculated.Theaimistogetminimalnumberofpeoplebutlargeenoughtopredicttheeffectsofthe drug. Totestfortoxicity,youcollecteverykindofbodyfluidsalivaurine,blood,etc.Evengases. Radioactivelabelingmoleculeslettoknowhowbehavesmoleculeandhowaremetabolized PreclinalstudiesThenewdrugistestedonanimalsfortoxicitiesandbiologicalefect InvestigationalNewDrugApplication(IND).BysubmitiinganIND,thecompanyasksforFDA permissiontotestadrugonhumans investigationisoftencarryoutinprisons Sometimesapostmarketingresearchisdone.Permissionsmaybewithdrawnwhensomethinggoes wrongandthedrughassometoxicactivity,whichturnedouttoappearafterplacingitinthe market.Itusuallyhappenswhensomethingwasn'tnoticedinthetrials. Aquicktourthroughtheapprovalprocess: FoodandDrugAdministration.TheFDAmandateistoprotectthepublichealthbyensuringthat thedrugsmedicaldevicesanddiagnosticsusedbythepublicaresafeandeffective.TogaintheFDA approval,acompanysponsoringanewdrugisrequiredtocompleteastrict,expensiveandlengthy drugapprovalprocessdictatedbytheFDA.Thisprocessincludes: Phase1ClinicalTrails(Safety).Inthistrial,alimitednumberofsubjects(2080)areenrolled,the trialisgearedtowardestablishingasaledosagerangeandlearninghowthedrugactsinthebody.

Phase2CTUpto200patientsareenrolledinthistrialwhichisdesignedtoprovideevidenceof efficacyandoptimaldose markersofdiseaseproteins/metaboliteswhichconcentrationisrelatedwithphaseofdisease for10compoundsinclinicaltrials8arereject. optimalterapeuticdoseneedtobeestablished.Thereisnogoodrelationsbetweenhumanand animaldosebecauseofdifferencesinspeedofmetabolism areaofskinisconnectedwithspeedofmetabolism PhaseI/IIclinicaltrials.AcombinationofphaseIandIItrials,aphaseI/IItrialaimstoevaluate toxicity,showindicationsofefficacyanddeterminetheoptimaldosage Phase3Pivotal,clinicaltrials(Efficacy).Asacontrolled,largescale,doubleblinded,multicenter study,thisisdesignedtoshowstasticallysignificantsafetyandefficacyandprovidesthebasisfor FDAapprovalofthedrug PhaseII/IIIClinicalTrialsSomeclinicaltrialsofnewtherapiesforlifethreateningindication combinephasesIIandIIIinanattempttoacceleratethedrugapprovalprocess NewDrugApplication(NDA)ThisdocumentissubmittedtotheFDA.IntheNDA,thecompany asksforapprovalofchemicallysynthesizeddrugformarketingintheUnitedStatesandusesthe clinicaltrialsdatatoshowthatthedrugissafeandeffectivefortheproposeduse. Product/BiologicalLicenseApplication(PLA/BLA)Thecompanysubmitsthisdocumenttothe FDAtoseekapprovalofabiologicallyproduceddrugformarketingintheUnitedStatesanduses theclinicaltrialsdatatoshowthatthedrugissafeandeffectivefortheproposeduse. PremarketingApproval(PMA).ThroughthesubmissionofthisdocumenttotheFDA,thecompany asksforapprovalofadeviceformarketingintheUnitedStatesandusestheclinicaltrialsdatato showthatthedeviceissafeandeffectivefortheproposeduse. FDAAdvisorycommittee.TheFDAadvisorycommitteeconsistsofanoutsidegroupassembledby theFDA(9independentexperts)whomeettodiscusstheclinicaltrialsdataandtovoteonwhether torecommendapprovalofthedrug. FDAapprovalgrantspermissiontomarketandselladrugintheUnitedStates. Libraryofcompoundswhichalreadyexist. naturalextracts combinatorialchemistry Therewere200companiesthatspecializedonlyinsynthesisingahugeamountofdifferent compounds(inlate90s). Librariesstoredrugsevenfor50years. Whatiscombinatorialchemistry.Everythingstartedfromaroboticsystemfromsynthesising peptides.Synthesisrobotsareusedincombinatorialchemistry.Stryer(molekularnywzrycias 70wstryerzez2007r=105s.):whatwearedoingweconnectaminoacid(acidgroup)tothe

chloromethylgroupofPS. 1)AtfirstCendaminoacidwithblockedaminogroupisattachedtosolidphase. 2)AminoendofnewaminoacidisblockedbytBoc[tBocaminoacid]andcarboxyendofthesame aminoacidisactivatedbyDCC. 3)aminogroupofaminoacid[1)]isunblockedandjointosolidphase Wecanobtainalmost100%yieldandalmost100%purepeptides.Containterswitheachaminoacids arechanged. ForsynthesiseDNAandRNAthesameprocedure A1A1A2A3 A2A2A1A3 A3A3A1A3 Afteraddinginstepsamixtureoftwoaminoacidstoone,weobtaintwodipeptides,thenfour,then eightandsoon.Theaimwastoobtainanenormousnumberofcompoundsandcheckifthereare activestructures.Torecognizetheactivestructure,eachindividualpartofpolymerhasaunique radioactivemark.Eachpolymerhasaspecificsignal.Bydecodingthesignalyoucanidentifywhat kindofcompoundisit. Splitandmixmethod. Mixtureofvariouscompoundsistakenandcheckedhowitworksonabiologicalsystem. LECTURE4 Classificationofdrugsbasedontheirgrossbiologicaleffect. 1. CNSagentsonpsychopharmaceuticals(CentralNervousSystem) Antipsychoticagents Anticonvulsants Analgesis Antiparkinsonianagents Antidepresants Anxiolytics Sedativehypnotics Drugsforcognitivedisorders Stimulants 2. Pharmacodynamicagents Antihypertensiveagents Calciummodulators Antieschemic(antistroke)drugs Antiulcergastrointestinalagents Antithrombotics Pulmaryandantiallergicagents Antiarrhythmics

Antiglaucomaagents Vasolidators Antianginals 3. Chemotherapeuticagents Antimicrobials Antifungals Anticancer,Antineoplastics Antiparasites 4. Metabolicdiseasesandendocrimefunction Contraceptives Antiandrogens Antiinflammatories Dermatologicals Antiatherosclerotics Antirheumaticsandautoimmunediseasedrugs Antiobesitydrugs Anotherclassification: 1. Drugsenzymeinhibitors Biochemistry,2005,44(15),5561 Therearebetalactameswhicharecountedasonedrug,butinthemarketthere'smuchmore betalactams.

317enzymeinhibitors,therealnumberis,however,about1000 Thereare71enzymesinhibitedbythesedrugs.48ofthemarehumanenzymes.Itmeansthatinthe stateofdiseasethosehumanenzymeshavepathologicalactivities.13arebacterial,5virusesand1 parasite. 65%ofthedrugsundergoreactions....(jakie...?) Cellwallsprotectandgiveashapeofbacteria.Thewholemetabolicpathwayforproducingthecell wallsisspecificofbacteria.Inhibitionofenzymeresponsibleforcellwallgrowingisverysafeway offightingwithbacteriadiseases.It'sbecausewedon'thavesuchenzymesthatproducecellwalls. Antibioticslikepenicillininterferewithcrosslinksinbacterialcellwalls.Thosecrosslinksare madeofpeptides. In1930Bayerdiscoveredthatadyecalledprontosilprotectsmicefrominfections.Invivothe compoundwasmetabolisedtoamideofpaminosulphonicacid.Itwasexplainedafterafewyears sulphonamideisandanalogueofpaminobenzoicacid.Itissubstrateofforsynthesisof tetrahydropholicacid.Bacteriasynthesiseitbuttheyneedforitpaminobenzoicacid.When sulphonamideisputinthepositionofpaminobenzoicacidthesynthesisisstopped.Itcausesthat bacterialDNAcannotbeproducedanymore.Howevermanybecteriaareresistanttosulphonamide nowadays.Todaytheyproducemoleculesthathelptocapturethismoleculeandtakeitoutofthe cell.Buteventodaysulphoneamidesareused. Therearemanycompoundssimilartosulphonamideinwhich,theamidenitrogenhasdifferent

substituents.Thesecompoundsarediuretic.Theyworkgoodagainsthypertension.Lesswaterin organismlowerBP. Anothergroupofcompoundssimilartosulphonamidehasathiodiazolering.Thosedrugsareused fortreatingdiabetics. Penicillin ThisdrugstructurecorrespondstothecarbonylintheacylDalanylDanalinethatacylatesthe activesiteserine,and,therefore,penicillinsalsocouldacylatethetranspeptidaseserineresidue. Amazingsmallconcentrationofpenicillincankillbacteria1mg/L.

penicillin benzylopenicylina(penicylinaG,penicylinakrystaliczna) fenoksymetylopenicylina(penicylinav,vcylina) BacteriahaveanenzymewhichracemizesLalaninetomixtureofLandDalanine.Whenwefind inhibitoritprobablywon'tbetoxicbecausewedon'thavesuchenzymesinourorganism.Instryer aminoacidsynthesisandaminoaciddegradation. Someaminoacidslikecycloserinewherefoundinnature.Cycloserineisstillusedfortuberculosis nowadays(despitethefactthatitisquitetoxic).DcycloserineandOcarbamoyleDserinearevery goodinhibitorsofAlanine(LD)racemase. DalaDalaconnectspartsofcellwalls.TranspeptidasetreatspenicillinlikeDalaDalagroupand theenzymaticreactionofcreatingacellwallisstopped.Blactamringopensandconnectsto transpeptidase. 6aminopenicillicacid(6PA)isobtainedbychemicalmeans.Throughanacyllationprocesswe addadifferentsubstituenttotheaminogroup.Todaypenicillinisasemisyntheticdrug.Weuse biotechnologyandpenicillinamidase.Inwasfoundinbacteriawhichwasresistanttopenicillin(the firstmechanismofdefendingfrompenicillin).Early70s. 2. Drugsinteractingwithreceptors 3. DrugsinteractingwithDNAandRNA MICMinimalInhibitoricConcentration MDRProteinMultiDrugResistantProtein LECTURE5 Differencesincellbuildingmakesneedofsomanydifferentpenicillincompounds. Bacteriadevelopedashieldthatcouldprotectthemfrombetalactams.Oneofsuchprotecting

systemswaspenicillinase.Betalactamaseattacksbetalactamringsandopensit(stopsthebacteria killingability).Betalactamaseworkedwithmostofbetalactams(notonlypenicillin). Therearesomemoremechanisms.Moregeneralwayforbacteriatogetresistanceisso called,multidrugresistanceprotein.Theproteinhasabilitytocatchmoleculesofdrugsand transportitoutsidethecell.Similarmechanismcanbefoundincancercellstheycanbecome resistanttocisplatininthesameway. Modifyingdrugstructureisawaytodealwithbacteriaprotectingsystems.Afteraddingacid chloridetotheaminogroupofpenillinyoucanobtainanamidederivativeofpenicillin.100sof differentderivativesofpenicillinweresynthesised(theycanbefoundontheweb). Usuallyverysmalldosesofantibioticsareneeded(forinstance0.006mg/L). Itiscrucialtofindoutwhatkindofbacteriacausedaninfection.Itdoesn'tmakeanysense togiveanykindofantibiotic.Differentantibioticsareactiveforsomecertainkindsofbacteria(for other,theyaren't). Cephalosporinsbasedonoriginalmoleculesthatwerediscoveredearlier.Theoriginalmolecule didn'tworkproperlyinthebeginningbecauseitcouldn'tpassthroughthecellularwallofbacteria. Thatiswhyonegroupwaseliminatedfromthestructureandtheresultwas7aminocephalosporic acid(7ACA).7ACAturnedouttoworkverygood.Cephalosporinsareoneofthemostoftenused antibioticsnowadays.Namesstarticwith'cepha'meansthatitisacephalosporin.Someofthe cephalosporinsaremuchmoreactiveandmuchbetterthanpenicillin(lessamountofthedrugis neededfortreatment).However,therearemanystrainsofbacteria(especiallyinhospitals)thatare resistanttothoseantibiotics.

Otherbetalactametypesofantibioticswerediscovered.Forexample:Norcordicine(it'snotuse clinicallynowadaysunstableandnotveryactive),Thienamycine. In1950sanothergroupofantibioticswasdiscoveredVancomycine.Itactedexactlylikebeta lactams.Itstoppedsynthesisofbacterialcellularwall.VancomycineformsacomplextoDalaD alaintheactiveplace(ithasapocketthatbindsDalaDala).Vancomycinishydrofobicandit couldpenatratequiteeasilytheplaceswherecellularwallissynthesised. Aminoglycosidetypeofantibiotics.Usedfortuberculosis

Tetracyclines.Orallyactive(pills). Poliensantybotics. Betalactamesarenotactiveorally,becausethebetalactameringisunstableatlowpH,instomach. Usuallyinjections. Todaycompoundslikepenicillinwouldbeneverintroducedtothemarket10%ofpopulationhave immuneallergicreactiontopenicillin.Thereactionisduetofastreactionofpenicillinwith albumin.Albuminstartstoberecognisedasaforeignproteinandisattacked. Inourbodythereareabout1,5kgofdifferentbacterias.Bacterialfloraisusuallykilledby antibioticsandourbodyisn'tresistanttofungus.Itisverydifficulttogetridoffungus,especiallyin caseofchildren. IHIBITORSOFPROTEOLYTICENZYME Activesiteresiduesandgeneralproteaseinhibitors. Alltheseenzymeshydrolasespecificproteinsinspecificplaces.Specificitypocketsarevery importantinthoseenzymes.10%ofhumangenesareproteolyticenzymes.Essentially,thetotal reactionrequiresattackofwatermoleculeonacarbonylgroup.Afterthattheproteinundergoes hydrolysisinacertainplace.DirectattackofwaterisincaseAspandmetaloproteinases(Zn,Fe). Incaseofserineproteinases,cysteinwatercomeslater. Howtodesigninhibitorsofsuchenzymesinarationalway?Inourmoleculewecanintroduce elements(X)whichshouldbeagoodkillerZn(incaseofmetaloproteinase,forex.SHbindsZn verystrongly).Substratehasnoabilitytoinhibitenzymebecauseitbindstoenzymeweakly.When weaddXweobtainavery,verygoodinhibitor.Whenwebuildamoleculesimilartotransition stateithasgotverystronginhibitionactivity. Metaloproteinasesarezincdependant. DRUGDESIGN Bloodpresureby1960shypertensionwasn'tconsideredasadisease.However,itwasfoundthat there'sanobviousconnectionbetweenhypertension,heartattacksandstrokes.AngiotensinI. Angiontensinconvertingenzyme.Thecrystalstructureofthisenzymeisnotknoweventtoday.The enzymeiscarboxydipeptidaseremovesaminoacids. Contractionofbloodvesselscausesthatthepreasureismuchhigher.Whenweinhibitthe AngiotensinIIthebloodvesselsstoptocontract.ProlinwithCH2Sattheend(insteadofCOOH) wasaverygoodinhibitorofAngiotensinIIbecauseitcouldbindstronglytozincbyS.All compoundswithSHsmellandtasteverybad.Itookalongtimetogetridofthesmellandtaste. Manymodificationsofthedrugwereprepared.Scientistswherelookingforstructureswiththe sameactivitythatcouldstayinhumanbodylonger.Itwasanexplosionofrationaldrugdesign. Involvementofalltheenzymewasfound,theylookedforleadstructure,theymodifiedthestructure toobtainagoodinhibitor.....

Latermanycompaniesappliedthesameapproachundestandmechanismofdisease,identify crucialenzyme,trytofindleadstructureassoonaspossible,trytomodifythestructuretoobtaina gooddrug. Captoprillucylanalog,peopledidntwanttotakeitcauseofsmellandtasteofSHgroup.Itwas morehydrophobicandstatelongerinbody. LECTURE6 Rationaldesign captoprilworksonlyon60%ofpopulation(angiotensit2synthesisinhibitor). OtherdrugsabletolowerBP: Calciumchannelblockers.Heartisapump.IfthepumpisweakertheBPislowered.Heart actionisslower diureticdrugs Whenenzymeisconsideredasadrugtarget.Potentialpainkillerconnectedwithinhibitorof enkephalinase.Pharmacophoreforenkephalinandmorphinisverysimilar.Enkephalin (pentapeptides)workforaveryshorttime.Thosepentapeptidesarehydrolysedveryfast.Enzyme thatisresponsibleforthehydrolysisiscalledenkephalinase.Findinginhibitoroftheenzymewould enabletoproducestrongpainkillers(mixtureofenkephalinandtheinhibitor). GoodinhibitorofenkephalinaseisThiorphan(withSHgroup).

Drugsprotectingfrombloodcoagulation(formationofbloodclots).Bloodclotscancausebrain death. Undersurgeonsknifethebloodmaycoagulateandclotsmaybefromed.Sothat,anticoagulants mustbeinjectedduringsurgeries.Afterclosingwound,thecoagulatingabilitymustbeback. Anticoagulants.Incoagulationcascade,thrombinplaysthefinalrole.Itformspolymerlikesolid materialsandclosesholescausedbyinjury.Whenyouinhibitthrombin,youcanstopcoagulation. Manyscientistsconsiderinhibitorsforfactor10andfactor9(earlierstepsofcoagulation),because theyprefernottoinhibitthefinalenzyme. Weuseheparinnowadays.Itisisolatedfromcowlounge.Theproblemwithitisthatwhenitis injected,theanticoagulanteffectsustainsformanyhours(usuallyabout12).Therestillisaneedfor ashortacting,goodanticoagulant.

Heparinlonglastingtill12h

Heparinisbindingtofreeantitrombin(naturalendogenousinhibitor).Togethertheyworkstronger thanjustfreeantitrombin. Collagenasesareenzymesthatbreakthepeptidebondsincollagen(MMP).Theyassistin destroyingextracellularstructuresinpathogenesisofbacteria. Inhibitorsfortheseenzymesmighthavestoppedgrowthoftumors. ItwasbelievedthatMatrixMetaloProteinasesinhibitorsstopsgrowthoftumor.Howeveritturned outtobeoneofthebiggestpharmaceuticaldisaster.Newgenerationofthesedrugsmaystop growthoftumors. Asparthylproteinases. RhinoviruscausesAIDS.HIVdoesn'tkill.Anopportunisticinfectionisthekiller.Marcksuggested veryeffectivedrugforAIDS.HIVattackscellresponsibleforhumanimmunesystem. Livecycleofvirus.Firstlyitgoesinsidethecell.WhenitisinsidetheRNAisreleased.Tobeable toformacopyofthevirus,theRNAcodeneedtobeconnectedtotheDNAofcell.Fromthe momentvirusRNAissynthesised(newcopies).Newcopiesformmatureviruses.Transformation ofRNAtoDNAiscausedbyanenzymewhichisinthevirus.Itiscalledreversetranscriptase transcriptsviralRNAtoviralDNA.ViralDNAisinjectedintocellDNAandtheviralRNAcopies areproduced.Whenalargenumberofvirusaresynthesisedduringtheprocess(RNAparts), proteinsareneededtoformcapsules.Proteolyticenzymeisresponsibleforprocessingproteinsof capsules.Theseezymescleave,proforms(RNAparts)toactiveforms(maturevirus).Tocreate maturevirustwoenzymesareneededversetranscriptase,andaspartylproteinase(capsules). ReversetranscriptaseinhibitorsgivesomeprotectionfromAIDS.However,theyareweakanti AIDSdrugs.That'swhypeoplestartedtolookforaspartylproteinasesinhibitors.Bymeansof combinatorialchemistrysomeverystronginhibitorsofHIVaspartylproteinasewerefound. Ifcoumpoundisatransitionstateanalogueitbindstotheactivecentreofanenzymeverystrongly. Mixtureofreversetranscriptaseandaspartylproteinasesinhibitorsisaverystrongantivirusdrug. Itpreventsthevirusfromspreading.Itdoesn'teliminatetheviruspatientisalwaysinfectedbutit stopsthereplication. Drugswhichdidn'tactasgoodBPloweringagents,turnedouttobeagoodbasisforcreatingdrugs thatcouldinhibitreplicationofviruses.

Mixtureof3compounds(differentstructuresofinhibitors)preventedthevirusfrommutating.In suchacasevirusmustbecomeresistant3inhibitors.Itisimpossiblefrommathematicalpointof view.Probabilityofsuchmutationisextremelysmall.Italsowasagoodstepforcompaniesonce developeddrugmixture,doesn'tneedtobechangedforalongtime. Rhinovirusesarenottoxictous.Almostallviruseshaveenzymeresponsibleforcapsuleformation. CrixivanMerckclassicalaspartylproteinasesinhibitor.Usedinmixturewithreversetranscriptase inhibitor. Reversetranscriptaseinhibitorshaveanazidogroup(azydek)Zidovudine HepatitisCthevirusislivinginhepatocytes,andspreadingslowly.HepatitisCproteaseisthe enzymewhichisresponsibleforcapsuleformation. Herpes(wirusopryszczki) Exampleswhatenzymeisatarget,whatcompoundsareunderdevelopment. Membraneisfromfattyacidderevatives.Fattyacidsynthase;Theinhibitoroffattyacidsynthase musthavealongchain.AgoodinhibitorisCeruleninitkillscancercells(withepoxyring) SHwhichisintheactivecentremayinhibittheenzyme. LECTURE7 Levelofcholesterolisregulatedinorganism.Sometimescellularmembranescontainover20%of cholesterol.Weobtainitfromfoodandourbodyissynthesisedinbody.There'sasystemwhich regulatesthelevelofcholesterolitcheckshowmuchcholesterolisgotfromfoodandsynthesises therest(whichisneeded).Pathologicalsituationiswhencholesterolisproducedinatoobig amount.Thenitcrystallisesinbloodvesselandcanleadtoheartattack.Adrugforthisdiseaseis calledLovastatin.Itisamemberofclasscalledstatics.Itisaninhibitorof3hydroxy3methyl glutarylCoAreductase(CoAcoenzymeA).Ki=6.4*10^10.Veryefficientinhibitorwhichstops biosynthesisofcholesterolinliver.Neverthelessabaddietcanleadtoanincreasedlevelof cholesterolinspiteofusingthedrug. Itisalsoverybadwhenyouhavedeficiencyofcholesterol(becauseofthefactthatitisapartof membranes). Inhibitorsofkinases MostofinhibitorsreportedinliteratureareresponsibleforbindingofATP,becauseofthepresence oftheATPbindingdomain.Theyarenotspecificcauseofwholeclassofthisenzymeshasthis domain. TherearealsoseverallgoodinhibitorsofenzymecalledDPPIV(dipeptidylpeptidaseIV).This enzymeisrepsonsiblefordiabetesII(disregulationofglukagonmetabolism).DPPIVis responsibleforactivationofglucagon.Glucagonisusedveryfastinolderorganisms.Ihhibitorsof DPPIVcauseinactivationofthisenzymeanddecreaselevelofglukagon.Thisinhibitorsare peptides,theyarenotstableinbloodcauseoftheproteinasesinfluence. Anthraxveryfastinfection.ThebacteriareleasesamixtureofproteinsandsocalledAnthrax

toxinZincproteinases(anproteolyticenzyme).Somedrugsinhibitthismetaloproteinasesvery efficiently.Itworksgoodinvivo(atmouse).Hydroxylderevativeofamidebindszincinthe enzyme.Ithasasimplestructure.In2or2yearsaverygoodinhibitorwasdevelopedanditcanbe anantidoteforAnthraxtoxin. Secondbiologicalweaponcalled(Clostridiumbotulinum).Itproducesbotulinneurotoxin.Adose ofseveralnanogramsperkgofhumanbodyisseverelytoxic.Thetoxinisveryactiveagainst acetocholinesteraze.Itgoestotheheartandtheactionofheartisstopped.Itisanolderbiological weapon,that'swhyantibodiesaredeveloped.Howeverthisprotectionisveryexpensiveandit cannotbeprovidedformillionsofpeople. DrugsinterferingwithDNA InourcellswehavesynthesisofDNA(acopyofgeneticmaterialiscreated) synth(replication)DNARNAissynthesised(transcription)proteinsynthesis(translation). Theprocesscanbestoppedintwoofthesteps(markedwith) Manyanticancerdrugsworklikethat.Cancercellsworkthesameasnormalcells,usethesame metabolicpathways(therearenospecificpathways).Wecannotinhibitorsandantibioticslikein caseofbacteria.Theonlydifferenceisthespeedofgrowth.Cancercellsaregrowingveryfast. Normalcellsgrowmuchslower.IfwehaveaninhibitorofsynthesisofDNAitwouldcause enormousharmtocancercells.Itwouldalsoharmnormalcellsbutnosomuch,becausenormal cellsgrowmuchslower.Thetherapeuticindexofanticancerdrugsisverysmall.Thedifference betweentoxicaldoseandtherapeuticdoseisverysmall(fromdefinitiontheanticancerdrugsare toxic). Chromosome9andchromosome22. Chromosome9Ageneandkinasewhichisresponsiblefornormalcellgrow Chromosome22agenebcr duringmutation(aberration)agenefromch.9istransferredtoch.22bcrabl.Itcausesunlimited growofwhitecellsandleadstoleukemia.ProductofageneBcrablisauniqueproteinwhich doesn'tnaturallyoccurinourorganism.Gleevecisthechimerickinaseinhibitor,widleyusedin therapy. Mustardgas,iperyteduringWorldWarI.Verytoxic,causesverypainfulwounds.Therewasa situationwhensomesailorssurvivedafteraniperyteatack.Itturnedoutthattheyhadaverylaw numberofwhitecellsinblood. Adoctorwonderedhowtolowerthenumberofwhitecellsinchildrens'organisms.Hefounda paperaboutiperyteandusedsmallamountsofittolowerthenumberofwhitecells.Eventoday certainkindsofleukemiacanbecuredwithderevativesofmustardgas.Allthederevativeshave betachloroethylenegroups.ThemechanismofthedrugsstopmakinganextracopyofDNA. ItbindstonucleobasesinDNAwiththetwochlorines(chlorinesareeliminatedfromthestructure) That'showreplicationofDNAisstoppedanditisimpossibletoproducenewwhitecells.It alkylatesDNA. Oneoftheiperytederivativeismechloroamine(chloromethine)guanineandmakesconnections betweentheminthesameDNAmolecule.Itstopstranscriptionandtranslation.Thecancercells devidestoofasttoremovethissubstanceitmakesmechloroaminegoodanticancerdrug.

Trofosfamidandifosfamiddruginwhichtheaminegroup(frommustardgas)issubstitutedwitha ringwhichcontainsphosphorus(P=Ogroup).TheyreactwithDNAexactlyinthesameway. Thisdrugsactssimilartomechloroamine. ThioTEPAworksexactlythesame.

Estersofmethanosulphonicacid. (siejdtu,siejdtu,siejdtu,prawda?) Themechanismofalkylationisexactlythesamedoublehelix,twoadeninebaseswhichare alkylated(onthebothsidesofthehelix). Nitrosoureaderevativesalsoakylating(crosslinkofDNA) Derevativesofcisplatin Cisplatinworksinasimilarway.Twobases(AorG)arebindedinthesamepartofthechainof DNAbyplatin.Chlorineatomsarereleasedandtwoaminegroupsstayoverthebindingofcis platin. InPolandloungeandbreastcancerisdangers.Thefirstthingwhichdoctorsareaskingthemselves canwetreatthiscancerwithcisplatin.Only30%ofpatientshavecancerwhichissensitivetocis platin.Howevermostofthepatientshavecancerwhichisresistanttocisplatin.Multidrug resistanceproteinprotectscancercellsfromcisplatin. Awellknowderevativeifcisplatiniscarboplatin.Asecondgenerationdrug.

Doctorshavebereallycarefullwithusingcisplatin.Cisplatincouldin6monthsinduceresistance toallalkylatingdrugs. Chloroambucilandmelfalanaveryolddrugsusedfortreatingcancer.There'salotofexperience withusingthisdrugsanddoctorsuseitveryoften. Chloroambucil

LECTURE9 Histaminedecarboksylazedhistydine;itsaninflamatoryfactor;relasingofhistaminecausethe decresingofpHinstomach;antihistaminedrugsandantiinflamatorydrugsareusedtogether; Examplesofantihistaminedrugs: cytometidin mepryamine antyazamine NonsteroidalantyinflamatorydrugsNSAID: inhibitorsofthefirststepprostaglandinsynthesis; prostaglandinsynthaseisacomplexofenzymeslocatedincellmembrane;thiscomplex formsachaneltroughwihicharachidonicacidistransferredandfirststepofprostaglandin synthesisoccurs; foreg.AspirinbindstoOHgroupinsidethechanelandinhibitsitscyclooxygenase(COX) propertiestransferofarachidonicacidandprostaglandinsynthesisdoesn'toccur; ExsamplesofNSAID: Aspirineacetylsalicilicacid:antiinflamatory;bigdosescouldcauseliverdamage;

paracetamol:antipain,antifeverproperties

ibuprophene:antipain

Naproxen Celebrex:inhibitorofCOX

ASPIRINSYNTHESIS

LECTURE10 PCRpolymerasechainreaction DNA+somethingwhatiscalledvector.Weintroducethisparttobacterialcodeandbacteriastarts toproduceacertainprotein.Sometimeswealsouseinsectcellswhichinsomecasesworkbetter. Sometimesmammalianexpressionsystemisalsoworkikngforcertainproteins. Weneedtoknowsomethingabouttheproteinweneedtohaveatleast10firstresidueswhich correspondstoabout30nucleotides.Usually1520nucleotidesareinthebegginingofacode. Thispartwillfindimmediatellythegeneresponsibleforthesynthesisoftheprotein.Nextweadd polymeraseenzyme.WerepeatitseveraltimestoobtainmorecopiesoffreshsynthesizedDNAof theprotein.Eachtimeweobtaintwicemorecopies1>2>4>8>16>32.Wecanobtainmany copies. Exampleofhumanrecombinantproteinsusedasdrugs: Linsuline FactorIXmedicineforHemofiliaBformerlyisolatedfromblood humangrowthfactor Interferonalpha3,interferongamma1,humaninsuline(cukrzyca),FactorVIII(Hemofilia A) erytropoetine glucagone Nowwedon'tuseproteinsisolatedfromhumanbloodbecauseofthepossibilityofaninfection. Purposesofgenemodifications: delayingthemaruration antifreezingproteins increasingstrachproduction increasingferritinamount

Cellculturesusedforproducingdrugs Transgeniccellsareveryimportantforcompaniesbecause,theycanproducemanydrugsinaquite cheapway. PlantswhicharesubjectstogeneticmodificationarecalledGMO.Wechangethecharacteristicof thefoodbyintroducingadditionalgenes.ThesegenesneedtobeproducedbyPCRandcellsare infectedwiththevirus(DNA).Wehavetopushcellstoincludesomecertaingenestothegenom. Forexamplewecanobtainplantsresistanttofungusorinsects,viruses.Themassproductionall aroundtheworldisduetogeneticalmodifications.Tomatos(forinstance)havegenesforpectin synthesismultiplied.Tomatosfruitsareverystablebecauseofthefact. Plantsresistanttospecialherbicidescanbealsoproduced.Allotherplantscanbekilled.Agood exampleiscornandsoy. Geneticallymodifiedproteinsinmedicinalchemistry.Wecanproducegeneticallymodifiedsalad whichcanworkasavaxine.Ifcertainbacterialorvirusproteinscouldbeusedforvaxination,we canintroducesthosegenestothegenomsoftheplantsweeat.Processofvaxinationinstreyer. Theprocessofproductionofgeneticallymodifiedorganism...(nicniepowiedzialwkocuotym) Vaxination Theprocessofdevelopingresistancytoadiseasetakesalongtime21days.Bacteria(orvirus) duringthistimeinourbodyessentialyhasnoenemy.Beforethe21daybacteriamaykillus.Ifwe getintouchwithacertaindangerousinvaderbeforearealinfectionourimmunesystemreactsvery fast.Forvaxineyoudonotuseawholeorganism.Sometimesitisenoughtouseafragmentofan organismoracertainprotein.Oursystemlearnstorecognizethesmallfragment.Aproteincanbe usedasavaxine.Antigeneisproduced. Processofproducingvirusvaxines (ICELLCULTURE)preparingabreedingandreplicatingthecellsreplicatingvirusesand putingtheminsidecellsgatheringandseparatingcells (IIPURIFYINGinactivationwithinthesteps)preeliminarypurifyingandincreasing concentrationmainpuryfingfinalpuryfyingduringpuryfingprocessthevirusis inactivated(withtheformaldehyde) (IIICONFECTIONING)formulatingandstabilizingpreparingpotrions(liofilization) packing Copingwithgeneticdiseases Invivomodificationinjectingavirusthatcouldchangethegeneticcode.Verydangeroussome experimentsmadesomeyearsago,showedthattheviruscanbecomedangerousforourorganisms. Exvivomodificationcellsaremodificatedinlaboratory.Cellsaretakenfromtheorganismand modifiedinlaboratoryandtheninjectedbacktoorganism. Steamcells! Steamcellscangotoanyorganandrebuilt.Therearemanyexperimentsusingsteamcellsfor rebuldingbrainafterstrokes.Theymaybefoundinbonenarrow.

Waystointroductiongenesintocells: chemical:liposmoes;precipitationwiththecalciumphosphate physical:microinjection;electroporation;particlebombardment biological:viral(retroandadenoviruses);notviralligand/receptor

LECTURE8 Anticancerdrugs Mitomycyny(MitomycinoC) Intercalators+Aliclatingdrugs(EthidiumBromide,Doxorubicyna) Antimetabolite(Analogusofpurynsandpirymidynsfluorpouracyliswidelyused, floksurydyna,allopurinol,tioguamina) RNAintercalators siRNA+ideaprzypomniesobie Animetabolite(Indoksyurydyne,Bromowinylo2deoksyurydyna,Azydotymidyna(AZT)) Forherpex(Aciklowir,Gancyklowir)

Metotreksatverystrong,toxicanticancerdrug

Antiviralcompound

Developmentphaseofthevirus 1.Adsorptionphase

2.Penetrationphase 3.Eclipsephase 4.Replicationphase 5.Synthesisofvirusproteins 6.Maturationphase 7.Releasephase LECTURE9 PCRpolymerasechainreaction

cykloalkilominederivatives(2,3step) antimetabolitsofnucleotydes tiosernikarbazonderivatives(Metisozon)(5,6step) guanidynderivatives

DNA+somethingwhatiscalledvector.Weintroducethisparttobacterialcodeandbacteriastarts toproduceacertainprotein.Sometimeswealsouseinsectcellswhichinsomecasesworkbetter. Sometimesmammalianexpressionsystemisalsoworkikngforcertainproteins. Weneedtoknowsomethingabouttheproteinweneedtohaveatleast10firstresidueswhich correspondstoabout30nucleotides.Usually1520nucleotidesareinthebegginingofacode. Thispartwillfindimmediatellythegeneresponsibleforthesynthesisoftheprotein.Nextweadd polymeraseenzyme.WerepeatitseveraltimestoobtainmorecopiesoffreshsynthesizedDNAof theprotein.Eachtimeweobtaintwicemorecopies1>2>4>8>16>32.Wecanobtainmany copies. Exampleofhumanrecombinantproteinsusedasdrugs FactorIXmedicineforHemofiliaBformerlyisolatedfromblood Nowwedon'tuseproteinsisolatedfromhumanbloodbecauseofthepossibilityofaninfection. Interferonalpha3,interferongamma1,humaninsuline(cukrzyca),FactorVIII(HemofiliaA) Cellculturesusedforproducingdrugs Transgeniccellsareveryimportantforcompaniesbecause,theycanproducemanydrugsinaquite cheapway. PlantswhicharesubjectstogeneticmodificationarecalledGMO.Wechangethecharacteristicof thefoodbyintroducingadditionalgenes.ThesegenesneedtobeproducedbyPCRandcellsare infectedwiththevirus(DNA).Wehavetopushcellstoincludesomecertaingenestothegenom. Forexamplewecanobtainplantsresistanttofungusorinsects,viruses.Themassproductionall aroundtheworldisduetogeneticalmodifications.Tomatos(forinstance)havegenesforpectin synthesismultiplied.Tomatosfruitsareverystablebecauseofthefact. Plantsresistanttospecialherbicidescanbealsoproduced.Allotherplantscanbekilled.Agoo exampleiscorn. Geneticallymodifiedproteinsinmedicinalchemistry.Wecanproducegeneticallymodifiedsalad whichcanworkasavaxine.Ifcertainbacterialorvirusproteinscouldbeusedforvaxination,we canintroducesthosegenestothegenomsoftheplantsweeat.Processofvaxinationinstreyer.

Theprocessofproductionofgeneticallymodifiedorganism...(nicniepowiedzialwkocuotym) Vaxination Theprocessofdevelopingresistancytoadiseasetakesalongtime21days.Bacteria(orvirus) duringthistimeinourbodyessentialyhasnoenemy.Beforethe21daybacteriamaykillus.Ifwe getintouchwithacertaindangerousinvaderbeforearealinfectionourimmunesystemreactsvery fast.Forvaxineyoudonotuseawholeorganism.Sometimesitisenoughtouseafragmentofan organismoracertainprotein.Oursystemlearnstorecognizethesmallfragment.Aproteincanbe usedasavaxine.Antigeneisproduced. Processofproducingvirusvaxines (ICELLCULTURE)preparingabreedingandreplicatingthecellsreplicatingvirusesand putingtheminsidecellsgatheringandseparatingcells (IIPURIFYINGinactivationwithinthesteps)preeliminarypurifyingandincreasing concentrationmainpuryfingfinalpuryfying (IIICONFECTIONING)formulatingandstabilizingpreparingpotrions(liofilization) packing Copingwithgeneticdiseases Invivomodificationinjectingavirusthatcouldchangethegeneticcode.Verydangeroussome experimentsmadesomeyearsago,showedthattheviruscanbecomedangerousforourorganisms. Exvivomodificationcellsaremodificatedinlaboratory.Steamcells! Steamcellscangotoanyorganandrebuilt.Therearemanyexperimentsusingsteamcellsfor rebuldingbrainafterstrokes. LECTURE11 Biotechnologyinmedicinalchemistry. Exvivotrials: Oneofgreatestdevelopmentisgenetherapy.Cellswithgeneticdefectaretakenoutfromhuman bodyandinlabspeopletrytofixthegeneticdefect.Thentheygrowthecellsandafterthat,fixed cellsareinjectedbacktothebody.Insomecasesitisaverygoodtherapy. 254phenotypesandtheycannotdifferentiate(skincellscannotbecomelivercellshepatocytes). Steamcellscandifferentiateintoanyothertypeofcellstheymayregenarateorgans.Adultbody stillproducesasmallamountofsteamcellsandscientistslearnhowtousethem.Greatpotential liesintheresearch. Theprocesswhencellsaretakenout,modifiedandputbackinthebodyiscalledanexvivo modification.Thekeyissueistheintroductionofanewgenetothecell. Liposomsmaybeusedtoputsomethingintothemandthentheycanserveasatransporterand bringwantedmaterialdirectlytothecell. Othermethodsaremicroinjectionandelectroporation.Donewithmicroscope,materialisinjected toonecell.Inthiscasestabilityofexpressionisshort.

Biologicalprocessesforintroductionofnewgenesintothecells.Viruses(f.ex.AAV)areusedhere weintroducespecialgenesinthem(nottotallyselectiveonlypartially).Specialproteinsthat connectwithDNAcanbeused.Nonvirusbiologicalmethodsareselective. Tissueengineering Certaintissuecanbegrownupexvivo.Themostpopularisgrowingbonetissue.Apartofthe brokenboneistakenanditisgrowninlaboratory.Thisissoeasybecausebonesaremostlybuilt frominorganicmaterials.Aftergrowingtheboneistakenandimplantedintothebody. ArtificialskinwasinventedinBoston.Ittookthemalmost18yearstogettheproduct.Itisvery difficulttoproduceskininlaboratory.Itisnotactuallyskin,butwhenitisputonbodyitactsasa basetorebuilttherealskin. Animalsastherapeuticpurpose. Organsfromanimalsareusuallyrejected.Xenographusuallyarerejected(transplantologyfromone toanotherspecies)Howevergeneticalmodificationmayhelpinintroducingsomeanimalorgans. Milkofsomemammalianmaybeusedasystemtoproductcertainproteins.Glycosylationupper synthesisofproteinsproteinisglycosylated,afewmoleculesofsugaronthesurfacehelptomake theproteinwatersoluble.Proteinsexpressedinbacteriaareglycosylatedinadifferentwaythan mammalian.That'swhyproducingproteinsfrombacteriacausesmanyproblems.Anewprocessof productingproteinsinanimalsmilkworksverygoodandisagoodalternativetousingbacteriafor growingproteins.Thecertainproteinwewanttoobtainshouldbeproducedonlyinmilk.Itmustbe introducedtogenomnexttolactoalbumingene. Monoclonalantibodies. Antibodiesareproducedbyourimmunesystem(ingeneral).Theproductioniscausedbyan infection.Theantibodiesrecognizesaverycharacteristicpartofthevirusorbacteriaandbindstoit. Thenthebacteriaarekilled.Antibodiescanalsobindandneutralizeanyprotein.Ifwehavesome pathologicalproteinorproteinresponsibleforadisease,wecanfindanantibodyforit,binditand deactivate.Inlate80sitwasagoldenboomintheUSconnectedtoantibodiestechnology.However thegreathopeoffindingverygood,selectivedidn'teverleadtoabigtechnology. PolyclonalantibodiescomefromdifferentclonesofBcells,thesequencesoftheantibodiesfrom differentBcellsdifferesabit,howevertheyworkinthesameway. AntibodieswhicharetotallythesameproducedbyonetypeofBcellsarecalledmonoclonal antibodies.Theycomefromisolatingonecellwhichisclonedthen.Thecellsproducethe monoclonalantibodies.Humanantibodiesderivedinmicewereinjectedtohumans.Despitethefact that99%oftheantibodywashumanlike,thesideeffectwasveryserious.Theywillneverworkasa gooddrugbecauseofsideeffects.Howevertherearemanydiseasesforwhichtherearenoother drugs.Theseterriblesideeffectsareacceptedbecausethere'snootheralternative.Sometimes chemotherapyiscombinedwithmonoclonalantibodiesandtheyhaveasynergiceffectsometimes (forararekindofcancer).

Ourimmunesystemproducesallthetimeabout10^12differentkindsofantibodies.Itisimportant toeatalotofmeataftereating0,5ofsteaktheamountofantibodiesinorganismgrowstwice. diagnostics Doctorsacceleratestheresistanceofbacteriatoantibiotics.Nospecificantibioticsareappliedfor specifickindofbacteria.Usuallyabroadworkingantibioticisgiven... Diagnosticsisveryimportant ELISA(enzymelinkedimmunosorbentassay) Fordiagnosticsyoudon'tneedmonoclonalalsopolyclonalmaybeused. Anenzymeisbindcovalentlytoantibodywhichrecognisespecificprotein.Lateryouputasubstrate fortheenzyme(enzymehorseradishperoxidaseusually).Theintensityofsubstratefluorescence helpssometimesdetermineconcentrationofexaminedprotein.Asocalledsandwichmethod enzymeisnotbindtotheantibodieconnecteddirectlywiththepathologicalproteinbutitisbindto

anotherantibodies.Themethodismoresensitive. Divalentantigeneanantibodyisbindtotheviruscapsuleintwoplaces(forexampleatbothsides ofthecapsulewithsamestructure).Thismethodisverysensitiveforcertainviruses. Antidigoxinpolyclonalantibody(antidote)(przeciwnaparstnicy) digoxinadrugwhichisuseddurignheartattacks.Itisveryeasytooverdosethedrug.During heartattackdoctorshavenotimetocalculatethedoseofthedrugnottokillthepatient.When doctorsseethattheinjectedtomuch,thepolyclonalantibodiesareinjectedtosavethepatientslife. Howeverthedigoxincannotbeusedagainever. Bindingaradioactivemetalradiationcanbereleasedselectivelyinthetumorcells.Thedamageis limitedmainlytothecancercells.Metalisbindedtopolyclonalantibodies.Themetalisbindlikein EDTA.Howevercancerisdyingfromnecrosis.Ifyoukillthecancercellstofast,averystrong inflamationmaybecaused(alsoaimmuneresponse). LECTURE12 Prodrugsbecomeactivewhenmetabolised.Thetransformationfromnotactiveformtoan activeonetakesplaceinourbodybecauseofpHgradientinstomach. Sometimesdrugsarenotstable. Theconcentrationofmostofdrugsisveryhighatthebeginningandthatitgoesdownveryfast. Therearesomedrugswhichreleasetheactivestructureveryslowly.Theallowtohaveconstant concentrationofacertaindruginbloodforquitealongtime.Sometimesprodrugsareproducedto releaseactivemoleculesonlyinacertaintissuewherewewantthedrugtowork.Whiletheprodrug meetsthetissueinorganismtheactivemoleculeisreleasedinsidethetissue. Betalactamsneedthreecarboxylgroups,whenabacterialinfectionisinbrainitisverydifficultto treatsuchaninfectionwithantibiotics.That'swhyabetalactamisconnectedtoaderevativeof piridine(nmethylonicotinicacidderevatives)andwhenitmeetstheinfectedtissuetheantibioticis releasedanditcankillthebacteria. CaptoprilhasSHgroupandgivesodourtothedrug.Somederevativescanbedoneandtheycan workasaprodrugforexampleesterswithaceticacid. Prednisolonisnotreallygoodsolubleinwater.Anestercanbedonewithsubsinylacid. Ingeneralprodrugscanbeconstructedinsuchawayputthreeelementstogether. Threeelementarprodrugs:carrierlinkerdrug>(enzyme)carrier+linkerdrug>(spontanieusly) linker+drug Maindrug+polymer(polyacid,polylysine,polylacticacid)+something Forinstanceaspirinprodrugispreparedinsuchaway. Polylysineisaverygoodpolymerwhichcanbeboundtoadrug.Ifwehaveaspecificcancerinone certainkindoftissue,metatrexatboundtopolylysineisanidealdrugthatdestroyscancercells.It hasbigselectivityandverystrongactivity.

Methodsofdrugapplication. Manybloodvesselsareundertounge.Thatswhysomedrugsmaybeputundertounge.That'show nitroglycerineisimmediatelydistributedinthewholebodyandliveristhelastorgantowhich nitroglycerinecomes.Itisveryimportant,becauselivermetabolisesalmosteverything.Especially drugswithOHgroup.Livermakeshydrofobicdrugshydrophilic.Whendrugbecomessolublein wateritisremovedbykidneys. Injectingdrugsinveinsdistributesthedrugsveryfastoverthebody.Sometimeslessthanaminute. Intramuscularinjectionsalsoquitefast.Forexampleinsuline. Inhalationputtingdrugstoloungesasanaerosol.Inloungestherearealotofbloodvessels.Parts ofeverydrugthataretobeputbyinhalationshouldbeverysmall.Everythingwhatislargerthan (forex.)50nmshouldbeeliminated. Penetratingdrugsthroughskinisquitedifficult,howevertherearesomeagentsthathelpdrugsto penetrateskinandgettoblood. Phenytoinanticonvulsantdrug. Newpillsmadewithlactosekilledpeople.Thedrugwasverysafe,howeverchangingformulaeof thepillmadethedistributioneasier.Tobigamountofthecompoundinbloodwasverydangerous. Treatingcancer AchillesheelofcancercancercellsgetenergyfromATP,fromglucose.80%ofcanceruses glucosetoobtainenergyfromATP.Glucosederevativewithradioactivefluorineisgivento patients.Gammaradiationcomingfromradioactivefluorinehelptomeasurehowbigisthetumor. Itisthebestwayformeasuringtumours.Thereareonlyproblemswithsolidtumours.Posytron tomographyisused. Dietwithoutcarbohydrateshelpstomaketumourssmaller(upto40%).Someresearchwasdoneon miceonlydietcanhelpincuringcancer. Thelaststageofcancerisloosingweight.Tumourproducesglucosefromaminoacids.Tumourcan pushthewholebodyjusttosupplyglucose.Thebiggestproblemisthatsometimeswedon'tneedto eatcarbohydratestoproduceglucose. Toinhibitprocessofproductionglucosebyliverwehavetousenetformine(netformina),glucofage. Theprocessofproductionofglucoseiscalledgluconeogenesis.Peoplewhohavediabesis2are gettingcancerafewtimesmoreoften(34timesmoreoften). Inhinitionofgluconeogenesisinliverhasaverygoodanticanceractivity. Glucosecanbeproducedfromlacticacid. Braincannotworkonfattyacid(albuminthathelpstotransportfattyacidscannotgoacrossthe barriertobrain).Ourbraineatsmostlyglucose.Lacticacidalsopenetratesbraintissue.Shortlength fattyacidalsoworkasresourcesforbrain. PETPositronEmissionTomograph