doktorska disertacija
Andreja Kovač (Avtor), Stanislav Gobec (Mentor), Dušanka Janežič (Član komisije za zagovor)

Povzetek

Pojav multi-rezistentnih sevov patogenih bakterij (kot so npr. S. aureus, E. faecium in P. aeruginosa), ki so odporne na več protimikrobnih učinkovin hkrati (MDR = Multidrug resistant bacteria), predstavlja resen problem v sodobnem zdravstvu. Iskanje novih tarč in sinteza protibakterijskih zdravilnih učinkovin z delovanjem na rezistentne bakterije je zato neizogibno v bitki zoper infekcijske bolezni. Ena od pomembnejših tarčnih mest za razvoj novih protibakterijskih učinkovin je še vedno bakterijski peptidoglikan, ki daje bakterijskim celicam obliko in jih varuje pred visokim osmotskim pritiskom. Sestavljen je iz mreže polisaharidnih verig, ki so prečno povezane s kratkimi polipeptidi. Vpletanje v njegovo sintezo ali strukturo vodi do lize celic in posledično do propada bakterij. Biosinteza peptidoglikana je kompleksen proces, ki vključuje okrog 20 reakcij in poteka v citoplazmi, na notranji ter na zunanji strani celične membrane. Pri tem sodelujejo številni encimi, ki predstavljajo potencialna tarčna mesta za razvoj novih protimikrobnih učinkovin. V zadnjem desetletju je vse več raziskav usmerjenih v znotrajcelične citoplazemske faze biosinteze peptidoglikana, med katerimi zavzemajo pomembno mesto Mur ligaze. Gre za štiri od ATP odvisne ligaze (MurC-MurF), ki katalizirajo zaporedno pripenjanje aminokislinskih ostankov na D-laktoilni del peptidoglikanskega prekurzorja UDP-N-acetilmuraminske kisline. Poleg Mur ligaz sodelujejo pri sintezi peptidoglikana še številni drugi pomembni encimi, med njimi tudi D-lanin:Dalanin ligaza (Ddl), ki iz dveh D-alaninov naredi D-Ala-D-Ala dipeptid, ki je substrat za MurF. Peptidoglikan se med rastjo in delitvijo bakterijskih celic stalno obnavlja in preoblikuje, pri čemer poleg omenjenih ligaz sodeluje še murein peptidna ligaza (UDP-Nacetilmuramat: L-alanil-γ-D-glutamil-mezo-diaminopimelat ligaza ali Mpl). Ta encim je sicer neesencialen za preživetje bakterij, vendar zelo pomemben pri recikliranju peptidoglikana, ker lahko v enem koraku pripne tripeptid L-alanil-γ-D-glutamil-mezo-diaminopimelat na UDP-Nacetilmuraminsko kislino.V sklopu raziskav doktorske naloge smo se osredotočili na encime MurC-F, DdlB in Mpl. Medtem ko je o Mpl-u v literaturi le malo podatkov, pa so za ostale encime poleg reakcijskih mehanizmov in specifičnosti, znane še tridimenzionalne strukture apoencimov, oziroma kompleksov encimov s substrati in produkti reakcije, kar nam omogoča racionalno načrtovanje novih zaviralcev Mur ligaz s potencialnim protibakterijskim učinkom. V okviru raziskovalnega dela smo najprej vpeljali v naše laboratorije in vitro test z malahitno zelenim za določanje encimske aktivnosti. Test temelji na osnovi določanja fosfata, ki se sprosti pri encimsko katalizirani reakciji in nam omogoča ovrednotenje inhibitorne aktivnosti potencialnih inhibitorjev Mur ligaz in Ddl. Iskanja inhibitorjev smo se najprej lotili z virtualnim rešetanjem NCI-jeve (National Cancer Institute, Ameriški nacionalni inštitut za raziskovanje raka) banke spojin in z naključnim rešetanjem banke spojin Fakultete za kemijo. Pri virtualnem rešetanju smo uporabili dostopne kristalne strukture encimov MurD (E.coli), MurF (S. pneumonie) in DdlB (E. coli) ter računalniške programe za virtualno rešetanje visoke zmogljivosti eHits 6.0 in AutoDock 4.0, ki nam s svojimi cenilnimi funkcijami omogočijo, da in vitro ovrednotimo le manjšo serijo tistih spojin, ki jim program napove najmočnejšo vezavo. Dobili smo nekaj strukturno povsem novih inhibitorjev DdlB in MurD z IC50 v nizkem mikromolarnem območju. Nekateri so poleg inihibitornega imeli tudi protibakterijsko delovanje. S preučevanjem encimke kinetike smo ugotovili, da je večina DdlB inhibitorjev kompetitivna napram ATP. Nove inhibitorje DdlB diazendikaboksamidnega tipa pa smo odkrili z naključnim rešetanjem domače banke spojin. Z uporabo peptidomimetičnega pristopa in poznavanja reakcijskih mehanizmov, ki pri ligazah MurC-F in DdlB poteka preko tetraedričnega prehodnega stanja smo načrtovali in sintetizirali nove spojine s hidroksimetilkarbonilnim skeletom. Ker murein peptidna ligaza (Mpl) uspešno vgrajuje tripeptid L-Ala-γ-D-Glu-m-A2pm na prekurzor peptidoglikana UDP-N-acetilmuraminsko kislino, smo sintetizirali serijo tripeptidov z osnovnim skeletom L-Ala-γ-D-Glu-Xaa, pri čemer smo na tretjem mestu uporabili različne aminokisline (npr. L-norleucin, L-norvalin, L-diaminobutirno kislino, L-2- aminoheptanojsko kislino, L-alanin, L-glutaminsko kislino). Čeprav encim ni ključen za biosintezo peptidoglikana, bi z vključitvijo lažnih tripeptidov s pomočjo Mpl lahko vplivali na končno sestavo peptidoglikana, ki bi bila zaradi neustrezne zgradbe nestabilna in tako smrtonosna za bakterijo. Dobljeni rezultati so dobro izhodišče za optimizacijo in nadaljnji razvoj, predvsem inhibitorjev encima DdlB.

Ključne besede

bakterijska rezistenca;sinteze;protibakaterijske učinkovine;inhibitorji;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.08 - Doktorska disertacija
Organizacija: UL FFA - Fakulteta za farmacijo
Založnik: [A. Kovač]
UDK: 543.057
COBISS: 2598769 Povezava se bo odprla v novem oknu
Št. ogledov: 211
Št. prenosov: 37
Ocena: 0 (0 glasov)
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Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Discovery of new inhibitors of bacterial ligases by virtual screening and synthesis of substrate analogues
Sekundarni povzetek: Appearance of multidrug resistant strains of pathogenic bacteria (MDR = Multidrug resistant bacteria) is a serious medical issue in modern healthcare. Battle against infectious diseases caused by drug-resistant bacteria therefore inevitable involves searching for new bacterial targets and the development of new antimicrobial agents. Peptidoglycan, an essential component of the bacterial cell wall which provides the structural integrity necessary for bacterial cells to resist internal osmotic pressure, remains one of essential and well validated targets for antibacterial therapy. Net-like structure of peptidoglycan consists of polysaccharide chains interconnected with short polypeptides. Interference in the synthesis or structure of peptidoglycan results in cell lysis and eventually bacterial death. Biosythesis of peptidoglycan is a complex process which involves around 20 reactions catalyzed by different enzymes that represent targets for development of new antibacterial agents. It occurs in the cythoplasm, on the inner side of bacterial cell membrane and on the outer cell membrane. In last decades more and more efforts have been made toward early cytoplasmic stages of biosynthesis, where Mur ligases play an important role. Mur ligases which consists of four enzymes (MurC-F), assemble the final intracellular peptidoglycan precursor UDP-MurNAc-pentapeptide by successive addition of L-Ala, D-Glu, m-A2pm or L-Lys, and D-Ala-D-Ala to D-lactoyl part of peptidoglycan precursor UDP-Nacetylmuramic acid. Beside Mur ligases other important enzymes are involved in the synthesis of peptidoglycan. One of them is D-alanine:D-alanine (Ddl), which is responsible for supplying the MurF substrate, D-alanyl-D-alanine. Another ligase, murein peptide ligase (UDP-N-acetylmuramate:L-alanyl-γ-D-glutamyl-meso-diaminopimelate ligase or. Mpl), is responsible for constant remodeling of peptidoglycan during bacterial cell maturation and division. This enzyme is not essential for bacterial growth itself but since it is able to add tripeptide L-alanyl-γ-D-glutamyl-meso-diaminopimelate onto UDP-N-acetylmuramic acid in one step it is very important in recycling of peptidoglycan. In this thesis we focused our attention on MurC-F, DdlB and Mpl enzymes. There is little known in the literature about Mpl, whereas reaction mechanisms, specificity of enzymes and tridimensional structures of apoenzymes or complexes with substrates and products are known for other ligases. That will enable us to design new inhibitors of Mur ligases and DdlB with potential antibacterial activity. In the context of the research work, we first introduced in our laboratories in vitro assay with malachite green for the determination of enzyme activity. The assay is based on the determination of free phosphate released in the enzyme-catalyzed reaction and allows us to evaluate the inhibitory activity of potential Mur and DdlB inhibitors. First attempt in finding inhibitors was screening of inhouse bank of compounds from Faculty of chemistry. Diazenedicarboxamide inhibitors of DdlB were discovered in that manner. Later virtual screenig was performed on available crystal structures of MurD (E.coli), MurF (S. pneumonie) and DdlB (E. coli) enzymes using computational tools for highthroughput virtual screening eHits 6.0 and AutoDock 4.0 and NCI diversity set (National Cancer Institute bank of compounds). Only high ranked compounds with strong predicted binding afinity were evaluated in vitro. New inhibitors with novel scaffolds of DdlB and MurD with IC50 in low micromolar range were discovered. Some inhibitors had also promising antibacterial activities against Gram-positive and Gram-negative bacteria. Kinetic experiments revealed that most of the DdlB inhibitors are ATP competitive. Compounds with hydroximethylcarbonyl fragment were designed and synthesized using peptidomimetic approach. Common reaction mechanisms of MurC-F and DdlB ligases via tetrahedral transition-state provided the starting point for new inhibitors. Since murein peptide ligase (Mpl) succesfuly adds tripeptide L-Ala-γ-D-Glu-meso-A2pm onto peptidoglycan precursor UDP-N-acetylmuramic acid we synthesized series of tripeptides with core structure L-Ala-γ-D-Glu-Xaa. Amino acid on the third position was altered using Lnorleucine, L-norvaline, L-diaminobutiric acid, L-2-minoheptanoic acid, L-alanine, Lglutamic acid etc. Although the enzyme is not essential for biosynthesis of peptidoglycan, incorporation of false tripeptides with Mpl would affect final composition and cross-linking of peptidoglycan which could lead to bacterial cell death. Obtained results reveal good starting point for optimization and further development of inhibitors, especially inhibitors of DdlB.
Vrsta dela (COBISS): Doktorska disertacija
Komentar na gradivo: Univ. Ljubljana, Fak. za farmacijo
Strani: 174 str.
ID: 12895810