Razvoj peptidnih inhibitorjev lipaz in fosfolipaz z metodo bakteriofagnega prikaza : doktorska disertacija

Lunder, Mojca (avtor), Kreft, Samo (mentor)

Biološke knjižnice naključnih peptidov, predstavljenih na površini organizmov, nam omogočajo sistematično proučevanje interakcij ciljnih makromolekul. Peptidni modulatorji terapevtsko zanimivih ciljnih proteinov imajo v sodobnem načrtovanju učinkovin vse pomembnejšo vlogo. V prvem delu smo vpeljali, razvili in optimirali metodologijo bioloških kombinatoričnih knjižnic z uporabo streptavidina kot modelne ciljne makromolekule. V drugem delu pa smo jo uporabili kot orodje za identifikacijo strukturno in funkcionalno optimalnih peptidnih ligandov izbranih ciljnih makromolekul. Primerjalno smo ovrednotili dva tipa bioloških knjižnic, bakterijsko in bakteriofagno predstavitveno knjižnico. Na podlagi lastnih eksperimentov in podatkov, objavljenih v literaturi, smo zaključili, da so bakteriofagne predstavitvene knjižnice sicer tehnično zahtevnejše, vendar v večini primerov vodijo v uspešnejšo selekcijo. Za rešetanje in ovrednotenje posameznih bakteriofagnih klonov smo primerjali dve metodi, encimskoimunski test na trdnem nosilcu in površinsko plazmonsko resonanco. Obe metodi je mogoče uspešno uporabiti za določanje vezavnih lastnosti bakteriofagnih klonov, izbira pa temelji na naravi in količini ciljne makromolekule ter nenazadnje na opremi, ki nam je na voljo. Predvsem kadar je količina ciljne makromolekule omejujoč dejavnik, je metoda izbora površinska plazmonska resonanca, saj raznoliki senzoski čipi omogočajo pritrditev katerekoli makromolekule, ne glede na njene fizikalno-kemijske lastnosti. V tehnologijo bakterifagnega prikaza smo vpeljali uporabo ultrazvoka, ki pri eluciji pripomore tako k pretrganju interakcij med tarčo in peptidom na bakteriofagu, kot tudi desorpciji tarčnega proteina skupaj z bakteriofagom s trdnega nosilca. Uvedba tega inovativnega postopka v afinitetne selekcije bo brez dvoma prispevala k razvoju tehnologije bakteriofagnih predstavitvenih knjižnic. Tehnologijo bioloških peptidnih knjižnic smo uporabili za razvoj novih ligandov in potencialnih inhibitorjev encimov, ki sodelujejo pri razgradnji in absorpciji maščob v prebavnem traktu (pankreasne lipaze, pankreasne fosfolipaze A2). Uporabili smo knjižnico naključnih, cikličnih heptapeptidov in linearnih dodekapetidov izraženih na bakteriofagih. Naredili smo več neodvisnih selekcij, pri katerih smo spreminjali načine spiranja in elucije. Inhibicijo pankreasne lipaze smo dosegli s peptidom D23 (navidezna konstanta inhibicije 16 μM). Ligandi pankreasne fosfolipaze A2 in sorodne fosfolipaze, amoditoksina C, ki smo jih izolirali z različnimi selekcijskimi protokoli, jasno kažejo afiniteto do obeh encimov, neodvisno od ciljne molekule uporabljene pri selekciji. Navkljub vezavi na tarčna proteina, pa peptidi ne vplivajo na njuno aktivnost. Peptidi, pridobljeni s to metodologijo, so ligandi določenih mest ciljnega proteina in lahko predstavljajo spojine vodnice pri razvoju novih učinkovin. Njihova uporabna vrednost je predvsem v sposobnosti selektivnih interakcij s ciljnimi proteinskimi makromolekulami.

• Jezik: Slovenski jezik
• Ključne besede: peptidne knjižnice, zdravljenje debelosti, farmakologija, bakteriofagna knjižnica, bakterijska knjižnica
• Vrsta gradiva: Doktorska disertacija
• Tipologija: 2.08 - Doktorska disertacija
• Organizacija: FFA - Fakulteta za farmacijo
• Kraj izida: Ljubljana
• Založnik: [M. Lunder]
• Leto izida: 2008
• Št. strani: 102 str.
• PID: 20.500.12556/RUL-127019
• UDK: 577.115
• COBISS.SI-ID: 2277233
• Datum objave v RUL: 13.05.2021

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Development of peptide inhibitors of lipases and phospholipases by phage display technology

Izvleček

Libraries of random peptides displayed on the surface of biological systems are an essential tool that enables systematic study of target molecule interactions. Peptide modulators of therapeutically interesting target proteins are gaining an increasingly important role in drug discovery and development. First part comprises of introduction, development and optimization of biological display libraries with the use of a model target molecule, streptavidin. The second part describes the use of such libraries and optimized procedures in a search of novel peptide ligands of selected targets. Bacterial and phage display libraries were compared for their efficiency in the search of model target protein (streptavidin) binding motif. Under similar conditions, phage display library, although technically more demanding, proved to be convincingly better. Efficiency and convenience of enzyme linked immunosorbent assay and surface plasmon resonance were compared for determining the binding properties of peptide-displaying phage clones using streptavidin as a model protein target. Both methods can be successfully used for affinity ranking. The choice therefore depends on the nature and the amount of target protein and the equipment available. Versatile sensor chips enable immobilization of almost any target molecule, regardless of their physiochemical properties and when there is only a small amount of target molecule available, surface plasmon resonance would be a method of choice. An improved non-specific elution strategy was introduced to the phage display technology. This procedure employs ultrasound to break the interaction of high affinity clones with the target and also to detach the target molecule from the immobilization surface. This new approach is able to surmount the incapability of other commonly used non-specific elution techniques and represents an important improvement in a search for novel specific ligands. Phage display technology was used to develop new ligands and potential inhibitors of lipid digestion and absorption enzymes (pancreatic lipase, pancreatic phospholipase A2) in gastrointestinal tract. A random, cyclic heptapetide and a linear dodecapeptide phage display library were used. Several independent selections were performed, differing in washing and elution steps. Inhibition of pancreatic lipase was achieved by peptide D23 (apparent inhibition constant of 16 μM). Peptide ligands of pancreatic phospholipase A2 and structurally related ammodytoxin C, selected by different selection protocols, clearly indicate affinity towards both enzymes, regardless which of the two proteins was used as a target in the selection procedure. Despite clear affinity, none of the peptides had inhibitory activity in vitro.Peptides selected and discovered by screening biological libraries target only a few sites on a given protein, often associated with biological activity. These peptides are modulators of protein function and are a starting point in drug development, their main advantage being the ability of highly specific interaction with protein macromolecules.