Načrtovanje, sinteza in vrednotenje aciliranih analogov tripeptida Gly-L-Ser-D-Glu kot modulatorjev prirojenega imunskega sistema

Klančič, Veronika (avtor), Jakopin, Žiga (mentor)

Receptor NOD2 je pomembna komponenta prirojenega imunskega sistema in je zato zanimiva tarča za iskanje novih imunomodulatorjev. NOD2 sicer spada med znotrajcelične receptorje za prepoznavo vzorcev, ki zaznavajo metabolite peptidoglikana - ta se nahaja v celični steni tako Gram pozitivnih kot Gram negativnih bakterij. Minimalni strukturni fragment, ki aktivira NOD2 in posledično izkazuje imunostimulatorne učinke, je muramil dipeptid, ki so ga razvijali kot obetaven adjuvans. Adjuvansi so namreč pomembne komponente cepiv, saj z njimi dosežemo boljši učinek cepiva – torej večji titer protiteles, ob tem pa potrebujemo tudi manjše količine antigenov. Agoniste NOD2 lahko potencialno uporabimo ne samo kot adjuvanse za cepiva temveč tudi v imunoterapiji rakavih obolenj kot alternativo klasičnemu zdravljenju, za zdravljenje okužb v kombinaciji z antibiotiki in pri zdravljenju nekaterih vnetnih bolezni. V magistrski nalogi smo načrtovali, sintetizirali in ovrednotili acilirane analoge Gly-L-Ser-D-Glu kot potencialne agoniste receptorja NOD2. Izhajali smo iz raziskav o dezmuramilpeptidnih mimetikih muramil dipeptida, osnovanih na tripeptidu Gly-L-Val-D-Glu, v strukturi katerih je namesto N-acetilmuraminske kisline vgrajena trans-ferulična kislina kot aromatski glikomimetik. Glavni cilj naloge je bil ugotoviti, kako zamenjava centralnega L-valina z L-serinom vpliva na njegovo NOD2 agonistično aktivnost. Nato pa smo sintetizirali tudi različne lipofilne acilirane analoge, da bi videli vpliv spremembe strukture in lipofilnosti na sposobnost aktivacije NOD2 Končnim spojinam smo biološko ovrednotili sposobnost aktivacije NOD2 na celični liniji HEK293-NOD2, ki ima pretirano izražen gen za NOD2. Spojina 4, s strukturo Gly-L-Ser-D-Glu, v kateri je na serinsko OH skupino vezana lipofilna benzilna skupina, je pokazala najvišjo aktivnost od vseh testiranih spojin, zato smo njeno imunomodulatorno delovanje še dodatno ovrednotili s funkcijskimi testi. Preverili smo njen učinek na citotoksično sposobnost mononuklearnih celic iz periferne krvi napram tumorskim celicam, a je eksperiment pokazal, da spojina v 10 μM koncentraciji na to ni imela vpliva. Nato smo preverili, kako spojina 4 vpliva na izražanje nekaterih označevalcev iz monocitov pridobljenih dendritičnih celic, ki so najpomembnejše celice za doseganje adjuvantnega delovanja. Ugotovili smo, da je spojina 4 značilno povečala izražanje označevalcev CD80, CD86 in HLA-DR, s čimer je nekoliko pospešila zorenje dendritičnih celic.

• Jezik: Slovenski jezik
• Ključne besede: Receptor NOD2, agonisti NOD2, muramil dipeptid, dezmuramilpeptidi, adjuvans
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2020
• PID: 20.500.12556/RUL-119772
• Datum objave v RUL: 11.09.2020

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Design, synthesis and evaluation of acylated Gly-L-Ser-D-Glu analogs as innate immune system modulators

Izvleček

NOD2 receptor is an important component of the innate immune system and therefore constitutes a valuable target for the research of new immunomodulators. NOD2 receptors belong to a group of intracellular pattern recognition receptors that detect metabolites of peptidoglycan, which is a part of the cell wall of Gram positive and Gram negative bacteria. The minimal structural fragment that activates NOD2 and consequently exhibits immunostimulatory effects is muramyl dipeptide, which has been developed as a promising adjuvant. Adjuvants are important components of vaccines and improve their efficacy – an increased antibody titer while such vaccines usually also require smaller amounts of antigens. NOD2 agonists can potentially also be used in cancer immunotherapy as an alternative to classical treatment, for treating infections in combination with antibiotics and in treatment of some inflammatory diseases. In this Master's thesis, we designed, synthesised and evaluated acylated analogues of Gly-L-Ser-D-Glu as potential NOD2 receptor agonists. Our plan was based on recent studies of muramyl dipeptide mimetics, specifically from desmuramyl peptide, in which a trans-ferulic acid was used as an aromatic glycomimetic instead of N-acetylmuramic acid. The main goal was to establish how the replacement of the central L-valine with L-serine affects the NOD2 agonistic activity of compounds. We also synthesised diverse lipophilic analogues to investigate the effect of structure and lipophilicity alterations have on their ability to activate NOD2. Final compounds were evaluated for the ability to activate NOD2 on HEK293-NOD2 cell line with overexpressed gene for NOD2. Compound 4 based on a Gly-L-Ser-D-Glu core structure carrying a lipophilic benzyl group attached to the serine OH group showed the highest activity of all tested compounds, therefore its immunomodulatory activity was additionally evaluated in functional assays. We examined its effect on cytotoxic ability of peripheral blood mononuclear cells against tumor cells and revealed the compound lacked activity at the concentration of 10 μM. Further, we evaluated the effect of compound 4 on the expression of specific surface markers, important for achieving adjuvant activity, present on monocyte-derived dendritic cells. Compound 4 was found to significantly increase the expression of CD80, CD86 and HLA-DR markers, thereby moderately facilitating dendritic cell maturation.

• Ključne besede: NOD2 receptor, NOD2 agonists, muramyl dipeptide, desmuramylpeptides, adjuvant