Proučevanje vpliva zamenjave glicina z aromatskimi in heteroaromatskimi aminokislinami na agonistično aktivnost dezmuramilpeptidov na nukleotid-vezočo oligomerizacijsko domeno vsebujoč protein 2

Bele, Klemen (author), Jakopin, Žiga (mentor), Janež, Špela (comentor)

Receptor z nukleotid-vezočo oligomerizacijsko domeno 2 (NOD2), ki se nahaja predvsem v imunskih celicah, spada v družino receptorjev za prepoznavo vzorcev (PRR) in je eden pomembnejših receptorjev za prepoznavanje patogenih mikroorganizmov v našem telesu. Receptor NOD2 kot osnovni ligand prepozna muramil dipeptid (MDP), ki je del celične stene bakterij. Ob vezavi liganda se sprožita tako prirojeni kot pridobljeni imunski sistem, zato imajo agonisti NOD2 velik terapevtski potencial kot adjuvansi v cepivih pa tudi kot imunoterapevtiki za zdravljenje raka. V zadnjih letih so odkrili tudi imunoregulatorno vlogo agonistov NOD2, zaradi česar bi se lahko v prihodnosti uporabljali v terapiji nevrodegenerativnih bolezni, kot sta multipla skleroza in Alzheimerjeva demenca. Trenutno je na evropskem farmacevtskem tržišču registriran mifamurtid, ki se v določenih primerih uporablja za zdravljenje operabilnega nerazsejanega osteosarkoma. V sklopu magistrske naloge smo želeli izboljšati agonistično aktivnost dezmuramilnih derivatov MDP na NOD2. Izhajali smo iz spojine vodnice, v strukturo katere smo namesto aminokisline glicin uvedli različno orientirane aromatske in heteroaromatske aminokisline, s tem pa vpeljali različno velike lipofilne elemente in sintetizirali šest končnih spojin. Z reporterskim testom na celicah HEK-Blue hNOD2 smo preverili njihovo sposobnost aktivacije NOD2, hkrati pa smo preverili tudi njihovo citotoksičnost. Rezultati so pokazali, da so končne spojine z D-orientiranimi aromatskimi aminokislinami bolj aktivne kot njihovi analogi z L-konfiguracijo na kiralnem centru, kot najbolj optimalna aminokislina pa se je izkazal homofenilalanin. Spojina 14, ki v strukturi vsebuje D-homofenilalanin, namreč izkazuje NOD2 agonistično delovanje že v femtomolarnem koncentracijskem področju (EC50 = 16 fM), kar predstavlja izboljšanje jakosti za več kot pet velikostnih razredov v primerjavi z do tedaj najmočnejšim dezmuramilpeptidom pa tudi muramilpeptidom in je daleč najmočnejši sintezni agonist NOD2 do sedaj.

• Language: Slovenian
• Keywords: Receptor NOD2, agonisti NOD2, dezmuramilpeptidi, odnos med strukturo in delovanjem, imunski sistem
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-158747
• Publication date in RUL: 20.06.2024

Secondary language

• Language: English
• Title: Investigating the impact of glycine replacement with aromatic and heteroaromatic amino acids on nucleotide-binding oligomerization domain protein 2 agonistic activity of desmuramylpeptides

Abstract

The nucleotide-binding oligomerisation domain 2 receptor (NOD2), found mainly in immune cells, belongs to the pattern recognition receptor (PRR) family and is one of the most important receptors for detection of pathogenic microorganisms in our body. NOD2 recognises muramyl dipeptide (MDP), which is part of the bacterial cell wall, as its primary ligand. Binding of the ligand to the NOD2 receptor triggers both the innate and acquired immune system, and therefore NOD2 agonists have great therapeutic potential as adjuvants in vaccines as well as immunotherapeutics for the treatment of cancer. In recent years, researchers have also discovered an immunoregulatory role for NOD2 agonists, which could lead to future applications in the therapy of neurodegenerative diseases such as multiple sclerosis and Alzheimer's dementia. Currently, mifamurtide is registered on the European pharmaceutical market and is used in certain cases for the treatment of operable unresectable osteosarcoma. In the scope of the Master's thesis, we aimed to improve the agonist activity of MDP desmuramyl derivatives on NOD2. Starting from the lead compound, we introduced differently oriented aromatic and heteroaromatic amino acids instead of the amino acid glycine into its structure, thereby introducing lipophilic elements of different sizes thus synthesising six final compounds. Their ability to activate NOD2 was verified by a reporter assay on HEK-Blue hNOD2 cells, and their cytotoxicity was also tested. The results showed that final compounds with D-oriented aromatic amino acids were more active than their analogues with an L-configuration at the chiral centre, and homophenylalanine was found to be the most optimal amino acid. Indeed, compound 14, which contains D-homophenylalanine in its structure, exhibits NOD2 agonist activity in the femtomolar concentration range (EC50 = 16 fM), which represents an improvement in potency of more than five orders of magnitude over the most potent desmuramylpeptides to date, as well as muramylpeptides, and is by far the most potent synthetic NOD2 agonist to date.

• Keywords: NOD2 receptor, NOD2 agonists, desmuramylpeptides, structure-activity relationship, immune system