Načrtovanje, sinteza in vrednotenje novih zaviralcev in himernih razgrajevalcev človeškega galektina-8 : raziskovalni podatki, obravnavani v doktorskem delu

Purić, Edvin (author), Anderluh, Marko (mentor)

Galektin-8 igra pomembno vlogo v prirojenem in pridobljenem imunskem odzivu, saj je vključen v širok spekter fizioloških procesov in pri številnih boleznih, zaradi česar je zanimiva tarča za zelo močne in selektivne zaviralce galektina-8N. V našem delu smo zasnovali knjižnice spojin, začenši z znanim zaviralcem galektina-8N, ki ima nizko mikromolarno afiniteto za galektin-8N in rahlo selektivnost v primerjavi z galektinom-3, ki ima podobno vezavno mesto kot galektin-8N. Z našo strategijo smo uspeli pripraviti prve zaviralce galektina-8N z vrednostmi Kd v nanomolarnem območju in nekoliko izboljšano selektivnostjo do galektina-3 v primerjavi s spojino vodnico. Izotermna titracijska kalorimetrija (ITC) je pokazala pomembne razlike v entalpijskih in entropijskih prispevkih k vezavi in zelo zanimiv termodinamski odtis izbranih zaviralcev galektina-8N. Pridobili smo kristalno strukturo 2-O-propargilnega derivata v kompleksu z N-končno domeno galektina-8, ki nakazuje na interakcijo med acetilenskim delom kokristaliziranega liganda in gvanidinskim delom stranske verige Arg45. Nadaljnji kvantno-mehanski izračuni so potrdili novo vrsto interakcije, ki do zdaj še ni bila opisana in jo lahko označimo kot nekanonično kation-π interakcijo. Poleg tega smo dokazali, da je test fluorescenčne polarizacije zanesljiva metoda za rešetanje in se lahko uporablja pri rešetanju visoke zmogljivosti zaviralcev galektina-8N. Hkrati smo pripravili novo fluorescentno sondo, ki zahteva manj sinteznih korakov v primerjavi s tetrasaharidno LNnT sondo in se lahko alternativno uporablja pri rešetanju zaviralcev galektina-8N. Skupaj s partnerji z Univerze v Firencah smo uspešno zasnovali BODIPY sondo in dokazali njeno uporabnost pri preučevanju lektinov v testih na osnovi fluorescence. Kot prvi smo načrtovali, sintetizirali in biološko ovrednotili himerni razgrajevalec (PROTAC) galektina-8, ki je povzročil razgradnjo galektina-3 in -8 v celicah raka dojke. Nasprotno pa nobena od načrtovanih molekul PROTAC ni vplivala na ravni galektina-3 in -8 v celicah HUVEC. Rezultati testa angiogeneze in vitro so pokazali spremembe v tvorbi cevk, medtem ko popolno zaviranje angiogeneze ni bilo doseženo.

• Language: Slovenian
• Keywords: galektini, galektin-8, zaviralci galektina-8, imunski odziv, tumor, test fluorescenčne polarizacije, himerni razgrajevalci
• Typology: 2.20 - Complete scientific database of research data
• Time coverage: 2021-2025
• Organization: FFA - Faculty of Pharmacy
• Year: 2025
• PID: 20.500.12556/RUL-171959
• Data col. methods: Experiment: Laboratory
• Publication date in RUL: 05.09.2025

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: English
• Title: Design, synthesis and evaluation of novel inhibitors and proteolysis-targeting chimeras for human galectin-8 : research data underlying the doctoral dissertation

Abstract

Galectin-8 plays an important role in innate and adaptive immune responses, as it is involved in a wide variety of physiological processes and in a number of diseases, making it an interesting target for highly potent and selective galectin-8N inhibitors. In our work, we have designed compound libraries, starting from a known galectin-8N inhibitor, which has a low micromolar affinity for galectin-8N and slight selectivity towards galectin-3, which has a similar binding site to galectin-8N. Our strategy led to the first inhibitors of galectin-8N with Kd values in the nanomolar inhibition range and slightly improved selectivity towards galectin-3, compared to the lead compound. Isothermal titration calorimetry (ITC) revealed important differences in enthalpic and entropic contributions to the binding and indicated a very interesting thermodynamic footprint of the selected galectin-8N inhibitors. We solved a crystal structure of the 2-O-propargyl derivative in complex with the N-terminal domain of galectin-8, showing a close contact between the acetylene moiety of the co-crystallised ligand and the guanidine moiety of the Arg45 side chain. Further quantum mechanical calculations confirmed a new type of interaction, which has not been previously identified and can be characterised as a non-canonical cation-π interaction. Besides, we proved that the fluorescence polarisation assay is a reliable screening method and can be used for high-throughput screening of galectin-8N inhibitors. A new fluorescent probe was designed, which requires fewer synthesis steps compared to a tetrasaccharide LNnT probe and can be alternatively used in screening of galectin-8N inhibitors. Together with partners from the University of Florence, we have successfully designed BODIPY probe and demonstrated its usefulness in studying lectins in fluorescence-based assays. Finally, we were the first to design, synthesize and biologically evaluate chimeric degrader (PROTAC) for galectin-8, which induced galectin-3 and -8 degradation in breast cancer cells. Conversely, none of the designed PROTACs were able to affect galectin-3 and -8 levels in HUVEC cells. The results of the in vitro angiogenesis assay showed changes in tube formation, while the complete inhibition of angiogenesis has not been achieved.

• Keywords: galectins, galectin-8, galectin-8 inhibitors, immune response, tumor, fluorescence polarization assay, PROTAC molecules

Projects

• Funder: ARIS - Slovenian Research and Innovation Agency
• Project number: P1-0208
• Name: Farmacevtska kemija: načrtovanje, sinteza in vrednotenje učinkovin