20.500.12556/RUL-171959 Načrtovanje, sinteza in vrednotenje novih zaviralcev in himernih razgrajevalcev človeškega galektina-8 raziskovalni podatki, obravnavani v doktorskem delu Design, synthesis and evaluation of novel inhibitors and proteolysis-targeting chimeras for human galectin-8 research data underlying the doctoral dissertation 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. 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. galektini galektin-8 zaviralci galektina-8 imunski odziv tumor test fluorescenčne polarizacije himerni razgrajevalci galectins galectin-8 galectin-8 inhibitors immune response tumor fluorescence polarization assay PROTAC molecules true false false Slovenski jezik Angleški jezik 2021-2025 Neznano 2025-09-04 13:41:55 2025-09-05 15:13:13 2025-09-06 04:51:18 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2026-09-29 Readme.txt Readme.txt Readme 1 1CB94DBE1645BE4BD3B82090C72E11C2 08a53a103884b89d022548c67f5edc31fdb1d9e28402f262e544fabf8b4580d3 b2ef75e0-8984-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=215577 Files.rar Files.rar Files 1 4D55657B0C0F169B87329E98BB125401 140d781aab42be9b0932b0f16760e943058632579aba823e4ef69cac8cd51f9c f13175bf-8989-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=215670 Fakulteta za farmacijo Poskus: Laboratorij 0 0 0