Strukturno podprto načrtovanje, sinteza in biološko vrednotenje novih zaviralcev O-β-N-acetilglukozaminil transferaze : doctoral dissertation

Loi, Elena Maria (author), Anderluh, Marko (mentor), Pieters, Roland J. (comentor)

Thousands of nuclear and cytosolic proteins are subjected to post-translational modification at serine or threonine residues by O-linked ß-N-acetylglucosamine (O-GlcNAc). This unique type of glycosylation, known as O-GlcNAcylation, does not result in complex glycans but is rather cycled on and off proteins by the two enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Notably, these enzymes work in an interplay with phosphorylation regulating a wide variety of biological processes, including gene expression, cell cycle progression, epigenetics, and stress response. O-GlcNAcylation is thus critical in maintaining cell homeostasis, and its dysregulation is linked with severe human pathologies like cancer, diabetes, and cardiovascular and neurodegenerative diseases. Hence, the key enzymes OGT and OGA have both been proposed as potential therapeutic targets for these conditions. For instance, an increase in O-GlcNAcylation levels and OGT expression can be observed in many human cancers, and different studies have shown that OGT inhibition decreases cancer cell proliferation. Although the available data are promising, the exact role of O-GlcNAcyation in the pathogenesis of tumors and other conditions is far from being well understood. One of the main obstacles to studying this critical post-translational modification is the limited availability of potent and cell-permeable OGT inhibitors. Namely, most inhibitors are structurally related to the glycosyl donor uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) or the enzymatic reaction byproduct, UDP, so they are usually not cell-permeable nor selective. Only in recent years the first series of potent small molecules OGT inhibitors, known as OSMIs, has been developed, but the compounds are not suitable for in vivo studies. Our work within the scope of this dissertation focused on filling this gap and providing the field with new chemical tools to study OGT's role in different biological systems. In particular, we employed four different strategies to design novel OGT inhibitors. We began by developing two new libraries of fragment-like inhibitors based on the 2-hydroxyquinoline uridine mimetic scaffold discovered by our group in a previous study. The compounds were active in vitro at micromolar concentrations, and the best hit showed an IC50 value of 13 µM. Since the libraries were screened with two different biochemical assays, our data also pointed out that special care should be taken when testing UDP mimics with the commercial UDP Glo? assay, as false positives may occur. Our second approach consisted in performing virtual screening of an extensive library of drug-like molecules. This allowed us to identify novel uridine mimetic chemotypes and select the most promising commercially available hits for in vitro screening. Notably, some of the tested molecules inhibited the enzyme at micromolar concentrations, and the best hit displayed an IC50 value of 7 µM, which places it amongst the most potent OGT inhibitors reported to date. Thus, the compound was additionally explored by developing a focused library of its derivatives to study its SAR. The inhibitor was also tested in cell-based assays but unfortunately was not active at micromolar concentrations. Since the first nanomolar OGT inhibitors became available while we were working on our project, we decided to resynthesize the most potent one, OSMI-4, and use it to study O-GlcNAcylation in different human cancer cell lines. Our results revealed that the compound's metabolic stability varies between cell lines and could be improved by replacing a labile ethyl ester featured in the inhibitor's structure with more stable functional groups. We therefore designed and synthesized a library of amide and amine-based OSMI-4 derivatives and demonstrated that ester replacement does not significantly affect the compound's potency. Notably, while the cell permeability of the molecules is still being investigated, their stability to hydrolysis by plasma esterases has greatly improved. Hence, we believe this strategy will help us modulate the inhibitors' drug properties and might eventually yield the first potent chemical probe suitable for in vivo studies. Finally, we presented a new potentially druggable pocket near OGT's active site. The pocket surrounds Asp554, which is thought to be involved in the enzymatic catalysis and could thus represent a target for OGT inhibition. Our approach consisted in designing a small series of fragments using various computational tools, followed by their synthesis and in vitro screening. One fragment showed promising results by inhibiting the enzyme with an IC50 value of 1.4 mM. However, since the binding site of these compounds still needs to be experimentally confirmed, x-ray crystallographic data of the ligand-protein complex should be acquired to help design more potent inhibitors.

• Language: English
• Keywords: O-GlcNAcylation, OGT, OGA, OGT inhibitors, posttranslational, modifications, computational drug design, virtual screening, ester hydrolysis, metabolic stability
• Work type: Dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [L. ELena Maria]
• Year: 2023
• Number of pages: 185 f.
• PID: 20.500.12556/RUL-144929
• UDC: 615.4:54:616-00(043.3)
• COBISS.SI-ID: 146223619
• Publication date in RUL: 23.03.2023

Secondary language

• Language: Slovenian
• Title: Structure-based design, synthesis and biological evaluation of novel O-β-N-acetylglucosaminyltransferase inhibitors

Abstract

Na tisoče jedrskih in citosolnih proteinov je podvrženo posttranslacijski modifikaciji na serinskih ali treoninskih ostankih z O-β-N-acetilglukozaminom (O-GlcNAc). Ta edinstvena vrsta glikozilacije, znana kot O-GlcNAciliranje, ne povzroči pripenjanja kompleksnih glikanov, ampak gre za ciklično pripenjanje in odstranjevanje monosaharidnega ostanka (GlcNAc), kar katalizirata dva encima: O-GlcNAc transferaza (OGT) in O-GlcNAcaza (OGA). Delovanje obeh encimov je prepleteno s fosforilacijo na istih aminokislinskih ostankih in uravnava široko paleto bioloških procesov, vključno z izražanjem genov, napredovanjem celičnega cikla, epigenetiko in odzivom na stres. O-GlcNAciliranje je tako ključnega pomena pri vzdrževanju celične homeostaze, njena disregulacija pa je povezana s hudimi človeškimi patološkimi stanji, kot so rak, sladkorna bolezen ter kardiovaskularne in nevrodegenerativne bolezni. To nakazuje, da sta encima OGT in OGA potencialni tarči za zdravljenje omenjenih bolezni. Na primer, povečanje ravni O-GlcNAciliranja in izražanja OGT je mogoče opaziti pri številnih vrstah raka pri ljudeh, saj so različne študije pokazale, da zaviranje OGT zmanjša proliferacijo rakavih celic. Čeprav so razpoložljivi podatki o OGT kot potencialni tarči za zdravilne učinkovine obetavni, še zdaleč ne razumemo natančne vloge O-GlcNAciliranja v patogenezi tumorjev in drugih stanj. Ena od glavnih ovir pri preučevanju te posttranslacijske modifikacije je omejena razpoložljivost močnih in celično prepustnih zaviralcev OGT. Večino zaviralcev so načrtovali na osnovi substrata glikozil-donorja uridin difosfat N-acetilglukozamina (UDP-GlcNAc) ali stranskega produkta encimske reakcije UDP, zato običajno slednji niso celično prepustni niti selektivni za OGT. Šele v zadnjih letih je bila razvita prva serija močnih zaviralcev OGT z majhnimi molekulami, znanih kot OSMI, vendar spojine niso primerne za študije in vivo, saj so v esterski obliki nestabilne v plazmi in celicah z izraženimi esterazami. V okviru te disertacije smo se osredotočili na zapolnitev te vrzeli z načrtovanjem presnovno stabilnih zaviralcev OGT za preučevanje vloge OGT v različnih bioloških sistemih. Uporabili smo štiri različne strategije za oblikovanje novih zaviralcev OGT. Začeli smo z razvojem dveh novih knjižnic fragmentom-podobnih zaviralcev z 2-hidroksikinolinom kot osnovnim ogrodjem, ki posnema uridin pri interakcijah z vezavnim mestom. Spojine so bile učinkoviti zaviralci OGT in vitro pri mikromolarnih koncentracijah, najboljši zadetek pa je pokazal vrednost IC50 13 μM. Potencialne zaviralce OGT smo testirali z dvema različnima biokemičnima testoma, pri čemer smo ugotovili, da je potrebna posebna previdnost pri preskušanju mimetikov UDP s komercialnim testom UDP Glo␢, saj ta lahko generira lažno pozitivne rezultate. Naš drugi pristop je temeljil na virtualnem rešetanju obsežne knjižnice spojin podobnih učinkovinam. To nam je omogočilo identifikacijo novih kemotipov, ki posnemajo uridin, in izbiro najbolj obetavnih komercialno dostopnih zadetkov za testiranja in vitro. Najmočnejši odkriti zaviralci OGT so zavirali encim pri nizkih mikromolarnih koncentracijah, najboljši zadetek pa je pokazal vrednost IC50 7 μM, kar ga uvršča med najmočnejše zaviralce OGT. Na osnovi tega zadetka smo načrtovali in pripravili ožjo knjižnico spojin, da bi preučili odnos med strukturo in delovanjem te serije. Najmočnejši zadetek iz te serije spojin je bil testiran tudi v celičnih testih, vendar žal ni bil aktiven pri uporabljenih mikromolarnih koncentracijah. Med potekom našega projekta so S. Walker in sodelavci objavili prve nanomolarne zaviralce OGT, zato smo se odločili ponovno sintetizirati najmočnejšega med njimi (OSMI-4) in ga uporabiti za preučevanje O-GlcNAciliranja v različnih človeških rakavih celičnih linijah. Naši rezultati so pokazali, da se presnovna stabilnost spojine razlikuje med celičnimi linijami in bi jo lahko izboljšali z zamenjavo labilnega etilnega estra v strukturi zaviralca s stabilnejšimi funkcionalnimi skupinami. Zato smo oblikovali in sintetizirali knjižnico derivatov OSMI-4 z amidnimi in aminskimi strukturami ter dokazali, da zamenjava estra ne vpliva bistveno na zaviralno jakost spojine. Medtem ko celično prepustnost molekul še preiskujemo, se je njihova stabilnost na hidrolizo s plazemskimi esterazami močno izboljšala. Zato verjamemo, da bomo s to strategijo razvili prve zaviralce OGT primerne za študije in vivo. Nazadnje smo predstavili nov žep v bližini aktivnega mesta OGT. Žep obdaja Asp554 s prostim karboksilatom, za katerega se domneva, da je vključen v encimsko katalizo, zato bi ta žep lahko uporabili za načrtovanje novih zaviralcev OGT. Naš pristop je bil sestavljen iz oblikovanja majhne serije fragmentov z uporabo različnih računalniških orodij, čemur je sledila njihova sinteza in in vitro testiranje. En fragment je pokazal obetavne rezultate z zaviranjem encima z vrednostjo IC50 1,4 mM. Ker pa je treba vezavno mesto teh spojin še eksperimentalno potrditi, je treba pridobiti rentgenske kristalografske podatke kompleksa ligand-protein, da bi na osnovi tega oblikovali močnejše zaviralce OGT.

• Keywords: O-GlcNAciliranje, OGT, OGA, zaviralci OGT, posttranslacijske modifikacije, računalniško podprto načrtovanje učinkovin, virtualno rešetanje, hidroliza estrov, metabolna stabilnost, farmacevtska kemija, bolezni