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Design and synthesis of Covalent Kinase Inhibitors Utilising Novel Electrophiles

dc.contributor.advisorVan Otterlo, Willemen_ZA
dc.contributor.advisorPelly, Stephenen_ZA
dc.contributor.authorHodson, Luke Ericen_ZA
dc.contributor.otherStellenbosch University. Faculty of Science. Dept. of Chemistry and Polymer Science.en_ZA
dc.date.accessioned2019-08-06T07:54:32Z
dc.date.accessioned2019-12-11T06:42:11Z
dc.date.available2020-02-07T03:00:08Z
dc.date.issued2019-12
dc.identifier.urihttp://hdl.handle.net/10019.1/106993
dc.descriptionThesis(PhD)--Stellenbosch University, 2019.en_ZA
dc.description.abstractENGLISH ABSTRACT: Cancer is the collective name given to an extensive group of diseases which is mainly characterised by the uncontrolled growth of abnormal cells that either ignore or evade biological processes or display enhanced biological processes. Cancer is the second leading cause of death worldwide, claiming an estimated 9 million lives and accounting for 1 in 6 deaths in 2016. In a post-genomic era, rapid advances in molecular cancer biology and genomics has led to the identification of defining characteristics and traits of this complex disease, known as the hallmarks of cancer. Consequently, the last two decades has seen a shift from the use of conventional chemotherapy to the era of targeted cancer therapy. Epidermal growth factor receptor (EGFR) is a transmembrane receptor which belongs to the ErbB/HER family of protein-tyrosine kinases. Owing to its expansive role in signal transduction pathways, EGFR regulates a host of essential aspects of cellular life, including division, growth and apoptosis. Oncogenic activation of this receptor, resulting in overexpression or hyperactivation, has been implicated in a variety of malignancies. To overcome this, researchers make use of tyrosine kinase inhibitors (TKIs) to effectively inhibit the receptor and cease uncontrolled cellular growth. The past 16 years has seen the development of four generations of EGFR inhibitors attempting to overcome the emergence of multiple resistance mechanisms which are brought about by point mutations within the enzyme. Most recently, the emergence of resistance mediated by the EGFR-C797S mutation in 2015 has presented a challenging therapeutic endeavour. To date, no major breakthroughs have been achieved in targeting this clinically relevant mutant variant. As such, the research efforts documented within this thesis focus on the development of new therapeutic strategies to overcome this resistance mutation. The first of these approaches entails the synthesis of highly potent reversible and irreversible EGFR inhibitors. Building on a previously identified lead compound, and drawing inspiration from dual EGFR/HER2 inhibitors, we incorporated several glycol chain functionalities at strategic positions to introduce favourable reversible interactions within the active site of the enzyme. Furthermore, studies on the effects of derivatisation and interchange of other structural elements, such as the solubilising group and acryl amide electrophile, were undertaken. Departing from traditional inhibition methods, our second strategy sought the re-establishment of irreversible inhibition by targeting the EGFR catalytic Lys745 residue with novel electrophiles. To induce covalent bond formation, inhibitors containing 1,4-dicarbonyl warheads, able to undergo Paal-Knorr pyrrole formation with the Lys745 residue, were synthesised. Furthermore, potential reversible and irreversible interactions with the catalytic lysine residue were investigated with inhibitors containing nitrile and reactive sulfonyl fluoride moieties. Lastly, we pursued covalent bond formation of the mutated Ser797 residue - the main mechanism by which resistance is conferred in the EGFR-C797S mutant variant. To accomplish this, inhibitors containing a multitude of reactive serine-targeting electrophiles were synthesised, including nitrogen-, boron-, phosphorous-, sulphur- and fluorine-containing warheads. Biochemical and cellular evaluation of the 48 final compounds synthesised during this research project provided encouraging results. This included picomolar IC50 values for biochemical inhibition of the clinically relevant EGFR-L858R/T790M/C797S triple mutant variant of the enzyme by the synthesised reversible inhibitors. However, these results were unfortunately not reflected in the cellular-based assays. Additionally, several inhibitors containing the conventional acrylamide electrophile exhibited low nanomolar and picomolar range activity against the EGFR-L858R/T790M double mutant in a biochemical and cellular setting respectively, alluding to irreversible inhibition of the enzyme. A crystal structure of the most efficacious irreversible inhibitor complexed with the cSrc-T338M/S345C surrogate was obtained, providing confirmation of covalent bond formation with the Cys797 residue and reinforcing our postulates regarding the observed trends in activity. Finally, several lead compounds were appropriately evaluated and shown to be effective dual EGFR/HER2 inhibitors. The use of covalent mass spectrometry experiments revealed a reversible binding mode for all inhibitors targeting the catalytic Lys745 and mutant Ser797 residue. However, several functional groups were identified for their ability to create favourable interactions with these residues, as reflected in their effective biochemical and cellular activity profiles against the EGFR-C797S mutant. This included the 1,4-dialkene-, boronic ester-, vinyl sulfonyl fluoride-, imidazole- and nitrile-bearing compounds. Ultimately, novel synthetic methodology was established en route towards these inhibitors and their respective electrophiles. Highlights include development of a new and convenient one-pot installation of an acetamide- or propanamide-imidazole functional group to aniline derivatives utilising 1,1’-carbonyldiimidazole and the corresponding α-, β-halo-carboxylic acid. Furthermore, optimised methods were derived for future access to thevariety of electrophiles and heterocyclic scaffolds synthesised throughout this research project.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Kanker is die kollektiewe naam wat gegee word aan 'n uitgebreide groep siektes, wat hoofsaaklik gekenmerk word deur die onbeheerde groei van abnormale selle. Hierdie selle vermy, of bevat negatiewe of verbeterde biologiese prosesse. Kanker is die wêreld se tweede grootste oorsaak van dood, wat ongeveer 9 miljoen lewens geeis het in 2016. Dit reken vir 1 uit 6 van die totale jaarlikse sterftes wêreldwyd. In 'n post-genomiese era het vinnige vooruitgang in molekulêre kankerbiologie en genomika gelei tot die identifisering van kenmerkende vermoëns van hierdie komplekse siekte. Gevolglik het die afgelope twee dekades 'n verskuiwing vanaf konvensionele chemoterapie na die era van geteikende kankerterapie gesien. Epidermale groeifaktor reseptor (EGFR) is 'n transmembrane reseptor wat behoort aan die ErbB/HER familie van proteïen-tyrosien kinases. As gevolg van sy uitgebreide rol in seintransduksiepaaie, reguleer EGFR verskeie noodsaaklike aspekte van sellulêre lewe, insluitend verdeling, groei en apoptose. Onkogene aktivering van hierdie reseptor, wat tot ooruitdrukking of hiperaktivering lei, is in 'n verskeidenheid maligniteite betrokke. Navorsers gebruik tyrosienkinase-inhibeerders (TKI) om die reseptor effektief te inhibeer en onbeheerde sellulêre groei te staak. Deur die afgelope 16 jaar is vier generasies EGFR-inhibeerders ontwikkel wat die opkoms van meervoudige weerstandsmeganismes probeer oorkom. Hierdie meganismes word deur puntmutasies binne die ensiem veroorsaak. Die onlangse opkoms van weerstand in 2015, wat deur die EGFR-C797S-mutasie veroorsaak is, het 'n uitdagende terapeutiese poging aangewend. Geen groot deurbrake om hierdie klinies relevante mutantvariant te oorkom is nie tot op datum bereik nie. Die navorsingspogings in hierdie proefskrif fokus op die ontwikkeling van nuwe terapeutiese strategieë om hierdie weerstandsmutasie te oorkom. Die sintese van hoe-kragtige omkeerbare en onomkeerbare EGFR-inhibeerders was die eerste van hierdie benaderings. Hierdie werk was gebaseer op 'n voorheen geïdentifiseerde suksevolle inhibeerder. Verskeie glikkol-funksionaliteite, wat by strategiese posisies ingevoer is om gunstige omkeerbare interaksies binne die aktiewe plek van die ensiem voor te stel, was geinspireer van dubbele EGFR/HER2-inhibeerders. Verdere studies was onderneem oor die gevolge van derivatisering en verwisseling van ander strukturele elemente, soos die oplosbare groep en akrielamiedelektrofil. Ons tweede strategie, wat afwyk van tradisionele inhibisiemetodes, het gesoek na die herstelling van onomkeerbare inhibisie deur die EGFR katalitiese lysine 745 residu met nuwe elektrofiele. Om kovalente bindingsvorming te veroorsaak was inhibeerders met 1,4-dikarbonyl-koppe gesintetiseer wat Paal-Knorr-pyrrolvorming kon ondergaan met die Lys745 residu. Verder is potensiële omkeerbare en onomkeerbare interaksies met die katalitiese lysienresidu ondersoek met inhibeerders wat nitril- en reaktiewe sulfonielfluorieddele bevat. Laastens het ons kovalente binding vorming nagestreef met die gemuteerde serine 797 residu - die hoofmeganisme van weerstand in die EGFR-C797S mutant variant. Inhibeerders wat 'n verskeidenheid reaktiewe seriene-teiken-elektrofiele bevat, insluitende boron-, fosfor-, swael- en fluoorbevattende hoofkoppies, was gesintetiseer om dit te bereik. Bemoedigende resultate was opgelewer in die biochemiese en sellulêre evaluering van die 48 finale inhibeerders, wat tydens hierdie navorsingsprojek gesintetiseer is. Dit sluit in pikomolêre IC50 waardes vir biochemiese inhibisie van die klinies relevante EGFR-L858R/T790M/C797S triple mutant variant van die ensiem deur die gesintetiseerde omkeerbare inhibeerders. Hierdie resultate is egter ongelukkig nie weerspieël in die sel-gebaseerde toetse nie. Daarbenewens het verskeie inhibeerders wat die konvensionele akrylamiedelektrofil bevat, lae nanomolêre en pikomolêre reeks aktiwiteit vertoon teen die EGFR-L858R/T790M dubbelmutant in ‘n onderskeidelik biochemiese en sellulêre omgewing. Hierdie resultate het die onomkeerbare inhibisie van die ensiem aangedui. 'N Kristalstruktuur was verkry van die mees effektiewe onomkeerbare inhibeerder gekompleks saam met die cSrc-T338M/S345C-surrogaat. Hierdie struktuur het die kovalente bindingvorming met die Cys797-residu beverstig en ons postulate oor die waargenome tendense in aktiwiteit ondersteun. Laastens was verskeie van die mees suksesvolle inhibeerders geëvalueer en getoon dat dit ‘n effektiewe dubbele EGFR/HER2 inhibeerder is. 'n Omkeerbare bindingsmodus deur die gebruik van kovalente massaspektrometrie-eksperimente was bewys vir alle inhibeerders wat die katalitiese Lys745 en mutant Ser797 residu geteiken het. Verskeie funksionele groepe is egter geïdentifiseer vir die vermoë om gunstige interaksies met hierdie residue te skep, soos wat weerspieël was in hul effektiewe biochemiese en sellulêre aktiwiteitsprofiele teen die EGFR-C797S mutant. Dit sluit in die 1,4-dialkeen-, boronester-, vinielsulfonylfluoried-, imidasool- en nitril-draende verbindings. Op pad na die sintese van hierdie inhibeerders en hul onderskeie elektrofiele is nuwe sintetiese metodologie gevestig. Hoogtepunte sluit in die ontwikkeling van 'n nuwe en gerieflike eenpot-installasie van 'n asetamied- of propanamiedimidasool funksionele groep na anilienderivate, wat gebruik maak van 1,1'-karbonyldiimidasool en die ooreenstemmende α-, β-halo-karboksielsuur. Geoptimaliseerde metodes was afgelei vir toekomstige toegang tot die verskeidenheid elektrofiele en heterosikliese strukture wat deur hierdie navorsingsprojek gesintetiseer word.af_ZA
dc.format.extent334 pages ; illustrationsen_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.subjectKinase Inhibitorsen_ZA
dc.subjectEpidermal Growth Factoren_ZA
dc.subjectIrreversible Inhibitorsen_ZA
dc.subjectCancer Therapeuticsen_ZA
dc.subjectDrug -- Designen_ZA
dc.subjectLung Canceren_ZA
dc.subjectElectrophilic Warheadsen_ZA
dc.titleDesign and synthesis of Covalent Kinase Inhibitors Utilising Novel Electrophilesen_ZA
dc.typeThesisen_ZA
dc.description.versionDoctorate
dc.rights.holderStellenbosch Uinversityen_ZA
dc.embargo.terms2020-02-07


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