TY - GEN
T1 - Modulation of Protein-Protein Interactions with Pre-Structured Peptides
AU - Maas, Marijn
PY - 2022/9/14
Y1 - 2022/9/14
N2 - Protein-protein interactions regulate biochemical processes associated with health and diseaseand are generally considered ‘undruggable’ due to the large and undefined nature of the surfacesof the participating proteins. Development of new chemical technologies which allow for thestudy thereof would open the door towards the rational design of potent and specific probes ortherapeutics which target specific PPI networks involved with key processes involved in healthand disease. Pre-structured peptides have surfaced as potent tools to study or inhibit proteincomplex formation. The focus of this PhD dissertation is to gain insight into the importance ofpeptide conformational flexibility towards facilitating recognition by their binding partnersthrough pre-structuring of the peptide backbone. In chapter 3 we set out to develop lactam-based i,i + 4 pre-structured hACE2 peptides targeting the SARS-CoV-2 spike protein. Inhibition of the Spike protein RBD-hACE2 complexformation was observed through in vitro assays with pre-structured hACE221-55A36K-F40E peptide (IC50: 3.6 μM, Kd: 2.1 μM), suggesting that hACE2 peptides could form the basis forthe development of anti-COVID-19 therapeutics.In chapter 4 we report on the catalytic specificity of SETD3 towards pre-structured i,i + 3 stapled β-actin peptides. Limiting the conformation flexibility of β-actin results in decreased SETD3-catalyzed Nτ-His73 methylation. Moreover, none of the pre-structured βactin peptides acted as competitive inhibitors of SETD3. These findings suggest that reduced β-actin backbone flexibility prevents optimal protein-protein interactions between the substrate and enzyme.Chapter 5 explores the catalytic selectivity of PAD2 and PAD4 towards pre-structured histone tails. Select histone peptides pre-structured through i,i+3 stapling were efficiently citrullinated by PAD2, and were not accepted as substrates for histone methyl- or acetyl transferases. i,i+3 stapled histone peptides were identified as competitive inhibitors of PAD2 (IC50 = 4.0 μM) and PAD4 (IC50 = 8.1 μM).In chapter 6, thermodynamic analysis with ITC illustrates that pre-structuring of the Nterminal histone 3 tail bearing a trimethyllysine mark can significantly improve recognition byepigenetic readers BPTFPHD and JMJD2ATTD through enhanced entropy components. In contrast, recognition by JARID1APHD3 is impaired through unfavorable enthalpic and entropic components. This suggests that there is a level of selectivity between readers of H3K4me3 for peptides with different levels of conformational freedom.
AB - Protein-protein interactions regulate biochemical processes associated with health and diseaseand are generally considered ‘undruggable’ due to the large and undefined nature of the surfacesof the participating proteins. Development of new chemical technologies which allow for thestudy thereof would open the door towards the rational design of potent and specific probes ortherapeutics which target specific PPI networks involved with key processes involved in healthand disease. Pre-structured peptides have surfaced as potent tools to study or inhibit proteincomplex formation. The focus of this PhD dissertation is to gain insight into the importance ofpeptide conformational flexibility towards facilitating recognition by their binding partnersthrough pre-structuring of the peptide backbone. In chapter 3 we set out to develop lactam-based i,i + 4 pre-structured hACE2 peptides targeting the SARS-CoV-2 spike protein. Inhibition of the Spike protein RBD-hACE2 complexformation was observed through in vitro assays with pre-structured hACE221-55A36K-F40E peptide (IC50: 3.6 μM, Kd: 2.1 μM), suggesting that hACE2 peptides could form the basis forthe development of anti-COVID-19 therapeutics.In chapter 4 we report on the catalytic specificity of SETD3 towards pre-structured i,i + 3 stapled β-actin peptides. Limiting the conformation flexibility of β-actin results in decreased SETD3-catalyzed Nτ-His73 methylation. Moreover, none of the pre-structured βactin peptides acted as competitive inhibitors of SETD3. These findings suggest that reduced β-actin backbone flexibility prevents optimal protein-protein interactions between the substrate and enzyme.Chapter 5 explores the catalytic selectivity of PAD2 and PAD4 towards pre-structured histone tails. Select histone peptides pre-structured through i,i+3 stapling were efficiently citrullinated by PAD2, and were not accepted as substrates for histone methyl- or acetyl transferases. i,i+3 stapled histone peptides were identified as competitive inhibitors of PAD2 (IC50 = 4.0 μM) and PAD4 (IC50 = 8.1 μM).In chapter 6, thermodynamic analysis with ITC illustrates that pre-structuring of the Nterminal histone 3 tail bearing a trimethyllysine mark can significantly improve recognition byepigenetic readers BPTFPHD and JMJD2ATTD through enhanced entropy components. In contrast, recognition by JARID1APHD3 is impaired through unfavorable enthalpic and entropic components. This suggests that there is a level of selectivity between readers of H3K4me3 for peptides with different levels of conformational freedom.
U2 - 10.21996/r8dn-6c84
DO - 10.21996/r8dn-6c84
M3 - Ph.D. thesis
PB - Syddansk Universitet. Det Naturvidenskabelige Fakultet
ER -