Thesis advisor: Jianman Gao / The interaction between the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the human angiotensin-converting enzyme 2 (hACE2) receptor is an advantageous target for the development of therapies for COVID-19. We used an anti spike receptor binding domain (S RBD) antibody (AM122) to competitively elute phage binding to the S RBD in phage display screening to identify a novel peptide that binds the S protein and hACE2 interaction. We identified a peptide sequence (P1: CPLEYHTC) as a possible hit, and the KD was determined to be 2.667 μM, indicating the potential of this peptide sequence as a therapeutic agent. However, we found no inhibition of the spike protein and hACE2 receptor interaction, suggesting that the peptide may not directly bind to the hACE2 binding site on S RBD. Although further studies are needed, the competitive elution method in phage display screening appears to be an effective method for elucidating onsite peptide sequences that target protein-protein interactions (PPIs). / Thesis (BA) — Boston College, 2023. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Chemistry.

No description available.

Cystic Fibrosis (CF) is an autosomal recessive disease that arises from mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The deletion of phenylalanine-508 (ΔF508-CFTR) is the most prevalent CF mutation and results in a misfolded protein that fails to exit the endoplasmic reticulum (ER). Previous studies demonstrated that mutation of a di-arginine based ER retention motif (R553AR555) in the first nucleotide binding domain (NBD1) rescues the trafficking defect of ΔF508-CFTR. We hypothesized that if the R553AR555 motif mediates retention of the ΔF508-CFTR protein, peptides that mimic this motif should antagonize mistrafficking mediated by aberrant exposure of the endogenous R553AR555 motif. We generated a peptide bearing the R553AR555 motif (CF-RXR) and conjugated it to the cell penetrating peptide Tat (CPP-CF-RXR) to facilitate intracellular delivery and investigated its efficacy in rescuing the mistrafficking and function of ΔF508-CFTR. Using a variety of biochemical and functional assays we demonstrate that the CPP-CF-RXR peptide is effective at increasing surface expression of ΔF508-CFTR in baby hamster kidney (BHK) and human embryonic kidney (HEK) cell lines. Furthermore, the increased surface expression is accompanied by an increase in its functional expression as a chloride channel. Using Ussing chamber assays, we demonstrate that the CPP-CF-RXR peptide improved ΔF508-CFTR channel function in respiratory epithelial tissues obtained from CF patients. Additionally, we investigated the effects of small molecules on mediating biosynthetic rescue of a ΔF508-CFTR construct bearing the additional mutations R553K and R555K (ΔFRK-CFTR) to inactivate the R553AR555 motif. Interestingly, mutation of the R553AR555 motif exerts an additive effect with correctors VRT-325 and Corrector 4a. Taken together, our data suggests that abnormal accessibility of the RXR motif present in NBD1 is a key determinant of the mistrafficking of the major CF causing mutant.

Cystic Fibrosis (CF) is an autosomal recessive disease that arises from mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The deletion of phenylalanine-508 (ΔF508-CFTR) is the most prevalent CF mutation and results in a misfolded protein that fails to exit the endoplasmic reticulum (ER). Previous studies demonstrated that mutation of a di-arginine based ER retention motif (R553AR555) in the first nucleotide binding domain (NBD1) rescues the trafficking defect of ΔF508-CFTR. We hypothesized that if the R553AR555 motif mediates retention of the ΔF508-CFTR protein, peptides that mimic this motif should antagonize mistrafficking mediated by aberrant exposure of the endogenous R553AR555 motif. We generated a peptide bearing the R553AR555 motif (CF-RXR) and conjugated it to the cell penetrating peptide Tat (CPP-CF-RXR) to facilitate intracellular delivery and investigated its efficacy in rescuing the mistrafficking and function of ΔF508-CFTR. Using a variety of biochemical and functional assays we demonstrate that the CPP-CF-RXR peptide is effective at increasing surface expression of ΔF508-CFTR in baby hamster kidney (BHK) and human embryonic kidney (HEK) cell lines. Furthermore, the increased surface expression is accompanied by an increase in its functional expression as a chloride channel. Using Ussing chamber assays, we demonstrate that the CPP-CF-RXR peptide improved ΔF508-CFTR channel function in respiratory epithelial tissues obtained from CF patients. Additionally, we investigated the effects of small molecules on mediating biosynthetic rescue of a ΔF508-CFTR construct bearing the additional mutations R553K and R555K (ΔFRK-CFTR) to inactivate the R553AR555 motif. Interestingly, mutation of the R553AR555 motif exerts an additive effect with correctors VRT-325 and Corrector 4a. Taken together, our data suggests that abnormal accessibility of the RXR motif present in NBD1 is a key determinant of the mistrafficking of the major CF causing mutant.

Voltage-gated sodium channels are composed of pore-forming α-subunits, and modulatory and multifunctional associated β subunits. While much of the field of cardiac electrophysiology and pathology has focused on treating and preventing cardiac arrhythmias by targeting the α subunit, there is also evidence that targeting the β subunits, particularly SCN1B, the gene that encodes β1 and an alternatively spliced variant β1B, has therapeutic potential. The first attempt at targeting the β1 subunit was with the generation of and treatment with an SCN1B Ig domain mimetic peptide βadp1. Here we describe further investigation into the function and mode-of-action of both βadp1 and novel peptides derived from the original βadp1 sequence. We find that in a heterologous expression system βadp1 initially disrupts β1-mediated trans-homophilic adhesion, but after approximately 30 hours eventually increases adhesion. Novel mimetic dimers increase β1 adhesion up to 48 hours post-treatment. Furthermore, it appears that βadp1 may increase β1 adhesion by upregulating the intramembrane proteolysis of β1, a process which has important downstream implications and effects on translation. Despite these exciting findings, we were unable to translate them into a primary culture of cardiac cells with endogenous expression of β1 because we found that both neonatal rat cardiomyocytes and isolated adult mouse cardiomyocytes do not express β1 at detectable levels, whereas they do appear to express β1B. In summary, we show exciting findings on the function and mode-of-action of SCN1B mimetic peptides and their therapeutic potential in targeting the β1 subunit, but further work is needed to determine the translatability of our findings to in vivo models and eventually to humans. / Doctor of Philosophy / Voltage-gated sodium channels have two main parts: the pore-forming α-subunits and the modulatory β subunits. Most research in heart function and issues has focused on fixing problems with the α subunit. However, there's evidence that working on the β subunits, specifically the SCN1B gene that makes β1 and another version called β1B, could be helpful. Previously, researchers used a peptide that is designed exactly like a part of β1, called βadp1, to target the β1 subunit. In our study, we explore more about how βadp1 works and test new peptides based on βadp1. We found that βadp1 initially disrupts trans-homophilic adhesion, where 2 β1 subunits interact with each other across the space between 2 cells, but after about 30 hours, it actually increases adhesion. New mimetic dimers also boost adhesion up to 48 hours later. It seems like βadp1 might enhance adhesion by triggering a process called intramembrane proteolysis of β1, which has important effects on translation. Despite these exciting findings, we couldn't confirm the presence of this protein in heart cells because we discovered that certain heart cells don't have enough β1, although they do have β1B. In conclusion, our study shows promising results about how SCN1B mimetic peptides work and their potential for treating arrhythmia. However, more research is needed to see if these findings apply to real-life situations and eventually to help people with cardiac conduction abnormalities.