Tumors of neuroectodermal origin are among the most aggressive and treatment-refractory forms of human cancer. While such tumors arise from a variety of defects, two key targets are the transcription factors p53 and OLIG2. We have developed stabilized peptides to study and target deregulated p53 and OLIG2 pathways in neuroectodermal cancers. PUMA (p53-upregulated modulator of apoptosis) is a BH3-only member of the BCL-2 protein family that regulates apoptosis in response to p53-dependent and p53-independent stress signals. The specific interactions that mediate the pro-apoptotic activity of PUMA remain controversial. We generated stabilized alpha-helices of BCL-2 domains (SAHB) peptides modeled after the BH3 effector domain of PUMA. Structural analyses determined that PUMA SAHB contacts BAX at both the N-terminal \(\alpha1/\alpha6\) trigger site and the canonical BH3 binding pocket, binding events that functionally activate BAX. Notably, both PUMA SAHB and PUMA protein pull-downs identified anti- and pro-apoptotic binding partners in a cellular context. As PUMA has been implicated in driving apoptosis in multiple neural cell types, we further demonstrated that treatment of neuroblastoma cell lines with a cell-permeable PUMA SAHB analog triggered dose-dependent apoptosis. Together, we find that the PUMA BH3 domain activates apoptosis through multimodal interactions with BCL-2 family proteins, and its mimetics may serve as prototype therapeutics in tumors of neural origin. Whereas suppression of p53 signaling and apoptosis are features of diverse tumor types, the basic helix-loop-helix (bHLH) transcription factor OLIG2 is selectively overexpressed in gliomas. Early in development, OLIG2 is responsible for maintaining progenitor cells in a replication-competent state. Tumor stem cells are believed to co-opt this OLIG2 functionality to continually repopulate glial tumors. To achieve its transcriptional function, OLIG2 must dimerize via its bHLH domain. Stabilized alpha-helices of OLIG2 (SAH-OLIG2) peptides of the OLIG2 bHLH domain were generated in an effort to disrupt this pathologic dimerization. While helical stabilization of several SAH-OLIG2 peptides was achieved, specific engagement and disruption of the native bHLH dimer did not occur, informing alternative design strategies for future targeting efforts. These studies underscored the importance of interrogating the OLIG2 dimeric structure and catalyzed the discovery of candidate OLIG2 interaction partners for therapeutic targeting.

Macrocyclic peptides have been identified as key backbones in several biologically active compounds. They have been considered as great inspiration in the development of novel cyclic scaffolds in medicinal chemistry, notably in the introduction of α-helically constricted stapled peptides with the ability to mimic biologically relevant α-helices. DNA-binding transcription factors often bind their DNA promoter through an α-helix, making a parallel with stapled peptides as inhibitors. Despite this relevant feature, DNA-binding stapled peptides are highly unrepresented in the literature, as will be discussed here in a review. We also further expand this area of research with a study of DNA binding stapled peptide ana-logues with the goal of optimizing and investigating the DNA binding and antivirulence of an RpoN-based stapled peptide. Cyclic depsipeptides are highly biologically active natural product molecules however their synthesis can be challenging with the presence of a macrolactone. Due to this complexity, solid phase peptide synthesis strategies have been utilized to access peptide intermediates that can be synthetically macrocyclized using solution phase or on-resin approaches via macrolactam or macrolactone formation. A representative number of total syntheses in the literature is reviewed. Furthermore, we describe here the chemical total synthesis and chemoenzymatic synthesis of seongsanamide E cyclic depesipeptide via thioesterase medi-ated macrolactonization. Cyclic depsipeptides also play major roles in their producing organisms, notably siderophores capable of chelating and transporting iron. The biosynthesis of fungal siderophores is poorly explored, such as the iterative mechanism of oligomeric compound fusarinine C. We explore the synthesis of the previously never synthesized fusarinine C monomer to be utilized as a di-domain inhibitor of the adenylating-thiolation domains of the non-ribosomal peptide synthetase (NRPS) SidD.

With the increasing threat of multidrug resistant bacteria, there is a growing need to invent new drug classes that combat untreatable infections. Small molecule antibiotics have been successful in the past, but humanity is now losing the arms race against previously treatable pathogens. However, the number of clinically approved drugs targeting traditionally undruggable targets in bacteria remains low. New targets of complex protein-target interactions must be targeted for future pharmacological development. In an effort to create clinically viable biologics, the Verdine lab has developed a class of therapeutics called hydrocarbon stapled α-helical peptides; these peptides are known to affect protein-protein interactions by retaining secondary structure in vivo. Although this class of molecules has been extensively researched in cancer and viral therapies, there has been little work in bacteria due to the proposed endocytic method of entry. Moreover, DNA-binding stapled peptides have not been extensively investigated due the complexities in designing a peptide with gene selectivity. In an attempt to study peptides in bacteria, two stapled peptides based on the RpoN domain of σ54 and the FtsZ C-terminus have been synthesized. σ 54 is a DNA-binding co-factor of RNA polymerase (RNAP) and has been shown to regulate virulence and nitrogen and carbon metabolism. FtsZ is the structural unit of the contractile Z-ring that induces cell division. By designing stapled α-helical peptides to target these untraditional PPIs, we anticipate that these molecules may be used for future antimicrobial pharmacological development that treat multidrug resistant bacteria.

<p>Basic leucine zipper (bZIP) transcription factors can have an oncogenic role in cancer development. Nuclear factor erythroid related 2-factor 2 (Nrf2) is a bZIP transcription factor that traditionally is thought of as a cellular protector. In normal cells, Nrf2 is only activated after exposure to reactive oxygen species or electrophiles and induces expression of antioxidant and detoxification genes. However, in many cancers (<i>e.g.,</i> lung, pancreatic, and breast) Nrf2 is constitutively activated and is associated with poor overall survival and intrinsic resistance to anticancer therapies. Nrf2 heterodimerizes with transcription factors small musculoaponeurotic fibrosarcoma Maf (sMAF) proteins (e.g., MafG) in the nucleus and binds DNA, inducing transcription of Nrf2 target genes, conferring chemotherapeutic resistance to cancer cells. c-Myc another bZIP transcription factor is often overexpressed in a variety of cancers and acts like a protooncogenic transcriptional regulator. Mutations that drive abnormal MYC expression are the most common cariogenic event in tumor progression. c-Myc heterodimerizes with Max, its obligate bHLH-LZ heterodimerization partner, to form an active transcriptional state and induces DNA transcription. Hence inhibiting the interaction between c-Myc-Max and Nrf2-MafG will not only prevent bZIP heterodimerization but also DNA binding and downstream functions of c-Myc and Nrf2 that promote carcinogenesis. Stapled peptides, with their ability to target large surface area interactions, have shown promise for specifically inhibiting protein-protein interactions. Stapled peptides have improved cell permeability and oral bioavailability when compared to biologics. We have designed and synthesized stapled peptide for Nrf2/MafG interaction inhibition and stapled peptides for c-Myc/Max heterodimerization inhibition. Nrf2/MafG inhibition using the synthesized stapled peptide N1S, was demonstrated by luciferase and fluorescence polarization assays. Overall, we hypothesize that stapled peptides will be an effective therapeutic strategy resulting in decreased chemotherapeutic resistance and cancer cell proliferation.</p>

Protein-protein interactions (PPIs) are responsible for the regulation of a variety of important functions within living organisms. Compounds which can selectively modulate aberrant PPIs are novel therapeutic candidates for treating human diseases. Whilst PPIs have traditionally been considered as "undruggable", research in this area has led to the emergence of several effective methodologies for targeting PPIs. One such methodology is peptide stapling, which involves constraining a short peptide into its native alpha-helical form by forming a covalent link between two of its amino acid side-chains. The Sondheimer dialkyne reagent has previously been used in strain-promoted double-click cycloadditions with diazidopeptides to generate stapled peptides that are capable of inhibiting PPIs. However, the Sondheimer dialkyne suffers from poor water-solubility; it decomposes rapidly in aqueous solutions which limits its application in biological systems. This dissertation describes the design and synthesis of new substituted variants of the Sondheimer dialkyne with increased solubility and stability, that are suitable for application in strain promoted double click peptide stapling. In total, ten different derivatives were generated; of these, a meta-trimethylammonium substituted variant was found to have particularly high water-solubility and aqueous stability, as well as high azide reactivity. The substituted Sondheimer dialkynes were applied to the strain promoted double click stapling of p53-based diazido peptides in an effort to generate stapled peptide-based inhibitors of the oncogenic p53 MDM2 PPI, a validated target for anticancer therapeutics. Three stapled peptides were found to have inhibitory activity, thus demonstrating the utility of the novel dialkynes in the preparation of PPI inhibitors. The functionalised stapled peptide formed from a meta-fluoro substituted Sondheimer dialkyne was found to be the most potent inhibitor. All ortho-substituted Sondheimer dialkynes were found to be unreactive, whereas those with a meta-trimethylammonium substituent were highly reactive when compared to other meta-substituted dialkynes. These patterns in azide reactivity could be explained through X-ray crystallographic studies and density functional theory calculations.

A limited number of drug targets can be exploited by conventional drug-like compounds as the vast majority of disease-associated targets are involved in protein-protein interactions (PPI). PPI targets possess binding surfaces that lack a well-defined hydrophobic pocket amenable for binding to small drug-like compounds. A new class of therapeutics that has shown great potential at modulating PPI are macrocyclic peptides, particularly for their ability to bind to large and topologically complex protein surfaces as well as their potential to access intracellular targets. However, the efficiency of macrocyclic peptides at mediating PPIs and permeating cell membranes is conformation dependent. Here, I describe the role of peptide conformation on target recognition using three clinically relevant PPI targets: the Kelch like ECH Associated Protein-1 (KEAP1), (Chapter Two and Chapter Three); the RET receptor tyrosine kinase (Chapter Four); and β-catenin (Chapter Five). Guided by published X-ray crystal structures, peptides derived from PPI epitopes were designed and structurally constrained to mimic the conformation of the natural PPI recognition motif. In Chapter Two, I report the development of a cyclic heptapeptide derived from the transcription factor Nuclear Factor (Erythroid-derived 2)-Like 2 (Nrf2) with similar affinity for KEAP1 as native Nrf2 through conformational optimization of a linear Nrf2-derived heptapeptide. Efforts to improve the potency and physicochemical properties of the cyclic heptapeptide are discussed in Chapter Three. In Chapter Four, I describe the design of dimeric peptides as tool compounds to investigate the mechanism by which the interaction between glial cell-line derived neurotrophic factor family ligands (GFLs) and GPI-linked co-receptors, GFRα, induce RET signaling. These peptides were derived from the β-sheet regions of GFLs, GDNF and ART, that interact with GFRα1 and GFRα3, respectively. Peptide cyclization and the introduction of a β-turn promoting motif yielded GFL mimetic peptides with stronger affinity for GFRα. Lastly, Chapter Five focuses on exploring the scope of i, i+4 carbamate and amino-staples as a novel peptide stapling system to stabilize α-helical peptides. An axin-derived α-helical peptide that disrupts the β-catenin/TCF4 interaction was used as a model to determine the effect of peptide α-helical stabilization on binding affinity for β-catenin. / 2023-07-07T00:00:00Z

L’intérêt des peptides comme des médicaments potentiels est en constante augmentation. Des stratégies ont été développées pour améliorer la sélectivité, l’activité, et la stabilité des peptides vis-à-vis de la protéolyse. Dans ce mémoire de thèse, deux nouvelles modifications de peptides sont proposées.Dans le premier chapitre, nous présentons une nouvelle application de la réaction de transfert O-N acylique pour la synthèse de peptides agrafés (ou ‘stapled peptides’). L’introduction d’une ‘agrafe’ dans un peptide est un moyen de stabiliser une structure secondaire hélicoïdale en établissant un pont entre les résidus appropriés des chaînes latérales. Dans notre cas, l'agrafe est formée par un O-acyl isodipeptide. La liaison ester peut être convertie en liaison amide par un transfert O-N acylique. Cette stratégie permet une amélioration de la solubilité d’un peptide hydrophobe agrafé, avant son réarrangement à pH neutre.Dans le dernier chapitre, nous avons développé une méthodologie nouvelle pour la dimérisation de peptides non protégés. Cette méthode repose sur la formation de liaisons siloxane entre des peptides hybrides portant chacun un groupement dimethylchlorosilane. Nous avons ainsi dimérisé une séquence dérivée de la protéine p53, impliquée dans l’apoptose. A titre de comparaison, cette même séquence a été dimérisée en utilisant la cycloaddition d’Huisgen entre deux peptides modifiés possédant un azoture ou un alcyne en position N-terminale. Enfin, plusieurs dimères de la séquence du GHRP-6 (growth hormone releasing peptide) ont été synthétisés, avec des bras dimethylhydroxysilane placés à différentes positions. L’homodimérisation a été effectuée dans l'eau à pH neutre. / The interest in peptides as potential drug candidates was revived and is increasing constantly. Strategies have been developed to improve their selectivity and activity, and their stability toward proteolysis. In this thesis, two new peptide modifications are proposed.In the first chapter, we present a new application of the O-N acyl transfer reaction for the synthesis of stapled peptides. Peptide ‘stapling’ is a way of stabilizing secondary helical structure by establishment of a bridge between the side chains of suitable residues. In our case, the staple is formed by an O-acyl isopeptide which can be converted into amide bond by acyl migration. This strategy allows an improved solubility of the stapled hydrophobic peptide prior to rearrangement at neutral pH.In the last chapter, we developed also a new methodology for the dimerization of unprotected peptides. This method is based on siloxane bond formation between hybrid dimethylhydroxysilane peptides. A peptide derived from the tumor suppressor protein p53 was dimerized in water, at neutral pH using this methodology. The method was compared with the homodimerization carried out by Cu(I) azide-alkyne cycloaddition (CuAAC). For that purpose, p53 peptide derivative was synthesized with azide and alkyne linkers at the N-terminus. At last, several homodimers of growth hormone releasing hexapeptide (GHRP-6) were synthesized, with dimethylhydroxy silane linkers placed at various positions.

The human papillomavirus (HPV) has been a “companion” of humanity for as long as humanity has existed. The migration of peoples around the globe has given rise to more than 170 different types of the virus, which cause a variety of conditions. All five genera of HPV infect epithelial cells in the body, but only the Alphapapillomaviruses infect the genital mucosa. Most infections are benign and typically regress to subclinical within two years, but persistent infections can cause precancerous lesions. HPV types 16 and 18 are among the highest risk and account for the majority of cervical cancer, and more than 90% of all other HPV-related cancers. While the two vaccines, Gardasil and Cervarix, have been successfully implemented in the US market and some European and Asian countries, complete world penetrance has been burdened by multiple factors, including financial constraints and social norms. Treatments for established papillomas are invasive (cryosurgery, conization, etc.) and advanced malignant HPV-related tumors have been targeted with chemo- and radiotherapy with varied success. The high morbidity and long-term effects of current treatment options make clear the need for easy-to-administer, low-cost therapies, which can specifically treat both early and advanced HPV-associated cancers.<div><br></div><div>The hallmark of HPV tumors is the inactivation of p53, an evasion strategy key to the progression of HPV- derived cancers. Through an interaction between the viral protein E6 and the E3 ubiquitin ligase E6AP, p53 is polyubiquitinated and targeted for proteasomal degradation, allowing infected cells to bypass their own defense mechanisms. This work explores interruption of the association between E6 and E6AP as an opportunity to combat the infection and resulting malignancies.<br></div><div><br></div><div>In the first part of this project, disruption of the E6-E6AP interactions is pursued through the development of helical stabilized peptidomimetics of the LxxLL motif, which E6AP uses for E6 recognition and binding. Several reports have indicated that targeting the E6 binding groove is a viable means for disrupting the interaction. However, reported peptides were not cell permeable or optimized for α-helicity and proteolytic resistance (for reference, the LxxLL motif is an α-helix when bound). To address this challenge a peptide stabilization strategy was applied, which uses an all-hydrocarbon chain to connect two non-adjacent residues and enforce α-helicity. Results from in silico simulations and biochemical assay with these new stapled peptides showed that affinity for E6, α-helicity, and cell permeability can all be improved with the installment of the proper staple.<br></div><div><br></div><div>The second question examined by this work is whether fragment-based drug design can be successfully employed to derive new small-molecule inhibitors of the formation of the E6-E6AP complex. From a design perspective, the significant challenge was to define discreet binding hot-spots capable of accommodating fragments with reasonable affinity, which can then be linked together into a complete ligand. Using existing structural knowledge of the E6 protein and computational hot-spot searching tools, three previously-unidentified regions (sub-pockets) on E6 were discovered, which are near but not directly engaged by either the E6AP motif or p53. Using high-throughput in silico and biochemical screening, three sets of sub-pocket specific fragments were defined and elaborated into larger molecules with two different scaffolds. As a result, the work herein presents a stepwise approach to targeting the E6-E6AP protein-protein interaction – the discovery of new binding hot spots, the identification of site-specific fragments, and the design of complete molecules with versatile scaffold.<br></div>