The generation of high affinity, selective, and in vivo-stable peptide-based drugs is currently a major challenge in the field of drug development. Technologies exist that permit the generation of a vast diversity of chemical and conformational space and an example of such a technology is mRNA display, which utilizes protein translation machinery to produce a wide array of polypeptides starting from a combinatorial library of mRNA templates. The intention of this research was to bridge mRNA display to a reconstituted translation system using protein synthesis using recombinant elements (PURE) system for a new drug discovery platform. We hypothesized that it is possible to generate mRNA-peptidomimetic fusions using reconstituted translation system and chemo-enzymatically charged tRNAs, to incorporate unnatural amino acids into mRNA-peptidomimetic fusions. Upon demonstating that the reconstituted system was functional, we have synthesized hexapeptide fusion products containing four alanine residues and one biocytin residue. Fusions were assayed using urea-PAGE in the presence of streptavidin which allowed for unambiguous evaluation of the full length fusion fraction. It was determined that overall more fusion product was generated with template that codes for biocytin early in the coding sequence, but that the percent of biocytin-containing product stays similar regardless of the biocytin place in the coding region. We have also found that the change in template untranslated region length does not improve incorporation of biocytin in dipeptide fusions within the tested range. Finally, after first unsuccessful attempts to make sarcosine hexapeptide fusions, we investigated the effect of magnesium ion concentration on the translation reaction. As a result of four series of experiments performed involving both alanine and sarcosine fusion synthesis in parallel, we concluded that an increase in magnesium concentration from 5 mM to 20 mM coincided with enabling of the reconstituted system in making hexapeptide fusions with sarcosine in a significantly high number of cases. This research work arises from the need to enable a new drug discovery tool that will allow both synthesis and affinity maturation of peptide-based compounds. It represents our pioneering efforts to develop a new technology and ultimately help bring to existence compounds of significant therapeutic value.

The generation of high affinity, selective, and in vivo-stable peptide-based drugs is currently a major challenge in the field of drug development. Technologies exist that permit the generation of a vast diversity of chemical and conformational space and an example of such a technology is mRNA display, which utilizes protein translation machinery to produce a wide array of polypeptides starting from a combinatorial library of mRNA templates. The intention of this research was to bridge mRNA display to a reconstituted translation system using protein synthesis using recombinant elements (PURE) system for a new drug discovery platform. We hypothesized that it is possible to generate mRNA-peptidomimetic fusions using reconstituted translation system and chemo-enzymatically charged tRNAs, to incorporate unnatural amino acids into mRNA-peptidomimetic fusions. Upon demonstating that the reconstituted system was functional, we have synthesized hexapeptide fusion products containing four alanine residues and one biocytin residue. Fusions were assayed using urea-PAGE in the presence of streptavidin which allowed for unambiguous evaluation of the full length fusion fraction. It was determined that overall more fusion product was generated with template that codes for biocytin early in the coding sequence, but that the percent of biocytin-containing product stays similar regardless of the biocytin place in the coding region. We have also found that the change in template untranslated region length does not improve incorporation of biocytin in dipeptide fusions within the tested range. Finally, after first unsuccessful attempts to make sarcosine hexapeptide fusions, we investigated the effect of magnesium ion concentration on the translation reaction. As a result of four series of experiments performed involving both alanine and sarcosine fusion synthesis in parallel, we concluded that an increase in magnesium concentration from 5 mM to 20 mM coincided with enabling of the reconstituted system in making hexapeptide fusions with sarcosine in a significantly high number of cases. This research work arises from the need to enable a new drug discovery tool that will allow both synthesis and affinity maturation of peptide-based compounds. It represents our pioneering efforts to develop a new technology and ultimately help bring to existence compounds of significant therapeutic value.

The generation of high affinity, selective, and in vivo-stable peptide-based drugs is currently a major challenge in the field of drug development. Technologies exist that permit the generation of a vast diversity of chemical and conformational space and an example of such a technology is mRNA display, which utilizes protein translation machinery to produce a wide array of polypeptides starting from a combinatorial library of mRNA templates. The intention of this research was to bridge mRNA display to a reconstituted translation system using protein synthesis using recombinant elements (PURE) system for a new drug discovery platform. We hypothesized that it is possible to generate mRNA-peptidomimetic fusions using reconstituted translation system and chemo-enzymatically charged tRNAs, to incorporate unnatural amino acids into mRNA-peptidomimetic fusions. Upon demonstating that the reconstituted system was functional, we have synthesized hexapeptide fusion products containing four alanine residues and one biocytin residue. Fusions were assayed using urea-PAGE in the presence of streptavidin which allowed for unambiguous evaluation of the full length fusion fraction. It was determined that overall more fusion product was generated with template that codes for biocytin early in the coding sequence, but that the percent of biocytin-containing product stays similar regardless of the biocytin place in the coding region. We have also found that the change in template untranslated region length does not improve incorporation of biocytin in dipeptide fusions within the tested range. Finally, after first unsuccessful attempts to make sarcosine hexapeptide fusions, we investigated the effect of magnesium ion concentration on the translation reaction. As a result of four series of experiments performed involving both alanine and sarcosine fusion synthesis in parallel, we concluded that an increase in magnesium concentration from 5 mM to 20 mM coincided with enabling of the reconstituted system in making hexapeptide fusions with sarcosine in a significantly high number of cases. This research work arises from the need to enable a new drug discovery tool that will allow both synthesis and affinity maturation of peptide-based compounds. It represents our pioneering efforts to develop a new technology and ultimately help bring to existence compounds of significant therapeutic value. / Pharmaceutical Sciences, Faculty of / Graduate

The tumour suppressor p53 is essential for genome stability and loss of its function can lead to human cancer. The functional roles of p53 are regulated by a variety of mechanisms, some of which are not well understood. However, the murine double minute 2 (MDM2) protein, a major negative regulator of p53, has been found to be overexpressed in many human cancer cell lines in which p53 was not mutated; thus establishing MDM2 as a target for cancer therapeutics. MDM2 is defined as both an oncoprotein and an E3-ubiquitin ligase; its interactions with p53 are controlled through multiple domains, providing different possible pathways to inhibit MDM2 and therefore reactivate p53 function. Previous work undertaken in the Ball laboratory has shown that the MDM2 RING domain plays a critical role in p53 ubiquitination; thus screening for modulation of its activity by small molecules could provide new leads for the inhibition of the E3 ligase activity of MDM2. The MDM2 RING domain was cloned, expressed and purified so that it could be studied using a series of in vitro experiments. The generation of a library of short (12-mer) peptides as potential inhibitors of the MDM2 RING domain was investigated using phage display against His-tagged RING protein to screen the peptide ligands. In order to study the specificity of these peptides towards MDM2 (res. 396-491 and 396-479) compared with MDM4 and BRCA1, the MDM4 RING domain (res. 395-490 and 395-478) and BRCA1 (res. 1-304) domain were expressed and purified for further characterisation. A small selection of peptides was isolated and their binding affinity and activity as MDM2 inhibitors evaluated by in vitro ELISA, affinity chromatography and ubiquitination assays. One peptide in particular, KCCYFETHMPRH, was found to bind to MDM2 and was able to inhibit MDM2-mediated ubiquitination of p53 in vitro. Preliminary optimisation of this peptide by alanine scan revealed a peptide with a 2-fold increased potency. Since peptides provide comparatively weak therapeutic leads due to a combination of poor cellular uptake and susceptibility to cleavage by proteases, cyclic peptidomimetics based upon this lead were developed using side-chain to side-chain cyclisation. These peptidomimetics were successfully generated by the synthesis and incorporation of novel N-propargylated glycine and N-azidoalkyl glycine building blocks into a peptide sequence by Solid Phase Peptide Synthesis (SPPS). Following a Copper-catalysed Azide-Alkyne Cycloaddition (CuAAC) on solid phase or in solution, these peptoid-peptide hybrids were isolated, purified and characterised.

Nature has gifted peptides as important modulators in the human body, but these types of molecules often have not been favored when we were looking for therapeutic agents. The poor bioavailability, fast degradation and until recent high manufacturing costs of some bioactive peptides lowered their potential usage in the health industry. Under these circumstances, unnatural amino acids were developed as indispensible tools providing enormous support to peptide science. By incorporating proper unnatural amino acids into a peptide or protein, we now can significantly improve peptide's or protein's half-life, cell permeability, bio-distribution, etc. In addition, their potency and receptor/acceptor selectivity could also be enhanced. Site-specific modifications of peptides and proteins under physiological conditions with the use of unnatural amino acids also have been made easier with the advance of biotechnology. Therefore, my research described in this dissertation contributes to the efforts in the development of novel unnatural amino acids. In particular, I have focused on novel methods in the synthesis of anti beta-functionalized gamma,delta-unsaturated amino acids. These amino acids have special interests in peptide chemistry: they can provide conformational constraints to the peptide 3D structures; the beta-functionalization allows the introduction of pharmaceutically interesting side chain groups; and the terminal double bond which is orthogonal to peptide synthesis provides access to further chemical modifications. Two general methodologies for the synthesis of both racemic and optically active anti beta-functionalized gamma,delta--unsaturated amino acids were developed by using the thio-Claisen rearrangement (TCR) reaction. Excellent diastereoselectivies and enantioselectivities were obtained when C2-symmetric chiral auxiliaries were selected to control the stereochemistry outcome. The mechanism and the scope of the TCR reaction were also studied, showing unique advantages in the preparation of these biological interesting amino acids.Another effort of developing angiotensin II type 1 (AT1) receptor biased peptide ligands is also documented in this dissertation. The AT1 receptor is a 7-transmembrane G-protein coupled receptor, which recent researches have shown could be activated through a beta-arrestins only, but G-protein independent, pathway. We synthesized 12 analogs of Sar1,Ile4,Ile8-AngII (SII), and tested them in biological assays, and obtained valuable information for further "perfect" biased ligands design.

No description available.

Current cancer therapies have low specificity for tumor cells and have serious toxic side effects. Targeting drugs to the cancer cells can help improve the outcome of existing cancer therapies. In recent years, a number of peptides have been identified by peptide phage display for targeting different tumor types. Peptides identified from the phage display for targeting cancer cells can be further improved for specific binding and metabolic stability by chemical manipulation of their structures. The aims of this work were: (i) to develop a peptide array-whole cell binding assay for screening peptides with specific binding affinity for cancer cells (ii) design of novel peptidomimetics to improve their properties as drug candidates. First, peptide arrays based on the lead peptide sequences NGR and p160 were designed and synthesized. A direct peptide-cell binding assay using CyQUANT dye allowed identification of several new peptides with higher affinity for MDA-MB-435 and MCF-7 cancer cells compared to the wild type p160. These peptides did not recognize the normal endothelial HUVEC cells. Three p160 peptide analogues, namely, 11 (RGDPAYQGRFL), 18 (WXEAAYQRFL), and 40 (WXEPAYQRKL), that displayed highest affinity for the cancer cells were manually synthesized and labelled with FITC. The binding ability of these peptides was confirmed using fluorescence imaging and flow cytometry. The results confirmed the high and specific affinity of peptides 11 and 18 for the cancer cells. The peptide array-cell binding assay established in this study is not only useful for the identification of cancer targeting peptides. It can also be used for the generation of diagnostic tools for cancer. Secondly, two new classes of -peptides, 3- and 2-peptides derived from L-Asp and L-Dap monomers, respectively, were synthesized. The methodology allowed independent buildup of the -peptide backbone and the introduction of sequential side chain substitutions. It is shown that / mixed peptide increases target recognition and retains the proteolytic stability. Moreover, -peptides impart no cytotoxicity, which will expand their potential application in the design of biologically active peptides. As a result, these compounds represent good candidates for new drugs and as tools to gain further insight into protein folding and molecular recognition processes. / Pharmaceutical Sciences

Background: Conventional chemotherapeutics target bulk tumour cells and generally leave cancer stem cell (CSC) populations unaffected. Recent literature characterized the presence and the role of CSC in several types of solid tumors, including colorectal cancer. Colorectal CSCs (CCSCs) display enhanced WNT/β-catenin pathway activity, sustaining self-renewal and tumor-initiating capacity. Thus, CCSCs are crucial for tumour recurrence and metastasis. As one of the main contributors to sustained self-renewal activity in CCSCs, enhanced formation of β-catenin/CBP complex is fostering transactivation of canonical WNT target genes such as c-myc. However, maintenance of healthy intestinal stem cells also dependents on the canonical WNT pathway. Thus, selective targeting CCSCs while sparring normal intestinal cells is still a significant challenge. Interestingly, Sam68 is a key mediator of the interaction between β-catenin and CBP. It has been reported as a “druggable” target to selectively disrupt β-catenin/CBP in CSCs. Indeed, CWP232228 successfully targets CSCs in AML by facilitating Sam68/CBP complex formation, and consequently lowering the abundance of β-catenin/CBP complexes. CWP232228 was clinically tested on multiple human cancers. Unfortunately, such clinical trials were halted due to unknown causes, and limited information was released on clinical safety and benefits. Consequently, developing more potent pharmacological modulators of Sam68/CBP complex formation is still highly relevant to eradicate CCSCs. Here we describe the discovery and characterization of a new CWP analog, known as YB-0158, which displays enhanced potency and neoplastic selectivity against CCSC. Methods and Results: Following the confirmation that ICG/CWP class of compounds bind to Sam68 in CSCs, I used in silico docking methods to screen for CWP analogs having high predicted affinity for Sam68 Cterminal proline-rich domain. Using high content imaging techniques, I confirmed our top candidate (YB-0158) as more potent vs. CWP parent molecule to compromise cell growth, to induce loss of pluripotency, and to increase Sam68 nuclear localization in a surrogate model of human CSCs. YB-0158 also displayed enhanced selective toxicity in colorectal cancer models vs. normal intestinal epithelium progenitor cells. Moreover, I confirmed that YB-0158 exert negative impact on cancer cell growth by inducing apoptosis and reducing proliferation. Lentiviral-based knockdowns explicitly displayed decrease in drug effectivity in the absence of Sam68, reinforcing the essential role of Sam68 mediating ICG-001/CWP response in CSCs. I demonstrated that Sam68 expression is enriched in tumor-initiating cell fractions derived from primary colorectal tumor tissues vs. bulk heterogeneous tumor organoids. Therefore, YB-0158 showed striking efficacy at supressing tumor-initiation activity in a patient-based serial organoid formation assay. Finally, YB-0158 eradicated CSCs activity in vivo as demonstrated by a syngeneic mouse-to-mouse serial transplantation assay. Conclusion: Overall, YB-0158 is a novel analog of CWP232228 with superior potency to target CCSCs activity through facilitation of Sam68 nuclear localization, thus reducing the interaction frequency between CBP and β-catenin.

No description available.

Peptidergic G Protein-Coupled Receptors (GPCRs) play a role in many of the most important biological functions, and the ability to modulate the activity of these critical proteins has tremendous potential to increase our understanding of biology and allow the development of new therapeutics. In some cases this knowledge will point towards the importance of interconnected proteins of the same or different classes, such as kinases, which interact in a complex and dynamic network in vivo. Understanding these systems will be crucial for addressing unmet therapeutic needs, and new chemical structures may be important at every step of the process.Our contribution to this pursuit includes the development of new ligands for the melanocortin receptors based on a bicyclic or tricyclic core structure. These were designed to be peptidomimetics, built from amino acids to leverage the accumulated knowledge of the group but with properties that complement those of peptides. Most of the molecules in this series bind to the melanocortin receptors, and many with significant selectivity. Some are selective for the MC5R, which may allow further study of this widely distributed but largely unexplored subtype. Others bind preferentially to the MC1R, a property which may be useful in the development of imaging agents targeting melanoma.Imaging using fluorescent probes can provide a tremendous amount of information in studies of receptor biology. With this in mind, we have developed new fluorescent ligands which bind to melanocortin receptors. These compounds use the previously discovered bicyclic template and incorporate the small organic fluorophores anthranilate and N-methylanthranilate.While these structures are in a sense bifunctional, as they exhibit both pharmacologic and fluorescent activity, other molecules may instead incorporate two different pharmacophores. We have synthesized designed multiple ligands (DMLs) of this type for the opioid and neurokinin receptors, as well as molecules which target both the opioid receptors and p38 MAP kinase. These structures merged known active ligands, such as fentanyl for the opioid activity, into one bifunctional molecule. In addition we have used our newly developed template to create a novel NK1R antagonist which may be part of the next generation of bifunctional ligands.