The field of Combinatorial chemistry was first introduced in the early 1980s. In 1984 Geysen et al1 reported the use of a 96-well plate multi pin system for the synthesis and selection of peptides. This method was later modified by synthesizing the peptides onto cellulose paper, allowing the production of “spot libraries”2 . These methods3–7 , among others played a crucial role in the development of the field of combinatorial chemistry. Their contributions, the development of high-throughput screening methods and the advancements made in analytical characterization3 , revolutionized the pharmaceutical industry, and paved the way for the development of diverse and unique methodology4,8–13. One such method is the One-Bead OneCompound (OBOC) combinatorial method developed by Kit Lam in 199114. OBOC uses Fmoc-chemistry coupled with solid-phase peptide synthesis (SPPS) and “split-mix synthesis” to efficiently generate large peptide libraries with extensive synthetic diversity contained on millions of beads. Each of which contains a single unique peptide sequence that can be analyzed using Edman microsequencing or mass spectrometry. Figure A-1 shows the stepwise procedure used for OBOC libraries. Upon completion of the final step each library is globally deprotected using 4-methylpiperdine and trifluoroacetic acid (TFA) before being prepared for screenings. Using this method with 19 reaction vessels to synthesize a pentapeptide library we can produce up to 2,476,099 (195 ) individual peptides14 . The diversity of these libraries is further increased through the utilization of unnatural amino acids and small molecules allowing us to synthesize libraries containing billions of possibilities. This is one of the traits that makes iii OBOC a unique and versatile method that has been used to identify novel ligands for targets against live tumor cells15, protein inhibition16,17, and molecular imaging16,18–21 . While this method does have it disadvantages the first two chapters will be used to discuss projects in which using this method has been advantageous. The final chapter will go into more detail about the limitations and methods that can be used to overcome these limitations. In chapter 1 we discuss the use of OBOC technology to discover short peptide dye pairs to be used as molecular probes. These genetically encoded small illuminants (GESI) can be fused to integral membrane proteins (IMPs) and utilized to monitor changes to those proteins’ environment, such as structural or post-translational modifications (PTMs). GESI are smaller iv (~2kDa) than frequently used fluorescent probes like green fluorescent protein (GFP), which allows their grafting into IMPs without interfering with their physiological properties. These peptides, discovered from screening several cyclic OBOC libraries with confocal microscopy22 using molecular rotor dye(MRD) such as bromocresol purple as probes. This novel GESI technology, once fully developed, will greatly expand the capabilities of the molecular imaging toolbox. Here we report the discovery of several GESI peptides that were successfully transfected between GFP domain and the transmembrane domain of platelet-derived growth factor receptor (PDGFR) using HEK293T cells. Using Bromocresol Purple (BCP) we were able to demonstrate the utility of the proposed GESI imaging system in living cells. In Chapter 2 we focus on developing peptide binders to an allosteric binding pocket adjacent to the RGD motif binding site on integrin αvβ3. Integrins are a family of transmembrane proteins viewed as potential therapeutic targets because of their integral function in various physiological and pathological pathways. One of the prototypical binding sites associated with some of these integrins is known as the RGD binding site, named for its high affinity for RGD tripeptide motif. Recent studies on the binding mechanism of αvβ3 with its ligand Fractalkine in the absence of its receptor, CX3CR1, has supported the hypothesis that there is an allosteric binding pocket located on the β subunit. This site is adjacent to the classical RGD binding site and contrary to this site, is most accessible when the integrin is in its low affinity conformation. This theory is shown to apply to other proteins and not just Fractalkine when solubilized αvβ3, in its inactive state, was tested against human secreted phospholipase A2 type IIA (sPLA2- IIA), which is known to bind this integrin as well. By targeting an allosteric binding site, we v hope to develop peptides that are more specific and have reduced overall off-target effects. In this study we designed and prepared 6 cyclic and linear OBOC peptide libraries. These libraries were then screened using cell and protein binding assays with K562(+αvβ3) cells and a GST fusion protein of Site 2 peptide (QPNDGQSHVGSDNHYSASTTM, residues 267–287of β3, C273 is changed to S). Positive hits from both screenings were validated using both previous methods and with soluble integrin αvβ3 integrin, yielding 5 lead peptides. Of these only one peptide, Peptide 10, showed binding to K562(+αvβ3) when analyzed using flow cytometry. Lastly, we access various methods that can be used to reduce the non-specific binding found in OBOC libraries. This non-specific binding is inherent in many library screening methods and can be addressed in a variety of ways. In the OBOC method, the following three approaches are often used either individually or in combination to increase the screening stringency so that true positive ligands with high affinity can be identified: (i) including soluble competing ligands in the screening buffer, (ii) decreasing the concentration of screening probes, and (iii) down substituting the peptide displayed on the resin surface. Additionally, compounds that show binding and read as positive hits on resin can fail to translate well in solution, yielding peptides that must undergo significant optimization to be viable. It is important to develop methods to minimize non-specific binding. Here we use bivalirudin, an FDA-approved anticoagulant, as a model ligand against thrombin, and systematically assess the contribution of stealth peptides, linkers, peptide substitution, and resins to non-specific binding of proteins to peptide-beads, which could be readily determined by pull-down assay followed by gel electrophoresis. vi We found that replacing 50% of the peptide ligand on the bead surface with zwitterionic stealth hexapeptide EKEKEK, or simply (EK)3 could greatly lower the undesirable nonspecific binding. This new information was incorporated into the design of a focused OBOC library, which was then screened against thrombin.