A great number of cellular and biological processes depend, at some level, on protein-protein interactions (PPI). Their manipulation with chemical compounds has provided a great potential for the discovery of new drugs. Despite the increasing demand for molecules able to interrupt specific PPI, the development of small PPI inhibitors is beset by a number of challenges such as the large size of the interaction interface. Based on the interface's nature, the ability to mimic protein secondary structures is very important to bind a protein and inhibit PPI. With their interesting peptidomimetic abilities and pharmacological properties, cyclic peptides are very promising templates to discover protein ligands and development new PPI inhibitors. To fully exploit the great diversity accessible with cyclic peptides, the one-bead-one-compound (OBOC) combinatorial method is certainly the most accessible and powerful approach. Unfortunately, the use of cyclic peptides in OBOC libraries is limited by difficulties in sequencing hit compounds after the screening. Lacking a free N-terminal amine, Edman degradation cannot be used on cyclic peptides and complicated fragmentation patterns are obtained by tandem mass spectrometry (MS/MS). In this regard we have designed and developed new convenient ring-opening approaches to prepare and decode OBOC cyclic peptide libraries. Our strategy was to introduce a cleavable residue in the macrocycle and as a linker to allow linearization of peptides and their release from the beads for sequencing by MS/MS. First, amino acid residues sensible to nucleophiles, ultraviolet irradiation or cyanogens bromide were introduced in a model cyclic peptide. Afterward, the most promising residues were used to design and develop tandem ring-opening/cleavage approaches to decode OBOC cyclic peptide libraries. In the first approach a methionine residue was introduced in the macrocycle and as a linker to allow a simultaneous ring-opening and cleavage from the beads upon treatment with cyanogens bromide. In the second approach, a photosensitive residue was used in the macrocycle and as a linker for a dual ring-opening/cleavage upon UV irradiation. The linear peptide generated by these approaches can be efficiently sequenced by tandem mass spectrometry. Finally, an OBOC library has been prepared and screened against the HIV-1 Nef protein to identify selective ligands. The development of these methodologies will prompt the use of macrocyclic compounds in OBOC libraries and be an important contribution in medicinal chemistry for the discovery of protein ligands and the development of PPI inhibitors.

The one-bead one-compound (OBOC) concept was first recognized by Lam et al. in 1991 in application of identifying small linear peptides that bind to anti-[beta]-endorphin antibody and streptavidin.1 This concept is based on the fact that combinatorial bead libraries, prepared via a "split synthesis" approach, contain single beads displaying only one type of compound although there may be up to 1013 copies of the same compound on a single 90-100 [mu]m diameter bead. OBOC libraries are not limited to linear peptides. This technique covers synthesis of libraries of cyclic peptides, peptoids, peptidomimetics, small molecules, etc. The technique further expanded from on-bead screening to in situ releasable assays, in which the portion of library compound are released from each bead in solution.2 The use of high-density microbead cassettes allowed both in situ releasable solution phase assays and efficient bead mobilization. The sequence of the peptides comprised of [alpha]-amino acids and have a free N-terminus can be identified by Edman chemistry. For peptides that are not sequenceable by Edman microsequencing technique, a biphasic approach was invented to generate topologically segregated bilayer beads with library compounds on the outer layer of each bead and the coding tags inside each bead.4 Over the last three decades, OBOC method has evolved tremendously, in the areas of solid-support, chemical encoding, synthetic chemistry, and high-throughput biochemical and cell-based screening. This thesis focuses on the various applications of OBOC illustrated by three different projects, each is explained in one chapter. Chapter 1 discusses the use of OBOC method for rapid discovery of illuminating peptides for instant detection of opioids in blood. Current approaches for opioid identification and quantification in body fluids include immunoassays and chromatographic methods (e.g., LC-MS, GC-MS) that require expensive instrumentation and extensive sample preparation. In this chapter we report the development of a portable morphine-sensitive fluorescence-based sensor chip to sensitively detect morphine in blood using a homogenous immunoassay without any washing steps. Morphine-sensitive illuminating peptides were identified by a high throughput OBOC combinatorial peptide library approach. The OBOC libraries contain a large number of random peptides with a molecular rotor dye, malachite green (MG), coupled to the amino group on the side chain of lysine at different positions of the peptides. The OBOC libraries were then screened for fluorescence activation under a laser scanning confocal microscope, using an anti-morphine monoclonal antibody as the screening probe, in the presence and absence of free morphine. Using this novel three-step fluorescent screening assay, we were able to identify peptide-beads that fluoresced in the presence of anti-morphine antibody, but lost fluorescence when free morphine was present. After positive beads were decoded using automatic Edman microsequencing, the morphine-sensitive illuminating peptides were synthesized in soluble form, functionalized with an azido group, and immobilized onto microfabricated PEG-array spots on a glass slide. This proof-of-concept platform can be used to develop fluorescence-based sensors against morphine. More importantly, this technology can also be applied to the discovery of other novel illuminating peptidic sensors for detection of illicit drugs and cancer biomarkers in body fluids. Chapter 2 discusses the use of OBOC method to discover short peptide-dye pairs that can be genetically fused to target integral membrane proteins (IMPs) and used as genetically encoded small illuminants (GESIs) for functional cellular imaging. In this chapter, we establish a methodology to create a toolbox of functional illuminants to tag cellular proteins and probe the spatiotemporal dynamics, activation and phosphorylation of these proteins in living cells. This method relies on the OBOC technology coupled with confocal microscopy to screen and identify short peptide(s) that activate fluorophores which can be used as GESIs for functional cellular imaging. As opposed to large protein-based fluorophores (e.g., green fluorescent protein, or GFP), these GESIs are small (~2kDa), allowing them to be readily inserted along the sequence of IMPs without interfering with their physiological functions. This research will assist in broadening the field of fluorescence-based molecular imaging, both in vitro and in vivo. As a proof of concept, we have genetically inserted our GESIs between GFP and the transmembrane domain of PDGFR, such that the resulting GFP-GESI fusion would be expressed and displayed on the surface of HEK293 cells. We then used bromocresol purple to demonstrate the utility of the proposed genetically encoded functional imaging system to probe model intrinsic membrane protein in living cells. Chapter 3 describes a novel approach to minimize non-specific binding of target protein during OBOC combinatorial library screening while identifying strong thrombin inhibitors with potential clinical use as anticoagulants. In this chapter, we assessed the viability of various linkers, resins, and percentage of the resin surface containing peptides to develop a robust screening method for OBOC libraries, such that non-specific binding and false-positive results can be minimized. Using bivalirudin, an FDA-approved anticoagulant as a model, we were able to develop a linker, resin and peptide composition combination that minimized non-specific binding as shown via gel electrophoresis. As a proof of concept, an OBOC library was subsequently designed incorporating these parameters and screened against thrombin which led to discovery of six hits. All the six peptides were then proved to be true positives. We were able to demonstrate that lowering the concentration of hit compound density on the surface of beads to 50% and coating the remaining 50% of the surface with stealth hexapeptide (EK)3 as a way to greatly eliminate nonspecific binding, thus maximizing the chance of true-positive bead identification. In addition, the positive sequences found through OBOC screening with thrombin, would be of interest as they may yield clinically useful anticoagulants.

Abstract: Cyclic peptides are widely produced in nature and possess a broad range of biological activities. Their enhanced proteolytic stability in vivo and improved receptor binding affinity/specificity makes them excellent drug candidates, molecular probes and targeting agents. In fact, many cyclic peptides are clinically used therapeutic agents. Combinatorial library approaches provide powerful tools for the rapid identification of compounds with desired properties from large pools in biological and biomedical studies. However, the synthesis and screening of cyclic peptide libraries in a combinatorial format has been challenging. To overcome the issue, we have successfully developed one-bead-two-compound (OBTC) libraries with a cyclic peptide displayed on the bead surface accessible for protein targets screening, while the bead interior contains the corresponding linear peptide served as an encoding tag for hit identification. The primary goal of my research is to identify novel biologically active cyclic peptides, beyond what nature has provided us. By applying cyclic peptide library approach, we have successfully identified high affinity ligands against various biological targets, including: extracellular protein receptors (human prolactin receptor), intracellular protein domains (the capsid domain of HIV-1 Gag polyprotein and calcineurin catalytic domain) and enzymes (Pin1 catalytic domains). In the meantime, we have continued to improve the methodologies associated with combinatorial chemistry. To facilitate the process and improve the screening results, such as avoiding false positives, we have developed many cyclic library approaches including libraries on different solid supports, reduced surface density libraries, high diversity libraries with different ring sizes and library compatible with rapid solution phase validation. These new approaches greatly facilitate the ligands discovery process. My final work focused on the intracellular delivery of cyclic peptides. Little is known about how cyclization would affect peptides membrane permeability and the results from existing studies are controversial. With a combination of biophysical approaches and cell based studies, we have found that cyclization has a dramatic effect on the cell permeation of peptides with certain residues. By applying the rules, we were able to design cell permeable cyclic peptide inhibitors against various intracellular protein targets. Our studies provide guiding principles for designing membrane penetrating cyclic peptidyl drugs.

Abstract: One-bead one-compound (OBOC) peptide libraries have been useful tools in the biomedical sciences. However, OBOC peptide libraries usually display the N-termini of peptides on the surface as conventional solid phase peptide synthesis proceeds in the C to N direction. While large combinatorial libraries of cyclic peptides can be synthesized by the split-and-pool synthesis method, the sequence determination has been a challenge. Also, peptide libraries with free C-termini face the same problem as well as the difficulty of synthesis in the N to C direction. We report here the development of cyclic peptide libraries and C-terminal peptide libraries for high-throughput screening and sequencing. TentaGel microbeads (90 um) were spatially segregated into outer and inner layers; cyclic peptides were displayed on the bead surface, whereas the inner core of each bead contained the corresponding linear encoding peptide. After screening of the cyclic peptide library, the identity of hit peptides was determined by sequencing the linear encoding peptides using a partial Edman degradation/mass spectrometry method. Using the same spatial segregation approach peptides were synthesized in the conventional C to N direction, with their C-termini attached to the support through an ester linkage on the bead surface but through an amide bond in the inner layer. The surface peptides were cyclized between N-terminal amine and a carboxyl group installed at a C-terminal linker sequence, while the internal peptides stayed in the linear form. Base hydrolysis of the ester linkage in the cyclic peptides exposed a free [alpha]-carboxyl group at the C-termini of the peptides attached to the resin via the N-termini. An inverted peptide library containing five random residues was synthesized and screened for binding to PDZ domains. The identity of the binding peptides was determined from the encoding peptides. Consensus recognition motifs were identified for the PDZ domains and representative peptides were individually synthesized and confirmed for binding to their cognate PDZ domains. These methods expanded the utility of OBOC peptide libraries by displaying peptides in different ways.

This volume provides an overview of modern and emerging methods for production, analysis, and utility of peptide libraries. Chapter focus on methods and techniques for synthesis, genetic expression, hybrid synthesis-expression, examples of modern utility of these libraries, de novo discovery of reactions, hybrid organic-inorganic materials and, emerging tools for the analysis of these libraries by method of genetic selection and next-generation sequencing. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Peptide Libraries: Methods and Protocols seeks to serve both professionals and novices with its well-honed methodologies.

During the course of evolution, an imbalance was created between the rate of vertebrate genetic adaptation and that of the lower forms of living organisms, such as bacteria and viruses. This imbalance has given the latter the advantage of generating, relatively quickly, molecules with unexpected structures and features that carry a threat to vertebrates. To compensate for their weakness, vertebrates have accelerated their own evolutionary processes, not at the level of whole organism, but in specialized cells containing the genes that code for antibody molecules or for T-cell receptors. That is, when an immediate requirement for molecules capable of specific interactions arose, nature has preferred to speed up the mode of Darwinian evolution in pref- ence to any other approach (such as the use of X-ray diffraction studies and computergraphic analysis). Recently, Darwinian rules have been adapted for test tube research, and the concept of selecting molecules having particular characteristics from r- dom pools has been realized in the form of various chemical and biological combinatorial libraries. While working with these libraries, we noticed the interesting fact that when combinatorial libraries of oligopeptides were allowed to interact with different selector proteins, only the actual binding sites of these proteins showed binding properties, whereas the rest of the p- tein surface seemed "inert. " This seemingly common feature of protein- having no extra potential binding sites--was probably selected during evolution in order to minimize nonspecific interactions with the surrounding milieu.

The continued successes of large- and small-scale genome sequencing projects are increasing the number of genomic targets available for drug d- covery at an exponential rate. In addition, a better understanding of molecular mechanisms—such as apoptosis, signal transduction, telomere control of ch- mosomes, cytoskeletal development, modulation of stress-related proteins, and cell surface display of antigens by the major histocompatibility complex m- ecules—has improved the probability of identifying the most promising genomic targets to counteract disease. As a result, developing and optimizing lead candidates for these targets and rapidly moving them into clinical trials is now a critical juncture in pharmaceutical research. Recent advances in com- natorial library synthesis, purification, and analysis techniques are not only increasing the numbers of compounds that can be tested against each specific genomic target, but are also speeding and improving the overall processes of lead discovery and optimization. There are two main approaches to combinatorial library production: p- allel chemical synthesis and split-and-mix chemical synthesis. These approaches can utilize solid- or solution-based synthetic methods, alone or in combination, although the majority of combinatorial library synthesis is still done on solid support. In a parallel synthesis, all the products are assembled separately in their own reaction vessels or microtiter plates. The array of rows and columns enables researchers to organize the building blocks to be c- bined, and provides an easy way to identify compounds in a particular well.