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.

With combinatorial chemistry millions of organic compounds can be produced simultaneously, quickly, and in most cases by automated procedures. These compound libraries are a cost-effective resource for the pharmaceutical industry in their search for biologically active lead structures. Furthermore simultaneous parallel synthesis of single peptides and peptide libraries solve the problem of the worldwide increasing demand for peptides. The synthetic methods described here in detail contribute to a forward-looking technology that has a high impact for industrial and academic research. Fast and efficient analytical techniques are essential for using the complicated product mixtures and detecting by-products. Various synthetic approaches and technologies, mass spectrometry, and screening assays are discussed extensively. This book is a must and an indispensible source of information for every researcher in this rapidly developing field, which spans organic synthesis, biochemistry, biotechnology, pharmaceutical, medicinal, and clinical chemistry.

Abstract: A method for the rapid identification of high-affinity ligands was used to study the specificity of the interaction between FHA2 domain of Rad53 and phosphotyrosyl peptide. A phosphotyrosyl (pY) peptide library containing completely randomized residues at positions -2 to +3 relative to the pY was synthesized on TentaGel resin, with a unique peptide sequence on each resin bead. The library was screened against the biotinylated FHA2 domains, and the beads that carry high-affinity ligands of the FHA2 domains were identified using an enzyme-linked assay. Peptide ladder sequencing of the hundreds of selected beads using MALDI-TOF revealed consensus sequences for FHA2 domains. A new method was developed to rapidly sequence support-bound peptides derived from combinatorial peptide library. Support-bound peptides isolated from one-bead-one-compound libraries are subjected to partial Edman degradation in the presence of an N-terminal blocking agent (5% phenyl isocyanate). Repetition of the degradation reaction results in a series of sequence-specific truncation products (a peptide ladder). The sequence of the full-length peptide is determined by MALDI-TOF analysis of the peptide ladder. During screening of combinatorial libraries for optimal enzyme substrates, the challenge is to differentiate a reaction product(s) from a complex mixture of substrates. We have overcome this problem by partially labeling the substrates with a heavier isotope (heavy/normal isotope = 1:1), so that each member of the substrate library appears as a doublet in ESI-MS spectrum whereas the products appear as singlets in the spectrum, allowing for their unambiguous identification. The strategy has been successfully demonstrated by peptide deformylase screening. This method was further perfected in the screening of the pY library against the catalytic domain of SHP-1. Limited treatment of the library with a PTP removed phosphoryl group from the most preferred substrates to generate products as singlet peaks, which were readily identified in ESI-FTICR-MS spectrum and sequenced by tandem mass spectrometry. Several selected peptides were individually synthesized and assayed against SHP-1 and the kinetic data confirmed the screening results.

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.

An auto-fluorescent bacterial display peptide library was used to isolate red blood cell (RBC) binding ligands which were used to attach nanoparticles to the RBC surface to develop novel long circulating drug delivery vehicles. Bacterial display was also employed to isolate tissue targeting ligands in vivo. Finally, multi-color FACS was used to quantitatively isolate highly-specific peptides and further optimize their specificity for a target antibody present at a 10,000 - 100,000 fold dilution in serum IgG. The method was employed to screen serum antibodies of patients with Celiac Disease to identify potential biomarker candidates. The method developed here enables quantitative screening and evolution of specificity, enhancing the current repertoire of screening methods available in protein engineering.