Doctor of Philosophy, The Ohio State University, 2022, Biochemistry Program, Ohio State

Many conjugation techniques have been developed for the site-specific conjugation of various moieties such as small molecules and peptides to proteins of interest to generate bioconjugates. The most convenient of these methods are those that target the protein's N- terminus. To this end, this dissertation will highlight the generation of a new method of generating N-terminal cysteinyl proteins for the use of site-specific labeling of the N- terminus of proteins with fluorophores and peptides using proline and generation of bicyclic peptide inhibitors against the NEMO-IKKβ interaction. Chapters 2 and 3 detail the development of this novel method. The method uses proline as the protecting group and a proline aminopeptidase to deprotect the N-terminal cysteine. First, the substrate specificity of the proline aminopeptidase was determined using combinatorial chemistry. This study gave a consensus sequence for the AsPAP. In chapter 3, we then cloned this peptide into the N-terminus of a model protein, RBDv2, with a (GSS)2 linker, making this protein an efficient substrate for the AsPAP. We derivatized the N-terminus of the protein with fluorescein and CPP12 using N-terminal cysteine-specific conjugation in high yield. These data showed that we could produce useful protein conjugates through the generation of N-terminal cysteines by deprotection of the N- terminal cysteine with AsPAP. Finally, chapter 4 highlights the optimization of bicyclic peptide inhibitors against the NEMO-IKKβ interaction. The NEMO-IKKβ interaction is a PPI of importance due to its role in inflammation and cancers. The Pei lab generated a novel bicyclic peptide inhibitor against this interaction with an IC50 of 1.0 μM. In this chapter, we developed a bicyclic peptide inhibitor with increased potency against this interaction.

Committee: Dehua Pei (Advisor); Jill Rafael-Fortney (Committee Member); Michael Freitas (Committee Member); Charles Bell (Committee Member) Subjects: Biochemistry; Organic Chemistry

Doctor of Philosophy, The Ohio State University, 2017, Chemistry

The purpose of this dissertation is to highlight three unique approaches towards discovering a catalytic treatment towards organophosphorus (OP) poisoning. All three potential approaches focus on developing catalytic treatment methods that focus on hydrolyzing OP nerve agents before they can inhibit acetylcholinesterase (AChE). AChE is a serine hydrolase which is responsible for hydrolyzing the neurotransmitter acetylcholine (ACh). AChE operates near diffusion control and can hydrolyze upwards of 25,000 ACh molecules every second. However, when AChE is inhibited by a nerve agent, an excess amount of ACh will build up at neurosynaptic gaps, thereby causing a cholinergic crisis. Once this occurs, a person will start to develop symptoms of muscle contractions, blurry vision, seizures and/or respiratory failure. An OP nerve agent has this effect because it is a structural analog to ACh; however, phosphylation of the active site is more difficult to reverse. Reactivation of AChE can occur by hydrolyzing the phosphylated enzyme with a nucleophile such as 2-PAM (often administered after OP exposure has occurred). Unfortunately, if this reactivation does not occur, the phosphylated enzyme will undergo a spontaneous dealkylation step (termed aging) to give a “dead” enzyme, which to date cannot be reactivated. The first therapeutic design focuses on the research and development of phosphorane haptens. These haptens are conjugated to some mutagen and administered into mice. This causes an immune response and can generate catalytic antibodies which are capable of hydrolyzing the nerve agent VX. In total, ten different haptens were synthesized, mimicking the hydrolysis transition state of VX, and all generated specific antibodies. Each titer of antibodies were then tested against authentic VX samples. The second approach focuses on the development of a combinatorial approach to synthesizing a random library of cyclic peptides. These cyclic peptides are meant to model the activ (open full item for complete abstract)

Committee: Christopher Hadad (Advisor); Jon Parquette (Committee Member); Psaras McGrier (Committee Member) Subjects: Chemistry

Doctor of Philosophy, The Ohio State University, 2014, Chemistry

Ras is a small family of proteins that are ubiquitously expressed and play critical roles in cell proliferation, differentiation and survival. Mutations in the Ras gene are present in approximately 30 % of human cancers. These mutations result in the functional activation of Ras due to the impairment of GTP hydrolysis. Despite half a century of extensive research, no therapeutics has been very successful in clinical trials. Most of the efforts to discover inhibitors of Ras have focused on small molecules. However, this approach may not be successful, as Ras does not contain any deep binding pockets ideal for small molecule binding. The mutant K-Ras G12V is prevalent in major neoplasms that result in a high level of mortality. Unlike G12C and G12D mutants that contain a reactive moiety (thiol and carboxyl groups respectively) that can be specifically targeted with covalent inhibitors, K-Ras G12V presents a much more challenging target. Since inhibition of Ras function requires a molecule that can compete with large effector proteins for binding to Ras, we chose to screen monocyclic and bicyclic peptide libraries to obtain binders and inhibitors against K-Ras G12V. These macrocycles occupy a unique chemical space between small molecules and antibodies and currently underexplored as inhibitors of Protein Protein Interactions (PPI's). The one bead two compound (OBTC) approach was used to design bicyclic and monocyclic libraries with the potential inhibitor displayed on the surface and the corresponding encoding tag for hit identification. We successfully incorporated unique features in the library such as the ability to selective cleave and screen cyclic peptides in solution. This feature enhances our screening procedure and reduces the time and expense associated with the screening process. The bicyclic library screening produced compounds that bound to K-Ras G12V with high affinity. Some of these inhibitors bound to the effector binding surface of Ras and inhibit (open full item for complete abstract)

Committee: Dehua Pei (Advisor); Jon Parquette (Committee Member); Mark Foster (Committee Member) Subjects: Biochemistry; Chemistry

Doctor of Philosophy, The Ohio State University, 2011, Biochemistry Program, Ohio State

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. (open full item for complete abstract)

Committee: Dehua Pei PhD (Advisor); Peng George Wang PhD (Committee Member); Ross E. Dalbey PhD (Committee Member) Subjects: Biochemistry; Biomedical Research; Chemistry

Master of Science, The Ohio State University, 2010, Biochemistry

The increased prevalence of drug-resistant bacteria is a global human health problem. RNA-protein (RNP) complexes, which play a vital role in various cellular processes, have merited scrutiny as candidate targets for novel antibacterials. RNase P, a catalytic RNP, is primarily responsible for the Mg2+-dependent removal of the 5'-leader in all precursor tRNAs (pre-tRNAs). Despite its essential and conserved primary function in tRNA biogenesis, the subunit composition of the RNase P holoenzyme varies in the three domains of life. All RNase P holoenzymes comprise an essential RNase P RNA (RPR) and a variable number of RNase P Protein (RPP) subunits: one in Bacteria, and at least four and nine in Archaea, and Eukarya, respectively. Towards design of new antibacterials, we have exploited the striking differences between the structure of RNase P in pathogenic bacteria and their eukaryotic hosts. In this study, we hypothesized that certain synthetic Arg-rich, β-hairpin peptides, previously designed to disrupt the assembly of viral protein-RNA complexes, would also serve as structural mimics of a highly conserved, Arg-rich helix in bacterial RPP and disrupt bacterial RNase P assembly. Indeed, some of these cyclic peptides exhibited low-micromolar MIC (Minimal Inhibitory Concentration) values when tested for their ability to inhibit the growth of Salmonella enterica serovar Typhimurium strain 14028 (hereafter referred as S. Typhimurium 14028). While these peptides consist of similar number of Arg residues, their inhibitory potency is probably a reflection of differences in their amino acid sequences. This selectivity highlights the importance of sequence/structure over number of charges. When crude extracts prepared from S. Typhimurium 14028 cells grown in the presence of P17 (a cyclic peptide, which exhibits a low-micromolar MIC value) were tested for pre-tRNA processing, we observed a three-fold decrease in RNase P activity. Northern analysis confirmed pre-tRNAGly and pre- (open full item for complete abstract)

Committee: Venkat Gopalan (Advisor); Edward Behrman (Committee Member); Brian Ahmer (Committee Member) Subjects: Biochemistry

Doctor of Philosophy, The Ohio State University, 2007, Chemistry

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 μm) 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 α-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 (open full item for complete abstract)

Committee: Dehua Pei (Advisor) Subjects: Chemistry, Biochemistry