Abnormal metabolism is frequently observed in cancer, where the reprogrammed metabolic activities support cancer cells' malignant processes by altering bioenergetics, enhancing biosynthesis, and maintaining redox balance. Understanding the mechanisms of this metabolic reprogramming calls for novel analytical tools for detecting metabolites. This dissertation presents our efforts in developing chemical methods for detecting metabolites. In the first chapter, we present a chemical approach to profile fatty acid uptake in single cells. We demonstrate a surface-based competition assay using azide-modified analogs to probe the fatty acid influx and surface-immobilized dendrimers with dibenzocyclooctyne (DBCO) groups for detection. By integrating this method onto a microfluidics-based multiplex protein analysis platform, we resolved the relationships between fatty acid influx, oncogenic signaling activities, and cell proliferation in single glioblastoma cells. In the second chapter, we present a magnetic particle-based high throughput screening protocol to remove resins with strong non-specific binding from a library. This on-resin preclear method can be used to screen metabolites binders from a peptide library. In the third chapter, we showcase the implementation of the library screening approach for identifying cyclic peptide-based probes for several intracellular metabolites.

Optical microscopy is an essential tool in biological research, drug discovery, and medical diagnosis. Yet it harbors limitations in resolution, penetration depth, photobleaching, and multiplexity. Various strategies, including new techniques and novel probes, are developed to address those drawbacks and improve performance. With that in mind, this dissertation presents our work of designing peptide-based probes for advanced optical bioimaging applications. Our first study reports a cyclic peptide identified from a library screening against terbium(III) (Tb3+) ions. It can bind to Tb3+ and enhance Tb3+ luminescence by 150 times through antenna effects. The second study describes a peptide-based linker for photoactive immunofluorescence with iterative cleavage (PACIFIC). It is compatible with both fixed cells and formalin-fixed paraffin-embedded (FFPE) tissue sections for highly multiplex protein imaging. In the final study, we demonstrate the application of PACIFIC integrated with live-cell imaging. By investigating dynamic properties and phenotypical features in a glioblastoma cell line, we obtain a more comprehensive understanding of the cell motility. Overall, our work showcases how peptide-based modalities can be explored for advanced optical imaging and expanded applications.

Macrocyclic peptides are proven binders against structureless proteins. The complexity incorporated in multiple cyclization within a peptide backbone demands demonstration in multifaceted binding activity. We introduce the applications of cyclic peptide and an outline of the various research projects we aim to develop around them. As a starting point, we demonstrate the scope of our bicyclic peptides as highly specific fluorescent reporter probes for MAPK/ERK signaling pathway, often aberrantly involved in cancer. Bicyclic peptides can be designed to be highly specific binders for homologous ERK1 and 2 and can signal its phosphorylation. A library of significant diversity can assist in discovering such probes. The transcription factor c-Myc is a proto-oncogene which is overexpressed in most known cancers. c-Myc is commonly considered “undruggable” due to its intrinsically disordered structure. We had previously discovered a bicyclic peptide that could effectively inhibit the proliferation of a c-Myc-dependent cell line. Its drawbacks as a therapeutic were addressed through a series of medicinal chemistry modifications. We also address cytotoxicity concerns, applicability across multiple cancer cell lines, and mechanistic details of suppressed proliferation through multiple optimization processes. Additionally, we explored the potential of peptides as miniature enzyme and the structural considerations necessary for designing such molecules.

Aberrant protein activities are frequently observed in various diseases, especially cancer, where these proteins can promote oncogenesis and tumor progression. Many of those abnormal activities involve protein-protein interactions (PPI), which are often critical to protein activation and signal transduction. Developing chemical probes that can specifically interrogate and modulate PPI is attractive, as those probes promise therapeutic benefits and a better understanding of the underlying disease mechanisms. This dissertation presents our work of developing cyclic peptide-based probes that target two proteins: matrix metalloproteinase 2 (MMP2) and extracellular signal-regulated kinase (ERK), both of which are heavily implicated in cancer. In the first project, we report a cyclic peptide that can specifically inhibit MMP2 proenzyme activation, which decreased cancer cell invasion and migration abilities. In the second project, we demonstrate the potential of using the MMP2-targeting cyclic peptide as an imaging agent. In the final project, we showcase the development of cyclic peptide probes that bind to specific epitopes on the ERK protein and report its phosphorylation status.

Peptides are one of the most promising platforms for drug development due to their biocompatibility, similarity, and diversity. Over the past few decades, it has been proven that peptides are practical tools in various therapeutic and diagnostic applications. Studies show that monocyclic peptides are capable of functioning as an effective alternative to complex molecules, such as antibodies or small molecules. In this dissertation, I describe projects that aimed at applying monocyclic peptides into the chemical biology field. The first chapter covers the screening procedures and results of a monocyclic peptide library in detail. Lipid A moiety was used as the screening target to demonstrate the therapeutic application of monocyclic peptides for LPS neutralization. Chapter 2 talks about the application of monocyclic peptides as imaging probes for protein activities in suspension cells. Digitonin, a cell membrane perforator will be introduced as the delivery mediator for the imaging probes. Analysis of AKT signaling activities on a single-cell level will be reported, including the demonstration of drug perturbation on the regulator proteins. Chapter 3 will cover the application of monocyclic peptides as molecular transporters. A small library of monocyclic peptides capable of penetrating the cell membrane will be introduced in this project. These hydroxyl-rich cycle peptides will demonstrate the diversity of cargo molecules into more than ten different cell lines. The mechanisms and fate of these monocyclic peptides will be discussed, providing a thorough explanation of the cell-penetrating process. The discovery of this novel group of peptides with non- positive charges can bring more insights into the cell-penetrating peptide field.

The research covered in this dissertation is described in six chapters. The first two chapters focus on the design of supramolecular nanovalves on mesoporous silica and silicon materials to form drug delivery systems. These systems are capable of autonomously releasing drug molecules in vitro. The third chapter describes size-selective nanovalves that are able to control the release of both large and small molecules. These nanovalves are constructed on mesoporous silica materials with 6.5 nm pores. The fourth chapter demonstrates two experimental methods, dynamic fluorescence anisotropy and rigidochromism spectroscopy, for probing the properties of the microenvironment inside the mesopores of mesoporous silica nanoparticles. In the fifth chapter of this dissertation, the design and operation of a chemical amplifier based on enzyme-encapsulated mesoporous silica are shown. This system employs a pH-responsive nanogate assembly to control the access to the encapsulated enzyme. It is the first example of a chemical amplifier that is activated by and also amplifies the same type of reaction. The last chapter describes the spectroscopic consequences of the excited state mixed valence in a binuclear copper complex using a neighboring orbital model. The copper complex represents a new class of excited state mixed valence compounds, where the coupling of two equivalent charge-baring units is mediated through a copper-iodine cluster. The result from the simple neighboring orbital approach is supported by resonance Raman spectroscopy and Gaussian calculations.

Tumor heterogeneity poses a great challenge for current therapeutics. The ability of cancer cells to adapt and resist drug treatment provides a particular difficulty in two areas: having a full understanding of the mechanisms in which they adapt and a full understanding of how to overcome those mechanisms. Peptide-based therapeutics show great potential in confronting these challenges as they are known to aid in targeting drug targets previously deemed “undruggable.” To date, many peptide drugs are commercially available. With this in mind, our studies focused on the utilization of peptides for therapeutic purposes and investigating how cancer cell heterogenous characteristics respond to peptide drug treatment. In our first study, we treated cancer cells of known low mutational load with an immunogenic peptide. This treatment ultimately flagged the cancer cells as “pathogen-infected,” and we aimed to elicit an immune response. The second study incorporated a cyclic peptide into a proteolysis targeting chimera (PROTAC) structure to aid in the degradation of a commonly hyperactive protein in cancers known as AKT. This study also shed some light on the adaptation mechanisms cells can use in response to this drug treatment. In our final study, we used another cyclic peptide and other metabolic inhibitors to examine how varying types of inhibition affect the heterogeneity of cellular movement in cancer cells. Each study showcases how peptide-based drugs serve as a valuable tool for treating and further understanding the complexities of cancer.