Directed Evolution of Stabilized Peptides with Bacterial Display

Interactions between proteins govern cellular and the body’s states, including aberrant interactions found in diseases such as in cancers and infections. Small molecule drugs are not ideal in targeting these interactions as their size generally prevents efficient blocking of contacts over large surface areas. Antibodies and related biologics have seen clinical success in the past few decades and can block large surfaces but are typically limited to extracellular targets. Intermediate-size peptides have the potential to bridge this gap, with the ability to target large surface areas inside the cell. Peptide stapling, by chemically linking two or more amino acid residues, can confer affinity improvements, resistance to degradation, and better biological transport properties. As such, stapled peptides show promise as next-generation therapeutics. Unfortunately, existing methods to screen sequence and stapling locations suffer from numerous disadvantages including limited search space, lack of real-time monitoring of selections, and difficulty in incorporating the non-canonical amino acids used for amino acid stapling. In this dissertation, I describe my research on stapled peptide discovery with bacterial incorporation of non-canonical amino acids. To screen stapled peptides of the type desired, we incorporated azidohomoalanine (AHA) into surface displayed peptides, enabling an in situ ‘click’ chemistry reaction to bridge two turns of an alpha helical (i, i+7) amino acid library for directed evolution. Using the p53-MDM2 interaction as a model target, we developed peptides that block MDM2 degradation of the tumor suppressor protein p53, an interaction that is dysregulated in a sizeable fraction of cancers. We generated and displayed a stapled peptide library on the bacterial cell surface with fixed residues for stabilization and binding requirements, while randomizing the remaining amino acids. After multiple rounds of selection, clones were sequenced and characterized. The dissociation constants of the peptide-MDM2 interaction were measured on both the bacterial cell surface by flow cytometry and in solution by bio-layer interferometry. The highest affinity variant, named SPD-M6-V1 with sequence VCDFXCYWNDLXGY (dissociation constant = 1.8 nM; X = azidohomoalanine) was selected for structural characterization by NMR spectroscopy, revealing a bicyclic disulfide and double click-constrained peptide. Sequencing showed that peptides with two cysteines were highly enriched, further suggesting that the MDM2-binding conformation was enforced with a disulfide bond. In addition, SPD-M6-V1 was the most protease-resistant peptide from the library that we tested. Next, we stapled the displayed peptide library with chemically distinct linkers and screened each library separately. We performed deep sequencing to better understand the relationship between amino acid sequence and linker identity in contributing to high affinity MDM2 binding. We found that both linker-specific and linker-agnostic (i.e. MDM2-specific) mutations were enhanced. Finally, we developed a dual-channel, sequential labeling selection strategy to discriminate between high-display, low-affinity peptides and low-display, high-affinity peptides, two categories that would ordinarily overlap in a typical one-color screen in the absence of an independent display marker. In summary, this thesis develops the chemical tools to screen libraries of stabilized peptides on the bacterial cell surface and applies these techniques to select stabilized alpha helices that disrupt the p53-MDM2 interaction.PHDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163094/1/tejasn_1.pd

Antigen specificity can be irrelevant to immunocytokine efficacy and biodistribution

Cytokine therapy can activate potent, sustained antitumor responses, but collateral toxicity often limits dosages. Although antibody–cytokine fusions (immunocytokines) have been designed with the intent to localize cytokine activity, systemic dose-limiting side effects are not fully ameliorated by attempted tumor targeting. Using the s.c. B16F10 melanoma model, we found that a nontoxic dose of IL-2 immunocytokine synergized with tumor-specific antibody to significantly enhance therapeutic outcomes compared with immunocytokine monotherapy, concomitant with increased tumor saturation and intratumoral cytokine responses. Examination of cell subset biodistribution showed that the immunocytokine associated mainly with IL-2R–expressing innate immune cells, with more bound immunocytokine present in systemic organs than the tumor microenvironment. More surprisingly, immunocytokine antigen specificity and Fcγ receptor interactions did not seem necessary for therapeutic efficacy or biodistribution patterns because immunocytokines with irrelevant specificity and/or inactive mutant Fc domains behaved similarly to tumor-specific immunocytokine. IL-2–IL-2R interactions, rather than antibody–antigen targeting, dictated immunocytokine localization; however, the lack of tumor targeting did not preclude successful antibody combination therapy. Mathematical modeling revealed immunocytokine size as another driver of antigen targeting efficiency. This work presents a safe, straightforward strategy for augmenting immunocytokine efficacy by supplementary antibody dosing and explores underappreciated factors that can subvert efforts to purposefully alter cytokine biodistribution.National Cancer Institute (U.S.) (Grant CA174795)National Science Foundation (U.S.). Graduate Research Fellowshi

A Helix-Stabilizing Linker Improves Subcutaneous Bioavailability of a Helical Peptide Independent of Linker Lipophilicity

Stabilized peptides address several limitations to peptide-based imaging agents and therapeutics such as poor stability and low affinity due to conformational flexibility. There is also active research in developing these compounds for intracellular drug targeting, and significant efforts have been invested to determine the effects of helix stabilization on intracellular delivery. However, much less is known about the impact on other pharmacokinetic parameters such as plasma clearance and bioavailability. We investigated the effect of different fluorescent helix-stabilizing linkers with varying lipophilicity on subcutaneous (sc) bioavailability using the glucagon-like peptide-1 (GLP-1) receptor ligand exendin as a model system. The stabilized peptides showed significantly higher protease resistance and increased bioavailability independent of linker hydrophilicity, and all subcutaneously delivered conjugates were able to successfully target the islets of Langerhans with high specificity. The lipophilic peptide variants had slower absorption and plasma clearance than their respective hydrophilic conjugates, and the absolute bioavailability was also lower likely due to the longer residence times in the skin. Their ease and efficiency make double-click helix stabilization chemistries a useful tool for increasing the bioavailability of peptide therapeutics, many of which suffer from rapid in vivo protease degradation. Helix stabilization using linkers of varying lipophilicity can further control sc absorption and clearance rates to customize plasma pharmacokinetics

Dual-Purpose Linker for Alpha Helix Stabilization and Imaging Agent Conjugation to Glucagon-Like Peptide‑1 Receptor Ligands

Peptides display many characteristics of efficient imaging agents such as rapid targeting, fast background clearance, and low non-specific cellular uptake. However, poor stability, low affinity, and loss of binding after labeling often preclude their use <i>in vivo</i>. Using glucagon-like peptide-1 receptor (GLP-1R) ligands exendin and GLP-1 as a model system, we designed a novel α-helix-stabilizing linker to simultaneously address these limitations. The stabilized and labeled peptides showed an increase in helicity, improved protease resistance, negligible loss or an improvement in binding affinity, and excellent <i>in vivo</i> targeting. The ease of incorporating azidohomoalanine in peptides and efficient reaction with the dialkyne linker enable this technique to potentially be used as a general method for labeling α helices. This strategy should be useful for imaging beta cells in diabetes research and in developing and testing other peptide targeting agents

The Anaerobic Fungi : Challenges and Opportunities for Industrial Lignocellulosic Biofuel Production

Lignocellulose is a promising feedstock for biofuel production as a renewable, carbohydrate-rich and globally abundant source of biomass. However, challenges faced include environmental and/or financial costs associated with typical lignocellulose pretreatments needed to overcome the natural recalcitrance of the material before conversion to biofuel. Anaerobic fungi are a group of underexplored microorganisms belonging to the early diverging phylum Neocallimastigomycota and are native to the intricately evolved digestive system of mammalian herbivores. Anaerobic fungi have promising potential for application in biofuel production processes due to the combination of their highly effective ability to hydrolyse lignocellulose and capability to convert this substrate to H2 and ethanol. Furthermore, they can produce volatile fatty acid precursors for subsequent biological conversion to H2 or CH4 by other microorganisms. The complex biological characteristics of their natural habitat are described, and these features are contextualised towards the development of suitable industrial systems for in vitro growth. Moreover, progress towards achieving that goal is reviewed in terms of process and genetic engineering. In addition, emerging opportunities are presented for the use of anaerobic fungi for lignocellulose pretreatment; dark fermentation; bioethanol production; and the potential for integration with methanogenesis, microbial electrolysis cells and photofermentation