Probing the Biological Interactions of a Therapeutic Small Peptide at the Tissue, Cellular, and Molecular Levels for Treating Retinal Diseases

Citation

Koo, Jin Mo (2025) Probing the Biological Interactions of a Therapeutic Small Peptide at the Tissue, Cellular, and Molecular Levels for Treating Retinal Diseases. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/kye5-xz39. https://resolver.caltech.edu/CaltechTHESIS:03292025-043938908

Abstract

Age-related macular degeneration (AMD) and diabetic retinopathy (DR) are the leading causes of blindness in developed countries, affecting the lives of millions and lowering their quality of life due to limited eyesight. Although these retinal diseases afflict a large portion of the senior population, the current standard of care, primarily antibody injection treatments, merely treats symptoms and fails to address the root causes of diseases.

A therapeutic hexapeptide, risuteganib (RSG), designed by Allegro Ophthalmics, LLC., has shown efficacy in treating both angiogenic and inflammatory retinopathies such as wet- and dry-AMD and diabetic macular edema (DME), a progressive form of DR. In the past, many studies sought to identify integrins that demonstrated specific binding affinities to RSG, based on the structural similarity of RSG to the well-known RGD motif. In contrast, Caltech decided to pursue unbiased research directions to unveil the mechanism of action (MOA) of RSG. Following studies from Caltech, UC Irvine, Johns Hopkins University, and Duke University revealed unanticipated features of RSG, suggesting that it has both anti-angiogenic and anti-inflammatory effects and that it can rescue compromised mitochondrial functions in cells under chemically induced oxidative stress. In this thesis, we conducted a series of investigations, progressing from the tissue level to the cellular level and then to the molecular level, to test a hypothesis that may unify these observations.

In Chapter 2, we developed a peptide-directed fluorescent staining method to identify the binding location of RSG-dye conjugate using aged BALB/c to recapitulate features of age-related retinal diseases. We identified crucial parameters that visualized replicable, RSG-specific labeling at the aged RPE layer. Findings from Chapter 2 and past studies laid the groundwork for the multi-layered investigations on the MOA of RSG in subsequent chapters, hinting at the cell type of interest, the need for appropriate stress to tissue or cell, and the importance of limiting RSG probes below clinical dosage. In collaboration with the Kenney lab at UC Irvine, we tested the protective effects of RSG in differentiated ARPE-19 cell model in response to chemically activated hypoxia-inducible factor 1 (HIF-1) signaling pathway (Chapter 3). Sub-clinical dosage of RSG (30 µM) demonstrated protective effects against chemically elevated HIF-1α leading to cell death and compromised mitochondrial membrane integrity. Further, RSG-dye conjugate localized in the mitochondria of differentiated ARPE-19 cells, where partial co-localization with mitochondrial protein, pyruvate dehydrogenase E1 α subunit (PDHA1), was observed. In Chapter 4, we developed a luminescence-based in vitro assay to quantify the effect of RSG on the activity of a mitochondrial kinase, pyruvate dehydrogenase kinase 1 (PDHK1). PDHK1 interacts with pyruvate dehydrogenase complex (PDC) which contains PDHA1. PDHK/PDC interaction governs the pyruvate decarboxylation process, thereby serving as a dynamic metabolism switch. Upregulation of PDHK1 decreases PDC activity, which subsequently increases glycolytic flux as an adaptive response against hypoxia, temporarily alleviating oxygen deficiency at the cost of total ATP production per mol glucose. When such response is prolonged in the RPE cells, energy deficiency and compromised homeostasis lead to cell death of not only RPE cells but other crucial retinal cells including the photoreceptor cells. Here, tested RSG concentrations (3.125, 6.25, and 12.5 µM) did not demonstrate significant inhibition of PDHK1 activity; nevertheless, the devised in vitro assay laid the foundation for a reliable, quantitative assessment of RSG or other inhibitors’ effects on the activity of PDHK1 and its isoforms. Chapter 5 summarized the findings in the previous chapters and suggested future studies that might aid in the continued search for the mechanism of action of RSG.

Thesis Files