Student Theses and Dissertations

Date of Award

2021

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Hang Laboratory

Abstract

The identification and functional characterization of specific metabolite–protein interactions remains a major challenge in chemical biology and drug development. The functionalization of native metabolites and synthetic small molecules with bio-orthogonal detection tags (alkyne, azide, and others) has afforded chemical reporters for the biochemical analysis of metabolically labelled proteins, whereas functionalization with photo-crosslinkers enables non-covalent metabolite–protein interactions to be captured. While we and many others have employed this approach to different metabolites and synthetic ligands, the installation of bio-orthogonal detection tags and photo-crosslinkers to more complex and diverse small molecules can be challenging and limiting. In this regard, activity-based protein profiling (ABPP) using active site–directed probes in combination with small molecule competition studies allows the discovery of potential substrates and inhibitors of selective enzyme families. Moreover, the development of proteome-wide labelling of select amino acid (cysteine and lysine) has revealed new sites in proteins for function modulation. However, some key protein classes and specific ligand-binding domains have been inaccessible by these methods. New activity-based probes and targeted chemical reporters of specific protein families are thus needed to characterize their endogenous and exogenous ligands for fundamental biology and drug discovery. In this thesis, we describe two projects aimed at developing chemical probes to assist in small molecule target engagement and target deconvolution studies. In Chapter 1, we provide an overview of the origin and evolution of ABPP and related chemical reporter–based chemical proteomics methods. We also summarise recent developments in label-free methods that work with underivatized molecules. In Chapter 2, we describe the development of a ligand-directed probe (alk-GW9662) for a subset of nuclear receptors (NRs). NRs are a family of ligand-activated transcription factors that regulate diverse physiological processes in animals and are key targets for therapeutic development. Recent studies implicated NRs in host–microbiota interactions and suggested that various microbiota-derived metabolites may serve as ligands for NRs. We demonstrate alk-GW9662’s utility to assess target engagement of candidate ligands of peroxisome proliferator–activated receptor (PPAR) γ. We also explore repurposing alk-GW9662. We profile its target proteins using chemical proteomics and demonstrate that the probe can be used in target engagement study of other proteins. Overall, our results suggest that alk-GW9662 may be useful for target engagement analysis of candidate PPAR ligands and offer a starting scaffold in developing probes for other proteins. In Chapter 3, we describe efforts towards the combined use of proteolysis-targeting chimeras (PROTACs) and chemical proteomics in target deconvolution studies. We describe the design and synthesis of PROTACs that recruit putative E3 ligases. Overall, the projects described herein underline the utility of chemical tools such as ligand-directed probes and PROTACs in target engagement and target deconvolution studies.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfilment of the Requirements for the degree of Doctor of Philosophy

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