Application of chemical biology for the discovery of inducers of autophagy

Abstract

Human genetics have unveiled many coding variants in proteins that are implicated in health and disease. Many of these targets require novel methods of correcting the phenotypes caused by the mutations, in which the advances in chemical biology offer promising approaches. Phenotypic screens combined with target deconvolutions provide new chemical starting points for therapeutic discovery, while uncovering under-appreciated regulators of biological pathways of interests. Chemical-inducers of proximity, on the other hand, confers new functionalities to small molecule depending on the recruited proteins, allowing for precise methods to modify a protein of interest. Autophagy, a cellular recycling process important for homeostasis and cell maintenance, has been implicated in a wide range of diseases. This dissertation details our efforts in employing the new tools in chemical biology to discover chemical starting points for developing autophagy inducers. Chapter 1 provides an introduction to the process of autophagy, and our motivations for modulating this pathway. A literature survey of the available chemical modulators of autophagy highlights the need for more selective targeting of the autophagy machinery. Chapter 2 describes our efforts to identify autophagy inducers using phenotypic screens. Employing a high throughput microscopy screen, we monitored LC3 puncta formation and identified BRD3217 and BRD3808 as autophagy inducers. We characterized the biological profiles of BRD3217 and BRD3808, and demonstrated that the compounds enhanced the host cells’ ability to clear intracellular bacteria in an autophagy-dependent manner. We performed medicinal chemistry to improve the potency of the compounds, and conducted target identification using a bead-base pulldown method and a photo-crosslinking probe. Unfortunately, our efforts did not result in meaningful increases in compound potency, and we did not identify putative targets for BRD3217. Using a diversity-oriented synthesis inspired photo-stereoprobe library from the Cravatt lab, we identified DBK-032A as a stereoselective inducer of autophagy. Integrating structure-activity relationship studies and chemical proteomics, we demonstrated DBK-032A modulate autophagy by engaging the mitochondrial serine protease CLPP. Chapter 3 describes an approach to screen for Chemical inducers of proximity (CIPs) and its application in identifying an autophagy potentiator. CIPs are molecules that recruit one protein to another and introduce new functionalities toward modulating protein states and activities. While CIP-mediated recruitment of E3 ligases is widely exploited for the development of degraders, other therapeutic modalities remain underexplored. We describe a non-degrader CIP-DNA-Encoded Library (CIP-DEL) that recruits FKBP12 to target proteins using non-traditional acyclic structures, with an emphasis on introducing stereochemically-diverse and rigid connectors to attach the combinatorial library. We deployed this strategy to modulate ATG16L1 T300A, which confers genetic susceptibility to Crohn’s disease, and identified a compound that stabilizes the variant protein against Caspase-3 cleavage in a FKBP12-independent manner. We demonstrate in cellular models that this compound potentiates autophagy, reverses the xenophagy defects and increased cytokine secretion characteristic of ATG16L1 T300A. This study provides a platform to access unexplored chemical space for CIP design to develop therapeutic modalities guided by human genetics.