Repurposing AMP-activated Protein Kinase with Bifunctional Small Molecules to Phosphorylate Non-native Substrates

Abstract

Nature uses induced proximity to carry out many cellular functions. Inspired by native processes, we can target desired biological processes using heterobifunctional small molecules to induce proximity, regardless of whether the substrate is native or a non-native substrate. For example, bifunctional small molecules have been designed to trigger ubiquitin-dependent proteasomal degradation of intracellular proteins. Since the discovery of proteolysis-targeting chimera (PROTACs) 20 years ago, the field of bifunctional small molecules to induce proximity has expanded very rapidly. In the first chapter of this thesis, I review the latest bifunctional modalities to induce degradation other than PROTACs, as well as bifunctional molecules that add or remove post-translational modifications (PTMs). For example, heterobifunctional modalities can change protein phosphorylation and glycosylation states to synthetically alter the biophysical properties of target proteins. I also describe some general design principles on how to create this innovative class of molecules, and review some of the techniques and assays to characterize and develop them.

In Chapter 2, I describe my foundational work that led to the development of the first phosphorylation-inducing chimeric small molecules (PHICS) from our lab. Small molecule protein modulators have conventionally been developed to inhibit enzyme activity, generally as active site competitors; however, new classes of small molecules that bestow new functions to enzymes via proximity-mediated reactivity are emerging. Native or neophosphorylation (phosphorylation not known to nature) of any given protein of interest (POI) can alter its structure and function, and we hypothesized that such modifications can be accomplished by small molecules that bring a kinase in proximity to the POI. PHICS enables kinases—for example, AMP-activated protein kinase (AMPK) or protein kinase C (PKC)– to phosphorylate target proteins that are not otherwise substrates of these kinases. PHICS are formed by conjugating binders of the kinase and the target protein, and exhibit several features of bifunctional molecules that were described in Chapter 1, including the hook effect, turnover, isoform specificity, dose and temporal control of phosphorylation, and proximity-dependent activity (i.e., linker length). Using PHICS with a rapid click chemistry conjugation platform, we were able to induce native and neo-phosphorylations of recombinant BRD4 by AMPK or PKC to demonstrate the proof of concept. Furthermore, PHICS induced a signaling-relevant phosphorylation of the target protein Bruton’s tyrosine kinase in cells.

In Chapter 3, I describe significant technological improvements in the PHICS platform, specifically with respect to AMPK-targeting molecules. Prior work was unable to demonstrate induced phosphorylation in endogenous target proteins, due to the limitations of the AMPK binder used for the first AMPK-PHICS which limited the ability to study biological repercussions of targeted protein phosphorylation. Furthermore, we could not induce phosphorylation without serum starvation conditions because the first AMPK binder we used was not sufficient to activate AMPK on its own. As AMPK is a master regulator of energy in the cell, inducing serum starvation in cells is a method of AMPK activation. Unfortunately, this requirement would significantly limit the therapeutic applications of AMPK-PHICS, thus I show that improved AMPK-PHICS can phosphorylate an endogenous protein target and induce phosphorylation without need for serum starvation. In addition, I show that this new generation of AMPK-PHICS are more cytotoxic to ibrutinib-resistant cells than the respective PROTAC and studied a potential mechanism of action. Ibrutinib is an FDA-approved inhibitor of Bruton’s tyrosine kinase (BTK), a driver of B-cell malignancies. Unfortunately, some cancer patients will develop resistance to ibrutinib, thus other therapeutic approaches must be developed to combat drug resistance in cancer. PROTACs have been explored as a method to address clinical limitations of conventional inhibitors.

Overall, this dissertation provides an overview of bifunctional molecules as a whole and their potential to transform human medicine through modulating the proteome in ways that were previously never explored, such as targeted induction of PTMs. The culmination of the work summarized herein lays the groundwork for the first phosphorylation-inducing chimeric small molecules and shows major technological platform improvements to expand the potential of this new modality, both as chemical probes and as next-generation therapeutics in the future.