Targeting the Undruggable Proteome – one probe at a time

by: Brandon Chan

Recent advances in utilizing chemoproteomic platforms for drug discovery poses an enormous potential for investigating new novel therapeutics. This greatly expands the scope of discovering promising treatments for diseases, particularly in the realm of targeting proteins for degradation. Much interest has been devoted to understanding this strategy, as the hallmark of many neurodegenerative diseases includes the accumulation of misfolded proteins and protein aggregates.

By targeting protein targets for degradation, protein clearance could mean the reduction in incidence, susceptibility or even a cure for a disease. Such targeted tagging of proteins for degradation could also be implicated in anticancer treatments where selective recruitment of cancer-associated proteins could be eliminated from cells. The overall strategy of this new drug discovery relies on a high throughput screening of chemical ligands that could bind against known E3 ubiquitin protein ligases (an integral component of the normal cell machinery to degrade misfolded and abnormal proteins from the system), also known as activity-based protein profiling (ABPP). Therefore, by fine tuning the specificity of E3 ligases to target proteins, an entire complex of disease proteins could be subsequently cleared and eliminate a diseased state.        

Activity-based protein profiling (ABPP) is a technique by which a small molecule chemical ligand could be used to interact with target proteins and probe potential protein interactions. A small probe is designed with three distinct regions: a chemically reactive group, a linker region, and a reporter group. The reactive group usually consists of an electrophilic (electron accepting) functional group such as an epoxide or disulfide which are able to react with common sites of modulation in proteins. The linker region attaches the reactive group to the reporter group, often diminishing the chances of cross-reactivity between the reporter and reactive groups. It typically consists of a chain of repeating carbon atoms bound to hydrogens or polyethylene glycol repeating units. Lastly, the reporter group is usually a fluorescent molecule, biotin or alkyne and azide functional groups, to trace reactivity with the target.

This probe is subsequently incubated with cell lysate, proteins or tissue samples to react with potential hit targets. Identification of target proteins is accomplished through gel electrophoresis (protein separation by size on an agarose gel matrix) followed by fluorescence imaging or by Western blotting (antibody-specific detection of fluorescence reporter). 

Traditional methods of drug discovery relies on binding pockets of target proteins for ligand binding. Drugs typically act as direct modulators at these binding pockets to disrupt or inhibit protein function. With advancements in chemoproteomic strategies, a new era of drug discovery utilizing targeted protein degradation presents an exciting frontier for medicine.

No longer are binding pockets necessary for new therapeutics. Instead, druggable hotspots on proteins could be identified with broad chemical screenings with a covalent ligand small molecule. These small molecules associate with E3 ligases (protein degradation machinery) which then recognizes protein targets and degrades them. Therapeutics harnessing this protein degradation pathway in cells therefore offers specificity in tagging proteins and helps tackle the current “undruggable proteome.”        

Previous studies have identified small molecule interactors for E3 ligases such as MDM2 and UBE3A, however a vast majority of E3 ubiquitin ligases remain largely unexplored. In this study, Nomura’s research group screened a cysteine-reactive covalent ligand library against IA-Rhodamine labelling of pure human RNF4, a known E3 ligase. Following gel-based ABPP, TRH 1-23, a covalent ligand showed promising interactions with RNF4. This interacting ligand was further optimized. It was found that an analogue of TRH 1-23, CCW 16, exhibited significant inhibition of IA-labelling of RNF4 even at the 1 uM levels, possibly indicating strong ligand association to the RNF4 target. A covalent docking model was performed showing that CCW 16 occupies a groove within RNF4 outside of zinc coordinating sites.   

To demonstrate the recruitment activity of CCW 16 for RNF4, a ligand analogue, CCW 28-3 was synthesized. This ligand combines and links together CCW 16 with JQ1 (a BET bromodomain family inhibitor) which is an inhibitor that binds to BRD4 and prevents enhancer mediated gene transcription. When introduced to 231MFP breast cancer cells, BRD4 degradation was observed within one hour in a dose-dependent manner. Inactivation or or downregulation of transcriptional regulator BRD4 has implications of inhibiting or slowing cancer progression.    

To establish a role for RNF4 and its link on to CCW 28-3 mediated degradation of BRD4, further experiments were performed. It was found that BRD4 degradation which was present in wildtype HeLa cells was absent in RNF4 knockout (KO) HeLa cells, supporting that RNF4 presence is necessary.  

This strategy to find new chemical entry points for lead compounds as drugs could target many proteins in the human body that once would not have been possible. Future aims stemming from this study according to the paper would be “to optimize the potency, selectivity, and cell permeability of CCW 16 for targeting RNF4 and to improve linker positioning and composition of CCW 28-3 to promote better degradation of protein substrates.” Overall, targeted protein degradation opens new doors to combating human diseases, a revolutionary platform to tap into reservoirs of novel cures and treatments.   

Image credit: CSIRO

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