Catalytic Protein Inhibitors

Introduction to catalytic protein inhibitors

Catalytic Protein Inhibitors
For a recent review of this field, see: Kodadek, T. (2024) “Catalytic protein inhibitors” Angew. Chem. Intl. Ed., 62, e202316726

For some of the most difficult protein targets it

will be extremely difficult, if not impossible, to develop reversible inhibitors with sufficient affinity and residence time to serve as a viable drug candidate. One strategy to increase the potency of a protein antagonist beyond further increasing its affinity is to make it catalytic. In this scheme, the protein ligand (red circle) is linked to some type of warhead (gray hexagon) that mediates the covalent modification of the target (blue). The small molecule is then free to disengage and repeat the catalytic cycle. In theory, catalysis could provide a huge increase in the apparent potency of the inhibitor.


Photo-Oxidative Catalytic Inhibitors

Photo-Oxidative Catalytic Inhibitors

Our laboratory published the first demonstration that catalytic inhibitors exhibit far greater potency than the corresponding stoichiometric inhibitor. We attached a Ru(II)(bpy)32+ moiety to a modest affinity peptoid ligand for the VEGF Receptor. When irradiated with blue light, the Ru complex catalyzes the efficient generation of singlet oxygen, which is highly reactive with proteins but has a short diffusion radius. This results in inactivation of proteins in the immediate proximity of the compound but does not result in significant “collateral damage”. The peptoid-Ru conjugate was a 900-fold more potent inhibitor when irradiated with blue light than in the dark, highlighting the power of catalytic inhibition. See: Lee, J., Udugamasooriya, D.G., Lim, H.-S. and Kodadek, T. (2010) “Potent and selective photo-inactivation of proteins with peptoid-ruthenium conjugates” Nature Chem. Biol. 6, 258-260


Protein Degradation

While Ru-ligand conjugates are powerful tools for protein inactivation in cultured cells, the requirement for blue light irradiation, which does not penetrate tissues well, limits its applications in drug development.

Recently, another strategy for catalytic protein inhibition has been developed that is better suited for drug development. This involves the use of chimeric compounds called PROTACs (proteolysis-targeting chimeras) or molecular glues that force the proximity of a target protein and an E3 Ubiquitin ligase. In favorable cases, this results in poly-Ubiquitylation of the protein and its subsequent degradation by the proteasome.

Protein Degradation

Ubiquitin-Independent Degraders

Ubiquitin-Independent Degraders

One the major projects in the laboratory is to develop a new class of catalytic protein degraders that function by delivering the target protein directly to the proteasome, thus bypassing the need for Ubiquitylation. This would be extremely advantageous for oncology applications, since the currently “highjackable” E3 ligases employed for protein degradation are not essential proteins. Thus, cancer cells can evade existing degraders by simply downregulating the expression of the E3. The proteasome is essential to all cells.

We have established a model cell line in which Rpn13, one of the Ubiquitin receptors of the proteasome, is linked to the HaloTag protein. This allows chloroalkane conjugates of target protein ligands to be employed as Ub-independent degraders of native proteins. We are using this system to probe important questions surrounding Ubiquitin-independent degradation. Another major effort is the discovery of small molecules that engage, but do not inhibit, the native proteasome. These compounds, none of which currently exist, are a pre-requisite for the development of Ubiquitin-independent degraders as drugs. 

see:

Balzarini, et al. (2023) “Chemically Induced Degradation of Native Proteins by Direct Recruitment to the 26S Proteasome” BioRxiv 2023.07.19.549534.


Development of Self-Assembling PROTACs

Another major limitation of PROTACs is their high molecular weight and other poor drug-like properties. To address this problem, we are developing Self-Assembling PROTACs (SAPTACs) in which the E3 Ubiquitin ligase and target protein ligands are can come together in a reversible fashion. This should allow the much smaller pieces of the PROTAC to pass through the cell membrane more readily but still allow the on-target assembly of the active PROTAC inside the cell. As part of this effort, we are interested in the development of new types of reversible covalent chemistry.

Development of Self-Assembling PROTACs

See: Gui, W. and Kodadek, T. (2023) “Reversible assembly of proteolysis targeting chimeras” ACS Chem. Biol., 18, 1582-1593.


De Novo Discovery of Molecular Glues From DNA-Encoded Libraries

Protein degradation is only one of a myriad of potential post-translational modifications (PTMs) that one might wish to force using small molecule molecular glues. A major current effort in the laboratory is to leverage our one bead one compound DNA-encoded library platform for the de novo discovery of such glues using functional screens.

De Novo Discovery of Molecular Glues From DNA-Encoded Libraries

See: Koesema, E., Roy, A., Paciaroni, N.P., Coito, C., Tokmina-Roszyk, M. and Kodadek, T. (2022) “Synthesis and screening of a DNA-encoded library of non-peptidic macrocycles” Angew. Chem. Intl. Ed. 61, e202116999.