Targeted Protein Degradation
Targeted Protein Degradation (TPD) is a new and exciting therapeutic modality. The ability to tag a protein of interest (POI) for rapid destruction is an important tool in the medicinal chemistry arsenal to address human disease. The development of TPD molecules is empirical and requires many different components to be optimized to achieve acceptable activity and properties. Our expertise in BioNMR, small molecule discovery, and ADC payload-linkers are all important assets in the optimization of TPD molecules from concept to development. The utility of BioNMR in TPD discovery was showcased by the development of Nutlins (MDM2 inhibitors) by our lead NMR scientist.1 We can accelerate and add value to your TPD program with our synthetic chemistry, linker design, BioNMR, bioconjugation and functional assay capabilities. Our services can be a la carte or all-inclusive.
Contact us to see how we can accelerate your targeted protein degradation programs.
Mechanism of Targeted Protein Degradation
Targeted Protein Degradation
Cells maintain protein homeostasis by regulating protein levels and destroying aberrant proteins by tagging them for destruction. Many diseases involve overexpression of wildtype or mutant proteins which drive the disease phenotype. Inhibiting or removing these pathways can restore normal function. In the past 10 years, there has been a growing interest in utilizing the degradation pathway to remove disease-causing proteins and this approach spans many therapeutic areas such as oncology, immunology and neurology. For example, a protein that is essential for cancer cell replication may be identified and a ligand developed that may have good binding but may not be effective enough at modulating the function of the protein to reverse the disease state. The addition of an appropriate linker and an effective E3 ligase recruiter to the inhibitor can organize the degradation tagging complex and consequently lead to the destruction of the protein in the proteasome. Once the protein is degraded, the TPD molecule is released and can undergo the cycle again, making the activity catalytic. In effect, the addition of E3 recruiters has increased the effectiveness of known small molecule inhibitors. While this may be conceptually simple, in practice finding the right linker that is flexible enough to allow the E3 complex and the POI to organize properly for tagging but rigid enough to help facilitate degradation is a delicate and difficult balance. Not all E3 ligase recruiters are optimal for all proteins and finding an appropriate linker, protein binder and E3 recruiter combination is not predictable a priori and must be found through rounds of empirical optimization. An active combination of all three components can result in rapid degradation of the POI and lead to new therapies for unmet medical needs.
NJ Bio’s Targeted Protein Degradation Design Process
NJ Bio has a variety of tools, expertise and skills at its disposal to accelerate TPD discovery.
BioNMR of Targeted Protein Degraders
Challenges in the design of protein degraders include optimizing the type of E3 ligase recruiter, the length and rigidity of the linker, and the POI binder. The POI binder must allow attachment of a bifunctional linker. The linker must adopt a conformation that facilitates the POI and the E3 ligase to be arranged in such a manner as to allow ubiquitination. All of this occurs in solution at physiological temperature, pH and buffer that is not reproducible by X-ray crystallography. BioNMR is the gold standard for solution phase elucidation of proteins. The utility of BioNMR was showcased by the development of MDM2 inhibitors (Nutlins) which then became E3 recruiters in TPD molecules.1 Expansion of BioNMR into TPD could yield important new TPD molecules. By using labeling techniques, we can observe which amino acids are involved in binding and use this information to help guide projects. Proteins and/or protein degraders can be labeled with non-radioactive nuclei such as deuterium, 15N and 13C. The usefulness of BioNMR is that the structure activity relationship determination is performed at physiological conditions and temperature.
At NJ Bio, we offer both sophisticated BioNMR services and standard NMR techniques that can help determine the natural conformation of the TPD at physiological conditions. TPDs usually include flexible linkers whose mobilities can make the active conformations less energetically attractive. Our advanced NMR techniques can elucidate the lowest-energy conformations to help guide linker design.
More on our BioNMR Capabilities
Targeted Protein Degrader Optimization
TPD molecules require optimization of the POI binder, the E3 ligase recruiter, and a tethering linker to bring the POI and E3 ligase proteins together optimally for efficient ubiquitination. Our experience with linker design for ADCs and other bioconjugates applies well to linker design of TPD molecules. Many of the same considerations apply, such as having an appropriate length, hydrophilicity, and rigidity to maximize the pharmacological effect. Like bioconjugate therapeutics, there are no rules or guidelines that can predict favorable constructs. These constructs are identified by a combination of empirical data, experience, an understanding of how molecules bind together, and a robust knowledge of protein-protein interactions.
At NJ Bio, our synthetic ability and experience in all types of linkers allows us to rapidly install a linkable group to a molecule without changing its binding properties. Incorporating linkers with different length, hydrophilicity and rigidity can be synthetically challenging. The breadth of our linker chemistry experience helps us accelerate programs by rapidly generating compounds for testing. Our approach is flexible and lets the data guide the path forward.
Targeted Protein Degrader Bioconjugates
A core strength at NJ Bio is our vast knowledge of linking molecules to small peptides, antibodies, or other targeting molecules. Our understanding of ADCs makes us unique in the ability to design TPD molecules for bioconjugation. Many TPD molecules bind and degrade their targets rapidly but do not have suitable properties in terms of membrane permeability, pharmacokinetics or non-specific toxicity. Attaching these powerful therapeutics to antibodies or other targeting moieties has led to increases in therapeutic index for chemotherapeutics.2 Experience in bioconjugation chemistry is key to designing a linker position on the protein degrader that can release the TPD molecule in the targeted cells selectively.
At NJ Bio, we understand the effects of hydrophilicity and conjugation technology and their impact on efficacy and toxicity.3 The fields of bioconjugation and TPDs can complement each other and help advance new therapeutics.
Our biological and bioconjugation teams can perform biochemical and whole-cell assays to determine inhibition, cell killing, and other important properties. We have performed DC50 assays to measure protein degradation kinetics specifically for degrader molecules. We perform assays in-house, which speeds up discovery programs. Our bioanalytical team is experienced in PK sample analysis by mass-spectrometry and/or ELISA to measure preclinical in vivo properties of TPD molecules.
More on our Functional Assays
Protein degraders are large molecules with higher molecular weights and longer syntheses than most traditional small molecule therapeutics. Having a scalable and robust route can be challenging with long synthetic sequences. Our familiarity, due to our work with ADC payload-linkers, with lengthy syntheses and scale-ups gives us the ability to perform multistep syntheses rapidly at a suitable scale to support pre-clinical packages. These syntheses are most often not linear and selecting the best time to assemble all the pieces together in the right order is critical for rapid scale-up and robustness.
At NJ Bio, we have incorporated both batch and flow chemistry for development processes, enabling us to have the widest range of tools available to tackle synthetic challenges. We allow the chemistry to dictate the technology used in the scale-up, which speeds up production and lowers risk.
(1) Fry, D. C.; Emerson, S. D.; Palme, S.; Vu, B. T.; Liu, C.-M.; Podlaski, F. NMR Structure of a Complex between MDM2 and a Small Molecule Inhibitor. Journal of Biomolecular NMR 2004, 30 (2), 163–173. https://doi.org/10.1023/B:JNMR.0000048856.84603.9b
(2) Pillow, T. H.; Adhikari, P.; Blake, R. A.; Chen, J.; del Rosario, G.; Deshmukh, G.; Figueroa, I.; Gascoigne, K. E.; Kamath, A. v.; Kaufman, S.; Kleinheinz, T.; Kozak, K. R.; Latifi, B.; Leipold, D. D.; Sing Li, C.; Li, R.; Mulvihill, M. M.; O’Donohue, A.; Rowntree, R. K.; Sadowsky, J. D.; Wai, J.; Wang, X.; Wu, C.; Xu, Z.; Yao, H.; Yu, S. F.; Zhang, D.; Zang, R.; Zhang, H.; Zhou, H.; Zhu, X.; Dragovich, P. S. Antibody Conjugation of a Chimeric BET Degrader Enables in Vivo Activity. ChemMedChem 2020, 15 (1), 17–25. https://doi.org/10.1002/cmdc.201900497
(3) Maneiro, M.; Forte, N.; Shchepinova, M. M.; Kounde, C. S.; Chudasama, V.; Baker, J. R.; Tate, E. W. Antibody–PROTAC Conjugates Enable HER2-Dependent Targeted Protein Degradation of BRD4. ACS Chemical Biology 2020, 15 (6), 1306–1312. https://doi.org/10.1021/acschembio.0c00285