Protein levels within a cell are not fixed and invariant. Rather, they represent the net result of two continuous and dynamic operations – protein synthesis and protein degradation. These two processes jointly determine a protein’s turnover rate, or the rate at which older proteins are replaced by newly synthesized ones. When the synthesis and degradation processes are balanced, protein levels remain stable over time. However, the two processes can also be independently modulated, enabling cells to response readily to internal or external stimuli by precisely altering levels of a relevant protein or proteins. To evaluate protein turnover, researchers rely on specialized techniques that typically involve labeling a subset of proteins and tracking their abundance over time. The kinetic profiles generated by these experiments provide critical information that can be used to guide drug discovery and development choices, including selection of an optimal target or modality, mode-of-action analysis, and evaluation of drug efficacy and safety.

In the canonical framework, DNA is transcribed into mRNA, which is then translated into proteins that serve their cellular function(s) before eventually being degraded through any of several regulated pathways. To this end, protein turnover or stability varies substantially across the proteome. At one extreme, lens crystallins in the human eye are synthesized only during development and persist throughout the life course without being subjected to turnover. At the other extreme, researchers have identified a number of human proteins with half-lives as short as 20 minutes. Protein degradation serves as a regulatory and quality control mechanism, and a number of cellular systems (most prominently, the ubiquitin-proteasome pathway and the lysosomal proteolysis pathway) target damaged or misfolded proteins for degradation. In fact, it is these same mechanisms that are leveraged by techniques such as PROTAC to enable targeted degradation of a protein of interest.

While ‘snapshot’ measurements can reveal levels of a given protein at a particular time, a more sophisticated approach is needed to interrogate the kinetics of protein degradation and resynthesis. These techniques typically leverage metabolic labeling, in which cells or organisms are provided with amino acids or other molecules that have been labeled, as with a radioactive or heavy stable isotope.  These analogs are incorporated in vivo into proteins or other biomolecules, after which they can be detected and traced throughout their lifespan. Performed under normal conditions, these assays provide crucial information about the dynamics of protein turnover in cells, tissues, and organisms. More elaborate experiments compare these baseline profiles to those following treatment with a drug or similar agent, providing valuable insights into the on- and off-targets of a particular drug, as well as the protein turnover changes induced by a drug or degrader. These approaches are even more powerful when applied on the proteomic scale to comprehensively evaluate the multifarious effects of a given stimulus or perturbation. In particular, simultaneous proteome-wide tracking of protein synthesis and degradation kinetics can be achieved by utilizing a combination of SILAC (Stable Isotope Labeling by Amino acids in Cell culture) and TMT (Tandem Mass Tag) labeling strategies followed by quantitative mass spectrometry-based analysis, commercially available as TurnoverScout.

Protein turnover data can be an invaluable resource to guide many stages of the drug discovery and development process, including the choice of therapeutic modality. Because newly synthesized proteins continually replace those that have been bound by a covalent inhibitor, effective covalent inhibition requires a slow re-synthesis rate relative to the inhibitor’s residence time. Similar constraints apply to the development of targeted protein degraders, such as PROTACs. Because these therapeutics act to degrade mature proteins, they are most effective against targets with slow rates of resynthesis, such that lower or less frequent dosing is sufficient to maintain target reduction. Alternatively, modalities that target RNA and the splicing process – such as small molecule splice modulators, explored in a previous blog post – are attractive therapeutic options for rapidly resynthesized targets. Characterization of target re-synthesis profiles can guide decisions about small-molecule dosing regimens and reveal whether target synthesis is up- or down-regulated by drug treatment, while knowledge of the re-synthesis rates of off-targets can further predict the likelihood of sustained side-effects in a clinical context. More broadly, analysis of protein turnover dynamics can be used to identify key mediators of biological responses, determine the regulatory mechanisms that govern signaling pathways, and characterize cellular response to injury.

In the context of drug discovery and development, it is important to expand beyond the static capture of protein levels to appreciate the broader context of dynamic synthesis and degradation processes. By using metabolic labeling techniques, researchers can better understand the kinetics underlying both homeostatic and stimulus-response conditions. These techniques are even more powerful when applied on the ‘omic’ scale, moving beyond a single target protein to comprehensively evaluate dynamics across the proteome. Today’s researchers can take advantage of existing commercial resources (such as the Protein Turnover Atlas) to access high-quality baseline turnover data that has already been generated, processed, and analyzed by expert scientists, or leverage services like TurnoverScout to evaluate turnover dynamics following stimulation, drug treatment, etc. These techniques can be applied to cell culture systems, as well as for the interrogation of in vivo dynamics in mice and other model organisms.  Ultimately, a comprehensive understanding of protein turnover dynamics is a critical component of any drug discovery and development program, enabling effective therapeutics to be developed quickly, efficiently, and safely.

To connect with our scientific team and explore how TurnoverScout, Protein Turnover Atlas, and our extensive catalog of screening and proteomic services can advance your research and development program, send us a message at http://momentum.bio/contact    

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