Limited Proteolysis (LiP) as Drug Target Deconvolution Strategy

The functionality of a protein is determined by its structure. Understanding how a protein changes its structure upon interaction with other proteins, drugs, small ligands or chemical derivatization is essential for an understanding of biological processes. Many different techniques have been developed for characterization of protein structure and conformational changes. However, most of them are only applicable to isolated, purified proteins or require labeling either of the protein or the small molecule and are only suitable for small molecules with high target-affinity (1, 2).   

Limited Proteolysis (LiP) has emerged as a label-free technology to identify and analyze changes in protein structures in un-purified lysates.  The underlying concept of LiP is based on proteins being subjected to proteolysis in the presence or absence of a compound. When a protein binds to a compound, it often undergoes a conformational change, and/or the binding site becomes inaccessible for proteolysis. Both effects change the pattern of proteolysis and lead to the formation of different peptides that can be characterized by mass spectrometry.

 

Limited Proteolysis Coupled with Mass Spectrometry (LiP-MS) Detects Protein Conformational Changes Induced by Compound Binding

The laboratory of Prof. Dr. Paola Picotti at the ETH in Zurich has developed a unique LiP protocol coupled to mass spectrometry (LiP-MS), which is able to address protein conformational changes induced by interaction with compounds for proteome-wide applications (3, 4, 5).

This LiP-MS technology is based on double protease digestion. The first protease step under native conditions generates site-specific cleavages in the protein that result in “conformotypic” peptides so-called because they are specific to the protein’s conformation at the time of cleavage. The second protease under denaturing conditions results in complete protein digestion and allows peptide identification and quantification via bottom-up proteomics.


LiP-MS for Target Deconvolution in More Complex Cellular Environments

This technology has shown promising results in yeast and bacteria in a large-scale proteome analysis. Biognosys has expanded upon this research for the analysis of more complex organisms, such as humans. As an organism’s proteome becomes more complex the number of potential target proteins that can interact with compound increases and leads to more “noise”. To overcome this analytical challenge Biognosys has introduced a dose-response approach: By demonstrating a correlation between protein conformational changes and the concentration of compound we have been able to increase the signal to noise ratio. This enables the reliable prediction of protein-compound interaction even in complex proteomic systems. Significant peptides are then identified via a positive correlation to a dose-response and high q-values. This is shown exemplarily in figure 1.

 




Detecting Site-specific Target-protein Interactions Through LiP-MS

Tacrolimus is a drug with immunosuppressive properties that has been used to decrease the risk of rejection of transplanted organs. Tacrolimus interacts with FKBP12 to induce T-cell inactivation (6). Using our LiP-MS technology we have compared FKBP12 protein lysate treated with tacrolimus or vehicle. With LiP-MS we have been able to identify conformotypic peptides induced by the interaction of tacrolimus with its target protein FKBP12. Applying the knowledge of the three-dimensional structure of the protein allows us to even specify the localization of the protein-drug interaction (manuscript in preparation).


 




LiP Can Be Deployed in a Variety of Biological Settings

To date, LiP-MS has been used to confirm or identify proteins interactions with 16 different molecules, experiments included:

 

  • Recombinant proteins, human cell lines, fungal cells, and insect cells;
  • Proteins that localize to the cytosol;
  • Protein classes including kinases, phosphatases, other enzymes;
  • Relative IC50 calculation of the target proteins.


 

References:

  1. Protein structure determination by x-ray crystallography. Ilari et al., 2008 Methods. Mol. Biol.
  2. Label-free technologies for target identification and validation. Li et al., 2016 Med. Chem. Commun.
  3. Probing protein structure by limited proteolysis. Fontana et al., 2004 Acta Biochim Pol.
  4. Global analysis of protein structural changes in complex proteomes. Feng et al., 2014 Nat Biotech.
  5. A Map of Protein-Metabolite Interactions Reveals Principles of Chemical Communication. Piazza et al., 2018 Cell.
  6. FKBP12 is the only FK506 binding protein mediating T-cell inhibition by immunosuppressant FK506, Xu, et.al-, 2002, Transplantation.


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