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New star in protein targeted degradation technology-LYTAC

2021-07-22
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Targeted protein degradation technology (Proteolysis TArgeting Chimeras, PROTAC) is an emerging hot drug development area following small molecule inhibitors (SMI) and monoclonal antibody (mAb) drugs. Compared with the latter two, PROTAC has unique advantages, being able to target numerous protein molecules that were previously considered “undruggable”. Hundreds of PROTAC molecules have been or are being developed so far, and many emerging startup companies have also emerged.

However, this PROTACs protein degradation technology also has its own limitations: in addition to the large molecule and poor PK, it uses the ubiquitin degradation system and can only target intracellular proteins. However, many potential drug targets are usually membrane proteins and secreted proteins. Membrane proteins and secreted proteins account for 40% of the total proteins, and extracellular proteins such as growth factors and cytokines can bind to receptors on the cell surface to trigger abnormal signal transduction in many diseases.

So, is there a technology that targets the degradation of extracellular proteins related to diseases?

Recently, a research team from Professor Bertozzi from Stanford University published a research paper in Nature entitled: Lysosome-targeting chimaeras for degradation of extracellular proteins [1]. In this study, a known agonist of the lysosomal targeting receptor (LTR) on the cell membrane is linked with antibodies or small molecule ligands to form LYTAC. LYTAC can endocytose the bound target protein into the lysosome for degradation. This technology can successfully degrade secreted proteins and membrane proteins, including EGFR, CD71, PD-L1 and so on.

 Picture source: Nature
Picture source: Nature

The choice of lysosomal targeting receptor (LTR) is the non-cation-dependent mannose-6-phosphate receptor (CI-M6PR). The receptor-ligand complex formed by the combination of lysosomal enzyme and M6PR is transported to the prolysosome. In the low pH environment, the receptor and the ligand are separated, and the lysosomal enzyme is transported to the lysosome. M6PR circulates back to the Golgi complex or plasma membrane.

Therefore, Cl-M6PR has been used for the delivery of enzyme replacement therapy for lysosomal storage disorders. The study links the known agonist of the lysosomal targeted receptor (LTR) on the cell membrane with antibodies or small molecule ligands to form LYTAC, which can be combined with LTR to internalize secreted and membrane proteins into the lysosome for degradation.

Studies have found that LYTACs can be used as biochemical probes to study receptor transport and protein degradation, and can mediate the degradation of secreted proteins and membrane proteins.

In order to develop a ligand suitable for CI-M6PR, it is studied to obtain the M6Pn glycopeptide through chemical synthesis and achieve the best CI-M6PR agonistic effect. To demonstrate the feasibility of CI-M6PR-driven LYTAC, the study designed an intracellular assay to measure the uptake of biotin-neutravidin (NA-647). The data shows that LYTACs mediate the uptake of NA-647 in different cell lines, proving the breadth of CI-M6PR targeting and the accuracy with which small molecules are responsible for targeting.

Next, study the high-throughput genetic screening technology based on CRISPR interference (CRISPRi) to identify the main target genes. Fluorescence-activated cell sorting (FACS) was used to isolate a cell population showing a significant reduction in NA-647 markers, and next-generation sequencing was performed to identify the over-expressed sgRNA in this population. The data indicate that the surface presentation of CI-M6PR is partially mediated by extracapsular complexes, and indicates that LYTAC can be used to study molecular pathways that regulate cell surface receptors.

The study also found that LYTAC-mediated target enrichment in lysosomes is related to target degradation. More broadly, conjugating a lysosomal targeting ligand to an antibody can reprogram the antibody to direct the degradation of extracellular antigens.

Can LYTAC be used to accelerate the degradation of extracellular membrane-bound proteins?

In principle, this requires the binding of surface-related proteins and CI-M6PR at the same time. The research first focused on EGFR (a known driver of cancer proliferation). LYTAC uses a blocker of EGFR that binds to M6Pn (ctx-M6Pn). The study observed substantial degradation of EGFR, but no changes in CI-M6PR levels were observed.

In addition, studies have shown that LYTAC, which has dual affinity for the target and CI-M6PR, can maintain high target specificity. EGFR degradation has also been observed in breast cancer and hepatocellular carcinoma cell lines.

Next, the study uses quantitative mass spectrometry to characterize the specificity of LYTAC degradation in the cell proteomic range. Although the exact mechanism of these changes remains to be elucidated, these data suggest that LYTAC may provide a means to monitor the cell’s response to the degradation of the target protein.

In order to understand the breadth of LYTAC’s effects, the study expanded the goal to several other membrane-associated proteins. For example, a therapeutic cancer target that is being developed into clinical trials-transferrin receptor 1 (CD71), which can circulate between the early endosome and the cell surface to avoid being transported to the lysosome for degradation. The experiment observed that CD71 under the action of LYTAC was degraded due to the recognition of M6Pn.

In addition, the study tested the degradation of PD-L1 (the driving force for cancer cell immune escape) by LYTAC. In order to accelerate the degradation of PD-L1, LYTAC must overcome the recycling pathway of PD-L1. Studies have found that LYTAC can surpass the endogenous circulation program of cell surface proteins and accelerate their lysosome degradation.

How does the chemically synthesized M6Pn ligand affect the antibody clearance rate in the body? The researchers injected ctx and ctx-M6Pn intraperitoneally into BALB/c mice, and measured the serum antibody level by Western blotting. The data indicates two types of removal: fast initial removal and slower, more permanent removal. Adjusting these two regimens is essential to adjust the in vivo efficacy of any individual combination of LYTAC and target protein.

In short, LYTAC can directly target extracellular and membrane-associated proteins for lysosomal degradation. The successful targeting of LYTAC is caused by a variety of factors, including the kinetics of endogenous protein transport and conversion, the number of lysosome surface localizations, and the lysozyme through clathrin-mediated endocytosis. Body sensitivity and stoichiometry associated with lysosomal targeting receptors.

Although LYTAC only used CI-M6PR in the experiment, it is not limited to this kind of LTR. By choosing other LTRs, it can reduce the possibility of drug resistance when targeting oncoproteins. Research has also shown that both small molecules and peptides can be used as protein-targeting binding agents in LYTAC. Adjusting the pharmacokinetic properties to control the targeted clearance and stoichiometry of LYTAC is still a key challenge for further applications for the effective degradation of membrane proteins. In the end, the study also proposed that through the chemical regulation and modularization of LYTAC, it will be able to better target the degradation of secreted proteins and membrane proteins, which is of great significance both in research and in potential treatment.

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