Harnessing the Intracellular Machinery to Degrade Cellular Proteins

Background

A countless number of proteins including growth factors receptors, and cytokines play a pivotal role during the onset and exacerbation of numerous human diseases including cancer, autoimmune and inflammatory disorders.¹

Of these proteins, extracellular and membrane-associated proteins represent around 40% of human proteome and they play diversified roles including signal transduction, cell adhesion, cell-cell interaction, and solute transport. Accordingly, the vast majority (ca. 60%) of current therapeutic approaches; such as small molecule inhibitors, oligonucleotide agents and monoclonal antibodies (mAbs), targets the membrane-associated proteins with the aim of altering the disease-associated downstream signalling pathway.² However, drug resistance, limited specificity and the inaccessibility of certain binding-pocket sites are the major limitations associated with the current therapies.

Targeted protein degradation: a new era of therapy

Cells dispose of an essential and precise machinery, called proteostasis, to control the correct protein function and expression at the right time and cellular location. Of course, erroneous protein misfolding occurs and it might lead to onset and progression of severe diseases. Once again, the proteostasis machinery intervenes to dispose misfolded protein via several cellular pathways responsible for protein degradation including ubiquitin-proteasome system (UPS) and autophagy.

Aware of the incredible therapeutic potential, during the last two decades, scientists have made tremendous effort to harness the natural cell disposal system and translate it into a sophisticated therapeutic platforms.

Targeted protein degradation (TPD) is a rapidly growing field of medicinal chemistry that has already revolutionized the drug discovery process and it is projected to positively impact the treatment of uncurable diseases. Pioneering examples of TPD includes a series of platforms, such as proteolysis-targeting chimaeras (PROTACs), molecular glues and small degraders, that allow for the rapid and irreversible depletion of specific disease causing proteins.

A typical PROTAC, among the most effective TPDs, includes a hetero -bifunctional constructs, connected via a suitable linker, where in one end a targeted molecule (peptide, antibody or small synthetic molecules) ligand binds to a protein of interest (POI) and at the other end a E3 ligase binder induces the ubiquitination and degradation of POI. By hijacking UPS, PROTACs has found successful application in diverse therapeutic areas including oncology, neuroscience, inflammation, dermatology, immunology, and respiratory diseases for the degradation of cellular metabolic enzymes, protein kinases, regulatory proteins, nuclear receptors, neurodegenerative related proteins.³

Degradation of protein target provides incredible advantages over traditional small molecule inhibitors. As such, a conventional inhibitor achieves its therapeutic goal via occupancy-driven mode herein impeding the receptor-ligand interaction and consequently preventing the downstream signalling cascade activation. However, given the complexity of the molecular pathways involved, cells frequently arise resistant mutations in response to prolonged small molecule inhibitors treatment.On the other hand, PROTACs, and in general any PTD platform, obtain a rapid and irreversible protein degradation at low dose, more sensitivity to drug-resistant targets and degradation of even well-known ‘undruggable’ proteins both enzymatic and non-enzymatic.⁴ After 2 decades, new PROTACs platform are continuously proposed and more than 30 targets have been successfully degraded so far.⁵

However, although PROTACs represent a cornerstone in medicinal chemistry, this technology involves the exploitation of intracellular protein degradation machinery and it is therefore fundamentally limited to proteins that contain cytosolic domains herein secreted and extracellular proteins are inaccessible to PROTACs.

LYTACs: specific degradation of extracellular targets

To overcome the aforementioned limitations, scientists recently developed a brand new technology: lysozyme targeting chimeras (LYTACs).

LYTACs twisted the idea and established as a revolutionary approach for the degradation of secreted and cell surface proteins by hijacking cell-surface lysosomal targeting proteins.⁶ In essence, the core component of LYTACs technology is the same as PROTACs: a hetero bifunctional molecule, with two distinctfunctional domains connected via a linker. On one end, a targeting motif which can be peptides, small molecule or antibody provides the specificity via the binding to the POI. On the other end, a motif recruits the targeted proteins to the lysosomal targeting receptors (LTR), which shuttles the whole construct into the lysosome for degradation. By binding to the POI and recruit to the lysosome mediated degradation, LYTACs allow for the degradation of both extracellular exposed and cell released proteins.

The first generation of LYTACs utilized the cation independent – mannose 6-phosphate receptor (CI-M6PR) for lysosome targeting, however its ubiquitous location raised concern about the overall safety prolifeof LYTACs.^7,8

With this in mind, a new generation of LYTACs harnesses the hepatic specific expression of the LTR asialogly co protein receptor (ASGPR) to induce the degradation of circulating and membrane-associated proteins including extracellular fluorescent avidin, ApoE4, EGFR and PD-L1exclusively in the liver and resulting more efficient when compared to competitive blocking approaches.^9,10

From a technical point of view, LYTAC simultaneously combines the CI-M6PR or ASGPR with the POI, the resulting ternary complex is engulfed and shuttled via the cell membrane receptor into the lysosome. Here, the acidic environment and lysosomal enzymes degrade the POI while functional CI-M6PR or ASGPR are recycled on the cellular surface for reuse.

Recent advances in targeted protein degradation

Explosive interest and expectations are growing around the TPD strategies and in particular LYTACs as their therapeutic value is started to uncover. Asexpected, the enormous medical potential of TPD has prompted a fast promotion action from investors and scientific community as well. Nowadays, an increasing number of pharmaceutical companies are pivoting their effort into R&D of TPD platforms development. In this respect, scientists have explored new sophisticated solutions to obtain major control over the PROTACs’ mechanism of action. Among these, it is worth of mentioning the so called photo-PROTACs, including PHOTAC sazo-PROTACs, here the presence of photo-sensitive blocking groups allow for a reversible photos witch ability of the whole PROTAC which action can be easily controlled via light activation.¹¹

Regarding LYTACs, as this technology is still in its infant stage, additional effort will be needed to streamline its synthesis andusage in different diseases, being today limited to oncology and immunotherapy for the majority.

In addition, while a lot of effort has been made into improving the specificity of actionof LYTACs, little has changed in the modality to develop the heterobifunctional scaffold which is limited tothe click chemistry or biotin/streptavidin systemto conjugate the LTR targeting moleculeto the POI binder in a modular fashion. With the intent to overcome important limitations including the unwanted immunogenicity, high production cost and low production yield, scientists at Jotbody, a biotech that aims to develop the next generation of single domain antibody based-therapeutic platform, is working on developing a new, efficient, robust and rapid conjugation method which may open the way for the third generation of LYTACs. At this stage, Jotbody did not disclose further details. To date, while at least 15 PROTACs candidates made it to clinical trials, no LYTACs are under clinical evaluation, in this respect, more preclinical studies are needed to explore the pharmacokinetics, pharmacodynamic and toxicity of LYTACs.

Conclusion

Most of current therapies works by physically binding to a specific target site and consequently blocking the function of that specific protein. However, many POIs lack a binding site for small molecules inhibitors thus resulting “undruggable” by using this strategy, and here it is where TPD platforms take over: indeed TPD obtain an efficient and potent removal of the POI and here in overcomes the tedious development for a chemical compound inhibitor. Preciseprotein targeting degradation is an exciting new therapeutic approach that is revolutionizing our knowledge of the druggable proteome; in this contest PROTACs and LYTACs are two complementary technologies: the former mainly targets intracellular proteins and the latter is dedicated to extracellular targets.

Two decades have passed since the first publication on PROTACs technology, and nowadays this field is still far from saturation. Both academic institutions and industry keep pivoting a lot of R&D effort toward generating innovative TPD approaches. However, lesson learnt from the past, the way for innovative therapeutic strategies to the patient is long and complicated, with this in mind, preclinical studies should focus on revealing less-explored aspects of the TPD in vivo behaviour such as toxicity, stability and biocompatibility.

This will be the year of truth for many TPD platforms with LYTACs as major protagonist. In this scenario, both pharmaceutical industries and capital investors are eagerly waiting that the first TPD candidate will finally find the FDA approval after more than 20 years of technical accumulation: the approval of one of these candidates will definitely give a boost to the entire TPD market.

References

1.     Brown KJ, Seol H, Pillai DK, Sankoorikal BJ, Formolo CA, Mac J, et al. The human secretome atlas initiative: implications in health and disease conditions. Biochim Biophys Acta 2013;1834(11):2454-61. doi: 10.1016/j.bbapap.2013.04.007
2.     Liger-Belair G. How many bubbles in your glass of bubbly? Journal of Physical Chemistry B 2014;118(11):3156-63. doi: 10.1021/jp500295e
3.     Wang Y, Jiang X, Feng F, Liu W, Sun H. Degradation of proteins by PROTACs and other strategies. Acta Pharm Sin B 2020;10(2):207-38. doi: 10.1016/j.apsb.2019.08.001
4.     Sun X, Gao H, Yang Y, He M, Wu Y, Song Y, et al. PROTACs: great opportunities for academia and industry. Signal Transduct Target Ther 2019;4:64. doi: 10.1038/s41392-019-0101-6
5.     Zou Y, Ma D, Wang Y. The PROTAC technology in drug development. Cell Biochem Funct 2019;37(1):21-30. doi: 10.1002/cbf.3369
6.     Paulk J. Lysosome-targeting chimeras evolve. Nat Chem Biol 2021;17(9):931-3. doi: 10.1038/s41589-021-00835-1
7.     Banik SM, Pedram K, Wisnovsky S, Ahn G, Riley NM, Bertozzi CR. Lysosome-targeting chimaeras for degradation of extracellular proteins. Nature 2020;584(7820):291-7. doi: 10.1038/s41586-020-2545-9
8.     Zhou Y, Teng P, Montgomery NT, Li X, Tang W. Development of Triantennary N-Acetylgalactosamine Conjugates as Degraders for Extracellular Proteins. ACS Cent Sci 2021;7(3):499-506. doi: 10.1021/acscentsci.1c00146
9.     Ahn G, Banik SM, Miller CL, Riley NM, Cochran JR, Bertozzi CR. LYTACs that engage the asialoglycoprotein receptor for targeted protein degradation. Nat Chem Biol 2021;17(9):937-46. doi: 10.1038/s41589-021-00770-1
10.     Caianiello DF, Zhang M, Ray JD, Howell RA, Swartzel JC, Branham EMJ, et al. Bifunctional small molecules that mediate the degradation of extracellular proteins. Nat Chem Biol 2021;17(9):947-53. doi: 10.1038/s41589-021-00851-1
11.     Xue G, Wang K, Zhou D, Zhong H, Pan Z. Light-Induced Protein Degradation with Photocaged PROTACs. J Am Chem Soc 2019;141(46):18370-4. doi: 10.1021/jacs.9b06422