PROTAC - BiopharmaDirect

PROTAC

About PROTAC

Proteolysis targeting chimeric molecules (PROTACs) are heterobifunctional small molecules with three chemical elements: a ligand binding to a target protein, a ligand binding to E3 ubiquitin ligase, and a linker for conjugating these two ligands. One of the small molecule ligands is designed to bind a disease target protein in the cell whilst the other binds with high affinity to an E3 ligase. In the cell, the PROTAC molecule seeks out and selectively binds to the disease target protein of interest and is then able to recruit a specific E3 ligase to form a ternary complex, holding both the disease target protein and the E3 ligase in close proximity. The E3 ligase then recruits an E2 conjugating enzyme to the ternary complex which is subsequently able to ubiquitinate the target protein. The E3 ligase can then recruit additional E2 molecules resulting in poly-ubiquitination of the target protein and flagging it for degradation. The PROTAC molecule dissociates from the target protein and initiates another cycle leaving the poly-ubiquitinated target protein to be degraded by the proteasome. This unique mode of action, which leads to depletion of disease target proteins from the cell, can drive distinct changes in cellular activity. PROTAC is a chemical knockdown strategy that degrades the target protein through the ubiquitin-proteasome system, which can expand the druggable space of the existing targets.

Figure 1 A summary of targeted proteins, ligands for target, ligand for E3 ubiquitin ligases, and recruited E3 ubiquitin ligases. (Yutian Zou, et al. 2018)

The PROTAC technology can be used to target varieties of proteins, including transcription factors, skeleton proteins, enzymes, and regulatory proteins. At present, PROTACs have been successfully employed in the degradation of different types of target proteins related to various diseases, including cancer, viral infection, immune disorders, and neurodegenerative diseases, which cannot be achieved by traditional therapy (inhibitor/activator).

PROTAC Evolution

Figure2 Time table describing the evolution of PROTACs (Mariell Pettersson, Craig M. Crews. 2019)

• Peptide-based PROTAC Technology

1) Initial PROTAC technologies were based on the short peptide sequence to recognize an E3 ubiquitin ligase.

2) Different peptides were examined to recruit E3 ubiquitin ligases including SCF complex, von‐Hippel‐Lindau ubiquitin ligase (VHL), and chaperon‐meditated autophage (CMA).

3) Peptide‐based PROTCs are difficult to permeate the cell membrane.

4) The size of the chimeric molecule could be recognized by immune system to produce antibodies.

• Small Molecule-based PROTAC Technology

1) This design of PROTAC took advantage of small molecule as a moiety to recognize E3 ubiquitin ligase.

2) Different sets of small molecules have been developed as the moiety of E3 ubiquitin ligases including SCF, VHL, cereblon (CRBN), mouse double minute 2 homologue (MDM2), anaphase‐promoting complex/cyclosome (APC/C), and cellular inhibitor of apoptosis protein 1 (cIAP1).

3) The small molecule–based PROTAC has been extended to the design of PROTACs against serine‐threonine kinase RIPK2, estrogen‐related receptor alpha(ERRα), the BET family proteins, oncogenic kinase BCR‐ABL, etc.

PROTAC Advantages

• Unique MOA, and do not require tight binding.
• Overcoming drug resistance of cancer.
• Eliminating both the enzymatic and nonenzymatic functions of kinase.
• Degrade the "undruggable" protein target (proteins that lack catalytic activity and/or have catalytic independent functions, like STAT3).
• Potential for improved selectivity and efficacy.
• Fast and reversible chemical knockdown strategy in vivo (suitable for the functional study of embryonic-lethal proteins in adult organisms).

PROTAC Chanllenges

• Degradation versus inhibition, safety concerns, and chemical/synthetic complexity.
• Until now, there is only one example of PROTAC reported for an "undruggable" target. More cases are needed to prove the advantages of PROTAC in "undruggable" targets in the future.
• "Molecular glue" represents the mechanism of stabilized protein-protein interactions through small molecule modulators of E3 ligases. It has been recently reported that some PROTACs may actually achieve target protein degradation via a mechanism that includes "molecular glue" or via "molecular glue" alone. How to distinguish between these two mechanisms and how to combine them to work together is one of the challenges for future research.
• Since PROTAC acts in a catalytic mode, traditional methods cannot accurately evaluate the pharmacokinetics (PK) and pharmacodynamics (PD) properties of PROTACs. Thus, more studies are urgently needed to establish PK and PD evaluation systems for PROTACs.
• How to quickly and effectively screen for target protein ligands that can be used in PROTACs, especially those targeting protein-protein interactions.
• How to understand the degradation activity, selectivity, and possible off-target effects (based on different targets, different cell lines, and different animal models) and how to rationally design PROTACs etc. are still unclear.
• Expand E3 ubiquitin ligase scope. The human genome encodes more than 600 E3 ubiquitin ligases. However, there are only very few E3 ligases (VHL, CRBN, cIAPs, and MDM2) used in the design of PROTACs.

Table 1 Reported cases applying PROTACs in the degradation of different types of target proteins

PROTAC Solutions

PROTAC solutions for TPD research and development cover molecule discovery, in vitro evaluation, and in vivo animal testing.

Figure 3 The Process of PROTAC Development

PROTAC Services

• Develop PROTAC molecules with a high degree of degradation selectivity.
• Schematic in vitro evaluation tests on solubility and chemical stability, cell permeability efficacy, and target degradation, etc.
• Ubiquitin Proteasome System Proteins and Assays
• Assays for Protein Degradation and Custom Degrader Services
• Toxicity and ADME assessments available plus custom assay design for in vivo animal experiments.
• TAG Degradation Platform (dTAG/aTAG). Better performance on dose tuneability, efficacy, reversibility, kinetics and selectivity compared with gene knockout/knockdown.
• Cell Line Construction (CRISPR Knock-in)

PROTAC Products

• Active Degraders Targeting BRD4, pVHL30, ALK, TRIM24, Cdk9, Cdk6, Cdk4/6, CRBN, BRD7/9, Multikinase, etc.
• Degrader Building Blocks
• Linkers, Crosslinkers
• E3 Ligase Ligands, Ligand-linker conjugate, Target ligands
• E3 Ligase and Target Proteins
• Bioactive Small Molecules
• dTAG/aTAG for Target Degradation and Validation
• VHL Spy Molecules for Ligand Discovery
• VHL-Related Products
• CRBN-Related Products
• Proteasome Inhibitors
• E3 Ubiquitin Ligase-Related Products

PROTAC Tool

UbiHub: a data hub for the explorers of ubiquitination pathways.

PROTAC Top Companies

Arvinas' leading programs are focused on high-value targets in cancer, including the androgen and estrogen receptors associated with prostate and breast cancers, respectively. Arvinas is to unlock the full potential of PROTAC protein degradation by developing robust pipeline and by partnering with the world's leading drug development companies.

Beginning with oncology, C4T is advancing multiple internal and partnered investigational degraders towards the clinic. C4T's balanced approach to target different cancers maximizes patient impact.

References:

• Hongying Gao, Xiuyun Sun, and Yu Rao. PROTAC Technology: Opportunities and Challenges. ACS Med. Chem. Lett. 2020, 11, 237–240.
• Yutian Zou, Danhui Ma, Yinyin Wang. The PROTAC technology in drug development. Cell Biochem Funct. 2019; 37:21–30.
• Scott J Hughes, Alessio Ciulli. Molecular recognition of ternary complexes: a new dimension in the structure-guided design of chemical degraders. Essays Biochem. 2017 Nov 8;61(5):505-516.