Main steps of PROTAC to achieve intracellular protein degradation
To achieve intracellular protein degradation requires multiple steps, such as cellular uptake, effective recruitment of E3 ligase, ubiquitination of target proteins, and recruitment of proteasomes.
As an emerging technology, PROTAC has completed the theoretical proof of its effectiveness in the laboratory and in the clinic. There are still some technical difficulties to be resolved in practicing specific steps.
- Molecular construction
As a technology with a short development history, PROTAC has accumulated less experience in optimizing its pharmacodynamics and pharmacokinetics, and has not yet formed a complete analysis system and design rules.
It is necessary to further understand the structural, biochemical, kinetic, and thermodynamic parameters of PROTAC in order to formulate applicable guidelines.
At present, about 270 of the 632 types of E3 ligases in the human proteome are considered related to the ubiquitin proteasome system, and currently less than 10 types (CRBN, VHL, IAP, MDM2, DCAF15, DCAF16, and RNF114) have been used in small molecule-induced degradation of the target protein. The main reason is that it is difficult to screen small molecule ligands with high affinity and specificity for E3 ligase.
Many documents reporting new E3 ligases and ways to improve affinity emerge as the PROTAC technology develops in recent years.
Large molecular weight, longer synthesis steps, and higher costs are the main problems to be fixed during PROTAC synthesis. The chemical properties of the two heads of PROTAC molecule E3RE and protein ligands are quite different that the synthesis is difficult, and the yield is low. Generally, the synthesis of PROTAC molecule is operable, and the complexity is mainly reflected in the high cost.
- Molecular permeable membrane
The relatively large molecular weight of PROTACs (generally between 700 and 1,200) and more polar molecular surfaces make their water solubility and oral availability relatively low, and the dosage is relatively large. For example, Arvinas clinical varieties commonly require larger dosage: ARV-110 (140mg), ARV-471 (120mg).
At present, there is no prediction model similar to “rule of five” for small molecules.
Some PROTACs show sufficient oral bioavailability despite these illegal properties. At present, scientists are trying to use computer simulation to define the description of compound absorption in this unique chemical space.
The “rule of five” is also called Lipinski rule, and its content is as follows:
A small molecule drug must have the following properties
- The molecular weight is less than 500.
- The number of hydrogen bond donors is less than 5.
- The number of hydrogen bond acceptors is less than 10.
- The fat-water distribution coefficient is less than 5.
- The number of rotatable keys does not exceed 10.
- Form a complex
Protein degradation does not only depend on the affinity of the binary target protein and the ligand, but on the activity of the triplet.
The conformation and position of the connection of PROTAC, the modification of the length and composition of the connection chain, the concentration, etc., will all have an impact on the activity of the triplet.
Taking the concentration of PROTAC as an example, PROTAC must form an effective ternary complex with the target protein and E3 enzyme to exert its drug effect. Experiments showed that when the concentration is high, the drug molecule will form a binary complex with the target protein or E3 enzyme respectively (hook effect).
- Protein ubiquitination and protease recognition
There are two mechanisms for resistance to traditional inhibitors: one is that the protein is mutated and has no binding ability with traditional small molecule inhibitors, the other is a stress mechanism that when the body feels the protein is reduced, it will produce more of this protein.
The inducing characteristics of PROTAC technology can overcome the second type of resistance caused by compensatory reactions. The potential of PROTAC to overcome the first resistance mechanism remains to be evaluated. PROTAC technology can cope with some mutations that render inhibitors ineffective.
According to the latest data from Arvinas, there are some AR mutations. The AR degradation agent ARV-110 can degrade T878 and H875, but there are also mutations. ARV-110 does not show degradation activity, such as L702H and AR-V7 mutations.
- Protein degradation
Unlike traditional small molecule drugs, there is currently no effective high-throughput screening technology for rapid and large-scale evaluation of the ability of PROTAC to degrade POI. It can only be achieved through methods such as cell viability screening or western blotting, which limits the development of PROTAC.
The screening efficiency can be improved through the DEL (DNA-encoded compound library) technology, and the specific method is being explored.
There is a risk of off-target for the PROTAC against low-affinity targets.
The expression profile of the used E3 ligase is relatively common, resulting in the relatively low tissue selectivity of the PROTAC molecule itself. In the future, more E3 ligases with tissue distribution specificity need to be developed.
After PROTAC molecule hijacks E3 ligase in large quantities, it may affect protein degradation in normal physiological processes.
In addition to the enzyme function, the target protein also has a scaffold function, which may lose after complete degradation.
Example of patent progress by degradation target
- Nuclear receptor
Nuclear receptors are an important signal transduction mechanism in cells, which play a key role in sensing stimulating ligands and regulating gene transcription in response to these ligands.
As current research shows, compared with antagonists, degradation of nuclear receptors through PROTAC strategy may be a more advantageous strategy. There have been many reports on the degradation of the androgen receptor (AR) mediated by PROTAC for the treatment of prostate cancer and the degradation of the estrogen receptor (ER) for the treatment of breast cancer.
- Kinase receptor
Kinases play a key role in regulating the activity of cellular proteins, but under abnormal conditions, kinases can promote tumor cell proliferation. Therefore, kinases are also the main targets of cancer drugs.
In order to overcome the problem of kinase inhibitor resistance and have the opportunity to target those kinases for which corresponding inhibitors have not yet been developed, targeted protein degradation (TPD) does not exert an inhibitory effect by binding to kinases, but directly destroy the disease-causing kinase.
- Error protein
The compound given in the Arvinas patent document uses VHL or CRBN binders to connect to the hyperphosphorylated Tau protein (PHF-Tau) through a linker.
The patent of the Dana-Farber Cancer Institute also uses both CRBN binder and VHL binder, and is also connected to a Tau binder based on [F-18]-T807.
Industry Latest Advances
- PROTAC that covalently combines POI and E3 while maintaining reversibility
Traditional PROTAC molecules form a ternary complex by forming non-covalent bonds with POI and E3. This method ensures the catalytic properties of PROTAC technology, but the relative strength of non-covalent bonding is different.
In follow-up research, scientists began to explore how to improve the strength of PROTAC target head binding through covalent bonding, but covalent bonding is often irreversible.
At present, in the laboratory, PROTAC molecules that can achieve covalent bonding while maintaining reversible catalytic properties have been synthesized.
- Research and improvement of oral availability
In recent years, the development of some drugs in druggability has gone beyond the scope described in Lipinski’s Ro5 or Veber’s rotatable bond/polar surface area guidelines, which usually have a molecular weight of 700 or more and have other attributes that violate the Lipinski/Veber rule. Despite these illegal properties, they still shows sufficient oral bioavailability and can be further developed as oral drugs.
Some research groups analyzed different bRo5 compound sets to use computer simulations to define the description of compound absorption in this unique chemical space. In terms of druggability, the best oral druggability is thalidomide and its derivatives combined with CRBN, followed by VHL binding ligand.
- New E3 ligases with better druggability have been continuously discovered
The E3 ligases used in traditional PROTAC molecules are mainly VHL and CRBN, and a few are IAP and MDM2. In recent years, new E3 ligases with drug-making potential have been continuously reported, including DCAF15, DCAF16, RNF114, RNF4, and KEAP14.
- Establish an open and accessible target kinase degradation data resource set (Cell)
In a study published in Cell by Eric S. Fischer, Nathanael S. Gray from the Dana-Farber Cancer Institute and the Taebo Sim team from the Korea Institute of Science and Technology, who sketched the first degradable kinase map and identified approximately 200 degradable kinases, greatly improving the 57 previously reported in the literature, and an open access data set has thereby been established.
In addition, the author also explored the key variables that affect the degradation efficiency of the targeted protein, and found that the molecule with the highest affinity for kinases is not the most effective for degradation. Other factors such as the formation of ternary complexes, the abundance of target protein expression, ubiquitin ligase, and the design of the connector are also very important.
- Expand the scope of E3 ligases that can be selected
In October 2020, Cullgen published an article on the discovery of TRK selective protein degradation agent candidates in the Journal of Medicinal Chemistry. The full name of TRK is neurotrophic factor receptor tyrosine kinase. The fusion protein produced after TRK mutation is found to be widely present in a variety of human malignancies, including lung cancer, colorectal cancer, and soft tissue sarcoma. In the article, Cullgen has developed two new TRK-targeted protein degradation agents CG416 and CG428, which are intended to solve the side effects and drug resistance problems in patients taking TRK inhibitors.
- Develop/optimize new degradant molecules
At present, most PROTACs belong to BiDAC, and C4 develops and studies MonoDAC. By combining with E3 ligase and forming a new region on E3 ligase, it can enhance the binding of E3 ligase to the target protein.
- Improve site accuracy
The development of other E3 ligases besides cereblon and VHL ligases can also expand the scope of PROTAC targeting sites. For example, when the target protein is a membrane protein, using E3 ligase targeting the membrane protein will be more effective than using E3 ligase located in the nucleus. Progenra is doing this, which uses its proprietary platform UbiPro to discover a series of E3 ligases and deubiquitinating enzymes (another group of enzymes in UPS). One of its products under development can target K-Ras, a common membrane protein target in cancer.
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