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PROTAC — Future of Drug Molecules from Modular Construction
Introduction:
Many traditional small molecule drugs competitively occupy the substrate binding site of target protein, and they could play an inhibitory (or exciting) role, such as nonsteroidal anti-inflammatory drug COX-2 inhibitor Paracetamol , lipid-lowering drug HMG-CoA reductase inhibitor Simvastatin , and many tinib anticancer drugs.
Nevertheless, more than 80% of protein targets cannot be developed into drugs with such a simple logic for complicated reasons [1].
Reasons could be: the protein itself lacks a corresponding binding cavity, the protein resides in the cell and drugs cannot reach there, the endogenous substrates show high intrinsic affinity and concentration, the pathogenic mechanism of protein does not depend on catalytic activity or protein-protein interaction, etc. For example, endogenous substrates of KRAS are GTP and GDP. Both of them have high concentrations in human body, and KRAS also strongly combines to them. The solution to this problem is irreversible covalent binding of KRAS-G12C mutant pocket published in Nature in 2013 by Shokat et al, which eventually brought Sotorasib (AMG-510) [2].
It is true that covalent inhibition is an effective way to target many undruggable targets, but its safety and bioactivity evaluation need to be optimized. In contrast, PROTACs not only solve the problem of undruggability but they also have other advantages compared to traditional drug targeting strategies.
PROTACs vs Traditional Small Molecule Drugs:
PROTAC, stands for Proteolysis-Targeting Chimeras, is a heterozygous bi-functional small molecule composed of three parts: Target protein ligands, Linker ligands, and E3 ligands, which exploit the ubiquitin-proteasome pathway to specifically degrade target proteins by bringing the target protein closer to the intracellular E3 ubiquitin ligase [3].
Protein degradation by PROTAC is an event-driven procedure. Each ligand connect to its corresponding target i.e. E3 ligand connect to E3 ligase and the other ligand connect to target protein simultaneously, so that optimum activity of PROTAC is achieved. If the PROTAC concentration is too high, the formation of binary complexes either of E3 ligase-PROTAC and Target protein-PROTAC will lead to decline in the efficiency of PROTAC because of low concentration of free protein and E3 ligase[4].
Fig1: Mechanism of PROTAC molecules [3]
After PROTAC molecules close the distance between two proteins, ubiquitin binding on E2 and E3 enzyme will be transferred to the target protein for K48 polymerization, and then labeled protein will be recognized and degraded by proteasome.
Compared to traditional small molecules which are designed according to mechanism of target protein, PROTAC ignores the mechanism of target protein and directly degrades the target protein by promoting its labeling with ubiquitin. In addition, due to its unique mechanism, PROTAC requires less effective concentration, often at the nanomolar level, that’s why special assays are needed to evaluate its safety and activity. However, conventional PROTAC drugs also face problems of less solubility and bioavailability. Below is a detailed comparison of PROATCs and traditional small molecules.
PROTACs
Traditional small molecules
Advantage
•Conventional ligands can function without competitive sites;
•Degradation of target protein, no drug resistance;
•Event-driven, low dose, low toxicity;
•Improve the druggability of many tumor-related intracellular proteins and nucleoproteins.
•Small molecular weight, good bioavailability and metabolic distribution;
•Competitive mechanism, mature evaluation of activity and safety.
Disadvantage
•Large molecular weight, poor bioavailability;
•Degradation mechanisms need to be based on high selectivity;
•Immature evaluation of activity and safety.
•Targeted to competitive site, screening results may be invalid.
•Drug resistance.
Figure 2: Comparison of PROTACs and traditional small molecules
Ligand for E3 Ligase:
E3 ligase is usually divided into different families such as RING, HECT, RBR, and each family includes many sub-categories [5]. Currently, there are more than 600 known E3 ligases, but there are only four commonly used Ligands for E3 ligase in PROTAC, named CRBN, VHL, IAP, MDM2. In addition, a few more E3 ligases are reported but not often used, like DCF15, RNF114, DCAF16, KEAP1, FEM1B, etc [6]. Below are the common E3 ligase ligands and their corresponding PROTACs.
Figure3. Common E3 ligases and their ligands [6]
E3 ligand type
Common Structure
Feature
PROTACs
CRBN ligand
Domine derivatives
Mostly used; Small molecular weight; Good druggability
The ubiquitin-proteasome pathway degrades target proteins such as cyclins, fusion-associated proteins, cell surface receptors, transcription factors, tumor suppressors, oncogene products, intracellular denatured proteins and abnormal proteins under stress. Currently, selection of PROTAC ligands is based on known inhibitors/agonists, and most of the targets are cancer-related proteins. This indicates that the research on PROTAC is still in early stage. The target protein ligands can be selected by virtual screening and high-throughput screening without considering whether it occupies competitive binding site or not. Likewise, target protein degraders for neurodegenerative diseases and auto-immune diseases can also be developed through this way.
On July 20, 2021, Nature Reviews Drug Discovery published an article using PROTACtability method to evaluate the ability of protein targets for obtaining corresponding PROTAC. It showed that kinases (MEK, KRAS, CDK and Bcr/Abl), transcription factors (such as p53, STAT, RAR, ER and AR), epigenetic factors (such as HDAC and BET bromine domain) and E3 ligase itself (such as MDM2) were good candidates for protacification [7].
Figure 4. Target evaluation of PROTACtability [7]
Linker:
Linker is used to connect E3 ligase ligand with target protein ligand. Chain length is the most important parameter for a linker. Too short chain length will hinder the formation of ternary complexes due to spatial collisions, whereas too long chain length will increase binding entropy [8].
The common linker is PEG or straight-chain alkane, and the connection is commonly established by esterification, amidation, and click reaction, etc. On this basis, PROTAC molecules are further modified to improve their overall performance by introducing aryl groups to increase hydrophobicity and rigidity, adding a connecting chain to limit molecular torsion, and using photoswitch groups to shield active sites in advance[9] . Each linker motifs have corresponding characteristics, as shown in the figure below:
Structure
Linker type
Key points
Alkyl/PEG
- High syntheic accessibility and commercial availability
- Enable fine-tuning of linker length
- Flexible
- Facilitates library synthesis
- High-yielding synthesis
- Potential H-bond interactions in the TC
CLIPTACs
- Assembled from lower MW precursors
- More favourable physical properties
- Compounds must be administered separately to avoid clicking
Photoswitches
- High spatiotemporal control
- May alleviate toxicity
- Continuous irradiation may be required if photostates are not bistable
Figure 5. Different linker motifs and their features [10]
In addition to linkers mentioned above, there are also special linkers which can connect two target protein ligands. In the article Rational Design and Synthesis of Novel Dual PROTACs for Simultaneous Degradation of EGFR and PARP, With the introduction of amino acids with three functional groups (threonine, serine, etc.) in the middle, PROTAC molecules can play a dual role of simultaneously degrading two target proteins [10].
Figure 6. Dual functional PROTAC molecules [10]
Summary:
PROTAC technology is in full bloom at present. It can solve the undruggability problem of many target proteins, but problems such as solubility, membrane permeability and selectivity are a matter of concern. Overcoming these problems may require the discovery of new E3 ligands and the innovation of linker motifs, which is both an opportunity and a challenge.
In addition to PROTAC, there are many similar technologies; such as LYTAC, AUTAC and ATTEC which degrade membrane proteins and endocytosis proteins, "molecular glue" which degrades IKZF protein using the same ubiquitin-proteasome pathway, and PAC drugs which attach PROTAC to antibodies based on antibody-conjugated drug technology. At present, they all provide a new direction for the development of PROTAC technology.
No matter what is ahead, roads are made by travelers. The modular construction of PROTAC shortens the discovery and design cycle of small molecule drugs, which can be called as a frontier technology in the field of small molecule drugs.
Products and services available from MCE
MCE is global leading supplier of research chemical and biochemical reagents. We are equipped with a strong technical team and state of the art equipment. For PROTAC products, we have rich experience in R&D and production.
MCE can provide scientists with Building blocks related to PROTAC. Currently, we have 3700+ PROTAC products online, and the number is increasing.
MCE keeps making breakthroughs and innovations, and can also provide customized PROTAC products. We can synthesize different types of PROTAC products according to customers' requirements, and ensure the effectiveness and consistency of products. In addition, we provide information on the structural characterization, physical and chemical properties and drug impurities of all customized products.
N-Descyclopropanecarbaldehyde Olaparib is an analogue of Olaparib containing DOTA moiety. N-Descyclopropanecarbaldehyde Olaparib is a CRBN-based ligand for synthesizing novel dual EGFR and PARP PROTAC, DP-C-4. N-Descyclopropanecarbaldehyde Olaparib can be radiolabeled F-18 or fluorophore for positron emission tomography (PET) or optical imaging in several types of tumor.
Pomalidomide 4'-alkylC5-acid is a synthesized E3 ligase ligand-linker conjugate that incorporates the Pomalidomide based cereblon ligand and a PEG linker used in PROTAC technology.
FAK ligand-Linker Conjugate 1 incorporates a ligand for FAK, and a PROTAC linker, which recruit E3 ligases (such as VHL, CRBN, MDM2, and IAP). FAK ligand-Linker Conjugate 1 can be extensively used for PROTAC-mediated protein degradation.
K-Ras ligand-Linker Conjugate 4 incorporates a ligand for K-Ras recruiting moiety, and a PROTAC linker, which recruit E3 ligases (such as VHL, CRBN, MDM2, and IAP). K-Ras ligand-Linker Conjugate 4 can be used in the synthesis of PROTAC K-Ras Degrader-1 (HY-129523), which is potent PROTAC K-Ras degrader that exhibits ≥70% degradation efficacy in SW1573 cells.
dBET6 is a highly potent, selective and cell-permeable PROTAC connected by ligands for Cereblon and BET, with an IC50 of 14 nM, and has antitumor activity.
PROTAC BRD4 degrader for PAC-1 (compound 5), a PROTAC-linker Conjugate for PAC, comprises the chimeric BET degrader GNE-987 and disulfide-containing linker.
