PROTAC technology: Small molecule drug research and development mass murderer is coming! (Ⅰ)


Judging from the global drug sales trend in recent years, biological drugs have begun to become popular, while sales of small molecular drugs are declining. Innovations in research and development for small molecules and the accompanied technologies are urgently needed.  C&EN News recently published an overview of PROTAC technology written by Lisa Jarvis, which describes the development of this new technology over the past 20 years. Dr. Craig Crews, a chemical biologist at Yale University, one of the pioneers of the PROTAC technology, also published a special issue on PROTAC technology in 2018 as the invited editor of the Journal of Pharmaceutical Chemistry of the American Chemical Society. The continued development of the PROTAC has attracted the attention of a number of international pharmaceutical giants.

Protein degradation targeted chimera (PROTAC) technique

Protein degradation targeting chimera (PROTAC) technology was derived from the Nobel Prize in the category of Chemistry. On October 6, 2004, the Royal Swedish Academy of Sciences announced that the Nobel Prize in Chemistry would be awarded to Israeli scientists Aaron Ciechanover, Avram Hershko and American scientist Irwin Rose, for their joint discovery of ubiquitin (Ub) regulated protein degradation.

Eukaryotic cells are trying to maintain appropriate protein levels, producing and degrading thousands of proteins at every moment. The key factor in maintaining protein balance is a small protein molecule called ubiquitin. When linked to proteins, ubiquitin causes the transportation of these proteins to proteasome for degradation.

Since the human genome was interpreted, researchers have been trying to target thousands of proteins that cause disease. It is estimated that only 10% of proteins can be regulated by small molecules, 10% by biological macromolecules on the cell surface, and up to 80% of proteins cannot be regulated by existing drugs. Targeted protein degradation is a new direction in the field of drug research and development. Small molecules that target protein degradation are designed into new type of drugs, and the function of traditional small molecules is to block the function of proteins. The role of protein targeted degradants is to completely degrade these proteins by feeding them into proteasome.

  • Ubiquitin and Ubiquitination

Ubiquitin is composed of 76 amino acid residues with a molecular weight of about 8.5 kDa. The name “ubiquitin” comes from its prevalence in eukaryotes (ubiquitin has not been found in prokaryotic cells): it has highly conserved sequences and exists in all known eukaryotes. The genes that encode ubiquitin in eukaryotes are arranged in Tandem repeat, which may be due to the need for large amounts of transcription to produce enough ubiquitin for cells. It has been suggested that ubiquitin is the slowest protein ever found to evolve. Ubiquitin contains eight different amino acid residues, which can form a complex polyubiquitin chain on the target protein.

Ubiquitination refers to the process that ubiquitin protein in cells under the action of a series of special enzymes, selecting and modifying target protein molecules, and form polyubiquitin chains with target proteins. These special enzymes include ubiquitin activating enzyme (E1), ubiquitin binding enzyme (E2), ubiquitin ligase (E3) and so on. This process is a three-enzyme cascade reaction, that is, three enzymes catalyzes a series of reactions. The whole process is also known as ubiquitin signaling pathway. It is worth mentioning that the ubiquitin process is also reversible, and ubiquitin can be removed from the ubiquitin chain by deubiquitinase (DUB) to form a reverse regulation. Ubiquitination plays an important role in protein localization, metabolism, function, regulation and degradation. Protein ubiquitination is a common post-translational modification in organisms. At the same time, it is involved in the regulation of almost all life activities, such as cell cycle, proliferation, apoptosis, differentiation, metastasis and so on. Ubiquitination is closely related to tumor, cardiovascular, autoimmune and other diseases. As one of the important achievements of biochemical research in recent years, ubiquitin has become a new target for the research and development of new drugs.

  • Induced protein degradation

Ubiquitin-proteasome system (UPS) is an important pathway for selective degradation of proteins. 26s proteasome is an ATP dependent proteolytic complex, which is composed of 20s core particles, 19s regulatory particles and 11s regulatory factors. The hollow structure of 20s cylinder has the hydrolysis activity of trypsin, chymotrypsin and glutamic-like peptide, which can cleave most of the peptide bonds.

Induced protein degradation is not a new concept. Clinically, many drugs have been unintentionally found to degrade target proteins: for example, antineoplastic drugs Fulvestrant and Tamoxifen can degrade estrogen receptors; Lena polyamines can specifically degrade transcription factors IKZF1 and IKZF3. However, these unexpected discoveries are not universal, nor were the chemical structure of the drugs designed.

  • Hormone receptor inhibitors

Selective estrogen receptor inhibitors such as Tamoxifen (Selective estrogen receptor modulator, SERM) were first approved by FDA in 1977 for the treatment of breast cancer patients with ER+. The upgraded SERM Fulvestrant (ICI82780) was discovered with the ability to degrade estrogen receptors. This was also the earliest reported prototype of induced degradation protein technology. Although fulvestrant has no oral activity and poor competition, it has become the only breast cancer drug to be approved for second-line Tamoxifen resistance (approved by FDA in 2002) due to its estrogen receptor degradation ability to overcome drug resistance. But no significant progress in attempts to improve the drug formation of Fulvestrant has been reported lately.

Similarly, attempts to find fulvestrant analogues targeting prostate cancer have continued on the selective targeting of androgen receptor degradant (SARDs).

At present, the small molecular degradants based on SARDs and SERDs are limited to estrogen and androgen receptors, so it is difficult to develop into a general technology platform.

  • Hydrophobic label HyT

In order to extend the concept of induced protein instability to a wider range of protein targets, Crews and other laboratories have recently developed a new technical platform: hydrophobic tagging (HyT). Similar to the surface hydrophobicity induced by Fulvestrant, the hydrophobic part (i.e. Adamantane or Boc3-Arg) is attached to the surface of the target protein in an attempt to simulate the state of the partially unfolded protein. The cytoplasmic unfolded protein response system was used to degrade the target.

The ability of Boc3-Arg to induce protein degradation was proved in 2012, and glutathione-S-transferase α1 (GST-α1) was targeted by covalent inhibitor Etacrynic acid and Boc3-Arg. Similarly, dihydrofolate reductase can be degraded by trimethoprim, a non-covalent inhibitor coupled to Boc3-Arg in a micromolar concentration.

Although the detailed mechanisms of molecular chaperones or other control pathways are still unknown, this targeted protein degradation pathway is known to occur independently of proteasome ubiquitin and ATP.

  • Peptide-based PROTAC Technology

In several proof-of-concept experiments first published in 2001, Crews Laboratory and collaborator Ray Deshaies reported the first batch of PROTACs bifunctional molecules, the ubiquitin-proteasome system, by recruiting E3 ligases to target proteins. It leads to neighborhood-induced ubiquitination and subsequent protein degradation. The initial technology is based on short peptide PROTAC technology, the successful targets include MetAP2, androgen receptor, aromatics receptor, PI3K and so on.

However, the activity of these peptides PROTAC is low and still stays in the range of micromoles. The main obstacle may be that the peptide technology platform has poor cell permeability. Fortunately, continuous improvement attempts have contributed to the development of PROTAC small molecule technology platforms with in vivo stability.

  • Small molecule PROTAC

The earliest report was in 2008, when Nutlin3, a small inhibitor of MDM2 and p53 from Roche was linked to the E3 ligase MDM2 to degrade androgen receptors. The concentration of cell activity reached micromoles, and later, after many attempts, it still could not further effectively improve its cell activity.

The next step is the PROTAC technology based on cIAP1 E3 ligase, using cIAP1 inhibitor bestatin, to cross-link small molecules with multiple target targets, including ER, AR, ATRA, TACC3 and so on. However, due to the low selectivity and activity of bestatin itself, the activity of many chimeric PROTAC small molecules is not high enough, and no candidates have yet to begin animal trials.

Recent efforts have been made to replace the binding peptide HIFa-1 of VHL E3 ligase with small molecular compounds. The breakthrough took place in 2015, and finally obtained a small molecule of PROTAC with Namore cell activity, which was used to degrade estrogen associated receptor α (ERR α), RIPK2 and so on. Animal models in vivo showed that the degradation of ERR α can occur in heart, kidney and tumor xenografts, and about 40% of the target proteins can be degraded.

In 2010, Handa and colleagues found that E3 ligase Cereblon (CRBN) was the main target of thalidomide protein. Considering the ability of phthalimide to bind to CRBN, Crews laboratories and others tried to use phthalimide as an E3 ligand to hijack CRBN to degrade the target protein. The binding of small molecule BRD4 binding part (OTX015) to Pomalidomide produces PROTAC which can solve the epigenetic regulator BRD4 in the decrease of picomole efficiency.

Compared with BRD4 inhibitors JQ1 and OTX015, CRBN-based PROTAC can inhibit the expression of c-myc more permanently by counteracting the increased expression of feedback BRD4 known to be associated with BRD4 inhibition. At the cellular level, this continuous inhibition leads to excellent anti-proliferation and apoptosis of Burkitt’s lymphoma cell line. In one study, the BRD4 inhibitor JQ1 binds to thalidomide derivatives, resulting in dBET1 energy. DBET1 also showed good activity in animal models of leukemia, and the pharmacology in preclinical models was also encouraging, which laid the foundation for human clinical trials.

In addition to the CRBN-based PROTAC, researchers also developed Pimore’s VHL-based pan-BET- targeting PROTAC, which showed excellent antiproliferative activity compared with BET inhibitors. Pan-BET- targeted PROTAC also showed activity against xenografts in 22RV-1CRPC mice, thus expanding the application of PROTAC technique in solid malignant tumors.