Release date:2022/8/22 17:39:53

Since 2001, heterofunctional molecules have attracted increasing attention. Among these heterofunctional molecules, PROTACs is the most successful representative. PROTAC is a heterobifunctional molecule, one end of the molecule is connected to the ligand that binds the target protein, the other end is connected to the ligand of E3 ligase, and the middle is connected by a suitable linker. Since Crews et al. proposed the concept of PROTAC (Proteolysis Targeting Chimeras) in 2001, its advantages of targeting "non-drugable proteins" have attracted the attention of scientists in the industry.

Although PROTACs open up a new avenue for small molecule drug design, it is not suitable for all proteins. In addition, the effects of PROTACs depend on specific E3 ligase subunits and are therefore influenced by E3 expression, which limits their application in specific cell types. Therefore, there is an urgent need to develop more heterofunctional molecules that recruit other effectors to complement the deficiencies of PROTACs. Other heterobifunctional molecules such as LYTACs, AUTACs, RIBOTACs, PHORCs/PhosTACs have also been expanded and developed to make up for the shortcomings of PROTAC by recruiting other effectors, leading to the rapid development of the field of Targeted Protein Degradation (TPD).

Recently, Qidong You's team from China Pharmaceutical University published a paper entitled "Beyond Proteolysis-Targeting Chimeric Molecules: Designing Heterobifunctional Molecules Based on Functional Effectors", which introduced the characteristics of various heterobifunctional molecules, analyzed the advantages and challenges. It provides effective insights for future development strategies in the field of TPD.

Heterobifunctional molecules have been reported to simultaneously bind two or more molecules through a linker and bring them into proximity to interaction. More and more heterobifunctional molecules are designed to target non-druggable targets or expand the scope of treatment. With the positive clinical treatment effect of Arvinas' ARV-110 and ARV-471, PROTAC has been a widely recognized and focused treatment. 

Although PROTAC opens a new avenue for small-molecule drug design, they are not applicable to all protein classes because the ubiquitin proteasome system (UPS) is restricted to the cytoplasm and nucleus. However, for some membrane proteins, such as multiple transmembrane proteins with complex membrane-embedded topology and lack of ligandable intracellular domains, PROTACs targeting is not applicable. In addition to degradation, heterobifunctional molecules such as phosphorylation-inducing chimeric small molecules (PHICS), protein phosphatase-recruiting chimeras/phosphorylation targeting chimeras (PHORCs/PhosTAC), and acetyltransferase-recruiting chimeras (AceTAGs) have been considered as degradable targets or modulators of post-translational modification (PTM). The following is a brief introduction to these molecular designs.

Lysosome-targeting chimeras (LYTACs)

Mechanism of action of LYTACs

Figure 1 Mechanism of action of LYTACs

Lysosome targeting chimeras (LYTACs) degrades target proteins via lysosomal pathway rather than common proteasome pathway.

In addition to the proteasome degradation system, the endonuclease/lysosome pathway is also an important protein degradation pathway. LYTAC, with an oligosaccharide peptide binding to the cell-surface transmembrane receptor CI-M6PR at one end and an antibody or small molecule targeting POI at the other, and the middle is connected by a linker. LYTAC is using lysosomes to act by lapping POI to LTR(lysosomal targeting receptor), inducing lysosomal mediated degradation. The regulation of substrate acquisition in this system mainly depends on LTR, such as CI-M6PR and ASGPR. To date, two LYTACs have been developed, based on CI-M6PR(M6PN-LyTACs) and ASGPR(GalNAC-LyTACs).

LYTACs have the advantage of targeting extracellular and membrane-associated POIs. LTR is widely expressed on the surface of most cells. To avoid LYTACs targeting cells that express only LTR but not the target protein, non-specific glycosyl-modified antibodies are rapidly cleared, improving selectivity, safety and controlling off-target pharmacokinetic clearance rate is also an urgent problem to be solved.

Autophagy-targeting chimeras (AUTACs)

 Mechanism of action of AUTACs

Figure 2 Mechanism of action of AUTACs 

The autophagy system plays a major role in lysosome-mediated degradation of intracellular materials, such as damaged organelles, intracellular debris, and other substrates. AUTAC selectively degrades POI by recruiting autophagosomes. Autophagy-related tags "stick" to POI ligands through linker, thereby recruiting autophagy-related pathway molecules. The first generation of AUTACs selected guanine derivatives (cGMP) as autophagy tags to induce endogenous S-guanylation. The level of EGFP-HT(POI) was decreased in CGMP-HTL-treated cells, which demonstrated the feasibility of AUTAC. Given the limitations of AUTAC triggered by ubiquitin-dependent mechanisms, recruitment pathways have been further improved. For example, autophagy-targeting chimeras (AUTOTACs) can directly bind the receptor p62 to POI and induce selective autophagy at POI.

AUTACs/AUTOTACs further expand the range of POIs to include aggregated proteins, intracellular debris, and even damaged organelles. AUTOTACs are applicable to a wide range of intracellular target proteins, and they can selectively degrade aggregated proteins.

However, the development of AUTAC still faces many challenges. For example, the efficiency of AUTAC still needs to be improved, and the degradation of POI takes more time than other chimeras. Furthermore, the issue of selectivity remains to be elucidated. The effect of AUTACs on the entire autophagy process in vivo remains to be explored. Some key mechanisms of autophagy remain unclear, making it difficult to elucidate the exact mechanism of AUTAC and expand the AUTAC/AUTOTAC platform to utilize other tags or receptors to mediate autophagy.

Ribonuclease-targeting chimeras (RIBOTACs)

Mechanism of action of RIBOTACs

Figure 3 Mechanism of action of RIBOTACs

Abnormal RNAs are associated with many diseases. There have been advanced methods for RNA oligonucleotide degradation such as RNAi, ASO, CRISPR molecular technology and so on. RIBOTACs, novel heterobifunctional molecules that degrade RNA, consist of RNA-targeting ligands, RNase-recruiting moieties, and a linker between the two, which act by recruiting endogenous RNases to specific RNAs, activating RNases, and inducing selective cleavage of target Rnas.

RIBOTACs offer an alternative strategy to address undruggable disease-causing proteins by modulating mRNA. RIBOTAC can target many types of non-coding RNAs and has been seen to affect more disease progression than protein degradation. However, low permeability is a big challenge. In addition, the design of highly selective RNA small molecule ligands is difficult and prone to off-target. RIBOTACs may not be suitable for RNAs that function in the nucleus.

Phosphorylation Targeting Chimeras (PhosTACs/PHORCs)

Heterobifunctional molecules can also promote the phosphorylation of target proteins. PROTAC pioneer Professor Craig Crews and his team developed a "Phosphorylation Targeting Chimeras" (PhosTAC) technology that can also specifically remove tau protein hyperphosphorylation and reduce Tau protein levels, which is expected to be used in the treatment of Alzheimer's disease and other Tau protein diseases. In his view, the PhosTAC molecule is expected to achieve better results than traditional kinase inhibitors.

Removal of tau phosphorylation using PhosTAC technology

Figure 4 Removal of tau phosphorylation using PhosTAC technology
(Source: Resources [2])

Yamazoe's team discovered that the first PHORCs were PP1 phosphatase recruited to the vicinity of AKT and EGFR, promoting the dephosphorylation of target proteins in cells. On this basis, the Crews team designed another phosphorylation targeting chimeras (PhosTACs) that recruit serine/threonine protein phosphatase 2A (PP2A) to dephosphorylate PDCD4 (programmed cell death 4) and Forkhead- box O3a (FOXO3a, transcription factor). PDCD4 and FOXO3a are potential anticancer targets with tumor suppressor functions, and their defective expression is associated with many types of cancer.

 Mechanism of action of PHORCs PhosTACs

Figure 5 Mechanism of action of PHORCs/PhosTACs

PHORCs have emerged as an effective tool to precisely modulate the function of target proteins by altering their phosphorylation status rather than their expression levels, which may avoid the side effects caused by other protein degradation techniques. Therefore, PHORCs can be used for the biological study of PTMs and the treatment of diseases caused by abnormally hyperphosphorylated targets. However, there are still many issues to be resolved during the development of PHORC, such as its mechanism still needs to be elucidated, dephosphorylation of non-native substrates, relative shortage of protein phosphatase ligands and their druggability (related to poor intracellular permeability and instability in the presence of cellular hydrolases), etc.

Phosphorylation-Inducing Chimeric Small Molecules (PHICS) 

Mechanism of action PHICS

Figure 6 Mechanism of action of PHICS

Kinases play an important role in regulating the phosphorylation state of substrates by transferring phosphate groups from ATP to protein substrates. PHICS consists of kinase activators, POI binders, and linkers that bring them closer by inducing substrate translocation, recruiting kinases to phosphorylate POIs.

PHICS has great potential for the precise treatment of hyperphosphorylation disease-causing proteins, such as the dephosphorylated tyrosine proteins in the Black Death. However, the efficacy and druggability of PHICS in vivo, how to maintain the balance of dephosphorylated or phosphorylated POIs, and the precise relationship between certain phosphorylation sites and disease progression remain to be verified.

Acetyltransferases recruiting heterobifunctional molecules (AceTAGs)

Mechanism of action of AceTAGs

Figure 7 Mechanism of action of AceTAGs

AceTAG, an acetylation labeling system, induces acetylation of POI through heterobifunctional molecules. The first AceTAG used a KAT inhibitor linked to the FKBP12 binding ligand. These heterobifunctional molecules modulate the distance between endogenous KAT and FKBP12-tagged POI, thereby inducing substrate acetylation.

The advantages of AceTAG technology are :(1) it can provide efficient and accurate acetylation regulator blanks; (2) AceTAGs can be used in POI lacking ligand when combined with DNA recombination technology, extending the scope of AceTAGs regulation; (3) Proximity induced acetylation can regulate the acetylation status of POI without directly interfering with the structure of POI or competing with other PTMS, which can analyze the acetylation function and explore the acetylation mechanism in the downstream signaling pathway. However, AceTAGs still have many optimizations, such as selective acetylation induced by acetylation sites and the applicability of heterobifunctional molecules recruited by KAT, etc.

The Challenges of Heterobifunctional Molecules

However, in the development of heterobifunctional molecules, the technology is not yet mature and faces many challenges.

1. More in-depth mechanistic studies are needed, mainly including the specificity of chimeric molecules for POI and the kinetics of chimeric molecule-induced ternary complex formation. Furthermore, the kinetic mechanisms during the formation of ternary complexes (between effectors, POIs, and chimeric molecules) remain unknown.

2. Develop high-affinity and selective ligands to enhance the activity of heterobifunctional molecules. Determining how to efficiently recruit effectors to mediate biological processes is one of the key challenges in designing new heterobifunctional molecules.

3. Medicinal properties. Most of the currently known heterobifunctional molecules exhibit poor PK profiles, solubility and cell permeability due to their large molecular weight. Therefore, it is necessary to optimize its structure, which is of great significance for clinical development.


Research Progress of Heterobifunctional Molecules

Figure 8 Research Progress of Heterobifunctional Molecules

The rapid development of PROTAC in the field of protein degradation has opened the door to heterobifunctional molecules. Heterobifunctional molecules regulate POI function by recruiting their upstream effectors, such as E3 ubiquitin ligases, endosomes/lysosomes, RNase L, protein phosphatases/kinases, and acetyltransferases, to accelerate their interactions. Heterobifunctional molecules fully enrich the concept of new drug design by narrowing the distance between "effector" and "POI", setting off a wave in the discovery of small molecule drugs.

Heterobifunctional molecules that recruit more endogenous effectors need to be developed to enrich targeted regulatory mechanisms, such as chaperone regulation, methylation, lipidation, immune checkpoints, and DNA synthesis. If POI degradation reaches an "everything is degradable" state in the future, there will surely be a large number of milestones in clinical disease treatment.

Heterofunctional PEG derivatives are often used as crosslinkers or spacers between two different chemical entities. The PEG main chain part of heterobifunctional PEG provides end groups with water solubility, biocompatibility and flexibility. As a reliable PEG supplier, Biopharma PEG supplies heterobifunctional PEG derivatives which can be used in the development and application of antibody-drug conjugates (ADC's).

[1] Hua L, Zhang Q, Zhu X, Wang R, You Q, Wang L.
Beyond Proteolysis-Targeting Chimeric Molecules: Designing Heterobifunctional Molecules Based on Functional Effectors.J Med Chem. 2022;65(12):8091-8112.
[2] Hu et al., (2023). Targeted Dephosphorylation of Tau by Phosphorylation Targeting Chimeras (PhosTACs) as a Therapeutic Modality. J. Am. Chem.,

Related articles:
[1] Summary of PROTAC Degraders in Clinical Trials
[2] Four Major Trends In The Development of PROTAC
[3] Focus On PROTAC: Summary Of Targets From 2001 To 2019
[4] PROTACs and Targeted Protein Degradation

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