The Development of Heterobifunctional Molecules

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The Development of Heterobifunctional
Molecules

Since Crews et al. proposed the concept of PROTAC (Proteolysis Targeting
Chimeras) in 2001, heterobifunctional molecules began to enter people's eyes. Its
advantages of targeting "non-drugable proteins" have attracted the attention of scientists
in the industry, but considering that the "big sister" PROTAC is not suitable for all proteins,
other heterobifunctional "sisters" 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

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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)

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

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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)

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

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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)

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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.

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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.

Protein phosphatase recruiting chimeras
(PHORCs/PhosTACs)

Figure 4 Mechanism of action of PHORCs/PhosTACs

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

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suppressor functions, and their defective expression is associated with many types of
cancer.

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.

Kinases recruiting chimeras (PHICS)

Figure 5 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

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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)

Figure 6 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

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as selective acetylation induced by acetylation sites and the applicability of
heterobifunctional molecules recruited by KAT, etc.

Conclusion

Figure 7 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.

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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.

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.

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two different chemical entities. The PEG main chain part of heterobifunctional PEG
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Reference:
[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.

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