Two major challenges of PROTACs: resistance and toxicity

Cancer is a serious threat to human health and life.
Studies have screened more than 600 proteins from more than 20,000 human proteins, confirming that they have important functions in the occurrence and progression of various cancers, including nearly 400 non-enzymes.
For proteins, using the current occupancy-driven drug development method, most of the non-enzymatic targets are called "non-drugable" targets
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The development of small molecule targeted protein degradation drugs (Targeted protein degradation, TPD) has made a lot of progress in recent years, including molecular glues and heterobifunctional degradation drugs, such as proteolysis-targeting chimeras (PROTACs)
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By directly degrading the target protein, it is possible to target proteins that were previously considered "unavailable"
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Heterobifunctional degradation drugs, one end is connected to the protein of interest (POI), and the other end is connected to E3 ligase
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The difference between PROTACs and traditional occupancy-driven drug development models (Current Opinion in Chemical Biology 2019, 50:111–119) However, similar to all drug development technologies, PROTACs also present considerable challenges
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For example, the lack of E3 ligase ligands, the evolutionary drug resistance such as tumor mutations, and the toxicity caused by non-specific effects
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Drug-resistant PROTACs are dependent on E3 ligase, and tumor cells develop drug resistance by impairing the function of E3 ligase components
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In fact, studies have shown that after prolonged treatment, tumor cells acquired resistance to VHL recruitment and CRBN recruitment PROTACs
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The acquired resistance to PROTACs is mainly caused by changes in the genome or significantly reduced expression of the CRBN gene or CUL2 gene, rather than point mutations in the residues involved in the binding of POI and E3 ligase
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CRBN genome deletion has also been found to be the main reason why myeloma cells are resistant to IMiDs
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Mutation or deletion of CUL2 gene in drug-resistant cells (Mol.
Cancer Ther.
18, 1302–1311 (2019).
Genome-wide CRISPR-Cas9 knockout screening based on three different E3 ligases CRL2VCHL, CRL4CRBN and CRL4DCAF15 showed that CRBN, VHL and The deletion of DCAF15 mediates resistance to PROTAC
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Studies have shown that tumors acquire resistance to PROTAC by mediating the mutation of their non-essential E3 ligase
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Using RNAi research, 8 E3 ligases for PROTAC, at least 5 kinds of tumor cell function is not essential
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Research shows that for GM CRBN-PROTACs VHL-PROTAC resistance and do not overlap, and thus offer different alternating PROTACs E3 ligase may be one way to overcome resistance
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toxicity Drug toxicity is the main factor limiting the effectiveness of therapeutic drugs
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In the field of TPD, the toxicity of immunomodulatory imide drugs (IMiDs) is a concern
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Thalidomide was approved to treat multiple myeloma in the late 1950s, but was banned after it was found to cause widespread (more than 10,000) serious birth defects.
It can also cause allergic rash, neutropenia, and arrhythmia.

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Some toxic effects are believed to be caused by the degradation of CRBN neosubstrates mediated by IMiDs
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MDM2 ligands such as nutlin3 were initially found to be MDM2 antagonists and p53 activators by inhibiting the MDM2-p53 interaction
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Although preclinical studies and early clinical trials have shown the efficacy of nutlin3 in the treatment of cancer, the dose-limiting target toxicity caused by p53 activation of p53 in normal cells is still a major challenge in advancing nutlin3 as a monotherapy
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Sulfonamides are DCAF15 ligands that can induce the degradation of RNA splicing factors such as RBM39, which is essential in many cell types; consistent with this, dose-limiting toxicity has been observed in clinical trials
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However, BIRC2 ligand bestatin and RNF114 ligand nimbolide both seem to have low toxicity and relatively positive safety in clinical applications
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In view of the current lack of E3 ligands and their limitations, the TPD field urgently needs to develop new E3 ligands with low toxicity
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Although no new substrates for VHL have been reported for E3 ligase, many new substrates for IMiD-CRBN complexes have been reported, many of which contain zinc acetylene finger motifs
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The translation termination factor GSPT1 may be the most worrying new substrate.
Since GSPT1 plays a key role in most cells, GSPT1 may be degraded by the PROTACs recruited to CRBN and has potential toxicity
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For the first time, it was reported that GSPT1 was inadvertently degraded by MI-389 (PROTAC recruited by CRBN, pankinase inhibitor sunitinib as a POI binder), while the expected target KIT was not degraded
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Due to the key role of GSPT1 in translation, degradation of GSPT1 by PROTAC recruited by CRBN may lead to degradation of POI as a secondary effect
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According to reports, CRBN recruiting PROTACs can degrade other new CRBN substrates
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For example, CDK4 and CDK6 PROTACs can degrade the lymphoid transcription factors IKZF1 and IKZF3, which are two new substrates for CRBN to recruit IMiDs.
The effect in killing mantle cell lymphoma cells is significantly higher than that of degrading CDK4 and CDK6 alone
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Therefore, it is crucial to determine the degradation profile of new substrates for CRBN recruiting PROTACs to rule out secondary drug reactions
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In addition, the new substrates RBM39 and CyclinK of DCAF15 and DDB1-CDK12 are indispensable in many cell types, respectively; therefore, it is important to determine the new substrate degradation profile of DCAF15 recruitment and DDB1-CDK12 recruitment of PROTACs
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The editor summarized the hope of being a target of "unable to make medicine", PROTACs have made great progress in the past two years, and they have also attracted the attention of the capital market
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Of course, in addition to the well-known advantages, PROTACs also have some challenges, such as drug resistance and toxicity
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The resistance of PROTACs is mainly caused by the decrease or deletion of the expression of E3 ligase and related genes, and the toxicity is mainly due to the effect of E3 ligase on new substrates, which degrade other molecules and cause uncontrollable side effects
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References 1.
Stacey-Lynn Paiva and Craig M Crews, Targeted protein degradation: elements of PROTAC design, Current Opinion in Chemical Biology 2019, 50:111–1192.
Behan, FM et al.
Prioritization of cancer therapeutic targets using CRISPR-Cas9 screens.
Nature 568, 511–516 (2019) 3.
Lai, AC & Crews, CM Induced protein degradation: an emerging drug discovery paradigm.
Nat.
Rev.
Drug Discov.
16, 101–114 (2017).
4.
Zhang , L.
, Riley-Gillis, B.
, Vijay, P.
& Shen, Y.
Acquired resistance to BET-PROTACs (proteolysis-targeting chimeras) caused by genomic alterations in core components of E3 ligase complexes.
Mol.
Cancer Ther.
18 , 1302–1311 (2019).
5.
Assi, R.
et al.
Final results of a phase 2, open-label study of indisulam, idarubicin, and cytarabine in patients with relapsed or refractory acute myeloid leukemia and high-risk myelodysplastic syndrome .
Cancer 124, 2758–2765 (2018).
6.
Fischer ES, Bohm K, Lydeard JR, Yang H, Stadler MB, Cavadini S, et al.
Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide.
Nature 2014; 512:49–53.
7.
Cardote TAF, Gadd MS, Ciulli A.
Crystal structure of the Cul2-Rbx1-EloBCVHL ubiquitin ligase complex.
Structure 2017;25:901–11.
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