Design, Synthesis and Evaluation of WD-repeat containing protein 5 (WDR5) degraders
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Abstract
Histone H3K4 methylation serves as post-translational hallmark of actively transcribed genes and is introduced by histone methyltransferases (HMT) and its regulatory scaffolding proteins. One of these is the WD-repeat containing protein 5 (WDR5) that has also been associated with controlling long non-coding RNAs and transcription factors including MYC. The wide influence of dysfunctional HMTs complexes and the typically upregulated MYC levels in diverse tumor types suggested WDR5 as an attractive drug target. Indeed, protein-protein interface inhibitors for two protein interaction interfaces on WDR5 have been developed. While such compounds only inhibit a subset of WDR5 interactions, chemically induced proteasomal degradation of WDR5 might represent an elegant way to target all oncogenic function. This study presents the design, synthesis and evaluation of two diverse WDR5 degrader series based on two WIN site binding scaffolds and shows that linker nature and length strongly influence degradation efficacy.
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This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/4746304.
COMMENTS ON THE PREPRINT BY Dölle, Adhikari et al.
Targeted protein degradation is a very active field at the moment. Many efforts in this area are focusing on transforming known ligands (binders, inhibitors) of proteins with a clear disease relevance into bifunctional (PROTAC-based) degrader molecules. Unlike the traditional antagonist/inhibitor based compounds in preclinical and clinical use that diminish (inhibit) activity of the target, these degrader molecules induce selective degradation of the target. Thus, they remove the target from the proteome. This type of pharmacological activity could be a real benefit when the target in question plays significant scaffolding roles, by …
This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/4746304.
COMMENTS ON THE PREPRINT BY Dölle, Adhikari et al.
Targeted protein degradation is a very active field at the moment. Many efforts in this area are focusing on transforming known ligands (binders, inhibitors) of proteins with a clear disease relevance into bifunctional (PROTAC-based) degrader molecules. Unlike the traditional antagonist/inhibitor based compounds in preclinical and clinical use that diminish (inhibit) activity of the target, these degrader molecules induce selective degradation of the target. Thus, they remove the target from the proteome. This type of pharmacological activity could be a real benefit when the target in question plays significant scaffolding roles, by engaging multiple binding partners using different regions and binding sites. In such a case, inhibiting individual protein-protein interactions would be highly impractical. However, if the target is degraded, all these PPIs would disappear together with the target!
Dölle, Adhikari et al. select one such target - WDR5, a protein that performs different scaffolding roles (i.e. binds different partners) in the context of epigenetic regulation. Because of this, WDR5 has been implicated as a target for drug development and couple of compounds that inhibit WDR5 mediated PPIs have been described. These compounds served here as a starting point for WDR5 selective degrader development. The authors used existing structures of WDR5 bound to the PPI inhibitors to identify surface exposed areas of the molecules that could be modified for degrader molecule development. In brief, each PROTAC (bifunctional degrader) includes a ligand for the target and a ligand that recruits an E3 ubiquitin ligase, connected via a linker. The linker is known to have an impact on the performance of PROTACs and the authors use three chemically distinct types of linkers (PEG based, aliphatic and aromatic). The nature of the E3 ligase is also a major factor that affects degraders' activity, and the authors start by incorporating ligands for cereblon (CRBN), VHL and MDM2. Altogether, they generate number of PROTAC-based degraders featuring different linkers and different ligases.
They describe detailed validation steps of their degraders which included:
- Measuring in vitro (biochemical) affinity between WDR5 and degrader molecules using ITC, showing Kd values in low nM. - They also tested binding via DSF
- The authors tested that degraders were cell permeable and that they engaged the target using BRET. This is an important step in validation as degrader molecules tend to be larger, leading to concerns that may have difficulty entering cells.
- They provide evidence that their degraders induce target degradation in cells, including under endogenous conditions. Importantly, they show that negative control compounds (always critical to have on hand) show no activity, and that inhibiting proteasome rescues observed degradation. Additionally, they confirm that mRNA levels of WDR5 did not change, thus further validating that the reason for decrease in protein levels is due to degradation. (They also include additional pieces of evidence that effects on WDR5 protein levels are degradation dependent)
- Also importantly, the authors show selectivity by quantitative proteomics and demonstrate that WDR5 is the only protein depleted out of more than 5800 identified after 9 hours of treatment (while treatment with individual ligands did not have this effect)
- Lastly, they show anti-proliferative effects in MV4-11 cells of their best performing degraders (these compounds were VHL-based PROTACs). However, the concentration needed for cellular effects was high (10uM). The authors then showed that this is due to low levels of VHL present. When they overexpressed VHL, the growth inhibitory activity improved.
Overall, the work is of high quality and includes appropriate steps for degrader validation. This gives high confidence that WDR5 degraders described in this work are useful as probes for WDR5 biology. For example, what happens to histone methylation once WDR5 is removed? Does removal of WDR5 lead to destabilization (or stabilization) of some of its binding partners (proteomics results suggest that this may not be the case, but would be interesting to dig deeper into this question)? What happens to transcription? What effect does this have on MYC activity (MYC family is known to engage with WDR5)? I am sure the authors and the community have these and many other questions in mind, and I look forward to seeing what new biology they and others can discover with this new generation of tool compounds in hand.
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