Peer review of: Enhancing intracellular accumulation and target engagement of PROTACs with reversible (2019)

Reviewers' comments:  

 

Reviewer #1 (Remarks to the Author):  

 

This is an interesting study of the effect of reversible covalent Protac ligands on protein degradation. 
The authors provide compelling data for the effectiveness of their RC-1 ligand in producing BTK 
degradation. I cannot comment on the underlying chemistry but the biological experiments have 
been carefully performed and generally well analysed (but see below).  

 

I do, however, have a few issues with some of the target engagement calculations and the 
determination of IC50 values using the NanoBRET ligand binding approach. According to Figure 4 the 
IC50 values cited in the Tables have been calculated from the concentration that reduces the 
NanoBRET signal by 50% - which is not correct. The IC50 value should be calculated from the specific 
component of binding after subtraction of non-specific binding. In Fig 4b it is clear that the inhibition 
curve obtained with RC-1 levels off significantly above zero binding. This non-displaceable binding is 
likely to represent non-specific binding. The IC50 will therefore be at lower concentrations than 
currently calculated. The same is true for Fig 4c and Fig 4d. Some information should be provided on 
the occupancy of the fixed concentration of fluorescent ligand used in the assay.  

 

Target Engagement IC50 is not “the compound concentration required to achieve 50% BTK target 
engagement measured using a NanoBRET BTK in-cell target engagement assay” as described in the 
Table legends. It is the concentration of compound required to inhibit binding of the fixed 
concentration of fluorescent probe. The IC50 will very much depend on the concentration of probe 
used in the experiments. A much clear explanation is required on this.  

 

I also do not fully understand what is meant by ‘normalised target engagement’ in Table 1 and 2. 
Occupancy (target engagement) is normally represented by [ligand]/([Ligand] + Kd). What do these 
normalised target engagement values represent in the Tables?  

 

I struggled to understand the fluorophore phase separation-based assay for imaging ternary complex 
formation. It looked to me to be a general measure of protein aggregation with the complexes being 
quite large (as judged by their size with respect to the scale bar). I failed to understand how this was 
a measure of ternary complex formation. Surely the spatial resolution is too low?  

 

 

 

Reviewer #2 (Remarks to the Author):  

 

In this manuscript, Guo et al. developed RC-1 as the first reversible covalent BTK PROTAC, which has 
high target occupancy and is effective both as an inhibitor and a degrader. They provided 
biochemical and cellular data to support their conclusion that RC-1 induces mechanism-based BTK 
degradation against both wild-type BTK and C481S mutant form.  

The authors compared RC-1 with reversible noncovalent degrader (RNC-1) and irreversible covalent 
degrader (IRC-1) and found RC-1 shows better activity in cells and suggested that reversible covalent 
degrader could be a good strategy for the development of PORTACS. In addition, they demonstrated 
that the enhanced cellular permeability is responsible for better cellular potencies for RC-1 than 
RNC-1 and ICR-1. Their study suggests that “introducing a cyano-acrylamide moiety can be a general 
strategy to enhance cellular permeability for PROTACs.” To test this idea further, they have designed 
FLT3 degraders and obtained similar results. Finally, they tested RC-1 in vivo, shows BTK degradation 
in spleen, which suggests reversible covalent BTK degraders may be useful for in vivo studies.  

 

Overall, this study was well done and is significant for the field of PROTACs. It is recommended for 
publication in Nature Communications with the following revisions.  

 

1. Since the most attractive point of the manuscript is “introducing a cyano-acrylamide moiety can 
be a general strategy to enhance cellular permeability for PROTACs”, it is more convincing to provide 
direct evidence about the improved cellular permeability for RC-1 over other compounds using the 
Caco-2 cell-permeability assay or other similar assas.  

 

2. It will be useful to make another reversible noncovalent compound as reference, which is directly 
reduce the double bond of RC-1. Compare its cell permeability with RC-1 may give us more 
information, such as whether the enhanced permeability is caused by adding lipophilic groups 
(cyano and di-methyl) or because of the reversible covalent ability.  

 

3. Have the authors compared the BTK degradation between Crews’s compound (such as MT802) 
and RC-1 in Moml-14 cell line or other cell lines? It will be useful to compare these two compounds, 
since MT802 is an optimal reversible noncovalent degrader.  

 

4. In the in vivo PD study, the drug was dosed for 7 days. The PD effect is not as good what has been 
observed in vitro. Since degradation of BTK is already profound within 24 hours in cells, what was 
the reason for the 7-day dosing and for the relative less profound degradation of BTK?  

 

 

 

Reviewer #3 (Remarks to the Author):  

 

This is a well-done and timely study on the effect of semi-reversible BTK PROTACs and showing that 
these molecules are able to degrade BTK in cells. They perform followup proteomic studies and also 
compare their PROTACs to other BTK PROTACs that have been previously synthesized. This study 
complements recent cysteine-targeting semi-reversible BTK PROTAC papers from Nir London's lab 
and Pfizer that have been recently posted on ChemRxiv and BioRxiv. This study should be published 
as is.