SJF1528

Reviewer notes:

• The biochemical potencies listed above are approximately that of lapatinib (Chembl lists the IC50 of lapatinib on EGFR and HER2 to be between 2 < IC50 < 10 nM depending on the assay format).  No in vitro biochemical potency was provided for the actual PROTAC.
• No ‘hook effect’ was observed with this PROTAC in OVCAR8 even at a top dose of 10 micromolar.
• Compound 5 in the article (the same warheads but with a linker extended by 1 extra ethylene glycol unit appears to be a more selective reagent, showing no degradation of HER2, unlike the compound shown here (Compound 1 in article).  While an EGFR DC50 for Compound 5 was not quantified, the potency by gel looks similar to Compound 1 (Figure 2A).
• Compound 2 is a diastereomer of the hydroxyproline of the VHL-binding motif and ablates VHL binding, making a quality negative control.
• Despite evidence of broad RNA expression of VHL, VHL has not been observed in all tissues at the protein-level (https://www.proteinatlas.org/ENSG00000134086-VHL/tissue), so a negative result may require quantification of VHL abundance for interpretation.
• See comment above about sensitivity of DC50 to EGFR mutational status. 

This probe is highly recommended for the purposes of studying the phenotypic outcomes of degradation of HER-2 (ERBB2), EGFR, L858R or Exon 19 del EGFR in cells. Other than these RTK targets, as a result of the highly selective kinase profile of lapatinib from which this probe is based, this is anticipated that the degradation probe is highly selective against other kinases or off-targets. In terms of its use in cells, the only disadvantage of this probe is that it does not discriminate between EGFR and HER-2 in its degradation characteristics. It should be noted that the pharmacokinetic properties of this probe have not been described and therefore additional experiments would need to be undertaken in order to conduct any in vivo studies.