From serendipity to essential protac profiling
The microcosmos of biological processes is complex and full of surprises.
This is also the case for a new preprint from Richert, Nลฏskovรก et al.ย While developing rabeprazole-thalidomide hybrids as targeted protein degraders, the authors observed rapid, reversible ATP depletion in cells. Surprisingly, this effect was attributed to PROTAC molecules with a certain molecular structure.
๐ ๐ฒ๐ฐ๐ต๐ฎ๐ป๐ถ๐๐บ
Mechanistic studies traced the effect to inhibition of mitochondrial complex I. Oxygen consumption was dose-dependently suppressed and restored by a complex II substrate, pinpointing mitochondrial complex I as the target.
Systematic structural modification of PROTACs revealed that mitochondrial complex I inhibition occurs only in the presence of long, linear molecules that bind in the elongated (~30 ร
) hydrophobic ubiquinone-binding tunnel.
๐๐น๐ถ๐ป๐ถ๐ฐ๐ฎ๐น ๐๐๐ฎ๐ด๐ฒ ๐ฃ๐ฅ๐ข๐ง๐๐๐ ๐ฎ๐น๐๐ผ ๐ถ๐ป๐ต๐ถ๐ฏ๐ถ๐ ๐บ๐ถ๐๐ผ๐ฐ๐ต๐ผ๐ป๐ฑ๐ฟ๐ถ๐ฎ๐น ๐ฐ๐ผ๐บ๐ฝ๐น๐ฒ๐
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The authors then screened 13 cereblon-recruiting PROTACs and found three clinical stage androgen receptor degraders to be potent inhibitors of mitochondrial complex I:
๐ธ ARV-110: cell-free CI assay pIC50 = 7.66 (22 nM), nearly equipotent with rotenone
๐ธ BMS-986365: CTG/2-DG pEC50 = 6.22 (0.60 ยตM)
๐ธ ARV-766: submicromolar activity with partial inhibition
๐ฆ๐ผ๐น๐๐๐ถ๐ผ๐ป
Structural modification by introducing:
๐ธ “bumps” (e.g., N-acylation of a piperidine linker nitrogen)
๐ธ “kinks” (e.g., using a 1,3-substituted benzimidazolone cereblon recruiter)
A 384-well compatible CTG/2-DG assay was developed to enable routine CI liability screening.
This allowed the authors to abolish mitochondrial complex I inhibition while preserving AR degradation potency in cell-based assays.
๐ง๐ต๐ฒ ๐ธ๐ฒ๐ ๐๐ฎ๐ธ๐ฒ๐ฎ๐๐ฎ๐
Mitochondrial complex I inhibition by PROTACs is not target- or recruiter-specific. It appears to be a general feature of long, linear, hydrophobic molecules, a common molecular architecture across bifunctional modalities. The authors propose rational geometric redesign as a generalizable mitigation strategy.
Are you going to implement this assay to your PROTAC development workflows? Let me know your thoughts!
Full preprint: https://lnkd.in/gWC2_jmk
Share your thoughts and leave your comment under my LinkedIn post here.
