THNAN69

THNAN69 is a selective degrader of LIMK2 over LIMK1. The PROTAC binds to both LIMK1 and LIMK2 (demonstrated by NanoBRET), but only degrades LIMK2 with a DC50 of 1 nM and a Dmax of 88% at 10 nM - a small Hook effect is observed at 10 uM, far above the recommended cellular concentration. At 100 nM, complete degradation of LIMK2 is observed after 40 hours. Proteomic assessment demonstrates potent degradation of LIMK2 after 6 h treatment, while LIMK1 is unaffected; however, the data provided does not indicate at what concentration the compound is being tested. Two off-targets (CYBA and DGUOK) are identified in the same experiment. Phospho-proteomics shows that phospho-CFLs are not down regulated. The degradation of LIMK2 by THNAN69 is demonstrated to be proteasome dependent (neddylation inhibitor MLN4924 ablates degradation). THNAN69-NC is a negative control than does not degrade LIMK2. No cytotoxicty was observed for THNAN69 or THNAN69-NC up to at least 10 uM. Would recommend use of THNAN69 and THNAN69-NC between 10-100 nM in cells.

THNAN69 is suitable for use as a probe in cellular assays. Its potential for in vivo applications remains to be determined pending additional data. With a DC50 of 1 nM and Dmax values of 88.2% at 10 nM and 97.6% at 100 nM, a concentration range of 10–100 nM is appropriate for initial cellular studies. The optimal concentration for each cell line should be determined empirically prior to selecting the final experimental dosage.