Fesik Lab
Targeted protein degradation (TPD) represents an emerging class of therapeutics that selectively degrades disease-relevant proteins.
For example, targeted degradation of oncoproteins in a tumor can inhibit tumor growth while minimizing the toxic side effects of traditional chemotherapy. The degradation relies on the endogenous mammalian ubiquitin ligase pathway, specifically through the use of proteolysis targeting chimera (PROTAC) molecules which recruit E3 ligases to target proteins for ubiquitination. However, TPD strategies are hindered by the limited number of E3 ligases with useful targets.
Fesik Lab
In a recent study from the , the authors leverage an NMR-based fragment screening protocol to identify new targets for E3 ligases. The work focused specifically on the E3 ligase Kelch-like protein 12 (KLHL12), which is overexpressed in cancer cell lines as compared to healthy tissue.
In summary, 13,824 fragments were screened for binding to KLHL12, 35 compounds showed significant shifts in the NMR spectra, and 15 of those had shifts corresponding to the KLHL12 binding pocket. Of these 15, Benzimidazole 1 was identified as the compound with the strongest binding affinity (150 碌M). To increase the binding affinity even further, a collection of close structural analogs to benzimidazole 1 were synthesized, characterized, and modified.
Eventually, the researchers created benzimidazole 7k, which was shown by X-ray crystallography to bind KLHL12 in a similar pose to known ligands. Furthermore, benzimidazole 7k bound KLHL12 with submicromolar affinity (0.33 碌M) as determined by surface plasmon resonance. Additional structural modifications were made in an effort to further increase binding affinity, namely by occupying more of the binding cleft, but few compounds matched the binding strength of benzimidazole 7k.
To determine the feasibility of benzimidazole derived compounds as PROTACS, the specificity of benzimidazole derivatives for KLHL12 was then assessed. Overlay of benzimidazole 7k-bound KLHL12 crystal structures with other KLHL family members indicated that poor binding pocket sequence homology and steric hinderance would likely prevent benzimidazole 7k from binding most other KLHL proteins. KLHL8, KLHL18, and KLHL19 had high degrees of sequence similarity, but fluorescence polarization anisotropy of benzimidazole 11a and KLHL19 indicated very weak binding.
Therefore, benzimidazole derived compounds likely have a high degree of specificity for KLHL12 within the KLHL family. Additionally, when tested against endogenous KLHL12 in HEK293 cells, benzimidazole derivatives were found to bind KLHL12 in both permeabilized and live cells with submicromolar affinities.
While the identification of tight-binding targets for the E3 ligase KLHL12 is only the first step towards a new TPD therapy, this study provides key methodological and structural insights into the development of PROTAC molecules and paves the way for future KLHL12-targeting drug design.
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