Macrocyclic peptides have emerged as promising drug candidates, filling the gap between small molecules and large biomolecules in drug discovery. The antiapoptotic protein myeloid cell leukemia 1 (MCL1) is crucial for numerous cancers, yet it presents challenges for selective targeting by traditional inhibitors. In this study, we identified a macrocyclic peptide, 5L1, that strongly binds to MCL1, with a dissociation constant (KD) of 7.1 nM. This peptide shows the potential to specifically inhibit the function of MCL1, and demonstrates effective antitumor activity against several blood tumor cell lines with the half maximal inhibitory concentration (IC50) values for cell-penetrating peptide-conjugated 5L1 in the range of 0.6 to 3 μM. Structural analysis revealed that it functions similarly to molecular glue, capable of binding to two MCL1 molecules simultaneously and inducing their homodimerization. This unique mechanism of action distinguishes it from traditional small-molecule MCL1 inhibitors, underscoring the potential of macrocyclic peptides functioning as molecular glues. Moreover, it inspires the development of highly selective inhibitors targeting MCL1 and other related targets with this glue-like mechanism.
Qing Yu,Zhiguo Hu,Yanwen Shen,Yihan Jiang,Peichen Pan,Tingjun Hou,Zhen-Qiang Pan,Jing Huang,Yi Sun
Cullin-RING E3 ligase (CRL) is the largest family of E3 ubiquitin ligase, responsible for ubiquitylation of ∼20% of cellular proteins. CRL plays an important role in many biological processes, particularly in cancers due to abnormal activation. CRL activation requires neddylation, an enzymatic cascade transferring small ubiquitin-like protein NEDD8 to a conserved lysine residue on cullin proteins. Recent studies have validated that neddylation is an attractive anticancer target. In this study, we report the establishment of an Alpha-Screen-based high throughput screen (HTS) assay for in vitro CUL5 neddylation, and screened a library of 17,000 compounds including FDA approved drugs, natural products and synthetic drug-like small-molecule compounds. Gossypol, a natural compound derived from cotton seed, was identified as an inhibitor of cullin neddylation. Biochemical studies showed that gossypol blocked neddylation of both CUL5 and CUL1 through direct binding to SAG-CUL5 or RBX1-CUL1 complex, and CUL5-H572 plays a key role for gossypol binding. On cellular level, gossypol inhibited cullin neddylation in a variety of cancer cell lines and selectively caused accumulation of NOXA and MCL1, the substrates of CUL5 and CUL1, respectively, in multiple cancer cell lines. Combination of gossypol with specific MCL1 inhibitor synergistically suppress growth of human cancer cells. Our study revealed a previously unknown anti-cancer mechanism of gossypol with potential to develop a new class of neddylation inhibitors.
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