Discovery Of Small-molecule Inhibitors Of Cullin Neddylation As A Novel Class Of Anticancer Agents

Cullin-RING E3 ligases(CRLs),the largest family of E3 ligases,consist of 4 subunits: a scaffold cullin,an adaptor,a substrate-receptor,and a RING protein.CRLs are responsible for the ubiquitylation of ?20% of the cellular proteins doomed for proteasome degradation.In the process,a CRL E3 recognizes and recruits the substrate and catalyzes the ligation reaction by transferring ubiquitin from an E2 to a lysine residue on the substrate.Multiple rounds of this reaction lead to substrate polyubiquitylation for proteasome degradation.By promoting the degradation of short-lived key signaling molecules,CRLs play the vital roles in many cellular biological processes,including cell cycle control,gene transcription,signal transduction,DNA replication and repair,and tumorigenesis.Overexpression of several CRL subunits have been found in multiple types of human cancers,which is associated with poor survival of cancer patients.Furthermore,genetic deletion of overexpressed CRL subunits significantly inhibits tumorigenesis in multiple mouse tumor models.Taken together,these preclinical studies strongly validated CRL E3 s as attractive anticancer targets.The activity of CRLs requires cullin neddylation,which,like ubiquitylation,is catalyzed by a three-step enzymatic cascade to attach ubiquitin-like protein NEDD8 to a lysine residue at the C-terminus of cullins.These three enzymes are NEDD8 activating enzyme(NAE),NEDD8 conjugating enzymes(UBE2F and UBE2M),and NEDD8 ligases.Thus,blockage of cullin neddylation would effectively inhibit the ligase activity of CRLs.Indeed,MLN4924,a small molecule inhibitor of NAE has been advanced to several Phase II clinical trials as a novel anti-cancer agent by effectively inactivating CRLs.Given MLN4924-induced cytotoxicity derived from blockage of entire neddylation by inhibiting NAE E1,we seek to discover the small molecule inhibitors that target NEDD8 E3 ligases with expected less cytotoxicity.To the end,we developed a novel Alpha Screen-based high-throughput screen to identify small-molecule inhibitors of CRL5 E3,starting from a simple in vitro CUL5 neddylation assay.In a pilot screen of 17 K compounds,including FDA-proved drugs,MRCT core-structures and natural products,we identified several candidate compounds and focused on gossypol,a natural product known to inhibit anti-apoptotic protein BCL2.We found that gossypol indeed inhibits cullin neddylation,through binding to the E3 complexes of both SAG-CUL5 and RBX1-CUL1.In multiple types of cancer cells,gossypol caused selective accumulation of NOXA and MCL1,the known substrate of CUL5 and CUL1,respectively.Synergetic effect of apoptosis induction was observed when gossypol was combined with a MCL1 inhibitor in cancer cells,revealing a rational combination of anti-cancer agents for effective cancer cell killing.In another larger scale high-throughput screen of Chem Bridge chemical library of 220 K compounds,we identified several inhibitors of cullin neddylation with distinctive structures.Extensive SAR(structure-activity relationship)study led to discovery of K310 as a specific inhibitor of CUL5 neddylation in both in vitro test-tube and in vivo cultured cells.Mechanistically,K310 binds and destabilizes the UBE2F-SAG-CUL5 complex to block cullin-5 neddylation.The future studies are directed to synthesize large quantities of K310 and its analogues for detailed characterization at the levels of biochemistry,cell biology and in vivo xenograft animal models,with an ultimate goal to discover a novel class of anticancer agents with high selectivity against CRL5 and low cytotoxicity.