Discovery Of Novel Neddylation Co-E3 DCN1 Inhibitors And The Anti-Cardiac Fibrotic Activity Evaluation

Heart failure with high morbidity and mortality has seriously threatened human health worldwide.The main pathological features of heart failure include cardiac remodeling and fibrosis.Recent years,although significant progress for heart failure treatment,there are no approved drugs for the treatment of cardiac fibrosis.CRLs are the largest family of ubiquitin E3 ligases and control the turnover of about 20%of mammalian cellular proteins.The activation of each CRL depends on neddylation of individual cullin protein.Neddylation,an ubiquitination-like modification,can conjugate NEDD8 to specific target proteins through a tripartite enzymatic cascade mediated by E1-E2-E3 enzymes,with cullins being the best-characterized substrates.Targeting neddylation has been regarded as a promising approach for disease treatment.Especially,E1 inhibitor MLN4924 and TAS4464 are in clinical stage for cancer therapy.Besides,the important roles of neddylation modification for heart and fibrotic diseases have been gradually disclosed,while targeting neddylation or critical proteins of this pathway for cardiac fibrosis treatment remains unknown.DCN1 is a vital co-E3ligase in the neddylation pathway.Targeting DCN1 could selectively inhibit cullin neddylation,which is valuable for exploring the biological functions of each CRL and may result in lower side effect.However,the development of DCN1 inhibitors is extremely challenging.To date,the chemotypes of the reported DCN1 inhibitors are not rich enough,and lots of efforts are still needed for improving their activity,selectivity and pharmacokinetic properties.Moreover,the reported DCN1 inhibitors,except for the treatment of cancers and liver injury,have been rarely investigated for other diseases.Therefore,there is considerable significance in developing novel DCN1inhibitors with strong inhibitory activity,high selectivity,low toxicity and drug-like properties,which not only can enrich the structural types,but also may be beneficial for exploring the potential biological functions.In the current study,we designed and synthesized two novel series of DCN1 inhibitors and evaluated their potential effects on the treatment of cardiac fibrosis in vivo and in vitro.PartⅠ:Discovery of novel 2-thiopyrimidine-based DCN1 inhibitors and their anti-cardiac fibrotic activity evaluation（1）Based on the docking modes of DCN1 inhibitor DC-2 reported by our lab in 2019,we conducted further structural optimizations and structure-activity relationship studies（SARs）using several drug design strategies,including structure-based drug design and bioisosterism etc.,yielding 77 novel 2-thiopyrimidine derivatives as DCN1 inhibitors,which were characterized by NMR and MS.（2）HTRF assay indicated that 14 derivatives with the IC₅₀ values ranging from 2 to 20 n M,displayed comparable or more potent biological activity on DCN1,compared with the positive control DC-2(IC₅₀=18.31±1.12 n M)and COV(IC₅₀=58.21±0.60n M).Especially,in comparison to DC-2,compound 76（IC50=9.55±0.98 n M）possessed stronger inhibitory effect,higher selectivity toward DCN-like proteins and weaker toxicity against cardiac fibroblasts（CFs）.Molecular docking,biolayer interferometry,NAE and co-immunoprecipitation assays confirmed that compound 76could specifically target DCN1 and interfere DCN1-UBC12 interaction.（3）Based on the expressions of DCN1 at the m RNA and protein levels in the activated CFs as well as the previous reports,we speculated that DCN1 may be involved in cardiac fibroblast activation.Thus,the potential effects of compound 76 on cardiac fibrosis and the possible mechanism next were explored.The results exhibited that molecule 76 could significantly inhibit the proliferation and migration of CFs induced by Ang II.Besides,compound 76 treatment or silencing DCN1 effectively relieved Ang II-induced cardiac fibroblast activation,which was related to the inhibition of neddylated cullin3 and its substrates Nrf2 and NQO1 accumulation.Overall,we identified a 2-thiopyrimidine-based DCN1 inhibitor 76 with potent activity,high selectivity and low toxicity,which could effectively reduce AngⅡ-induced CFs activation,suggesting the potential anti-cardiac fibrotic effect through targeting DCN1.PartⅡ:Discovery of novel 3-thiol-1,2,4-triazole derivatives as DCN1 inhibitors and their anti-cardiac fibrotic activity evaluation（1）Based on the hit HD1 identified by high-throughput virtual screening,we performed further structural optimization and SARs through multiple drug design methods,such as structure-based drug design and bioisosterism ect.,giving 67 new 3-thiol-1,2,4-triazole-based DCN1 inhibitors,characterized using NMR and MS.（2）HTRF assay indicated that 14 of the synthesized compounds showed robust inhibitory activity with the IC₅₀ value ranging from 2 to 20 n M.Particularly,compound HD2(IC₅₀=2.96±0.80 n M)were 6-folds and 20-folds more active than the positive control DC-2 and COV,respectively.Compared to DC-2,HD2 also revealed higher selectivity toward DCN-like proteins and lower toxicity against CFs.Molecular docking and molecular dynamics simulations exhibited that HD2 could well occupy the active pocket and form several critical interactions with DCN1.Cellular thermal shift assay（CETSA）presented that HD2 could specifically target intracellular DCN1and enhance its stability.（3）Next,we found that HD2 could effectively suppress the proliferation and migration of CFs induced by Ang II.Besides,HD2 possessed favorable pharmacokinetic properties,potential drug-like properties and good in vivo safety.More importantly,HD2 not only revealed beneficial effects on relieving Ang II-induced CFs activation,but also could effectively reduce isoproterenol（ISO）-induced cardiac remodeling and fibrosis,connected with the suppression of neddylated cullin3and its substrates Nrf2 accumulation.Together,3-mercapto-1,2,4-triazole derivative HD2 can serve as a highly potent and selective DCN1 inhibitor with low toxicity and drug-like properties,which displays anti-cardiac fibrotic effect in vitro and in vivo.In general,we have designed and synthesized 144 new DCN1 inhibitors and performed systematic SARs based on scientific and rational design strategies,which not only enriches the structural types of DCN1 inhibitors,but also shows the 2-thiopyrimidine derivative 76 and 3-mercapto-1,2,4-triazole derivative HD2 can serve as promising lead compounds for further optimization and development.Furthermore,we,for the first time,report the effects of targeting DCN1 on relieving cardiac fibrosis,probably offering a significant theoretical and experimental basis on developing DCN1inhibitors for cardiac diseases,such as heart failure.