Discovery And Evaluation Of Bioactive Ligands Targeting Nuclear Receptors Based On Molecular Stimulation Technologies

In the early phase of drug development,the application of molecular dynamics stimulation has emerged as a reliable,cost-effective and time-saving methods for the discovery of lead compounds.Glucocorticoid receptor(GR)and androgen receptor(AR)belong to classical nuclear receptors.The dysfunction of the two receptors is closely associated with the development of a series of diseases.Dozens of drugs targeting GR or AR have been developed,but these drugs are limited in clinical practice due to their adverse side effects or drug resistance.To overcome the limitations of these drugs,it is of great significance to develop novel bioactive ligands targeting AR or GR.Therefore,based on molecular dynamics simulation,structure-based virtual screen was applied in the discovery of bioactive ligands targeting the GR ligand binding site(GR LBD)and the AR DNA binding site(AR DBD).Finally,we successfully discovered several bioactive compounds with new scaffolds.And the research was mainly divided into two parts:Part I:to avoid the side effects of glucocorticoids(GCs)that are mainly related with the transactivation function of GR and low selectivity during long-term or high-dosage treatment,three different virtual screening methods based on molecular stimulation were carried out to explore novel selective GR modulators(SGRMs)as the following:(1)By combining conventional structure-based virtual screening and bioassays,HT-15,a prospective SGRM,was discovered.HT-15 could selectively act on the NF-?B/AP-1-mediated transrepression activity of GR and down-regulate the m RNA expression of pro-inflammation cytokines(NF-?B:IC₅₀=1.86?M;AP-1:IC₅₀=1.06?M)and show evidence of less side effects.(2)For the purpose of improving the hit ratio,we introduced Interaction Graph Net(IGN)developed by our research group into the pipeline of virtual screening.This is also a first try of IGN application.And then we identified a novel SGRM HP210(NF-?B:IC₅₀=2.32?M).Based on the chemical structure of HP210,we discovered HP210?b4 that had an effective transcriptional repression activity against NF-?B(NF-?B:IC₅₀=0.99?M).(3)However,the anti-inflammation activity of the newly discovered SGRMs above was relatively low when comparing with GCs.We speculated that a virtual screening based on the static crystal structure could not predict SGRMs accurately since the static crystal structures of GR LBD/modulator complex is most likely different from true conformations.To face the challenge,we analyzed the conformational transformation of GR LBD induced by different types of ligands with molecular dynamics simulation and markov model system.The results showed that various ligands had diverse impact on the folding pathway of helix 12(H12)of GR.Especially,GR modulators tented to induce H12 of GR moving from outward to inward showing an antagonistic conformation,narrowed the volume of surface exposed pocket-the activation function 2(AF2)region and perturbed the original steady structural state of AF2,resulting the AF2 not conducive to the binding of coactivator factors.Then a passive antagonistic conformation of GR LBD/AZD9567 was applied for a new round of virtual screening to identify potentially safer GR modulators.Six compounds targeting the GR LBD were identified.Therein HP-19 showed the best anti-inflammatory activity in NF-?B signaling pathway(IC₅₀=41±1.0 n M),which was comparable to that of Dex(NF-?B:IC₅₀=12 n M).Comparing with Dex and AZD9567,HP-19 did not induce the transactivation of GR,which might reduce side effects caused by GCs.Part II:AR antagonists,developed by targeting the ligand binding pocket(LBD)of AR for the treatment of prostate cancer,have encountered a major clinical challenge of drug resistance.Perturbing the binding of AR and the DNA sequence of androgen receptor response elements is an effective strategy to repress AR signaling pathway.Based on the hypothesis,a structure-based virtual screening by integrating multiple docking methods was employed to discover novel AR antagonists targeting AR DBD.We discovered a novel small-molecule inhibitor Cpd39 targeting AR DBD.In vitro assays,Cpd39inhibited AR transcriptional activity,effectively blocked the binding of AR DBD and DNA,and repressed the m RNA expression of specific genes regulated by AR and AR V7.It could serve as a starting point for further structural optimization.In summary,we successfully discovered several ligands with novel scaffolds by combining in silico methods and bioassays,including SGRMs(HT-15,HP210?b4 and HP-19)and a novel AR antagonist Cpd39.Our study provided new insights for the discovery of bioactive ligands targeting nuclear receptors.