Identification Of Important Residues At The ?/? Subunit Interface Of Class ?a RNA And Screening Of Small Molecular Inhibitors Targeting RRM1 For Cancer

Ribonucleotide reductase?RNR?is the only known enzyme which catalyzes the conversion of nucleotides substrates?NDPs or NTPs?to deoxynucleotides?d NDPs or d NTPs?,providing the cell with the monomeric building blocks required for DNA replication and repair.RNR is widely distributed in various species.RNR is essential for cell proliferation,thus it is one of attractive targets for antineoplastic,antibacterial and antiviral etc.Much remains unknown about RNR's catalytic mechanism,its conformational change and the holo-enzyme structure,and RNR inhibitors such as hydroxyurea and gemcitabine have the deficiency of poor target specificity,high toxicity and drug resistance.In order to elucidate the catalytic mechanism of RNR and provide ideas for designing novel high-efficiency and low-toxic small molecular inhibitors,this article mainly focused on the function of important residues at RNR subunit interface and screening of small molecular antineoplastic inhibitors targeting RRM1.The content of our research on the function of important residues at RNR subunit interface are as follows:1)Based on the docking model for the E.coli active?2?2 complex,we looked at charged residues at the?/?interface that might form subunit interaction and transfer information,and found that two groups of residues?E52-R329/R323 and E350-R639/R735?possibly formed interaction.Protein sequence alignment revealed that R329 in?2,E52 and E350 in?2 were conserved.2)The results of RNR activity assay,K_d measurements and N₃CDP showed that mutation of E52,R329 and R639 resulted in loss of activity and weakened subunit affinity?with the exception of E52Q-?2?,confirming that R329 in?2,E52 and E350 in?2 were essential for enzyme activity and subunit interaction.3)An RNR mutant with 2,3,5-trifluorotyrosine radical?F₃Y122·?replacing the stable Y122·in WT-?2,partly overcoming conformational gating,was placed in the E52Q background.Incubation of E52Q/F₃Y122·-?2 with WT-?2 resulted in formation of pathway-radical intermediates?Y356·and N·?and dCDP.The double mutant could overcome conformational gating and rescue the activity of E52Q,confirming that E52was essential for conformational gating of radical transfer.4)The results of K_d measurements,Pull-down assay,SEC and negative stain EM showed that the interaction of E52Q/F₃Y122·-?2 and WT-?2 during reaction was very strong and they formed an stable and asymmetric?2?2 complex.5)The mutants of R329 and R639 were also studied with F₃Y122·-?2.F₃Y122·-?2could rescue the activity of R329A-?2,R329Q-?2 and R639Q-?2 and increase the activity of R329K-?2,revealing that R329 and R639 played important roles in conformational gating.6)We made the mutants of the corresponding residues in the human RNR subunits and measured their activity.Mutation of E106-h RRM2,E363-h RRM2 and R324-h RRM1caused the decrease or loss of enzyme activity,indicating that these residues were also essential for human RNR enzyme activity.The content of our research on the screening of small molecular antineoplastic inhibitors targeting RRM1 are as follows:1)A method for determinating human RNR activity of various substrate based on PCR was established and optimized.The result of this method was consistent with HPLC and it could be used for high-throughput screening of various types of RNR inhibitors.2)We used the RRM1 catalytic site as ligand-binding pocket,carried out virtual screening of the Dutch SPECS database through various molecular docking techniques,and obtained 287 candidate small molecular compounds targeting RRM1 catalytic site.3)We detected the effects of compounds on the activity of recombinant RNR and found that 9 compounds?RM23,RM26,RM27,RM41,RN15,RN27,RN41,RN78 and RN81?inhibited RNR's activity and were more potent than HU.Their structure characteristics were diverse.Then we detected the effects of these 9 compounds on the proliferation of various tumor cells and found that RN27 and RN81 inhibited the proliferation of various tumor cells and had a broader spectrum of anti-tumor activity.The IC50 of RN81 was 5 to 10?M.4)Choosing RN81 as the hit,we found 19 homologs of RN81 from the SPECS database and the Chemdiv database.RN8106,RN8107,RN8112,RN8113 and RN8116 inhibited RNR enzyme activity and proliferation of various tumor cells by in vitro recombinant RNR activity assay and tumor cell proliferation assay.The activity of these compounds was comparable to or stronger than RN81's.5)RN81 and its three homologues,RN8106,RN8107 and RN8113,showed good docking morphology with RRM1 catalytic site.In the fluorescence quenching experiment,they quenched the fluorescence of RRM1,and the quenching intensity was concentration-dependent.These results proved RN81,RN8106,RN8107 and RN8113could interact with RRM1.6)In the colony formation assay,RN81 and its three homologs,RN8106,RN8107 and RN8113,reduced the formation of clones in three pancreatic cancer cells,confirming again that they inhibited the proliferation of pancreatic cancer cells.d Ns increased the clones and partially attenuated their inhibitory effects,indicating that they inhibited the proliferation of tumor cells by disturbing DNA synthesis.The results of flow cytometry,Ed U and WB showed that these four compounds caused S phase arrest,reduced DNA synthesis,but did not affect the protein level of RRM1 after treatment for 24 h in three pancreatic cancer cells.In the study of RNR's enzyme mechanism,we found that E52 in?2,R329 and R639 in?2,were critical for RNR enzyme activity,and proved that their mechanism was involved in subunit interaction and conformational gating.The corresponding residues such as E363,E106 and R324 were also essential for maintaining human RNR enzyme activity.These results enriched our understanding of RNR's catalytic process.The high proportion and stability of the?2?2 complex formed by E52Q/F3Y122·-?2 and WT-?2provided the possibility to obtain a structure of the active RNR holoenzyme with high resolution.In the research of RNR inhibitors,we obtained several non-nucleoside RRM1 inhibitors with broad anti-tumor spectrum,such as RN81,RN8106,RN8107 and RN8113.We proved that these four compounds inhibited the proliferation of pancreatic cancer cells by inhibiting DNA synthesis.The study of non-nucleoside RRM1 inhibitors provided a new strategy for the research and development of RNR inhibitors.