Design,Synthesis And Biological Activity Of Novel NAMPT Degraders

The emergence of small molecule inhibitors has reduced adverse reactions during drug treatment,while targeted protein degradation（TPD）technology,represented by PROTACs,provides a new strategy.It no longer only inhibits the POI,but completely eliminates it.Compared with traditional small molecular inhibitors,the advantages of PROTACs mainly include:（1）moderate binding affinity of POI ligands required for degradation;（2）catalytic profile and low concentration to take effect;（3）degrading undruggable targets;（4）avoiding drug resistance;（5）blocking both enzymatic and nonenzymatic activities of POI.Up to now,PROTAC has been applied in more than 70 targets.However,PROTAC also has its limitations,such as poor selectivity to tumor cells,the drug resistance induced by E3 ligase,and limited target range.Therefore,researchers have developed a series of new strategies for targeted protein degradation,including photo-controlled PROTACs,multi-target PROTACs,PROTAC conjugates,and lysosomal based degradation technologies such as ATTEC,AUTAC,LYTAC,and AUTOTAC,which compensate for the shortcomings of traditional PROTACs.Our group designed and synthesized NAMPT PROTAC with excellent antitumor activity in vitro and in vivo.During the research process,we found that it is still difficult to real-time detect and track the protein degradation.Currently,most assays for the evaluation of the protein degradation activity relied on Western-blot.More convenient tools for visualization would contribute to develop more efficient assays and facilitate better understanding the mechanism of protein degradation.In addition,we hope to further improve the accuracy and efficiency of PROTACs and explore the possibility of multi-channel degradation of NAMPT.Therefore,this dissertation proposes a new strategy for multi-dimensional NAMPT degradation:realize visual degradation,selective degradation and multi-channel autophagic degradation.1.Making Protein Degradation Visible:Discovery of Theranostic PROTACs for Detecting and Degrading NAMPTIn order to solve the problem that the degradation process can not be monitored in real time,11 target compounds were obtained by rational design on the basis of the fluorescent NAMPT small molecule inhibitor II-A1 found by virtual screening in the early stage.Most of the PROTACs retained the NAMPT inhibitory activity of the prototype compounds in vitro and compound II-B4 showed the best activity(NAMPT IC₅₀=41.9 n M).Besides,it also exerted excellent NAMPT-degrading activity and antitumor activity in vitro(A2780DC₅₀=8.4 n M,A2780 IC₅₀=12.1 n M)and had a stronger ability to down-regulate intracellular NAD⁺.Compared with II-A1,the fluorescence intensity of the compound II-B4 was improved by five times,meanwhile,the compound II-B4 showed more obvious environmental sensitivity and the fluorescence intensity is obviously improved after being combined with NAMPT protein.Moreover the fluorescence intensity reduced along with the degradation of the NAMPT protein.This result confirmed that compound II-B4 could simultaneously detect and degrade NAMPT protein in A2780 cells and could be used as a tool molecule to track the degradation process in real time.All the mice injected with compound II-A1 died after the ninth day,while the mice injected with II-B4 kept alive,proving its low toxicity.Compound II-B4 showed excellent antitumor activity（TGI=73%）in the A2780 nude mouse xenograft tumor model indicating that compound II-B4 showed lower toxicity and better in vivo antitumor efficacy than the inhibitor.In this work,the first class of NAMPT fluorescent PROTACs with integrated diagnosis and treatment was designed and the visualization of the degradation process was realized for the first time,which provided an efficient small molecule probe for the subsequent degradation mechanism research and target drug property exploration.In addition,it expanded a new research direction for PROTAC drug development.2.Controlling NAD⁺Level with Light:Optical Regulation of NAD⁺with Photo-responsive NAMPT PROTACsIn view of the fact that the existing PS-PROTACs could not avoid the inhibitory toxicity to normal cells,22 target compounds were designed based on the NAMPT agonistⅢ-3 and the NAMPT degraderⅢ-2.First,the NAMPT agonist-based PROTACs were proved to have the same degradation activity.Then,a series of PS-PROTACs were designed by introducing the light control element dimethylpyrazolazobenzene.Compound III-D1showed the strongest degradation activity and anti-tumor activity in vitro(A2780 DC₅₀=3n M,A2780 IC₅₀=7.6 n M)and had faster degradation rate,wider degradable concentration range and significantly improved PK properties compared with compound III-2.It showed excellent antitumor activity（TGI=75.1%）in A2780 nude mice xenograft model.CompoundⅢ-B3 also showed excellent degradation activity and antitumor activity in vitro(A2780 DC₅₀=217 n M,A2780 IC₅₀=122 n M).Besides,compoundⅢ-B3 as PS-PROTAC had a high rate of configuration transition and a long thermal relaxation half-life.The positive and negative regulation of the expression levels of NAMPT protein and NAD⁺in cells could be realized under the illumination of different wavelengths.In addition,compoundⅢ-B3 had excellent PK properties and it could significantly inhibit tumor growth at a dose of 5 mg/kg（TGI=80.8%,IV,QD）.Moreover,it could show different degrees of anti-tumor activity in vivo through illumination.More importantly,compoundⅢ-B3 did not show the toxicity of the NAMPT inhibitor structure.It could achieve more accurate degradation.In this work,we designed the first class of PS-PROTACs based on NAMPT agonists,which realized the reversible regulation of NAMPT protein and NAD⁺for the first time.It could be used as a tool molecule to promote the study of NAMPT protein and NAD⁺.Besides,it provided a new strategy for the design of PS-PROTACs.In addition,the excellent PK properties,in vivo anti-tumor activity and low toxicity of the compoundⅢ-B3 provide new strategies for the research and development of new drugs targeting NAMPT.3.Design and Mechanism Validation of Novel Degraders Based on Autophagy-Lysosome PathwayIn this study,the activity verification of the degrader IV-1 showed that the compound IV-1 exerted excellent anti-tumor activity by degrading NAMPT.The degradation mechanism research confirmed that the compound IV-1 could simultaneously bind to the LC3 protein and the NAMPT protein in the cell and induce the degradation of the NAMPT protein through the autophagy-lysosome pathway.Although the activity of the compound IV-1 was not superior to that of a previously designed NAMPT PROTAC,this work proved that NAMPT could also be effectively degraded through ALP,which provided an alternative strategy for degrading NAMPT.Compound 41 was an effective LC3 ligand,which provided a new tool molecule and example for the design of ATTEC degraders.However,since compound 41 is a highly active KSP inhibitor,further optimization was needed to improve its binding efficacy and selectivity to LC3 protein.In addition,this chapter also explored the BET AUTOTACs.Although the final degradation activity was not strong,the feasibility of this strategy was preliminarily proved and other p62 ligands were further explored in the future.In summary,this chapter provided design strategies and examples for novel targeted protein degradation based on the autophagy-lysosome pathway.