Structure-Based Design,Synthesis And Biological Evaluation Of HBV Capsid Assembly Modulators And Degraders

Hepatitis B virus(HBV)is an enveloped DNA virus that specifically infects hepatocytes and causes an immune response leading to acute or chronic hepatitis B(CHB),and CHB can progress to life-threatening diseases such as cirrhosis or hepatocellular carcinoma(HCC).HBV infection poses a significant global public health challenge.According to World Health Organization(WHO)estimates,there were approximately 296 million HBV-infected people worldwide by the end of 2019.The incidence of HBV infection continues to rise,with approximately 1.5 million new infections reported annually.China has the highest prevalence of HBV infection,with approximately 70 million HBV-infected people.Drugs currently approved for HBV infection are mainly divided into two categories:interferon and nucleos(t)ide analogues(NUCs).However,interferon has the disadvantages of serious adverse reactions and poor patient compliance,while NUCs are prone to rebound after drug withdrawal,and drug resistance has emerged.Neither of the above two types of drugs can meet the needs of clinical treatment.Therefore,it is an urgent task to develop efficient and low-toxic anti-HBV drugs acting on new targets.HBV capsid is a structural protein required for the formation of a mature virus particle.It is assembled from 90 or 120 core protein(Cp)dimers,wraps and protects the viral genome,and interacts with host proteins to regulate the viral replication cycle.The versatility of the HBV capsid makes it an attractive target for drug design.Currently,a variety of chemotypes of HBV capsid assembly modulators(CAMs)have been reported,among which heteroaryldihydropyrimidine(HAP)is one of the most widely and deeply studied scaffolds.HAP derivatives accelerate the assembly of Cp into abnormal capsids,which are subsequently degraded in hepatocytes.An important representative of HAP derivatives,GLS4 with excellent antiviral activity,is currently in phase III.However,GLS4 is rapidly metabolized in humans,so multiple doses alone cannot achieve anticipated blood concentrations to show the desired therapeutic effect.To address this problem,GLS4 was used in combination with 100 mg ritonavir in a clinical trial treatment regimen.Nevertheless,this approach introduces the risk of drug-drug interactions(DDIs)and potential adverse reactions due to the addition of nontherapeutic drugs like ritonavir.Moreover,GLS4 exhibits potent inhibition of the human etherà-go-go-related gene(hERG)-encoded channel(IC50=1.34 μM),which can potentially lead to QT interval prolongation and the subsequent development of torsades de pointes ventricular tachycardia.In severe cases,this condition may result in sudden death.In summary,the metabolic instability and strong hERG toxicity significantly limit the clinical application of GLS4.On the other hand,mutations accumulated in the HAP pocket predispose to drug resistance,and these mutations are rare in CAM-na(?)ve patients,but the incidence of mutations has risen rapidly in early clinical trials.Therefore,the potential drug resistance of HBV CAMs should not be underestimated and will become an important issue in the clinical treatment of CAMs in the future.In the second chapter of this dissertation,34 HAP derivatives were designed and synthesized to improve antiviral activity and drug-likeness properties using structure-based drug design and multi-site binding strategy.We replaced the metabolizable morpholinyl at the 6-position of the GLS4 dihydropyrimidine core with a piperazinyl to mitigate metabolic liabilities;introduced a carboxyl group to reduce the hERG toxicity;introduced a bulky hydrophobic group to occupy the solvent-exposed region and gain additional protein-ligand interactions.In vitro anti-HBV activity screening showed that Ⅰ-6a-25(EC50=0.020 ± 0.005μM)had potent antiviral activitie,which was superior to the positive control lamivudine(EC50=0.09 μM)and slightly weaker than the lead compound GLS4(EC50=0.007±0.003 μM).In addition,Ⅰ-6a-25 reduced intracellular core proteins and capsids,suggesting that its mechanism is similar to that of GLS4.Molecular docking revealed that the newly introduced 6-position substituent extended better toward the solvent-exposed region:forming a hydrogen bond with side chain O atom of Ser141 and hydrophobic interactions with Pro134,Thr125 and Pro138.Preliminary drug-likeness evaluation results showed that Ⅰ-6a-25 was superior to GLS4 in terms of water solubility(pH=2.0:374.81 μg/mL;pH=7.0:6.85 μg/mL;pH=7.4:25.48 μg/mL),in vitro metabolic stability(t1/2=108.2 min)and hERG inhibitory effect(72.66%at 10 μM),which was valuable for further research.Aiming at the potential drug resistance of CAMs,the proteolysis targeting chimera(PROTAC)technology was applied to HAP derivatives in the third chapter of this dissertation to exert stronger degradation and improve the antiviral activity against wild-type and mutant Cp and we have conducted an extensive exploration of HAP-PROTACs.For the E3 ubiquitin ligase ligand,we chose a CRBN ligand,lenalidomide;for the linker,we chose flexible alkyl or glycol chains ranging from 2 to 14 atoms in length;for the HBV Cp ligand,we chose the phase III candidate,GLS4.In addition to the positive HAP-PROTACs(group B),compounds without lenalidomide(group A)and compounds containing methylated lenalidomide(group C)were also designed as controls,and a total of 21 compounds were designed and synthesized.The results of antiviral activity in HepDES 19 cells showed that the most potent compound among PROTACs(group B)was Ⅱ-13a(EC50=0.60 ± 0.20 μM),which was comparable to the positive control lamivudine(EC50=0.40±0.00 μM).In HepAD38 cells,the cytotoxicity of HAP-PROTAC II-13a(CC50>100μM)was significantly lower than that of control compounds Ⅱ-2a(CC50=18.3 ± 6.0 μM)and GLS4(CC50=50.2 ± 0.8 μM).The representative PROTAC Ⅱ-13a and its control compound Ⅱ-2a were evaluated in concentration-dependent degradation experiments.The results showed that both Ⅱ-13a and Ⅱ-2a could reduce the content of HBV Cp and capsid in a dose-dependent manner.The degradation effect of Ⅱ-13a was stronger than that of II-2a,indicating that PROTACs can exert stronger degradation effects,which initially verified the rationality and feasibility of the application of PROTAC technology on HBV Cp.In summary,aming at the poor drug-likeness properties and potential resistance of HBV CAMs clinical candidates,we designed and synthesized two series of 55 target compounds using a combination of structural biology information,medicinal chemistry strategies,and protein degradation techniques.On the one hand,after activity evaluation and drug-likeness evaluation,a promising lead compound Ⅰ-6a-25 with improved drug-like properties and maintained antiviral activity was found.On the other hand,a novel HBV capsid degrader Ⅱ13a was discovered based on PROTAC technology,which provided a new idea for the development of anti-HBV drugs.