Target-based Design,Synthesis And Bioactivity Evaluation Of Novel HIV-1 RNase H Inhibitors

AIDS is a collection of symptoms known as acquired immunodeficiency syndrome,caused by infection with human immunodeficiency virus type 1(HIV-1),which seriously endangers human life and health.Clinical treatment of AIDS relies primarily on "Highly Active Antiretroviral Therapy"(HAART).HAART has proven to be largely effective in suppressing viral replication and greatly reduce the morbidity and mortality of infected people.However,since current antiviral therapy does not cure HIV infection,the required long duration of HAART is expected to eventually lead to the selection of resistant viral strains,as well as serious side effects,which would eventually limit the efficacy of existing drugs.Therefore,there is still a need for novel anti-HIV drugs with unique resistance profiles to provide more compatible options for existing HAART,particularly for those acting on novel targets or showing different mechanisms of action from those used by currently approved drugs,which are crucial to combat drug-resistant viruses.HIV-1 reverse transcriptase(RT)is a key enzyme in viral replication cycle by converting the viral genomic single-stranded RNA into double-stranded DNA.RT contains two distinct domains:a DNA polymerase domain,containing the active site of the RNA-dependent and DNA-dependent DNA polymerase activity,and an RNase H domain,involved in the hydrolysis of the RNA template in RNA/DNA heteroduplexes formed during reverse transcription.Currently,all approved anti-HIV drugs inhibiting the RT enzyme(nucleoside RT inhibitors(NRTIs)and non-nucleoside RT inhibitors(NNRTIs))target the DNA polymerase activity of the RT,while no inhibitor of RNase H function has entered the clinical trial stage.Since RNase H function plays a pivotal role in HIV-1 replication,it is of great scientific significance to develop RNase H inhibitors as anti-HIV drugs.At present,the reported HIV-1 RNase H inhibitors can be classified into active-site inhibitors and allosteric inhibitors according to their mechanisms of action.Notably,seeking RNase H active-site inhibitors is becoming an important topic in antiviral drug research.Most of the reported active site inhibitors have disadvantages such as poor selectivity,high cytotoxicity,poor membrane permeability and weak antiviral activity due to their own structural characteristics,thus limiting into the clinical stage.As such,researchers still need to endeavor to develop highly active and druggable anti-AIDS drugs targeting RNase H.In this thesis,in view of the shortcomings of existing HIV-1 RNase H inhibitors,and to find anti-HIV-1 lead compounds with novel structure and unique mechanism of action,we explored novel HIV RNase H inhibitors by using scaffold hopping approach and the privileged structure repurposing strategy on the basis of structural biological information of HIV RNase H and the pharmacophore model derived from the reported active site inhibitors.1.Design,synthesis and activity evaluation of dihydroxycoumarins as HIV-1 RNase H inhibitors based on scaffold hopping approachThe dihydroxycoumarin skeleton has proven to bind to the active site of RT RNase H domain and coordinate with two magnesium ion cofactors involved in catalytic functions at the active site,thereby exerting RNase H inhibitory activity.In our previous study,we found a dihydroxycoumarin derivative,which showed a low micromolar RNase H inhibitory activity(IC50=12.3μM)and significant antiviral activity in cell culture with an EC50 of 3.94 μM.These studies suggest that the dihydroxycoumarin skeleton is a privileged chemical scaffold for developing HIV-1 RNase H inhibitors with improved activity and druggability through structural optimization.Furthermore,our group focused on exploring the polyphenol metal-chelated skeleton and identified a novel double-winged galloyl derivative DF-36 as HIV-1 RNase H inhibitor.It exhibited RNase H inhibitory activity in the nanomolar range with an IC50 of 0.067 μM,although the compound was inactive in antiviral assays.The results of cell unidirectional permeability assay indicated that DF-36 showed poor cell permeability,which may result in the inability of DF-36 to penetrate the phospholipid bilayer and to exhibit antiviral activity.The membrane permeability of DF-36 was predicted by the membrane permeability module of Schrodinger Suites 2022-1.The results of this prediction showed that polyphenolic skeleton of DF-36 appears as the most important determinant contributing to the poor membrane permeability of the compound.Based on the above analysis,we selected DF-36 as the lead compound and transformed the polyphenol skeleton which mainly affects the membrane permeability into the privileged dihydroxycoumarin metal chelating core,while retaining the privileged "double-winged"substituents of DF-36.Finally,a series of 28 novel dihydroxycoumarin derivatives were obtained and expected to identified novel molecules with excellent RNase H inhibition and antiviral activity.The target compounds were tested for HIV-1 RNase H inhibitory activity by polyacrylamide gel electrophoresis.The results showed that most of the compounds exhibited RNase H inhibitory activity in the low micromolar range(IC50=3.7μM～17.9μM).The best compounds were Ⅰ-4 and I-27,and their IC50 values for inhibiting RNase H were 4.2μM and 3.7μM,respectively,but they were low potent than β-thujaplicinol(IC50=1.98 p.M)and DF-36(IC50<0.5μM).Molecular modeling suggests potential interactions between the dihydroxycoumarin chelating core(i.e.1-O-7-OH-8-OH chelating triad)and the two divalent metal cofactors(Mg2+),and a hydrogen bond was formed between the 7-position hydroxyl group and the side chain of His539,which would stabilize the inhibitor positioning near the four metal-chelating residues(Asp443,Glu478,Asp498 and Asp549).The two hydrophobic substituents of the piperazine ring are oriented towards different protein-solvent interfaces,thereby helping in the stabilization of the chelation geometry.Then,only one compound Ⅰ-1 showed weak anti-HIV-1 activity with EC50 of 117.32μM in MT-4 cell line by the MTT method.It is worth mentioning that three derivatives(Ⅰ-2,Ⅰ-5,Ⅰ-26)of this series showed certain inhibitory activity against HIV-2 strain,and their EC50 values were 175.53μM,132.28μM and 173.48μM,respectively.2.Discovery of novel HIV-1 RNase H inhibitors based on the privileged structure repurposing strategyAlthough medicinal chemists have worked hard for nearly two decades to discover more than a dozen structural types of HIV-1 RNase H inhibitors,and some compounds have even reached the inhibitory activity at the nanomolar level,no inhibitor has yet entered clinical research,probably due to their unique structural characteristics are not conducive to the physicochemical properties and druggability.Therefore,it is still urgently necessary to explore chelators with novel metal chelating cores as HIV-1 RNase H inhibitors.In order to improve the druggability of RNase H inhibitors,we focus on the approved metalloenzyme inhibitors.Influenza virus endonuclease and HIV-1 RNase H belong to metal-dependent proteins,their active sites have similar amino acid arrangements,and the enzyme active centers contain two divalent metal ions necessary for enzyme catalytic activity and have similar catalytic mechanisms.Current influenza endonuclease inhibitors have similar pharmacophore models with HIV-1 RNase H inhibitors,and their metal chelating groups mostly contain three-dimensional structures such as aliphatic rings and spiro rings,which may be conducive to improving physicochemical properties and drug-like profile of compounds.Therefore,we can refer to the structural characteristics of the approved influenza virus endonuclease inhibitor baloxavir to develop HIV-1 RNase H inhibitors.In this chapter,we repositioned the privileged metal chelating skeleton of baloxavir to the metal chelating region of the catalytic site of HIV-1 RNase H based on the privileged structure repositioning strategy.A series of 50 novel compounds were designed and synthesized as HIV-1 RNase H inhibitors using methylene as a flexible linker and different substituted benzene rings and biphenyl groups as narrow hydrophobic side chains.The results of HIV-1 RNase H inhibition assays showed that all the target compounds did not exhibit RNase H inhibitory activity under the test concentration.The catalytic process mediated by divalent metal ions is a dynamic process.Although the catalytic mechanism of metalloenzymes is very similar,small structure change of compounds may has a significant impact on their inhibitory activities,which may be an important reason for its lack of RNase H inhibitory activity.The results of RNase H inhibitory activity showed that the baloxavir tricycle was not a good pharmacophore for RNase H active site.In the infected MT-4 cell cultures,most of the compounds had high toxicity(CC50=0.76μM-31.73μM),and only two compounds(Ⅱ-9 and Ⅱ-17)had low micromolar antiviral activity with EC50 values of 22.6μM and 25.42μM,respectively.Although Ⅱ-9 and Ⅱ-17 do not have RNase H inhibitory activity,they show low micromolar antiviral activity.Considering that RNase H and integrase active sites have similar catalytic mechanisms,it is speculated that the target of compoundsⅡ-9 and Ⅱ-17 may be HIV integrase.