| AIDS is a malignant infectious disease caused by human immunodeficiency virus type-1(HIV-1)through infecting T cells,which leads to the destruction of immune function and causes opportunistic infection and tumor.Reverse transcriptase(RT)is an important target for the design of inhibitors due to its essential role in the life cycle of HIV-1.The nucleoside reverse transcriptase inhibitors(NRTIs)and non-nucleoside reverse transcriptase inhibitors(NNRTIs)targeting HIV-1 RT are important components of highly active antiretroviral therapy(HAART).Besides,NNRTIs has been the hot research filed of drug development because of its high efficiency and low resistance properties.However,the incidence of drug resistance is greatly increased and the side effects caused by long-term drug administration can not be ignored in the current clinical treatment of HIV-1 infection.Moreover,the existence of HIV latent reservoir makes it difficult to cure AIDS by HAART treatment completely.Therefore,it is still necessary to find new generation of high-efficiency inhibitors with less drug resistance by traditional drug design methods.At the same time,it is imperative to introduce new technologies and strategies to enrich the methods of drug discovery.In recent years,the crystal structure of a large number of small molecule-protein complexes has been resolved with the rapid development of structural biology.The elucidation of the action mode of small molecular drugs provides the theoretical basis for structure-based drug design(SBDD).At the same time,the progress of human genomics and the emergence of new technologies and strategies also increased the possibility for the cure of AIDS.According to the structural characteristics and adaptability of HIV-1 RT,this paper carried out structural diversity modification on the left hydrophobic region of HIV-1 RT and preliminary research based on the new strategies to find novel high-efficiency lead compounds with low resistance properties.Design,synthesis and activity evaluation of HIV-1 NNRTIs targeting the hydrophobic region of NNIBP.In this chapter,the second generation NNRTI(RPV)was used as the lead compound,with the aim to improve its pharmacological and physicochemical properties,including strong cytotoxicity(Michael receptor structure),poor solubility and drug resistance.By the investigation of a large number of RPV/RT complex crystal data,a systematic structural modification of the cyanovinyl group on the left wing of RPV extending to the hydrophobic region of NNIBP was carried out,with the the hope to make additional new interactions with the surrounding amino acid residues,which may guarantee the activity,reduce the cytotoxicity and increase the solubility compared to RPV.A total of 64 novel compounds divided into 3 series were designed and synthesized.MTT and ELISA methods were used to test the antiviral activity of target compounds in the cell culture and enzyme test respectively.Most of the newly synthesized compounds in series IA with alkynyl linker exhibit single to dozens of nanomolar activity against wild-type(WT)HIV-1 except IA-6b and IA-11b,EC50 of other compounds was between 5-63 nM,which were better than NVP.Among them,IA-6i and IA-61 turned out to be the most potent HIV-1 inhibitors with the same EC50 value of 3.0 nM against WT HIV-1,which were superior to those reference drugs NVP,DLV,AZT,and equivalent to EFV and ETV.In the case of HIV-1 mutant strains,compound IA-61 showed the strongest inhibitory activities,the EC50 values for K103N,E138K and RES056 were 10 nM,22 nM and 0.935 ?M respectively.In addition,compared with RPV(CC50=3.984 ?M),the cytotoxicity of 7 compounds reduced 1.2-6.3 fold,and were 2.3-11.4 fold lower than ETV(CC50=2.2 ?M),which initially reduced the toxicity of lead compounds.The compounds in series IB holding 1,2,3-triazole linker can inhibit WT HIV-1 with EC50 values in the range of 0.029?M-5.62 ?M.Compounds IB-4b(EC50(?B)=0.020 ?M,ECso(K103N)=0.043 ?M,CCso>241.52 ?M),IB-4c(EC50(?b)=0.013 ?M,EC50(K103N)=0.022 ?M,CC50>241.52 ?M)and IB-4g(EC50(?B)=0.014 ?M,EC50(K103N)=0.054 ?M,CC50=2.1 ?M)showed potent activity and low cytotoxicity,superior to efavirenz(EFV).In particular,IB-4b and IB-4c showed extremely low cytotoxicity and high selectivity(SI>10000),which were superior to all the reference drugs.Furthermore,the preliminary physicochemical properties and metabolic stability of representative compounds IB-4b,IB-4c and IB-4g were investigated to evaluate their drug-like properties.Most of the compounds in series IC-3a-1 exhibited submicromolar to nanomolar activity against WT HIV-1,with EC50 values in the range of 5-63 nM.Among them,IC-3a(EC50=0.06 ?M)showed the best activity,better than 3TC and NVP.The compounds in series IC-6a-1 were potent than that in series IC-3a-1 against WT HIV-1,which indicated that the enylpiperidine moiety has better accommodation in the NNIBP.Six compounds in series IC-6a-1 exhibited inhibition activity at the nanomolar level(WT)with EC50 values of 19-90 nM.In addition,all of the compounds showed inhibition activity against K103N and IC-6a turned out to be the most potent inhibitor with EC50 value of 50 nM.Some of the compounds also showed activity against L100I,Y181C and E138K.In general,the activity of these compounds decreased and the cytotoxicity increased.Design,synthesis and activity evaluation of HIV-1 NNRTIs based on "four point pharmacophore model".In this chapter,based on the classical four point pharmacophore model of DAPYs,K5a2 and 25a with thiophene pyrimidine privileged scaffold and IA-61 were selected as lead compounds.By targeting the highly conserved hydrophobic region in NNIBP,the drug design strategy based on crystal data and molecular hybridization were adopted to modify the lead compounds,with the hope of that the new synthesized molecules could fit in the four point pharmacophore model adequately,increasing the interaction with NNIBP.In total,27 compounds were designed and synthesized.Among the three compounds in series IIA,?A-6c(EC50=9.0 ?M,SI=1130)exhibited the best activity,far superior to NVP.Compared with the leading compound 25a(CC50=2.30 ?M),the cytotoxicity of ?A-6a-c(11.51 ?M,6.39 ?M,10.27 ?M)decreased significantly,which verified the original intention of our design.For the mutant RES056,the three compounds showed submicromolar activity and ?A-6b(EC50=153 nM)exhibited the best activity,which is better than NVP.In addition,the three compounds exhibited more potent activity than NVP(IC50=0.59?M)against HIV-1 RT and the IC50 values were 0.86,0.28 and 0.29 ?M respectively,which indicated that these compounds were classic RT inhibitors.Compounds in series IIB showed potent activity against WT HIV-1 and m ost of mutant strains,especially for K103N mutation,and the activity of severa 1 compounds against K103N was more potent than WT(RF<1).The EC50 v alues of IIB-5p for WT,L100I,K103N,Y181C,Y188L and E138K were 9.93,10.5,6.02,18.9,23.9 and 23.2 nM,respectively.In view of the superior acti vity of IIB-5p,the molecular dynamics simulation study was further carried ou t,which provided a reasonable explanation for the significant activity of IIB-5 p against HIV-1 mutant strains.The research of pharmacokinetic properties and acute toxicity indicated that ?B-5p had good PK property in rats with a mod erate clearance rate and a high bioavailability of 33.8%.Design,synthesis and activity evaluation of lead compounds against HIV-1 based on the new strategy.Nowadays,"shock and kill" strategy have attracted great attention to eliminate virus reservoir,HD AC inhibitors are the initial choice to activate the latent HIV reservoir,but most of the latent virus activators showed significant toxic and unobvious activation effect.Therefore,based on the analysis of the pharmacophores of HD AC inhibitors,we have constructed a small compound library with hydroxamic acid and amide as zinc ion chelating group(ZBG)by utilizing the strategies of computer-aided drug design,scaffold hopping and structural diversity oriented drug design.Through the modification of lipophilic group(CAP)and intermediate lipophilic chain(linker),three subseries of compounds were designed and synthesized.The results showed that six compounds exhibited obvious latent activation activity with EC50 values ranging from 4.71-15.28 ?M,IIIB-5a(EC50=8.53 ?M,CC50=183.38?M),?A-8d(EC50=7.17 ?M3 CC50>217.5 ?M),IIIC-8a(EC50=4.71?M,CC50=19.97 ?M)were the most active compounds in three subseries with different skeletons,and their anti-HIV latent reservoir activity was slightly weaker than SAHA(EC50=1.21 ?M,CC50=0.78 ?M)which was undergoing clinical study,but the cytotoxicity was far lower than SAHA.Therefore,the screened structure showed obvious advantages.In addition,the inhibition effect of synthesized compounds on HIV replication were considered and compounds in series IIIA-8a-d exhibited potent activity against WT HIV-1 at nanomolar level.In addition,IIIA-8d exhibited strong latent activation effect and potent inhibition activity against HIV-1,and further exploration of it will help to find a dual functional lead compound.In the second section of this chapter,PROTAC technology was firstly applied to the design of HIV-1 RT degrader,leading to several small molecules by conjugating DAPYs and pomalidomide,and the inhibition activity in cell culture and at enzyme level of representative compounds were preliminarily investigated.All the synthesized PROTAC molecules showed inhibition activity against WT HIV-1 at submicromolar level to micromole level and also exhibited strong inhibition activity against HIV-1 RT,the cytotoxicity of these compounds was very low especially.?G-3b(EC50=0.16 ?M,CC50>168.06 ?M)was the most potent inhibitor.Moreover,?E-4a,IIIF-3a,?F-3b,IIIG-3a and IIIG-3c showed submicromolar inhibitory activity against WT HIV-1,and the solubility of the representative compounds were tested and behaved well in the acidic,neutral and alkaline environment.To sum up,the second and third chapters in this paper focus on the scientific issues of high cytotoxicity and poor solubility of the lead compounds of HIV-1 NNRTIs.By utilizing the methods of target based rational drug design,molecular hybridization and computer-aided drug design,structure diversity modification of the listed drug RPV and the lead compound 25a found by our research group were conducted and 91 novel NNRTIs were designed and synthesized.Many lead compounds with high efficiency,low toxicity and improvement physicochemical properties were found through biological activity evaluation,solubility measurement,C YP enzyme inhibition test in vitro and preliminary drug-like evaluation.In the fourth chapter,as an exploratory study,48 novel compounds were designed and synthesized by using the latent activation and protein degradation strategies(PROTAC).In addition,the lead compounds with sub-micromolecular activity against WT HIV-1 and low cytotoxicity were obtained through further structural optimization and activity screening,which also opened a new field for the research and development of drugs for the treatment of AIDS. |
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