Structure-Based Design,Synthesis,and Biological Evaluation Of Novel HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors

Acquired immunodeficiency syndrome（AIDS）,caused by human immunodeficiency virus type 1（HIV-1）,is a chronic epidemic disease that seriously threatens human health.Reverse transcriptase（RT）plays a very crucial role in the viral replication cycle,on account of its unique function of reverse transcription of singlestranded RNA（ssRNA）into double-stranded DNA（dsDNA）.Therefore,RT has been considered as an attractive target for the development of anti-AIDS drugs due to the well-known biochemical mechanism,abundant structural biology information,and lack of human homologous enzymes.Notably,non-nucleoside reverse transcriptase inhibitors（NNRTIs）have attracted great attention as an essential component of combination antiretroviral therapy regimens,on account of their remarkable potency,low toxicity and high selectivity.However,the occurrence of drug-resistant strains and cross-resistance after longterm usage have inevitably compromised the clinical application of NNRTIs,which need to be further optimized:1)There is a low genetic barrier for viral resistance,while NNRTIs should be taken for life,leading to the rapid emergence of drug resistant mutants.These drug-resistance mutants have greatly reduced the therapeutic efficacy of approved NNRTIs;2)NNRTIs with similar scaffold structures all interact with their targets in the same binding mode,leading to the rapid development of cross-resistance to the drugs.Therefore,it is urgent to develop new NNRTIs with distinct structural scaffolds and favorable drug resistance profiles.1.Structure-based design,synthesis and biological evaluation of novel IAS-typed HIV-1NNRTIsIn order to discover novel NNRTIs with structural diversity,analyzing the cocrystal structures of WT RT-bound indolylarylsulfone derivatives（IAS）,it was found that the 2-amide substituent of the indole scaffold towards the entrance channel composed of L100,E138,and V179.Based on the drug design strategy of targeting the protein-solvent interface with Ⅱ-2 as the lead compound,a series of novel IAS derivatives were designed and synthesized by introducing aromatic substituents with different hydrogen bonds to the 2-amides to form additional interactions with the amino acids around the NNIBP entrance channel to enhance their anti-HIV-1 activity.The antiviral assay results in TZM-bl cells showed that all these compounds exhibited excellent potency against WT HIV-1(EC_{50（NL4-3）}=3.98-354 nM).Among them,cyanobenzene substituted derivatives ⅡA-4,ⅡA-5,ⅡA-6,and ⅡA-7 showed potent inhibitory activity against HIV-1（EC60（NL4-3）=3.98-12.6 nM）,in which the inhibitory activity of compound ⅡA-7(EC_{50（NL4-3）}=3.98 nM)was higher than that of the lead compound Ⅱ-2(EC_{50（NL4-3）}=4.12nM)and slightly weaker than that of rilpivirine(EC_{50（NL4-3）}=0.63nM).Besides,all compounds did not exhibit significant cytotoxicity at the concentration of 500 ng/mL.HIV-1 RT inhibition assay showed thatⅡA-7 displayed high affinity for WT HIV-1 RT with an IC₅₀ value of 0.011 μM and functioned as a typical NNRTI,which was comparable to that of efavirenz(IC₅₀=0.010μM)and etravirine(IC₅₀=0.011 μM),and better than that of Ⅱ-2(IC₅₀=0.018 μM).Molecular docking studies showed that there is an additional π-π stacking interaction between ⅡA-7 and Y181,which may account for its excellent activity.2.Design,synthesis and biological evaluation of novel DAPY-typed HIV-1 NNRTIs based on the covalent inhibition strategyCovalent inhibitors exert their biological functions by reacting irreversibly or reversibly with nucleophilic groups of proteins.The covalent binding strategy enables the discovery of highly efficient,selective,and long-acting chemical molecules.In this chapter,with the second-generation NNRTI drug etravirine as the lead compound,we introduce different Michael receptor groups into the 5 position of the pyrimidine scaffold as covalent warheads,and design a series of covalent inhibitors targeting the Y181C mutation to improve the inhibitory activity against the Y181 C-containing mutant.In addition,we also designed non-covalent structural analogues as controls.The antiviral assay results in MT-4 cells showed that most of the compounds exhibited potent inhibitory activity against WT HIV-1(EC_50（ⅢB）=1.45-42.5 nM).Among them,a covalent inhibitor ⅢA-5 showed excellent inhibitory activity against WT and Y181C mutant HIV-1(EC_50（ⅢB）=5.90 nM,EC_{50（Y181C）}=13.0 nM),and its inhibitory activity against WT HIV-1 was significantly better than that of nevirapine(EC_50（ⅢB）=155 nM)and comparable to that of efavirenz(EC_50（ⅢB）=4.02 nM)and etravirine(EC_50（ⅢB）=3.50 nM).The inhibitory activity of the covalent inhibitors against the Y181C mutant was better than that of etravirine(EC_{50（Y181C）}=20.9 nM),and its cytotoxicity(CC₅₀=6.79 μM)was also slightly lower than that of etravirine(CC₅₀=4.61 μM).In addition,the inhibitory activity of all covalent inhibitors against Y181C and K103N+Y181C mutants was better than that of their non-covalent inhibitors,proving that covalent inhibitors could indeed enhance the inhibitory activity against drug-resistant strains containing Y181C mutations.The enzyme inhibition results showed that all compounds exhibited good inhibitory activity against HIV-1 RT,demonstrating that their binding target was RT.The covalent docking results showed that ⅢA-5 formed a covalent bond with the Cys 181 side chain mercaptan of Y181 CRT through nucleophilic addition reaction,which rationalized its potent inhibitory activity against Y181C mutant.In summary,in this thesis,to address the intractable issues of drug resistance of approved NNRTIs:on the one hand,using the drug design strategy of targeting the protein-solvent interface,18 novel IAS-typed NNRTIs were designed and synthesized,and ⅡA-7 was discovered as the most potent compound,while the structure-activity relationships of IASs were fully explored;on the other hand,using the covalent binding strategy,11 DAPY-typed NNRTIs were designed and synthesized with etravirine as the lead,and ⅢA-5 was discovered as the potent covalent inhibitor with good inhibitory activity against Y181C mutant strain,which initially verified the feasibility of designing covalent NNRTIs.In conclusion,the structure-activity relationships exploration and molecular simulation studies in this thesis laid the foundation for the further structural modifications of NNRTIs.