Structure-and Mechanism-based Discovery Of Anti-HIV-1 Lead Compounds

Acquired immune deficiency syndrome(AIDS)mainly caused by human immunodeficiency virus type-1(HIV-1),continues to be a major leading pandemic disease worldwide.The clinical application of highly active anti-retroviral therapy(HAART)involving multidrug combinations dramatically decreased the morbidity and mortality resulted from HIV-1 infection,however,the long-term efficacy of HARRT are limited by the rapid emergence of drug-resistant HIV-1 strains and serious side effects.The reverse transcriptase(RT)of HIV-1 plays a crucial role in the replication of the virus and has been identified as a prime target for anti-HIV drug discovery.Based on their mechanism of action and structures,RT inhibitors are classified as nucleos(t)ide RT inhibitors(N(t)RTIs)and non-nucleoside RT inhibitors(NNRTIs),both of which are important components of HAART.Compared with other inhibitors,NNRTIs have gained an indispensable place in AIDS chemotherapy,owing to their unique antiviral potency,relatively low toxicity and high selectivity.However,its use in the first line treatment is usually hampered by high rate of adverse effects,poor pharmacokinetics profiles as well as inevitable emergence of HIV-1 resistant strains.Therefore,by targeting new binding site or exploiting novel chemical scaffolds,discovery of novel HIV-1 NNRTIs with high antiviral potency,favourable pharmacokinetic and reduced drug resistance profiles is urgently needed.The flexibility of RT,high mutability and allostery of NNRTIs binding pocket make it difficult to predict inhibitor-binding modes and to obtain novel NNRTIs via structure-based approach.Currently,the development of structural biology,molecular modeling and pharmacophore investigations reported in numerous literatures,which represent the ligand features that are involved in interactions with the target protein,as well as the space around the ligand occupied by the protein,will provide valuable information and assist in the discovery and modifications of NNRTIs.Under this situation,the second chapter described the design,synthesis and bioactivity evaluation of novel DAPY-like HIV-1 NNRTIs.These compounds were designed based on the "three-point pharmacophore" model of DAPY derivatives with the following structural modifications:1)various privileged fragments,such as morpholine or substituted piperazine,which are beneficial for the solubility and PK properties of the molecule were introduced on the right wing of DAPYs pointing to the tolerant region I to form additional interactions with the NNIBP,aiming at the identification of compounds with higher potency against wild-type(WT)and drug-resistant HIV-1 strains and better pharmacokinetic profiles(Series IA);2)on the other hand,phenylsulfonyl group which is an important fragment in indole aryl sulfone(IAS)derivatives was employed as the hydrophobic left wing of DAPY derivatives,and further SARs were discussed(Series IB).All the target compounds in Series IA showed anti-HIV(ⅢB)potency with EC50 values ranging from the sub-micromolar to nanomolar level,which were better than those of 3TC(EC50= 6.41 μM)and NVP(EC50 = 0.24 μM).Among them,IA-II3 was the best antiviral inhibitor(EC50 = 0.0035 μM)with potency same as that of ETR and 3-fold better than that of AZT(ECso = 0.011 μM).The SI value of IA-II3 was higher than 48,774.Moreover,IA-II3 displayed moderate activity against the K103N/Y181C double mutant HIV-1 strain(EC50 = 0.79 μM),as in the same level as that of EFV(EC50= 0.24 μM).In addition,IA-I6(IC50= 0.042 pM)was the best compound with potency more than 10-fold better than that of NVP(IC50= 0.595 μM)in the in vitro anti-RT assay.IA-II3 also exhibited remarkable RT inhibition activity(IC50 = 0.067μM).Nine compounds in Series IB displayed moderate anti-HIV-1 activity with EC50 values ranging from 1.48 μM to 48.9 μM(SI = 2 to 80).The EC50 values of the best two compounds IB-I1 and IB-II1 were 1.48 μM and 1.61 μM,respectively,which were much lower than that of ddI(EC50 = 76.0 μM),comparable to that of 3TC(EC50=2.54 μM),but inferior to those of the leads RS-80(EC50 = 0.0026 μM)and RPV(EC50= 0.0013μM).Further test showed a significant improvement in the solubility of IA-I6 and IA-II3 comparing to that of ETR.In conclusion,the strategies applied in the design of compounds in Series IA,i.e.,modification of the DAPYs by providing additional interactions with the tolerant region I of NNIBP to enhance the binding affinity with RT,thus improving the potency against WT and drug-resistant HIV-1 strains,is effective.And the introduction of privileged polar fragments made the whole molecule more soluble and might have better PK profiles.However,the modification of the hydrophobic part remarkably impaired their anti-HIV-1 potency.In the third chapter,based on the above described "three-point pharmacophore model",the fourth pharmacophore element was introduced to the 5,6-positions of the core ring of ETR,which formed the "four-point pharmacophore model".By the classical definition of drug design,the NNRTIs with the "four-point pharmacophore model" should maximally occupy the binding pocket,thereby interacting more effectively with the amino acids around the binding pocket.To improve the anti-drug resistance profiles of DAPY derivatives,based on the newly constructed pharmacophore model with the "multiple sites binding" feature,we designed a series of fused ring containing derivatives derived from DAPYs(Series IC)and N-substituted piperidine amine modified[1,2,4]pyrazolo[1,5-a]pyrimidine DAPY derivatives(Series ID).In vitro RT inhibitory activity showed that the IC50 values of 17 derivatives in Series IC were in the double-digit nanomolar level,ranging from 0.014 μM to 0.080μM,which were better than that of NVP(IC50 = 0.595 μM).Encouragingly,the bridgehead nitrogen pyrrolo[2,1-f][1,2,4]triazine IC-VI2 demonstrated the highest potency(IC50 = 0.014 μM),which was 42.5 times more potent than that of NVP,slightly better than that of RPV(IC50 = 0.022 μ),and in the same level as that of RDEA427(IC50 = 0.016μM).The newly synthesized compounds in Series ID were evaluated for their in vitro anti-HIV activity and cytotoxicity in MT-4 cells infected with the WT HIV-1(strainⅢB),K103N/Y181C double mutant HIV-1(strain RES056),as well as HIV-2(strain ROD).Among them,12 derivatives showed moderate to excellent activities with EC50 values ranging from 8.1 nM to 42 nM and SI values higher than 1000.The antiviral activities against HIV-ⅢB strain of ID-I4 and ID-I9 were superior to those of the reference drugs ddl,3TC,NVP and DLV(EC50 23198 nM,2239 nM,312 nM and 540 nM,respectively),comparable to those of AZT and EFV(EC50 = 7.1 nM and 6.3 nM,respectively),and in the same order of magnitude with that of ETR(EC50 = 1.8 nM).Besides,there are three compounds,namely,ID-I2,ID-I4 and ID-Is,with moderate inhibitory activity against the most common clinical double mutant strain RES056(K103N/Y181C)(EC50 = 6.4 μM,13 μM and 22μM,respectively),which were better than that of DLV(ECso>36 μM).Moreover,the water solubility of compound ID-I4 was greatly improved,compared with that of ETR.Thus,its pharmacokinetic properties were expected to be improved.The biological results showed that,the newly constructed "four point pharmacophore model" of DAPYs could be used in the drug design of back-up derivatives with "multiple sites binding" feature,as to effectively enhance the binding affinity with RT and therefore to improve anti-resistance profiles.In addition,the introduction of the substituted piperidine moiety into the right part of DAPYs around the tolerant region I greatly enhanced the solubility of the inhibitors,which is expected to improve their pharmacokinetic properties.Furthermore,IC-VI2 and ID-I4 can be used as lead compounds for further optimization.In the fourth chapter of this thesis,a clinical NNRTI drug candidate GW678248,and another NNRTI GA-40 which has a good inhibitory activity against K103N/Y181C double mutant HIV-1 RT,were selected as lead compounds for further modification.Based on the principles of molecular hybridization and bioisosterism,a series of pyridine oxyacetamide HIV-1 NNRTIs was designed and synthesized by extracting and combining the priviledged structures from these two leads.The antiviral results demonstrated that,only three compounds IIA-I1,IIA-Is and IIA-I10 showed weak inhibitory activity against HIV-1 ⅢB(EC50= 41.5μM,10.8μM and 8.18 μM,respectively).Furthermore,the initial structure-activity relationships were also summarized.In the fifth chapter,based on the principle of prodrug and twin drug design,we have designed and synthesized RDEA427-PB(phosphate prodrug of HIV-1 candidate drug RDEA427),RDEA427-CIC(carbonate prodrug of RDEA427)and the twin drug molecule III-7(connecting HIV-1 NRTI drug AZT and HIV-1 NCp7 inhibitor SAMT-lb via a disulfide bond).And the in vitro enzymatic assay showed that RDEA427-PB and RDEA427-CIC had similar anti-HIV-1 RT activity with IC50 values of 0.239 μM and 0.264 μM,which were slightly lower than that of RDEA427(IC50 = 0.016 μM)and preliminarily verified the reasonablity of the drug design.The half-life(t1/2)of RDEA427-CIC in human plasma is more than 2h and the prodrug can release a little of its active form RDEA427 in 2h.For this result,we speculate that the prodrug RDEA427-CIC may firstly generate the hydroxymethyl containing intermediate and no longer release RDEA427,or there are some other unclear metabolic pathways.Though the AZT-NCp7i twin drug molecule III-7 had slight anti-HIV RT activity with an IC50 value of 128 μM,it was expected to release the two active compounds in the infected cells under the reaction catalyzed by GSH,and then displayed anti-HIV activity with synergistic effects.Besides,the anti-HIV RT activity of III-7 also verified the reasonablity of the twin drug design through forming the disulfide bond.Currently,the cell-based anti-HIV assay,stability test and the drug release property study of these target compounds are in progress.Totally,more than 120 compounds which belong to multiple categories of novel anti-HIV agents were designed and synthesized.Based on the results from cellular and enzymatic activity tests,some compounds displayed high activities against HIV-1(RT)in nanomolar level,and some compounds were confirmed to be active against K103N/Y181C double mutant HIV-1 strain.Notably,compound IC-VI2 with a novel scaffold showed higher activity against HIV-1 RT than that of the latest lead compound RPV.In addition,the antiviral potency of IA-I6,IA-II3 and ID-I4 were in the same order with that of ETR,and also the solubility of them were much better than that of ETR,thus these compounds were expected to have improved pharmacokinetic profiles,which are worth further investigation and development.