Mechanism Of2’,3’-didehydro-3’-deoxy-4’-Ethynylthymidine (114) Resistance To HIV

Objective:The purpose of the study is focusing on mechanism of resistance to AZT related mutation type viruses.Method:The experiment contains two parts:in vivo and in vitro study. In vivo part, firstly, the plasmids expressing wild and mutated types included T69SSS, M41L, T215, M41L/T215Y, M41L/L210W/T215Y, T69SSS/L210W/T215Y and T69SSS/M41L/L210W/T215Y were constructed. Secondly, wild and mutated types of viruses were made. The wild and mutated types of viruses infected the TZM-bl luciferase expressing cell lines. IC50was tested and calculated.In vitro part, we invested enzyme kinetics and primer extension two parts. Firstly, wild and mutated type of EPH palsmids with M41L/T215Y, M41L/L210W/T215Y, T69SSS, T69SSS/L210W/T215Y and T69SSS/M41L/L210W/T215Y were constructed. Secondly, we purified reverse transcriptases by NI-NIA and FPLC methods. Thirdly, without ATP, compared the competitive activity with nature sustrate of dTTP and derivatives of AZT TP、D4T TP、114TP by competitive reaction. Forthly, AZT MP、D4T MP and114MP were contected on primer. With3.2mM ATP, compared the excision activity with different reverse transcriptases, whether they were the same mechanisms. Results:1. In vivo part:HIV replication model was successfully constructed by agrose gel eletrophoresis and sequence results.3TC, AZT,114and TDF were treated TZM-bl cells with infected wild type virus. AZT was the most potent inhitor, which was17times fold change with3TC,82times fold change with114, and42times fold change with TDF. M41L/L210W/T215Y and T69SSS/M41L/L210W/T215Y showed more resistance to3TC about2times fold change with wild type. The IC50increased126and1235times fold change with M41L/L210W/T215Y and T69SSS/M41L/L210W/T215Y mutations. The resistance increased20-48times fold change resistance to TDF with T69SSS and/or M41L> L210W> T215Y mutations. M41L and T215Y showed no resistance with114treated. The resistance increased13to25times fold change with M41L/T215Y, M41L/L210W/T215Y and T69SSS/M41L/L210W/T215Y mutated viruses. Mutated type viruses showed lower resistance to114than AZT and D4T.2. In vitro part, reverse trancriptases were successfully constructed by pharmacokinetics and sequence results. Kinetic study showed RT activity was4-8times fold changes with D/D. Compared with D/D and D/R, the Km values is lower than D/R, and same trend with IC50. Compared the characteristics of competitive reactions,114TP showed the lowest Ki values, AZT TP showed lower Ki values and D4T showed the highest Ki values. Ki value of114TP was2times fold change efficiency than AZT TP, and20times fold change than D4T. In primer extension experienment, without ATP, the23bp product was almost no change with wild type reverse transcriptase; with ATP, the23bp products decresed. If incoparated primer with AZT MP,114MP and D4T MP, the24bp product shows no change with wild type reverse transcriptases and decresed with M41L/L210W/T215Y and T69SSS/M41L/L210W/T215Y mutated type reverse transcriptases with ATP.Conclusion:1. HIV replication model and reverse transcriptases were successfully made with wide type and AZT related mutations.2. In vivo part:Compared with3TC, AZT,114and TDF, AZT is the most potent inhibitor to wild type virus, and114is the most potent inhibitor to mutated virus.3. In vitro part:The competitive reaction occurs at reverse transcription. There is no difference with wild and mutated virus without ATP. AZT MP,114MP and D4T MP are excised with ATP exised.4. Treated M41L/L210W/T215Y, T69SSS, T69SSS/L210W/T215Y and T69SSS/M41L/L210W/T215Y mutated virus with114, the mechanism is pyrophosphorolysis depended on ATP. Compared with vivo part,114is most potent inhibitor treated HIV.