Impedance method for detecting HIV-1 protease and screening for its inhibitors using ferrocene-peptide conjugate/Au nanoparticle/single-walled carbon nanotube modified electrode.
A highly sensitive screening assay based on electrochemical impedance spectroscopy (EIS) has been developed for detecting HIV-1 protease (PR) and subsequent evaluation of its corresponding inhibitors at picomolar levels. The assay format was based on the immobilization of the thiol terminated ferrocene(Fc)-pepstatin conjugate on a single-walled carbon nanotube/gold nanoparticle (SWCNT/AuNP) modified gold electrode. The alteration of the interfacial properties of electrodes upon HIV-1 PR and Fc-pepstatin conjugate interaction was traced by EIS. On the basis of the charge transfer resistance data obtained and using a mixed kinetic and diffusion model, this procedure was capable of detecting picomolar HIV-1 PR owing to the specific binding of this enzyme to Fc modified pepstatin. A competitive inhibition assay format was then performed using four potent HIV-1 PR inhibitors. The estimated inhibition constant ( K i) attested that lopinavir/ritonavir ( K i = 20 +/- 3 pM) and saquinavir ( K i = 57 +/- 8 pM) even at 10 pM competed strongly with pepstatin for effective binding to HIV-1 PR. Indinavir ( K i = 630 +/- 22 pM) only competed well with pepstatin at a much higher concentration (1 nM). No significant inhibitory effect was observed for the fosamprenavir ( K i =11 +/- 0.5 nM) as expected from this pro-drug. Such results agreed well with the values reported in the literature. This assay format is a definite asset for the expedited development of effective HIV-1 PR inhibitors with low molecular weights.
Mahmoud
Khaled A
KA
Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, Canada H4P 2R2.
Luong
John H T
JH
D000595:Amino Acid Sequence; D004353:Drug Evaluation, Preclinical; D017097:Electric Impedance; D004563:Electrochemistry; D004566:Electrodes; D005296:Ferrous Compounds / Q000378:metabolism*; D006046:Gold / Q000737:chemistry*; D016333:HIV Protease / Q000032:analysis* / Q000378:metabolism; D017320:HIV Protease Inhibitors / Q000032:analysis* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D053768:Metal Nanoparticles / Q000737:chemistry*; D000075163:Metallocenes; D037742:Nanotubes, Carbon / Q000737:chemistry*; D010436:Pepstatins / Q000737:chemistry / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013499:Surface Properties
D005296:Ferrous Compounds; D017320:HIV Protease Inhibitors; D000075163:Metallocenes; D037742:Nanotubes, Carbon; D010436:Pepstatins; D013438:Sulfhydryl Compounds; D006046:Gold; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1; C004998:ferrocene; C031375:pepstatin