Fig 1.
Development of LC-MS/MS methods to quantitate lopinavir, lopinavir M1, nelfinavir, nelfinavir M8, ritonavir, and hydroxy ritonavir in human liver microsomes.
MS/MS spectra of (A) lopinavir, (B) lopinavir M1, (C) nelfinavir, (D) nelfinavir M8, (E) ritonavir, (F) hydroxy ritonavir with ESI proton adducts [M+H]+ in positive mode. The y-axis shows intensity (CPS, count per second) and the x-axis shows the mass to charge ratio (m/z, amu).
Table 1.
Summary of optimized parameters and characteristics for positive mode LC—MS/MS detection of Protease inhibitors.
Fig 2.
Spectral binding of methamphetamine with CYP3A4.
(A) The spectral binding was performed at varying concentrations of methamphetamine from 1 μM to 2 mM. The graph was plotted using difference in absorbance at 390 and 416 nm vs. concentration of methamphetamine. The graph was fitted with hyperbolic equation using non-linear regression analysis in sigma plot 11. (B) Docking simulation of methamphetamine binding with CYP3A4 in two different binding modes. The heme, methamphetamine, and interacting amino acid residues of CYP3A4 are represented in red, green, and blue, respectively.
Table 2.
The KD and δAmax of all the PIs with and without methamphetamine.
Fig 3.
Spectral binding of type I PIs with CYP3A4 in the absence (filled circles) and presence (open circles) of methamphetamine.
(A-D) The spectral binding was performed at varying concentrations of atazanavir, nelfinavir, tipranavir and lopinavir. The KD and δAmax for each PI with and without methamphetamine are presented in Table 2. One way ANOVA with Dunnet’s post-hoc test was employed to calculate the statistical significance. A p-value <0.05 is indicated by * and <0.1 is indicated by #.
Fig 4.
Effect of methamphetamine on metabolism of PIs in CYP3A4 human liver microsomes.
Effect of methamphetamine on (A) lopinavir M1 formation from lopinavir, (B) nelfinavir M8 degradation and, (C) hydroxy ritonavir formation from ritonavir. One way ANOVA with Dunnet’s post-hoc test was employed to calculate the statistical significance and ** denotes p-value <0.01.
Fig 5.
Docking simulations of PIs in the absence and presence of methamphetamine.
Structures of (A) Atazanavir, (B) Nelfinavir, (C) Tipranavir and (D) Lopinavir with their preferred binding sites pointing towards the heme moiety of CYP3A4in the absence of methamphetamine. Docking simulations of (E) Atazanavir, (F) Nelfinavir, (G) Tipranavir and (H) Lopinavir with heme moiety of CYP3A4 in the presence of methamphetamine. R-methamphetamine in binding mode 1 was shown in cyan sticks. Docking was performed as described in Materials and Methods.
Table 3.
The statistical results of PIs docking into CYP3A4 active site in the presence of methamphetamine.
Fig 6.
Spectral binding of type II PIs with CYP3A4 in the absence (filled circles) and presence (open circles) of methamphetamine.
(A, B) The spectral binding was performed at varying concentrations of ritonavir and indinavir. The KD and δAmax for each PI with and without methamphetamine are presented in Table 2. One way ANOVA with Dunnet’s post-hoc test was employed to calculate the statistical significance. A p-value <0.05 is indicated by * and <0.1 is indicated by #.
Fig 7.
Docking simulations of Type II PIs in the absence and presence of methamphetamine.
Structures of (A) Ritonavir and (B) Indinavir with their preferred binding sites pointing towards the heme moiety of CYP3A4in the absence of methamphetamine. Docking simulations of (C) Ritonavir and (D) Indinavir with heme moiety of CYP3A4 in the presence of methamphetamine. R-methamphetamine in binding mode 1 was shown in cyan sticks. Docking was performed as described in Materials and Methods.