TABLE 2.
Dissociation half-lives and off rates of INSTIs from HIV-1 IN-DNA complexes
| INSTI | WTa |
G140S+Q148Ha |
||||
|---|---|---|---|---|---|---|
| Apparent t1/2 (h)b | koff (s−1) (×10−6)b | P valuec | Apparent t1/2 (h)b (fold change vs WT) | koff (s−1) (×10−6)b (fold change vs WT) | P valuec | |
| BIC | 163 ± 31 | 1.2 ± 0.3 | 5.7 ± 0.4 (29) | 34 ± 2 (0.04) | ||
| DTG | 96 ± 29 [71] | 2.2 ± 0.7 [2.7 ± 0.4] | 0.0019 | 1.9 ± 0.2 (51) [3.3] | 100 ± 9 (0.02) [58 ± 8] | 0.333 |
| RAL | 10 ± 2 [8.8] | 21 ± 6 [22 ± 2] | 0.0003 | ND [0.2] | ND [1,130] | ND |
| EVG | 3.3 ± 0.9 [2.7] | 62 ± 16 [71 ± 4] | <0.0001 | ND [ND] | ND [ND] | ND |
| Compound 1 | 155 ± 25 | 1.3 ± 0.2 | 0.594 | 4.8 ± 0.2 (32) | 40 ± 2 (0.03) | 0.2 |
| Compound 2 | 152 ± 35 | 1.3 ± 0.3 | 0.776 | 1.7 ± 0.2 (89) | 114 ± 12 (0.01) | 0.2 |
Values in square brackets are from reference 3. ND, not determined. The scintillation proximity assays for determination of HIV-1 INSTI and IN-DNA complex t1/2 values were conducted according to the protocol defined in Hightower et al. (3) but measured using the HIDEX Sense microplate reader (model 425-312, version 0.5.5.0; HIDEX, Tirku, Finland) and were maintained at 37°C. Recombinant WT and G140S+Q148H mutant HIV-1 IN enzymes containing an N-terminal 6-histidine tag (6His-IN) were purified as described in Jones et al. (22). INSTIs were tritiated by ViTrax (Placentia, CA) and had specific activities of 16.8 to 21.3 Ci/mmol. Streptavidin-coated scintillation proximity assay imaging beads (PerkinElmer, Boston, MA) were used, and oligonucleotides were obtained from Trilink (San Diego, CA), as described in Hightower et al. (3). The single exponential decay analysis was done as in Hightower et al., with the exception that we set background decay to 5%. The apparent dissociation rate constant was determined by curve fitting the competition binding phase after subtraction of 5% background to the 2-parameter single exponential decay equation: y = M(e−koff·t), where M is the relative binding measured at the first time point of a dissociation phase and koff is an apparent dissociation rate constant. The half-life, t1/2, of the complex of INSTI bound to IN-DNA was calculated according to the equation t1/2 = (ln 2)/koff and is a time needed for half of the complexes to dissociate to their individual components.
Average ± standard deviation of 50% effective dose from 5 to 9 experiments for BIC, DTG, RAL, and EVG and 2 experiments for compounds 1 and 2.
BIC versus other INSTI comparisons of log10(koff) are based on the exact Wilcoxon rank sum test.