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  International HIV and Hepatitis Drug Resistance Workshop
June 8-12, 2010,
Dubrovnik Croatia
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Strong Inhibition of Wild-type and Integrase Inhibitor (INI)-Resistant HIV Integrase (IN) Strand Transfer Reaction by the Novel INI S/GSK1349572, poster report: "S/GSK1349572 had stronger and faster binding to mutant IN than RAL"
 
 
  Reported by Jules Levin
International Drug Resistance Workshop, 8-12 June 2010, Dubrovnik, Croatia
 
Tomokazu Yoshinaga, Mikiko Kanamori-Koyama, Takahiro Seki, Kayo Ishida, Erika Akihisa, Masanori Kobayashi, Akihiko Sato, Tamio Fujiwara Shionogi, Osaka, Japan
 
Author Conclusion
 
·The next generation HIV-1 integrase inhibitor S/GSK1349572 has strong inhibitory activity not only against WT IN enzyme but also against the signature RAL/ELV resistant pathway mutants.
 
·S/GSK1349572 had stronger and faster binding to mutant IN than RAL.
 
·The in vitro enzyme assay data supports the different binding characteristics of S/GSK1349572 and potentially explains its better resistance profile.
 
Author Discussion
 
·IC50 values of S/GSK1349572, RAL and ELV with No Wash assay and 3xWash assay for wild-type were similar and consistent with cellular assay IC50s.
 
·Fold change (FC) of mutants with No Wash assay, 3xWash assay and HeLa-CD4 assay were roughly correlated each other.
 
·IC50s of RAL and ELV with 3xWash assay increased very much for double mutants which showed higher fold change in HeLa-CD4 assay. This might suggest that RAL and ELV had weaker affinity to the double mutant INs, consistent with the dissociation experiment data3.
 
·Pre-incubation time course experiment suggested that S/GSK1349572 and RAL bound to integrase slowly and reached steady state by 90 minutes. This slow binding of INI is consistent with previous observations1. ELV bound to WT faster than 572 and RAL in our experiments. (Fig. 2A and 3A)
 
·RAL bound to WT in a similar time course to S/GSK1349572 (Fig.2A). But RAL could not fully bind to N155H or E138K/Q148R as compared to wild-type even when pre-incubation time was prolonged (Fig.2B, C).
 
·ELV bound to N155H in a similar time course to WT, although N155H virus showed resistance to ELV in HeLa-CD4 assay. However, FC with 3xWash assay was closer to FC with HeLa-CD4 assay. These data suggested Koff of the compound was important as well as Kon.
 
·S/GSK1349572 dissociated from E92Q/N155H IN-DNA complex more readily than from WT or other mutant INIs by washing, though less readily than for RAL or ELV. S/GSK1349572 drug concentrations above the PA-IC90, however, are sufficient to inhibit the proliferation of E92Q/N155H virus.
 
·INI binding to the enzyme is a relatively slow process suggesting some structure changes involved to form a stable complex4.
 
ABSTRACT
 
Background: The in vitro resistance profile of S/GSK1349572 is distinct from that of raltegravir and elvitegravir; for example, signature mutations at Q148 and N155 negatively impact raltegravir (RAL) and elvitegravir (ELV) antiviral potency, but do not for S/GSK1349572. Herein, we describe activity of S/GSK1349572 against WT and mutant INs in an in vitro strand transfer assay.
 
Methods: Biotinylated donor DNA was immobilized on streptavidine-coated plate in complex with recombinant integrase protein. INI was added and pre-incubated for 60min at 30 degrees. Washing was used to remove unbound INI, then strand transfer reaction was initiated by adding digoxigenin labeled target DNA followed by 10 minutes incubation. Products from assays run with or without washing were measured by ELISA assay For time course experiments, INI pre-incubation time ranged from 5 to 90 minutes, following which strand transfer reaction was started by adding digoxigenin labeled target DNA. After 5 minutes reaction, products were measured by ELISA.
 
Results:The IC50 (4.2+/-1.5 nM) of S/GSK1349572 against WT IN was equivalent to RAL and ELV (6.4+/-2.8 and 4.9+/-0.8 nM respectively) even when free INIs were washed three times.
 
IC50s against E138K/Q148R enzyme, however, were substantially different: 6.2, 67 and 105 nM for S/GSK1349572, RAL, and ELV, respectively. These results were correlated with IC50s determined in a cell-based assay. In time course experiments, S/GSK1349572 and RAL IC50s reached the maximum inhibition at 90 minutes pre-incubation, and binding of these INI to WT IN seemed to follow the same time course. However, IC50 (nM) of RAL and S/GSK1349572 to E138K/Q148R was 132.1, and 28.2, or 82.9 and 6.6, or 101 and 4.5 at 5 min, 60 min and 90 min pre-incubation, respectively.
 
Discussion: Strand transfer reaction IC50s of S/GSK1349572 were well correlated with IC50s from cell based assay. S/GSK1349572 had stronger and faster binding to mutant IN than RAL. These results are complementary to IN-inhibitor dissociation experiments and suggest the importance of Kon as well as Koff to better understand resistance profiles of INIs.
 
Introduction
 
The in vitro resistance profile of S/GSK1349572 is distinct from that of RAL and ELV; for example, signature mutations at Q148 and N155 negatively impact RAL and ELV antiviral potency, but do not for S/GSK1349572. Herein, we describe activity of S/GSK1349572 against WT and mutant INs in an in vitro strand transfer assay.
 
Methods
 
Biotinylated donor DNA was immobilized on streptavidine-coated microplate, then recombinant integrase protein was added to make IN-DNA complex. After washing out unbound integrase, INI was added and pre-incubated for 60 minutes at 30 degrees. For No Wash assay, strand transfer reaction was initiated by adding digoxigenin labeled target DNA and incubated for 10 minutes at room temperature. For 3xWash assay, IN-DNA-INI complex was washed three times, then strand transfer reaction was conducted for 10 minutes by adding target DNA. Products from assay run with or without washing were measured by ELISA assay. For time course experiments, IN-DNA-INI pre-incubation time ranged from 5 to 90 minutes, and following reaction was with No Wash assay method but reaction time was 5 minutes.
 
Figure 1.Schematic of Two Assay Methods

RESULTS
 
Table 1. IC50 nM Values of S/GSK1349572, RAL and ELV in No Wash Assay and 3xWash Assay

The IC50 (4.2 nM) of S/GSK1349572 against WT IN was equivalent to RAL and ELV (6.4 and 4.9 nM respectively) even when free INIs were washed three times. IC50s against mutantenzymes, however, were substantially different for S/GSK1349572, RAL, and ELV, respectively. These results were consisted with fold changes determined in a cell-based assay See Table 2).
 
Table 2. Fold Change of IC50 vs Wild-type for S/GSK1349572, RAL and ELV in No Wash Assay, 3xWash Assay and HeLa-CD4 Assay*

Fold change of mutants inNo Wash assay, 3xWash assay and HeLa-CD4 assay were roughly correlated each other.
*Data of HeLa-CD4 assay were quoted from Seki et al., 2010, 17th CROI; Poster 5552.
 
ELV Figure 2. Comparison of the Effect of Pre-incubation Time of IN-DNA-INI Complex on Strand Transfer IC50 for S/GSK1349572 or RAL

In time course experiments, S/GSK1349572 and RAL IC50s for WT reached the maximum inhibition at 90 minutes pre-incubation, and binding of these INI to WT IN seemed to follow the same time course. S/GSK1349572 bound to N155H and E138K/Q148R mutant INs seemed to follow the same time course as WT. However, IC50s (nM) of RAL to N155H andE138K/Q148R did not reach the same level as that of WT even at 90 minutes pre-incubation
 
Figure 3. Comparison of the Effect of Pre-incubation Time of IN-DNA-INI Complex on Strand Transfer IC50 for S/GSK1349572 or ELV