The HIV-1 integrase catalyzes the integration of HIV-1 viral DNA (vDNA) into the host cell chromosome in a process, which is essential for viral replication through two independent reactions, 3'-processing (3'-P) and strand transfer (ST), catalyzed by IN. Deciphering the structural determinants of the interaction between integrase and its substrates and the kinetics of this interaction sheds light on the importance of inhibitors targeting the pre-integration IN*vDNA complex. This approach led to the identification of raltegravir (RAL) and elvitegravir (ELV), which turned out to be highly efficient inhibitors of ST. RAL, formerly known under the code MK-0518, is a new anti-HIV drug that obtained clinical approval in the United States under the name IsentressTM on October 12, 2007. ELV is still in clinical trials. However, these compounds nevertheless encounter resistance phenomenon. To date, no experimental data characterizing the RAL structure, structure of the HIV-1 IN and/or interactions of RAL with its targets, has been reported.First, we characterized the structural and conformational properties of RAL in different states ‒ the gas phase, in water solution and the solid state. Second, a detailed study allowed characterisation the RAL recognition by the viral targets ‒ IN and the vDNA, before and after the 3'-P. We found that RAL shows a broad spectrum of conformations and configurations in isolated state and/or associated with the target(s). The best docking poses and scores confirmed that the model representing IN*vDNA complex is a biologically relevant target of RAL. This result is consistent with the commonly accepted mechanism of RAL inhibition.Based on the docking results we suggested that the inhibition process may include, as a first step, the RAL recognition by the processed vDNA bound to a transient intermediate IN state. RAL coupled to vDNA shows an outside orientation of all oxygen atoms, excellent putative chelating agents of Mg2+ cations, which could facilitate the insertion of RAL into the active site. The conformational flexibility of RAL further allows the accommodation/adaptation of the inhibitor in a relatively large binding pocket of IN*vDNA pre-integration complex thus producing various RAL conformation. We believe that such variety of the RAL conformations contributing alternatively to the enzyme residue recognition may impact the selection of the clinically observed alternative resistance pathways to the drug.We also studied the recognition of the HIV-1 IN inhibitors from two different strains, B and CRF02_AG. Our in silico study showed that the sequence variations between CRF02_AG and B strains did not lead to any notable difference in the structural features of the enzyme and did not impact the susceptibility to the IN inhibitors. Our analysis of the resistance mutations effects showed that structure of the wild-type enzyme and mutants is almost identical. However, the resistance mutations significantly altered the specificity of the viral DNA recognition by IN.We performed molecular dynamics simulations of the native and mutated IN with a point mutation R228A localized in the C-terminal domain. The study of targets flexibility opens a very promising way, not only in terms of fundamental research, but also for the application of our concepts to the development of new generations of inhibitors targeting IN.
| Identifer | oai:union.ndltd.org:CCSD/oai:tel.archives-ouvertes.fr:tel-00905951 |
| Date | 26 October 2012 |
| Creators | Arora, Rohit |
| Publisher | École normale supérieure de Cachan - ENS Cachan |
| Source Sets | CCSD theses-EN-ligne, France |
| Language | English |
| Detected Language | English |
| Type | PhD thesis |
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