**4. Conclusions and perspectives**

**3.3. Raltegravir recognition by the HIV-1 targets**

figuration (Figure 11).

Applications

392

references herein.

modes of Mg2+ cations chelation were observed [74,82-84].

catalytic efficiency [87,88] and inhibitor binding [89-91].

DNA probed by experimental and computing techniques.

unpaired cytosine similarly to those observed in the DNA bases pair G-C.

The published docking studies report located within the active site of either unbound IN or IN•vDNA complex. Distinct poses of RAL representing different RAL configuration and

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

Our docking calculations of RAL onto each model evidenced that (i) the large binding pock‐ et delimited by the active site and the extended catalytic site loop in the unbound IN can accommodate RAL in distinct configurational/conformational states showing a lack of inter‐ action specificity between inhibitor and target; (ii) the well defined cavity formed by the ac‐ tive site, vDNA and shortened catalytic site loop provides a more optimised RAL binding site where the inhibitor is stabilised by coordination bonds with Mg2+ cations in the Z/Z-con‐

Additional stabilisation of RAL is provided by non-covalent interactions with the environ‐ ing residues of IN and the viral DNA bases. Based on our computing data we suggested ear‐ lier the stabilizing role of the vDNA in the inhibitors recognition by IN•vDNA preintegration complex [51]. It was experimentally evidenced that RAL potently binds only when IN is in a binary complex with vDNA [85], possibly binding to a transient intermedi‐ ate along the integration pathway [86]. Terminal bases of the viral DNA play a role in both

It was reported recently that unprocessed viral DNA could be the primary target of RAL [92]. This study is based on the PFV DNA and several oligonucleotides mimicking the HIV-1

To explore the role of the HIV-1 viral DNA in RAL recognition we docked RAL onto the non-cleaved and cleaved DNA (the terminal GT nucleotides were removed) [79]. We found that RAL docked onto the non-cleaved vDNA is positioned in the minor groove of the sub‐ strate. No stabilising interactions between the partners, RAL and vDNA, were observed. In contrast, in the processed (cleaved) vDNA the Z/Z isomer of RAL takes the place of the re‐ mote GT based and is stabilised by strong and specific H-bonds with the unpaired cytosine. These H-bonds characterize the high affinity and specific recognition between RAL and the

Based on the docking results we suggested that the inhibition process may include as a first step the RAL recognition by the processed viral DNA bound to a transient intermediate IN state. RAL coupled to vDNA shows an outside orientation of all oxygen atoms, excellent pu‐ tative 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/adapta‐ tion of the inhibitor in a relatively large binding pocket of IN•vDNA pre-integration com‐ plex thus producing various RAL docked conformation. We believe that such variety of RAL conformations contributing to the alternative enzyme residue recognition may impact the selection of the clinically observed alternative resistance pathways to the drug [29] and

The HIV-1 Integrase is an essential retroviral enzyme that covalently binds both ends of lin‐ ear viral DNA and inserts them into a cellular chromosome. The functions of this enzyme are based on the existence of specific attractive interactions between partner molecules or cofactors ‒ IN, viral DNA and Mg2+ cations. Structure-based drug development seeks to identify and use such interactions to design and optimize the competitive and specific mod‐ ulator of such functional interactions. Drug design and optimisation process require knowl‐ edge about interaction geometries and binding affinity contributing to molecular recognition that can be gleaned from crystallographic and modeling data.

We have resumed the available structural information related to the retroviral integrase. We used this data to generate biologically relevant HIV-1 targets ‒ the unbound IN, the viral DNA (vDNA) and the IN•vDNA complex ‒ which represent with a certain level of reliabili‐ ty, two different enzymatic states of the HIV-1 over the retroviral integration process.

and cellular proteins (IN/LEDGF) [97,98]. These alternative strategies represent rational and

The HIV-1 Integrase: Modeling and Beyond http://dx.doi.org/10.5772/52344 395

The authors thank Dr. E. Laine for valuable discussions and for editorial assistance, I. Chau‐ vot de Beauchêne and S. Abdel-Azeim for providing of illustrative materials. This work is funded by the Centre National de la Recherche Scientifique (CNRS), Ecole Normale Supér‐

BiMoDyM, LBPA, CNRS -ENS de Cachan, LabEx LERMIT, CEDEX Cachan, France

Data Bank. Nucleic Acids Research 2000 Jan 1;28(1):235-42.

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[3] Hare S, Vos AM, Clayton RF, Thuring JW, Cummings MD, Cherepanov P. Molecular mechanisms of retroviral integrase inhibition and the evolution of viral resistance. Proceedings of the National Academy of Sciences of the United States of America

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prospective directions in the HIV-1 integrase drug developement.

**Acknowledgement**

**Author details**

**References**

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Rohit Arora and Luba Tchertanov

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We have characterised the RAL binding, a very flexible molecule displaying the E/Z isomer‐ ism, to the active site of its HIV-1 targets which mimic the integrase states before and after the 3'-processing. The docked conformations represent a spectrum of possible conformation‐ al/configurational states. The best docking scores and poses confirm that the generated mod‐ el representing the IN•vDNA complex is the biologically relevant target of RAL, the strand transfer inhibitor. This finding is consistent with well-documented and commonly accepted inhibition mechanism of RAL, based on integral biological, biochemical and structural data.

RAL docking onto the IN•vDNA complex systematically generated the RAL chelated to Mg2+cations at the active site by the pharmacophore oxygen atoms. The identification of IN residues specifically interacting with RAL is likely a very difficult task and the exact modes of binding of this inhibitor remain a matter of debate. Most probably the flexible nature of RAL results in different conformations and the mode of binding may differ in terms of the interacting residues of the target, which trigger the alternative resistance phenomenon.

The identified RAL binding to the processed viral DNA shed light on a putative, even plau‐ sible, step of the RAL inhibition mechanism.

We have implemented dynamic properties to the HIV-1 targets characterisation, particular‐ ly, the internal protein collective motions and the global conformational transition. Such transitions play an essential role in the function of many proteins, but experiments do not provide the atomic details on the path followed in going from one end structure to the other. For the dimeric IN, the transition pathway between the unbound and bound to vDNA is not known, which limits information of the cooperative mechanism in this typical allosteric sys‐ tem, where both tertiary and quaternary changes are involved. Description of the IN inter‐ mediate conformations open a way to localise the allosteric pockets, which in turn can be selected as the putative binding sites for small molecules in a virtual screening protocol.

Novel drugs, targeted the HIV-1 Integrase, outcome mainly due to the rapid emergence of RAL analogues (for example, GS-9137 or elvitegravir, MK-2048 and S/GSK 1349572, current‐ ly under clinical trials [93]). The clinical trials of several RAL analogues (BMS-707035, GSK-364735) were suspended. All these molecules specifically suppress the IN ST reaction. We conceive that the future HIV-1 integrase drug development will be mainly oriented to design of inhibitors with a mechanism of action that differs from that of RAL and its ana‐ logues. Distinct conceptions are potentially conceivable: (i) Design of the allosteric inhibi‐ tors, able to recognize specifically the binding sites that differ from the IN active site. Inhibitor V-165, belonging to such type inhibitors, prevents IN binding with the viral DNA such blocking 3'-processing reaction [94]. (ii) Design of the protein-protein inhibitors (PPIs) acting on interaction interface between either viral components (the IN monomers upon multimerization process or sub-units of the IN•vDNA complex) [95,96], or between viral and cellular proteins (IN/LEDGF) [97,98]. These alternative strategies represent rational and prospective directions in the HIV-1 integrase drug developement.
