In-Silicodocking of HIV-1 integrase inhibitors reveals a novel drug type acting on an enzyme/DNA reaction intermediate
© Savarino; licensee BioMed Central Ltd. 2007
Received: 12 February 2007
Accepted: 20 March 2007
Published: 20 March 2007
HIV-1 integrase (IN) is an emerging drug target, as IN strand transfer inhibitors (INSTIs) are proving potent antiretroviral agents in clinical trials. One credible theory sees INSTIs as docking at the cellular (acceptor) DNA-binding site after IN forms a transitional complex with viral (donor) DNA. However, mapping of the DNA and INSTI binding sites within the IN catalytic core domain (CCD) has been uncertain.
Structural superimpositions were conducted using the SWISS PDB and Cn3D free software. Docking simulations of INSTIs were run by a widely validated genetic algorithm (GOLD).
Structural superimpositions suggested that a two-metal model for HIV-1 IN CCD in complex with small molecule, 1-(5-chloroindol-3-yl)-3-(tetrazoyl)-1,3-propandione-ene (5CITEP) could be used as a surrogate for an IN/viral DNA complex, because it allowed replication of contacts documented biochemically in viral DNA/IN complexes or displayed by a crystal structure of the IN-related enzyme Tn5 transposase in complex with transposable DNA. Docking simulations showed that the fitness of different compounds for the catalytic cavity of the IN/5CITEP complex significantly (P < 0.01) correlated with their 50% inhibitory concentrations (IC50s) in strand transfer assays in vitro. The amino acids involved in inhibitor binding matched those involved in drug resistance. Both metal binding and occupation of the putative viral DNA binding site by 5CITEP appeared to be important for optimal drug/ligand interactions. The docking site of INSTIs appeared to overlap with a putative acceptor DNA binding region adjacent to but distinct from the putative donor DNA binding site, and homologous to the nucleic acid binding site of RNAse H. Of note, some INSTIs such as 4,5-dihydroxypyrimidine carboxamides/N-Alkyl-5-hydroxypyrimidinone carboxamides, a highly promising drug class including raltegravir/MK-0518 (now in clinical trials), displayed interactions with IN reminiscent of those displayed by fungal molecules from Fusarium sp., shown in the 1990s to inhibit HIV-1 integration.
The 3D model presented here supports the idea that INSTIs dock at the putative acceptor DNA-binding site in a IN/viral DNA complex. This mechanism of enzyme inhibition, likely to be exploited by some natural products, might disclose future strategies for inhibition of nucleic acid-manipulating enzymes.
Inhibitors of the human immunodeficiency virus type 1 (HIV-1) integrase (IN) enzyme, represent a major advancement in AIDS research, showing potent antiretroviral effects in advanced clinical trials [1–4]. However, despite the decade-long studies in this field (reviewed in: ), several questions on the interactions of IN with its inhibitors have remained unanswered [1, 2]. These include: the docking site, possible interactions with metal ions and viral DNA, the amino acids involved in binding, the role of drug resistance mutations, and the conformations assumed by the inhibitors in complex with the enzyme. Elucidation of these issues is crucial, given the strict requirement of IN for insertion of proviral DNA into the cell genome, leading to retroviral latency and persistence during therapy .
Given the increasing importance of selective INSTIs for AIDS medicine and their novel mechanism acting upon a protein/DNA complex, some two-metal IN models were created by molecular modeling in an attempt to describe inhibitor binding in silico [12, 14]. However, the only docking study using a protein/DNA complex was conducted by Barreca et al.  As a surrogate platform, these authors employed a 3D structure of Tn5 transposase in complex with two metal ions and donor DNA. Other models are however necessary, since susceptibility of HIV-1 IN to INSTIs may be affected by few amino acid changes, as shown by drug-resistance mutation studies . Moreover, the available structures present the Tn5 enzyme in complex with the blunt-end reaction intermediate which is not produced by HIV-1 IN . On the other hand, theoretical structures of the HIV-1 IN in complex with donor DNA (obtained by molecular modelling and in-silico automated docking) [21, 22] can only hazardously be used as a platform to study inhibitor binding, in the absence of further validation. In-silico docking of INSTIs at these models would be the final step of a number of computational simulations (e.g. reconstruction of full-length IN, protein/DNA docking), thus harboring the risk of becoming extremely artificial. In the absence of suitable 3D models, reliable information on the interactions of IN with DNA and specific inhibitors is derived from cross-linking experiments . These studies, however, detected only few protein/DNA contacts and cannot furnish a full 3D view of the complex.
Using a 3D platform exploiting crystallographic data on IN CCD in complex with 5CITEP as a surrogate model for in-silico docking simulations of INSTIs, the present study provides a first view into an IN active site harbouring the new antiretrovirals. The computational procedures adopted here bypass artificial steps such as in-silico reconstruction of full-length IN and IN/DNA complexes, and are limited to one small-molecule inhibitor docking step, using a widely validated genetic algorithm. The docking solutions are in agreement with robust biochemical data in the literature and may disclose new insights into inhibition of an enzyme/substrate reaction intermediate.
Results and discussion
The Tn5 transposase/transposable DNA complex shows similarities with and differences from the HIV-1 IN/viral DNA interaction
When the Tn5 DNA was transposed onto the HIV-1 IN CCD structure, a close contact was observed between K159 and the phosphate immediately 5' to the 3' terminal nucleotide (Fig. 3B). One similar contact was described to occur with the phosphate immediately 5' to the 3' processing site of HIV-1 DNA , thus supporting the hypothesis that the 3' terminal portions of transposable DNA and HIV-1 3'processed (3'P)DNA occupy similar positions within the active sites of the two enzymes. This hypothesis is further supported by the overlap of the tetrazole ring of 5CITEP (a bioisoestere of the carboxylate anion) with the phosphate contacting K159 (Fig. 3B). Although Tn5 DNA and HIV-1 3'PDNA likely map to corresponding portions of the active sites of the two enzymes, transposable DNA per se cannot mimic HIV-1 DNA, because it is a blunt-end reaction intermediate which is not generated in the reactions catalyzed by HIV-1 IN (see Fig. 1). Moreover, the experimental data clearly reveal a loop-like structure at the 5' terminus, a likely product of 5' processing (Fig. 3A–B). Given these reasons, 5CITEP was, in the present study, preferred over transposable DNA as an HIV-1 DNA mimic. In line with this choice, a recent study  showed that the contact of Q148 (in the flexible loop) with 5CITEP, displayed by the crystal structure of Goldgur et al. , was reproducible in cross-linking experiments, and that a similar contact occurred with the 5' terminal portion of viral DNA, as well.
The nucleic acid binding site of Bacillus haloduransRNAse H likely corresponds to the cellular DNA binding site of HIV-1 IN
Tn5 transposase is not the only IN-related protein co-crystallized in complex with a nucleic acid. Crystal structures of RNAses H in complex with RNA/DNA hybrids have been published, as well [24, 25]. Of note, RNAses H are susceptible to inhibition by INSTI-related compounds . To further explore possible IN/DNA interactions, a structural alignment was performed between the IN crystal structure of Goldberg et al.  and an averaged crystal structure of Bacillus halodurans RNAse H in complex with an RNA/DNA substrate published by Nowotny et al. . This enzyme presents the advantage of being small and limited to the basic "RNAse H" fold, also displayed by part of the HIV-1 IN 3D architecture. The structural alignment shown in Fig. 3C involved 45 amino acids with the minimal RMSD (1.2 Å) at the level of those amino acids surrounding D71 and D132 in the RNAse H, corresponding to D64 and D116 of IN. The 3D similarities between HIV-1 IN and RNAses H have extensively been discussed in the literature [for a review, see: ].
When the RNA/DNA hybrid was transposed onto HIV-1 IN CCD, its projection mapped to a region within the catalytic cavity, bordering with, but distinct from the putative viral DNA-binding site, and delimited at either side by lysine residues (K136 and K159). The positive charges furnished by the metal(s) and the lysine residues are consistent with a DNA-binding region. This hypothesis is supported by structural alignments showing an overlap between a phosphate bridge of the RNA/DNA hybrid and a phosphate ion co-crystallized with HIV-1 IN by Cherepanov et al.  (Fig. 3D). Given: 1) the existence of a potential DNA-binding region adjacent to but distinct from the donor DNA-binding site in the IN catalytic site, and 2) the correspondence of this region to a well documented nucleic acid-binding site in a structurally-related enzyme (RNAse H), this region was hypothesized in the present study to be the acceptor DNA-binding site.
Transposition of 5CITEP to a two-metal integrase model replicates contacts with flexible loop residues, Y143 and E148
To generate a surrogate platform for predicting docking of INSTIs, a model of HIV-1 IN in complex with both putative metal ions was first prepared based on homology with Rous sarcoma virus (RSV) IN. For this purpose, a structure of RSV IN CCD in complex with two metal ions , was superimposed to the HIV-1 IN CCD crystal structure of Maignan et al. Similarly to all HIV-1 IN structures in complex with metals, the structure of Maignan et al. presents only one metal ion in the catalytic cavity, but, differently from other published HIV-1 IN structures, displays a well ordered catalytic triad . In one subunit of this structure (chain C), the flexible loop is present in its entirety and connects two CCD subunits in a dimer that may have biological significance, as the distance between the two active sites corresponds to 18 Å (see PDB: 1BL3), approximately one half turn of a Watson-Crick DNA helix (i.e., the distance at which the two antiparallel strands of acceptor DNA are simultaneously nicked during strand transfer [1, 2]). The structural superimposition between the HIV-1 IN CCD and the two-metal RSV IN CCD structure involved 104 amino acids with a RMSD of 0.24 Å between the α-carbons of the highly conserved catalytic triads (D64, D121 and E157 for RSV IN). The position of the metal ion between D64 and D116 of HIV-1 IN and the metal ion between D64 and D121 of RSV IN was approximately coincident (data not shown). Then, the metal ion between residues D64 and E157 of RSV IN (corresponding to D64 and E152) was transposed onto HIV-1 IN CCD, and the E152 side chain of HIV-1 IN was moved to metal-coordinating position (matching that of the equivalent residue in RSV IN).
In Silico docking fitness of HIV-1 integrase strand transfer inhibitors (INSTIs) for the catalytic cavity of integrase in complex with 5CITEP correlates with the in-vitroinhibitory potencies
The fitness scores obtained using the two-metal/IN-CCD/5CITEP complex are higher that those obtained by Barreca et al. (i.e., ~ 50) using the Tn5 transposase/DNA complex . This is not surprising, because INSTIs were developed using HIV-1 and not Tn5-based assays . On the other hand, the present study agrees with Barreca et al. that the acidic INSTIs have similar fitness in both the protonated and non-protonated form (data not shown).
These results allow the conclusion that occupation (by 5CITEP) of the putative donor DNA binding site is important for obtaining optimal docking of INSTIs, in line with a theory of Pommier et al. . Moreover, the good agreement between the experimental IC50 values and docking solutions supports the idea that the two-metal/IN-CCD/5CITEP complex could be used as a surrogate platform for in-silico screening of potential INSTIs.
Docking of integrase strand transfer inhibitors (INSTIs) reveals unexpected metal-binding modes
The metal-binding mode is an unexpected finding of the present study and is a major difference with the docking results of Barreca et al.  and those of Merck researchers . Both research teams described metal chelation through the "classic" pharmacophoric groups (i.e. a coplanar β-hydroxy keto group, to which Merck researchers add a lonely pair donor atom). Differences between the present study and that of Barreca et al. can of course be attributable to differences between IN and transposase. Differences with the Merck study are attributable to the fact that these authors manually drove the INSTIs into an uncomplexed IN active site . It is finally possible that both docking poses A and B coexist in vivo, given the alternative binding modes crystallographically documented for other classes of antiretroviral drugs.
Docking of integrase strand transfer inhibitors (INSTIs) is concordant with the drug resistance mutation profiles
To further validate the docking results, the close contacts of the INSTIs were related to well documented drug resistance mutations selected by the same inhibitors. In its best docking pose, diketo acid L-731,988 showed the carboxylate oriented towards T66, with possible hydrogen bonding (Fig. 6A). In agreement with this docking pose, T66I is a resistance mutation induced by L-731,988 which, alone, decreases diketo acid susceptibility by 6-fold . Hydrogen bonding was also possible with N155, mutation of which was shown to confer cross-resistance to diketo acids . S-1360, which induces drug resistance mutations similar to those selected by L-731,988 , also interacted with T66 (Fig. 6B). The best docking pose for L-870,812 clearly showed the carbonyl oxygen of the rotated carboxamide group directly pointing to the amide group of N155 (Fig. 6C), in perfect agreement with the drug resistance mutation N155H (i.e. the only known L-870,812-selected drug resistance mutation) . The best docking pose for L-870,810 showed the hydrophobic portion of the sulphonamide ring in Van-der-Waals contact with the F121 sidechain (Fig. 6D), in agreement with the primary L-870,810 resistance mutation F121Y . Van der Waals contacts were also possible with N155 and E92, mutations of which were shown to confer cross-resistance to this inhibitor [12, 38] (Fig. 6D). The best docking pose for GS-9137 clearly presented the isobutyl substituent on the quinolone oriented towards E92 (Fig. 6E). The hydroxyl in the isobutyl substituent replaced one of the water molecules through which E92 coordinates the metal ion between D64 and E152 (see PDB structure: 1BL3 in Ref. ). Of note, a primary mutation induced by GS-9137 is E92Q, which, alone, is capable of decreasing drug susceptibility by 33-fold . On the whole, the good agreement between the drug resistance mutation profiles and the docking poses represents a further validation of the results obtained.
Docking of integrase strand transfer inhibitors (INSTIs) maps to the putative acceptor DNA binding site
Previous studies showing a dependence of the inhibitory activity of INSTIs from the concentration of acceptor DNA led to the hypothesis that INSTIs dock at the acceptor DNA-binding site [1, 11]. If 1) this hypothesis were correct, and 2) the binding sites of INSTIs and acceptor DNA had correctly been predicted in the present study, then, structural superimpositions should result in an overlap between the docking solutions for INSTIs and the RNA/DNA hybrid in complex with B. halodurans RNAse H. Results showed that the docked INSTIs overlapped with the RNA/DNA hybrid when the IN/inhibitor complexes were superimposed to the B. halodurans RNAse H/substrate crystal structure (Fig. 6F). This result further supports the hypothesis of Pommier et al. that INSTIs dock at the acceptor DNA binding site of an IN/donor DNA complex .
Integrase inhibitors in clinical trials are bioisosteric to fungal molecules is terms of metal binding
Molecular docking techniques may produce biologically sound results also when applied to difficult drugs targets such as an enzyme/substrate reaction intermediate. Occupation by 5CITEP of the putative donor DNA-binding site shows docking of INSTIs at a putative acceptor DNA-binding site. If future crystallographic data should confirm a similar binding of INSTIs to a IN/donor DNA complex, INSTIs might represent one of the few known drug types acting on an enzyme reaction intermediate. Moreover, novel INSTIs interact in silico with the metal cofactors similarly to certain natural products such as Fusarium sp. mycotoxins. This similarity suggests that it is possible to identify natural products drug leads capable of dissecting two different steps of an enzymatic process. The presence of potential leads for drugs of this type in natural products should encourage further natural product screening and may disclose potential drug leads targeting other nucleic acid manipulating enzymes such as the reverse transcriptase-associated RNAse H.
3D structures were retrieved from the Protein Data Bank (PDB) , or from the U.S. National Center for Biotechnology Information (NCBI) website . To obtain structural alignments, the α-carbons of the highly conserved catalytic triads of HIV-1 IN and related enzymes were initially superimposed using the Swiss PDB Viewer (SPDBV) program (Swiss Institute of Bioinformatics) , which calculates the root-mean-square distance between the corresponding atoms using a least square algorithm. Using the default matrix embedded in the program (with open and extended gap penalties of 6 and 4, respectively) , the calculation was extended to neighboring atoms until the maximum number of aligned atoms with the lowest RMSD was obtained. The program Pymol (v0.99; DeLano Scientific LLC, S. Francisco, CA) (freely downloadable from: ) was used to visualize the superimposed structures. Structural alignments were double-checked using binary ASN1 files and the Cn3D program (version 4.1), downloadable from the NCBI website .
Generation of a two-metal/integrase model
The crystal structure of HIV-1 IN CCD solved by Maignan et al.  (PDB accession: 1BL3_C) was used as a basis for modeling the IN CCD in complex with two metal ions. Using SPDBV, this structure was superimposed to one crystal structure of RSV IN CCD (PDB: 1VSH), where two metal ions are present in the active site . Using the 'torsion' option embedded in the program, the E152 side chain was moved to metal-coordinating position (matching that of the equivalent residue in the RSV IN, E157). The position of the metal between D64 and E152 was deduced from the 3D coordinates of the corresponding metal in the aligned RSV IN.
The 3D structures of well characterized IN inhibitors including INSTIs in clinical trials were initially generated as pdb files using the CORINA web interface , on the basis of the SMILES strings published in the NCBI website . The program VEGA ZZ (University of Milan, Italy; freely available at: ) was adopted to assign the correct bond types. The compounds were considered in their keto-enol tautomeric form, since it has been clearly established that these molecules mainly exist in this form in solution (reviewed in: ). Moreover, both neutral and ionic forms were generated for the carboxylic acid and triazole groups of compounds. Using the default parameters in the VEGA program, force fields and charges were assigned according to AMBER and Gasteiger algorithms, respectively, and the molecules were energy-minimized by 50 cycles of conjugate gradients (CG). Minimization was stopped when the RMSD between two subsequent solutions was lower than 0.1 Å. Energy minimized ligands were then saved as mol files.
A surrogate platform for molecular docking of INSTIs was generated by transposing the 3D coordinates of 5CITEP in the structure of Goldgur et al.  onto the aforementioned two-metal model of HIV-1 IN CCD, after performing a structural alignment. Water molecules were discarded from the pdb file, and missing side chains were reconstructed using the option 'prepare file for docking programs' available at the WHAT-IF web interface . Hydrogens were added using VEGA. The structure was then subjected to energy minimization using the default settings of the SPDBV program, i.e. 20 cycles of steepest descent (SD), and minimization stopping when the Δ energy was below 0.05 kJ/mol. The protein file was eventually converted to mol2 format using Mercury (v. 1.4.2; Cambridge Crystallographic Data Centre (CCDC); freely downloadable from: ). Automated docking studies were then performed using the genetic algorithm GOLD (Genetic Optimization for Ligand Docking)  (v. 3.1; CCDC, Cambridge, UK), according to a protocol published by Barreca et al. . The algorithm had been previously validated and successfully tested on a data set of over 300 complexes extracted from the PDB . The program was further validated in the author's hands by obtaining docking poses for HIV-1 protease inhibitors lopinavir and ritonavir nearly identical to the structures co-crystallized in complex with the HIV-1 protease (RMSD < 0.2 Å; data not shown). The binding site was initially defined as all residues of the target within 10 Å from the metal atom coordinated by D64 and D116, and later automated cavity detection was used. GOLD score was chosen as fitness function and the standard default settings were used in all calculations. For each of the 10 independent genetic algorithm runs, a default maximum of 10,000 genetic operations was performed, using the default operator weights and a population size of 100 chromosomes. Default cutoff values of 2.5 Å for hydrogen bonds and 4 Å for Van der Waals interactions were employed. The two metal ions were set to allow hexavalent coordination according to a Mg2+ type (i.e. the metal thought to act as a co-factor in vivo). Carboxylate and carboxamide substituents on aromatic rings were allowed to rotate. Early termination was allowed for results differing by less than 1.5 Å in ligand all atom RMSD.
Post docking analysis was done using the program SILVER (CCDC, UK), in order to evidence close contacts such as hydrogen bonds and Van der Waals interactions.
Supported by "Special Project AIDS", Istituto Superiore di Sanità, Rome, Italy (intramural grant: BASTET: Bases for Assessment and Evaluation of Eradication Strategies), and by the Italian Ministry of Research and University, Rome, Italy (FIRB grant: Costruzione di un laboratorio nazionale per le resistenze agli antimicrobici). The author is thankful to: Sandro Norelli (MS), Rome, Italy, Liliana Calosso (MD), Pinerolo, Italy, and Daniela D'Ostilio (BSc), Rome, Italy, for help in the preparation of the figures and in editing the manuscript; Romolo Savarino (DEng), Vinovo, Italy, for mathematical advice; Massimo Ciccozzi (PhD.), Rome, Italy for critically reading the manuscript; Roberto Cauda (MD), Rome, Italy, for furnishing the GOLD program and helpful discussion on integrase inhibitors; and, finally, Giancarlo Majori (MD), Rome, Italy, and Antonio Cassone (MD, PhD), Rome, Italy, for logistic support.
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