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J. Biol. Chem., Vol. 275, Issue 28, 21460-21467, July 14, 2000

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Mechanism of Inhibition of HIV-1 Integrase by G-tetrad-forming Oligonucleotides in Vitro^*

Naijie Jing^§, Christophe Marchand^¶, Jie Liu, Rahul Mitra, Michael E. Hogan, and Yves Pommier^¶

From the  Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030 and the ^¶ Laboratory of Molecular Pharmacology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255

Received for publication, February 18, 2000, and in revised form, April 24, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The G-tetrad-forming oligonucleotides T30177 and T30695 have been identified as potent inhibitors of human immunodeficiency virus type 1 integrase (HIV-1 IN) activity (Rando, R. F., Ojwang, J., Elbaggari, A., Reyes, G. R., Tinder, R., McGrath, M. S., and Hogan, M. E. (1995) J. Biol. Chem. 270, 1754-1760; Mazumder, A., Neamati, N., Ojwang, J. O., Sunder, S., Rando, R. F., and Pommier, Y. (1996) Biochemistry 35, 13762-13771; Jing, N., and Hogan, M. E. (1998) J. Biol. Chem. 273, 34992-34999). To understand the inhibition of HIV-1 IN activity by the G-quartet inhibitors, we have designed the oligonucleotides T40215 and T40216, composed of three and four G-quartets with stem lengths of 19 and 24 Å, respectively. The fact that increasing the G-quartet stem length from 15 to 24 Å kept inhibition of HIV-1 IN activity unchanged suggests that the binding interaction occurs between a GTGT loop domain of the G-quartet inhibitors and a catalytic site of HIV-1 IN, referred to as a face-to-face interaction. Docking the NMR structure of T30695 (Jing and Hogan (1998)) into the x-ray structure of the core domain of HIV-1 IN, HIV-1 IN-(51-209) (Maignan, S., Guilloteau, J.-P., Qing, Z.-L., Clement-Mella, C., and Mikol, V. (1998) J. Mol. Biol. 282, 359-368), was performed using the GRAMM program. The statistical distributions of hydrogen bonding between HIV-1 IN and T30695 were obtained from the analyses of 1000 random docking structures. The docking results show a high probability of interaction between the GTGT loop residues of the G-quartet inhibitors and the catalytic site of HIV-1 IN, in agreement with the experimental observation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Combination therapy for AIDS, which uses two or more drugs simultaneously to inhibit human immunodeficiency virus (HIV)¹ replication, was developed to lower toxicity by decreasing the dosage of individual compounds. This approach reduces the risk of developing drug resistance and maintains synergistic antiviral activity (1). Although combination therapy can depress the HIV virus to undetectable levels in the blood of many HIV-positive patients, the HIV viruses within T cells are still fully capable of replicating and infecting other cells (2-4). The development of new agents against human immunodeficiency virus type 1 integrase (HIV-1 IN) (5), which may eliminate HIV-1 from intracellular sites, would be a major advance in the treatment of HIV infection.

A new class of oligonucleotides with only G and T residues in their sequences has been discovered to inhibit HIV-1 IN (6, 7). The analyses have been performed on the oligonucleotide 5'-G*TGGTGGGTGGGTGGG*T, synthesized with a phosphorothioate linkage at the two end G residues (G*) and referred to as T30177. This G-rich oligonucleotide is capable of forming a stable intramolecular G-quartet fold (6). IC₅₀ (50% inhibitory concentration) values of T30177 for HIV-1 IN are in the nanomolar range in vitro (7).

A 16-residue oligonucleotide (5'-G*GGTGGGTGGGTGGG*T) referred to as T30695 has been designed and synthesized to improve both the structural stability and inhibition of HIV-1 IN activity (8). Compared with T30177, T30695 forms an even more stable and orderly G-quartet fold. Our NMR and kinetic data demonstrated that in response to K⁺ binding, T30695 folds into a stable and symmetric G-tetrad complex (8-10). The folding is a two-step process, dependent on the nature of the alkaline metal ion. The first step of the process involves the coordination of one K⁺ ion, which competes with a Li⁺ ion to bind within the core of two G-quartets. The second step involves the binding of two additional K⁺ ions to the loop domains. NMR results have shown that in the absence of K⁺, T30695 forms an intramolecular fold with a pair of distorted G-quartets flanked by extended, unfolded loop domains (called the Li⁺ form). When coordinated with 3 K⁺ eq, T30695 folds into a more compact and symmetric structure with an ~15-Å width and a 15-Å length (called the K⁺ form). This structure resembles a cylinder with positive charges inside and negative charges on the outer surface (10). The IC₅₀ of T30695 for inhibition of HIV-1 IN without K⁺ (Li⁺ form structure) is 530 nM. In the presence of K⁺, the IC₅₀ of T30695 (K⁺ form structure) decreases from 530 to 31 nM, corresponding to a 20-fold increase in the inhibition of HIV-1 IN activity. Kinetic data demonstrated that this increase in inhibition of HIV-1 IN activity for T30695 is correlated with the folding of its loops into a stable, compact structure by the binding of 2 additional K⁺ eq. The loop structures of T30695 appear to play a key role in the inhibition of HIV-1 IN activity.

To investigate the binding interaction between the catalytic site of HIV-1 IN and the G-quartet inhibitors, we have designed G-tetrad-forming oligonucleotides with two, three, and four G-quartets. The lengths of these G-quartet stems are 15, 19, and 24 Å, respectively. The results demonstrated that these G-tetrad-forming oligonucleotides with different stem lengths have the same ability to inhibit HIV-1 IN activity in vitro. Based upon the experimental evidence and modeling study, we provide some critical information regarding the interaction and structure-activity between HIV-1 IN and the G-quartet inhibitors, which could be of benefit in the design of novel anti-HIV therapeutic drugs.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Oligonucleotide Synthesis-- All of the G-rich oligonucleotides were obtained from Midland Certified Reagent Co. (Midland, TX). These oligonucleotides were synthesized using cyanothyl phosphoramidite chemistry. After removal of the protecting groups by hydrolysis with concentrated ammonium hydroxide, the products were purified by anion-exchange high pressure liquid chromatography. The qualities of materials were then measured by mass spectroscopy. We used these oligonucleotides without further treatments.

Melting and Annealing Measurements-- Oligonucleotides at 5 µM strand equivalents (20 mM Li₃PO₄, pH 7) were heated to 90 °C for 5 min and then cooled at 4 °C for 30 min in the presence of KCl at a designated concentration. Subsequent to the incubation step, thermal denaturation profiles of the oligonucleotides were obtained over a range from 20 to 90 °C for melting and from 90 to 20 °C for annealing. Absorbance was monitored at 240 nm by a Hewlett-Packard 8452A diode array spectrophotometer using a Hewlett-Packard 89090A temperature regulator.

The thermal denaturation curves of the oligonucleotides were analyzed with an intramolecular folding equilibrium (8, 11): A(T) = (1  )A_rc + A_st,  = (0.5 + 0.5(K_eq  1)/((1  K_eq)² + 4K_eq)^1/2), and K_eq = exp((H⁰ + TS⁰)/RT), where K_eq is the constant for the random coils to folded oligonucleotide equilibrium,  is the fraction of folded strands, 1   is the fraction of random coils, A(T) is the absorbance at temperature T, A_rc is the absorbance when all strands are random coils, A_st is the absorbance when all strands are folded, and  is the cooperativity of the melting transition (referred to as the helix interruption constant) defined by  = exp(S_i/R), where S_i is in units/mol of interruption. In our analysis study, the values of  are in the range of 0.9-0.999, determined by an optimized fitting program. Values for T_m and G were obtained on the basis of the fitting procedure, which inputs the values of H⁰, S⁰, A_rc, and A_st, estimated from the experimental measurements, and then uses an optimized fitting program to search for the best fit.

Gel Electrophoresis-- The G-rich oligonucleotides, T30695 and its derivatives, plus 10 mM KCl in 20 mM Li₃PO₄, pH 7, were labeled with ³²P using a 5'-end labeling procedure and purified using Microspin G-25 columns. The oligonucleotide solution was heated at 90 °C for 5 min and then cooled at 4 °C for 30 min. 20% nondenaturing polyacrylamide gels containing 1× Tris/borate acid/EDTA buffer, 10% ammonium persulfate, and 30 µl of TEMED in 1× TBA buffer were precooled in a 4 °C cold room for 1 h. The prepared samples were run on 20% nondenaturing polyacrylamide gels in a 4 °C cold room for 6 h. Gels were stained in a 0.01% Stains-All/formamide solution.

Circular Dichroism-- CD spectra of the G-quartet oligonucleotides were obtained in 15 µM strand concentration plus 10 mM KCl in 20 mM Li₃PO₄, at pH 7, on a Jasco J-500A spectropolarimeter at 24 °C. Each spectrum represents five average scans. Data are presented in molar ellipticity (degrees·cm²·dmol¹).

HIV-1 IN Protein-- The pET-15b-IN-(1-288)/F185K/C280S plasmid was expressed in Escherichia coli strain BL21 as described previously (12) with the following modifications. Cells were grown in 1000 ml of LB medium (Digene, Beltsville, MD) containing 5 µg/ml ampicillin until an optical density of 0.8 was reached at 600 nm. Protein expression was induced for 3 h with the addition of 0.4 mM isopropyl--D-thiogalactopyranoside. Cells were harvested and resuspended in lysis buffer containing 25 mM HEPES, pH 7.5, 1 M NaCl, 5 mM imidazole, 2 mM -mercaptoethanol, and 0.3 mg/ml lysozyme. After 30 min on ice and sonication, lysed cells were centrifuged for 20 min at 30,000 × g, and the supernatant was applied to a nickel-Sepharose column. Integrase retained on the column was washed with buffer containing 25 mM HEPES, pH 7.5, 0.5 M NaCl, 2 mM -mercaptoethanol, and an increasing imidazole concentration from 20 to 250 mM. The protein was then eluted with the same buffer containing 750 mM imidazole and dialyzed overnight against 25 mM HEPES, pH 7.5, 1 M NaCl, 2 mM EDTA, 10 mM dithiothreitol, 2 mM -mercaptoethanol, 100 mM imidazole, and 10% glycerol.

Anti-HIV-1 IN Activity Assay-- The HIV-1 IN assays were performed as described previously (7) with the following modifications. In the 3'-processing and strand transfer assay, HIV-1 IN was preincubated at a final concentration of 400 nM with inhibitors for 15 min at 30 °C in a reaction buffer containing 25 mM MOPS, pH 7.2, 25 mM NaCl, 7.5 mM MnCl₂, 0.1 mg/ml bovine serum albumin, and 14.3 mM -mercaptoethanol. Then, a 5 nM concentration of the ³²P-5'-end-labeled oligonucleotide was added to a final volume of 10 µl, and incubation was continued for an additional 30 min. Reactions were quenched by addition of 5 µl of denaturing loading dye. Samples were loaded on a 20% (19:1) denaturing polyacrylamide gel.

Modeling Computation-- The molecular structures of T40215 and T40216 were built up and optimized under AMBER force field by INSIGHT II/DISCOVER. The optimization of the molecular structures followed this procedure: 1) 100 steps of conjugate gradient energy minimization; 2) 1000 steps of restrained molecular dynamics equilibration with a time step of 0.33 fs at 1000 K; 3) 1000 steps of restrained molecular dynamics equilibration with a time step of 0.1 fs at 300 K; and 4) 1000 steps of conjugate gradient energy minimization. The intramolecular hydrogen bonds of G-quartets were used as constrains for the molecular optimization.

The molecular structure of the core domain of HIV-1 IN, HIV-1 IN-(51-209) (14), was obtained from the Protein Data Bank. Next, we docked the NMR structure of T30695 (10) into this x-ray structure using the GRAMM docking program with a high resolution matching mode (15-18). This program is based on a geometry-based algorithm for predicting the structure of a possible complex between molecules of known structures. It can provide quantitative data related to the quality of the contact between the molecules. The intermolecular energy calculation relies on the well established correlation and Fourier transformation techniques used in the field of pattern recognition. The docking calculation by GRAMM predicts the structure of the complex formed between the two constituent molecules by using their atomic coordination, without any prior information as to their binding sites. To eliminate the terminal effects of the structure of HIV-1 IN-(51-209) in the docking calculation, the docking range of the calculation was set up from amino acids 60 to 160. 1000 different structures of an HIV-1 IN·T30695 complex were created by the calculations. The information on the binding interaction and hydrogen bond formation of each docking structure was analyzed. Based upon the hydrogen bond formation, the statistical possibilities of the residues of T30695 interacting with HIV-1 IN and of the amino acid residues of HIV-1 IN binding T30695 were plotted.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Structures of T30923, T40215, and T40216-- T30695 has been determined previously as an intramolecular G-quartet structure with two G-quartets in the center and two folded loop domains on the top and bottom (10). Upon coordination with 3 K⁺ ion eq, the structure of T30695 becomes symmetric and compact with a 15-Å width and a 15-Å length. The distance between the two central G-quartet planes is ~3.9 Å. Recently, T30695 and the thrombin-binding aptamer, which also forms an intramolecular G-quartet structure (19, 20), were further studied by nondenaturing gel electrophoresis (11). Similar migration further confirmed that T30695 forms an intramolecular G-quartet structure with the same structural size as the thrombin-binding aptamer. T30923 (5'-GGGTGGGTGGGTGGGT) has the same sequence as T30695 (Fig. 1). However, the two terminal phosphorothioate linkages of T30695 in the G¹ and G¹⁵ positions are replaced by two phosphodiester linkages. We previously showed that T30923 has the same structure and physical properties as T30695 (11).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Intramolecular G-quartet folding models. The formation of G-quartets is indicated by dashed lines. The two TGTG loop domains of T30923 are formed on the top and bottom of the pair of G-quartets.

To investigate the interactions between HIV-1 IN and the G-quartet oligonucleotides, T40215 (5'-(GGGGT)₄) and T40216 (5'-(GGGGGT)₄) were designed to form an intramolecular G-quartet structure, composed of three and four G-quartets with stem lengths of ~19 and 24 Å, respectively (Fig. 1). CD spectra were employed to demonstrate that T30923, T40215, and T40216 form the same molecular structure as T30695. The CD spectra of T30695, T30923, T40215, and T40216 are characterized by nonconservative spectra, with maxima at 264 and 210 nm and minima at 240 nm (Fig. 2), which demonstrated that all four oligonucleotides form the same G-quartet structure. Compared with the CD spectrum of T30695, it appears that a longer G-quartet stem corresponds to weaker CD ellipticity at 264 and 240 nm.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   CD spectra of T30695, T30923, T40215, and T40216. CD data were obtained with a 15 µM strand concentration in the presence of 10 mM KCl at 24 °C.

Further evidence to support intramolecular G-quartet formation for T30923, T40215, and T40216 was obtained from nondenaturing gel electrophoresis (Fig. 3A) and from melting and annealing measurements (Fig. 3B and Table I). Fig. 3A shows that the bands of T30923, T40215, and T40216 have the same migration compared with that of T30695. T30695 was used as a control since its molecular structure has been determined by NMR to form an intramolecular G-quartet structure in the presence of K⁺ (10). The migration rate on nondenaturing gels depends on the molecular structure of the G-quartet oligonucleotides. The same migration of all four G-quartet oligonucleotides demonstrates that they form the same molecular structure with a similar size. Therefore, T30923, T40215, and T40216 also form an intramolecular G-quartet structure in the presence of 10 mM KCl. However, electrophoresis cannot resolve the small difference in the length of these G-quartets from 15 to 24 Å.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   A, electrophoresis of G-quartet oligonucleotides T30695, T30923, T40215, and T40216 in the presence of 10.0 mM KCl on a nondenaturing gel running at 4 °C (see "Materials and Methods" for details); B, melting and annealing curves. The melting and annealing curves of T40215 were obtained at a 5 µM strand concentration from UV absorbance at a 240-nm wavelength. Tm values of melting and annealing measurements are 72.7 and 70.3 °C, determined by the midpoints of the transitions.

A melting curve corresponds to a disordered state, called hyperchromicity. The annealing curve measures the base pairing and stacking of the secondary structure, called hypochromicity. The two curves (heating and cooling) of the same oligonucleotides are expected to be identical if the oligonucleotide forms an intramolecular, self-associated G-quartet. Compared with the melting T_mH (where "H" is heating) values in Table I, the annealing T_mC (where "C" is cooling) values of all three oligonucleotides at 5 µM strand concentration have a slight shift to a lower temperature. The T values of T30923, T40215, and T40216 are 1.4, 2.4, and 3.4 °C, respectively, showing that the molecules with longer G-quartet stems have greater T values. Interestingly, the T values of T40215 at strand concentrations of 2 and 20 µM are 0 and 4.8 °C, respectively, which suggests that T may also depend on oligonucleotide concentration and that higher concentrations induce greater T. At 2 µM, there was no measurable temperature shift (T = 0) between the melting and annealing curves of T40215, which provides evidence for an intramolecular G-quartet formation. The slight temperature shifts observed at 5 and 20 µM were considered to be induced by the disruption of structure refolding caused by higher molecular aggregation. The same phenomenon also applied when the G-quartet stem length was increased.

The constant T_mH values of T40215 from 2 to 20 µM (72.7 °C) also confirm that this oligonucleotide form an intramolecular G-quartet structure. T_m depends on the oligonucleotide concentration when a G-quartet structure is formed by dimeric or tetrameric oligonucleotides. Higher oligonucleotide concentrations increase the possibility of forming dimeric or tetrameric G-quartet structures, corresponding to higher T_m. Only the T_m of an intramolecular G-quartet structure is independent of the concentration of oligonucleotides. The conclusion obtained from these results is consistent with that from Fig. 3A.

The ion binding stoichiometry of the G-quartet structures has been established previously (8). First, the thermal denaturation curves of T30923, T40215, and T40216 in 0.1, 0.5, 1.0, and 5.0 mM KCl were obtained from UV absorbance at 240 nm and then analyzed using a single-strand denaturation equilibrium (see "Materials and Methods" for details). The fitting procedure is to input the constants H⁰, S⁰, A_rc and A_st, which were estimated from the experimental data, and then to use an optimizing program to search for the best fit. As shown in Table II, the fitting coefficient for each melting curve is ~0.99 or higher. The slopes of the lines for T30923, T40215, and T40216 were 3.8, 5.1, and 6.5, respectively, obtained by fitting the data points of the calculated G⁰ versus log[K⁺] (Fig. 4). Based upon n = G⁰/2.3RTlog₁₀[K⁺] and k_slope = G⁰/log₁₀[K⁺], the n K⁺ ion eq released from unfolding was calculated as 2.8, 3.8, and 4.8 for T30923, T40215, and T40216, respectively. NMR data previously confirmed that T30695 coordinates three K⁺ ions. One is bound between the two G-quartets, and two additional K⁺ ions are bonded between a G-quartet and a neighboring TGTG loop domain: one at the top and the other at the bottom (9, 10). The n values obtained here indicate that T30923 also coordinates three K⁺ ions, whereas T40215 and T40216 coordinate four and five K⁺ ions between three and four G-quartets and two loop domains, respectively. These results confirm that T30923, T40215, and T40216 are composed of two, three, and four G-quartets, respectively, as shown in Fig. 5.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Plot of G0 values of T30923, T40215, and T40216 versus log10[KCl]. These data were fitted by a straight line yielding a slope (kslope = G0/log10[K+]) of 3.8, 5.1, and 6.5 for T30923, T40215, and T40216, respectively. According to the simple model of the transition between the folded and unfolded states for an intramolecular G-quartet (7), the values of the released K+ equivalent, n (=G0/2.3RTlog10[K+]), for T30923, T40215, and T40216 are ~2.8, 3.8, and 4.8, which correspond to the release of 3, 4, and 5 K+ ion eq.

View larger version (65K): [in this window] [in a new window]   Fig. 5.   Molecular structures of T30695, T40215, and T40216. The structure of T30695 was determined by NMR (10). The structures of T40215 and T40216 were obtained from molecular modeling (see "Materials and Methods" for details). T30923 has the same structure as T30695 (not shown). A, top view, which shows that the three structures have similar GTGT loop structures; B, side view, which shows different G-quartet stem lengths for the three oligonucleotides: T30695, T40215, and T40216 with two, three, and four G-quartets, respectively.

Inhibition of HIV-1 IN Activity by T30923, T40215, and T40216-- The inhibition of HIV-1 IN by G-quartet oligonucleotides has been measured in a dual assay for 3'-processing and strand transfer activities (7). As shown in Fig. 6A, T30923, T40215, and T40216 were tested for the effect on both 3'-processing and strand transfer using a 21-mer duplex oligonucleotide. The 3'-processing reaction cleaves the 3'-terminal dinucleotide to a 19-mer oligonucleotide. The strand transfer reaction then results in an integration of two oligonucleotides together, which links the precleaved 3'-end of one 19-mer oligonucleotide to another 21-mer noncleaved or 19-mer precleaved oligonucleotide (Fig. 6A). The strand transfer products yield larger molecular species with slower migration compared with the 21-mer substrate. The inhibition of HIV-1 IN activity by T30923, T40215, and T40216 is shown in Fig. 6B. Compared with control lanes 2 and 21 (with only DNA plus integrase), the intensities of the 19-mer oligonucleotide band and of the strand transfer product bands decreased when the concentrations of T30923, T40215, and T40216 were increased. This result demonstrated that the G-quartet oligonucleotides blocked HIV-1 IN to yield the products of 3'-processing and strand transfer. IC₅₀ values for inhibition of 3'-processing and strand transfer for T30923, T40215, and T40216 were obtained from plots of percentage inhibition versus drug concentration (Fig. 6C). IC₅₀ values for T30923, T40215, and T40216 were 70, 100, and 80 nM in 3'-processing and 85, 90, and 60 nM in strand transfer, respectively (Table III).

View larger version (38K): [in this window] [in a new window]   Fig. 6.   HIV-1 IN catalytic assay. A, a 21-mer 5'-labeled double-stranded oligonucleotide corresponding to the U5 end of the HIV-1 proviral DNA is reacted with purified HIV-1 IN. The initial step (3'-processing (3'-P)) involves cleavage of the 3'-terminal dinucleotide, resulting in a 19-mer labeled product. The second step (strand transfer (ST)) involves joining this recessed 3'-end to the 5'-end of an integrase-induced break in another identical oligonucleotide, which serves as a target DNA. Strand transfer can also take place in the lower strand of the duplex oligonucleotide (not shown). B, concentration-dependent inhibition of HIV-1 IN by T30923, T40215, and T40216. Lane 1, DNA alone; lanes 2 and 21, DNA plus integrase; lanes 3 and 9, 5 nM; lanes 4 and 10, 15 nM; lanes 5 and 11, 45 nM; lanes 6 and 12, 137 nM; lanes 7 and 13, 410 nM; lanes 8 and 14, 1230 nM; lane 15, 7.4 nM; lane 16, 22.2 nM; lane 17, 66.6 nM; lane 18, 200 nM; lane 19, 600 nM; lane 20, 1800 nM. STP, strand transfer products. C, graphs derived from quantification. Shown are the inhibition of the 3'-processing reaction (left panel) and the inhibition of the strand transfer reaction (right panel) by T30923 (), T40215 (), and T40216 ().

The results of the assay of anti-HIV IN activity demonstrated that all three G-quartet oligonucleotides had comparable ability to inhibit HIV-1 IN. However, based upon the structure determination (see above), T30923, T40215, and T40216 are composed of two, three, and four G-quartets with stem lengths of ~15, 19, and 24 Å, respectively. The same range of IC₅₀ values for the three oligonucleotides indicated that the increase in length of the G-quartet stem did not appear to disrupt the binding interaction between the catalytic sites of HIV-1 IN and the G-quartet oligonucleotides. Based on this observation, we propose that the binding interaction occurs between a catalytic site of HIV-1 IN and a GTGT loop domain of the G-quartet oligonucleotides, referred to as a face-to-face interaction. Previous NMR data have demonstrated that a GTGT loop domain of the G-tetrad-forming oligonucleotides folds into a plane when a K⁺ ion is bound between the loop domain and a neighbor G-quartet (8, 10). The folded loop domains largely increase the ability of the G-tetrad-forming oligonucleotides to inhibit HIV-1 IN activity because of an enhancement in face-to-face interaction. Therefore, an increase in the G-quartet stem without disrupting the loop domains should not affect the inhibition of HIV-1 IN. Also, the IC₅₀ values of all three G-quartet oligonucleotides are in the range of 50-100 nM, corresponding to a strong binding interaction between HIV-1 IN and the G-quartet oligonucleotides. This binding interaction largely blocks the catalytic activity of HIV-1 IN, which suggests that the size of a folded GTGT loop plane matches the active site of HIV-1 IN.

Molecular Model for the Interactions between HIV-1 IN and T30695-- Based on the structure-activity between HIV-1 IN and T30695 derivatives (this study and Refs. 6-8 and 10), we attempted to build a reasonable molecular model to describe the interactions between HIV-1 IN and the G-quartet oligonucleotides. As shown in Fig. 7, a molecular model of an HIV-1 IN·T30695 complex was obtained from the docking calculation. T30695 residues G¹, T⁸, G⁹, T¹⁶, and G¹⁰ were bound to the active site of the catalytic core domain of HIV-1 IN. Four hydrogen bonds were formed between T¹⁶ O-3' and Asp¹¹⁶ -CO, T⁸ O-2 and Glu¹⁵² -CO, G¹⁰ P²-O, and Lys¹⁵⁹ -NH, and G¹⁰ O-5' and Lys¹⁵⁹ -NH, showing a strong binding interaction between HIV-1 IN and G-quartet oligonucleotides. This complex displays a molecular model referred to as the face-to-face interaction.

View larger version (40K): [in this window] [in a new window]   Fig. 7.   Molecular structure of the HIV-1 IN·T30695 complex proposed to display the binding interaction between HIV-1 IN and T30695 in two different side views (A and B). Based upon the x-ray structure of the core domain (14) and the NMR structure of T30695 (10), the complex structure was obtained from docking calculation using the GRAMM program.

The histograms were plotted based upon the number of hydrogen bonds formed between T30695 and HIV-1 IN from 1000 random docking structures in the docking range of amino acids 60-160 (Fig. 8). Based upon the number of hydrogen bonds formed between HIV-1 IN and T30695 in the docking ranges, Fig. 8A shows the statistical distribution of the binding interaction in T30695 residues, and Fig. 8B shows the statistical distribution of the binding interaction in the catalytic domain of HIV-1 IN.

View larger version (30K): [in this window] [in a new window]   Fig. 8.   A, formation of hydrogen bonds versus residue of T30695. The plot shows the number of hydrogen bonds formed between each residue of T30695 and HIV-1 IN from the analysis of 1000 random docking structures in the docking range of residues 60-160. B, formation of hydrogen bonds versus amino acid residue of HIV-1 IN. The plot shows the number of hydrogen bonds formed between each amino acid residue of HIV-1 IN and T30695 from the analysis of 1000 random docking structures in the docking range of residues 60-160 (see "Materials and Methods" for details).

Interestingly, the histogram in Fig. 8A demonstrates that residues G¹, T⁸, G⁹, G¹⁰, and T¹⁶ have a much higher possibility of interacting with HIV-1 IN. Based upon the NMR structure of T30695, G¹, T⁸, G⁹, and T¹⁶ form one GTGT loop structure. The high possibility of the GTGT loop to interact with the catalytic domain of HIV-1 IN supports the proposed face-to-face interaction and provides an explanation for the observation that increasing the length of the G-quartet stem does not affect the anti-HIV IN ability in vitro. The statistical distributions are also in agreement with the experimental observation that the loop structure of G-quartet oligonucleotides plays a key role in anti-HIV-1 IN activity in vitro (10). The residue T⁸ demonstrated a high possibility of interacting with HIV-1 IN, which could be one of the critical factors for inhibition of HIV-1 IN in vitro, as observed in a previous study (11). The statistical distributions for the residues of T40215 and T40216 interacting with HIV-1 IN were also obtained from the analyses of 1000 different structures in the docking range of amino acid residues 60-160. The results demonstrate that the loop residues of both T40215 and T40216 have a much higher possibility of binding to the active site of the catalytic domain of HIV-1 IN (data not shown), consistent with the statistical distribution of T30695 residues.

The possibility of amino acid residues of HIV-1 IN binding T30695 in the active site is shown in Fig. 8B. In Fig. 8B, the high binding possibilities for amino acid residues 64-70, 116-119, 143-148, and 156-160 suggest that G-quartet oligonucleotides most probably inhibit enzyme function by binding to the active site, including Asp⁶⁴, Asp¹¹⁶, Glu¹⁵², and Lys¹⁵⁹. In addition, other amino acid residues such as His⁶⁷, Glu⁶⁹, Tyr¹⁴³, and Gln¹⁴⁸ also have a much high binding possibility. Their functions in the enzyme are still unclear. We also found that the amino acid residues with a high binding possibility such as Glu⁶⁹, Asp¹¹⁶, Tyr¹⁴³, and Gln¹⁴⁸ are located in loops of the molecular structure of the catalytic core domain.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

HIV-1 IN is composed of three functional and structural domains. Although all three domains are required for 3'-processing and strand transfer (21), the central core domain is directly involved in catalysis for the strand transfer reaction, as demonstrated by a disintegration assay (22). Crystal structures show that the catalytic core domain comprises a five-stranded -sheet structure flanked by helical regions (23-25). The catalytic core contains the three active residues (Asp⁶⁴, Asp¹¹⁶, and Glu¹⁵²) that form an active site required for enzyme activity and is referred to as the D,D-35-E motif. This motif can be found in all retroviral integrases (13, 26, 27). Recent experimental data and modeling demonstrate that the catalytic core domain residues Lys¹⁵⁶, Lys¹⁵⁹, and Lys¹⁶⁰ are also involved in the functional interaction of HIV-1 IN with DNA and mononucleotide inhibitors (28, 29). The N-terminal domain of HIV-1 IN is composed of four helices with a zinc coordination site (12) and is also required for full integrase activity (30-32). The C-terminal domain can bind DNA nonspecifically (33-35) and is composed of a five-stranded -barrel with an SH3-like fold (36, 37). However, the precise roles of the N- and C-terminal domains in the overall integration reaction are not well understood. No crystal structure has been identified for a full-length integrase thus far, although structures of all three HIV-1 IN domains have been determined individually (12, 19, 23-25, 36, 37, 40).

The details of the inhibition of HIV-1 IN activity by T30695 and its derivatives have not been structurally determined yet. However, several critical features for enzyme inhibition have been observed by several laboratories (6-8, 10, 38). An intramolecular G-quartet structure is required for the inhibition of HIV-1 IN activity. The loop residues of T30695 and its homologues are important for the inhibition of HIV-1 IN activity, as indicated by comparison of IC₅₀ values of T30695 with those of its derivatives with base substitution within the loops (7, 8). The results from a gel-based assay using several different integrase mutants demonstrate that T30695 homologues require the enzyme zinc finger region (in the N-terminal domain of integrase) for inhibitory activity. The zinc finger is considered to assist in stabilizing the binding to the G-tetrad inhibitor (7). Also, the structure-activity results (11) demonstrate a functional relationship between thermal stability and anti-HIV activity for the G-tetrad-forming oligonucleotides, which suggests that the anti-HIV activity of the G-quartet inhibitors depends on their structural stability. The inhibitors with higher thermal stability have a stronger ability to inhibit the HIV-1 IN activity in vitro and HIV-1 in cell culture. Although the G-quartet inhibitor T30177 was proposed to inhibit HIV-1 adsorption in cell cultures (39), the G-quartets show a strong inhibition of HIV-1 replication in cell cultures (11). Understanding the mechanism of interactions between HIV-1 IN and G-quartet inhibitors in vitro can be of benefit in improving anti-HIV drug design.

This study on the extension of G-quartet stems demonstrated that an increase in length of the G-quartet stem from 15 to 24 Å did not affect HIV-1 IN inhibition. The binding interaction between a G-quartet inhibitor and an HIV-1 IN monomer was believed to occur between a loop domain of the G-quartet inhibitor and a catalytic site of HIV-1 IN, referred to as a face-to-face interaction. This conclusion was also supported by statistical results obtained from molecular calculation. 1000 molecular structures of HIV-1 IN-(51-209)·T30695 complexes were created from a random docking calculation. The statistical distributions of hydrogen bond formation obtained from the large number of docking calculations provide the critical information that the binding interaction between HIV-1 IN and T30695 mostly occurs when the loop residues of T30695 bind to the active site of the catalytic core domain of HIV-1 IN. The statistical distributions also suggest that the strong inhibition of HIV-1 IN activity by G-quartet inhibitors in vitro (Fig. 6 and Table III) could correspond to the high possibility of the G-quartet inhibitors to bind to the active site of HIV-1 IN-(51-209). In the calculation, we identified some amino acid residues of the catalytic domain with a high possibility to interact with the G-quartet inhibitors, such as His⁶⁷, Glu⁶⁹, Tyr¹⁴³, and Gln¹⁴⁸, some of which could be critical factors for enzyme function.

The crystallized core domains of HIV-1 IN form a tight dimer (14, 23), HIV-1 IN was also proposed to function as a tetramer (13) or an octamer (25). Our molecular structure of an HIV-1 IN-(51-209)·T30695 complex was obtained from docking calculations. Compared with the molecular model of 5-N₃-AZTMP binding to HIV-1 IN-(50-212) (29), our model shows that the G-quartet inhibitor binds simultaneously to several key amino residues in the active site of HIV-1 IN, such as Asp⁶⁴, Asp¹¹⁶, Glu¹⁵², and Lys¹⁵⁹, covering a lager section of the enzyme catalytic site.

This report also provides an explanation for the previous observation (10) that a folded and unfolded GTGT loop domain of T30695 yielded a great difference in the ability to inhibit HIV-1 IN activity. IC₅₀ values for the folded and unfolded loop structures were 31 and 530 nM, respectively. We propose that this difference is because the unfolded loop structure largely disrupts the face-to-face interaction. A recent observation (11) showed that inhibition of HIV-1 IN activity by derivatives of T30695 with the substitution of 5-amino dU for T 5-methyl (CH₃) was decreased two to four times. The decrease was considered to be caused by the charge-charge interaction between the positively charged loop of the derivatives and the positive charge of the lysine residues in (or near) the binding site of HIV-1 IN, such as Lys¹⁵⁹ and Lys¹⁶⁰. The statistical distribution also shows a high possibility for an interaction between Lys¹⁵⁹ of HIV-1 IN and G-quartets. The face-to-face interaction also provides further evidence to support the functional correlation between structural stability and anti-HIV-1 IN activity of G-quartet oligonucleotides observed in vitro (11). Varying the structure of the loop domains of G-quartet oligonucleotides not only induces a decrease in structure stability, but also disrupts the face-to-face interaction. Critical information for AIDS therapeutic drug design is that disruption of the face-to-face interaction strongly influences the inhibition of HIV-1 IN activity. Therefore, searching for a loop structure with a better binding affinity will be one of the key steps for improving the efficiency of these compounds to inhibit HIV-1 IN activity.

	ACKNOWLEDGEMENTS

We thank Yongli Guan for obtaining Fig. 3A and Ilya A. Vakser for providing the GRAMM docking program.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM60153 (to N. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed. Tel.: 713-798-3685; Fax: 713-798-6033; E-mail: njing@bcm.tmc.edu.

Published, JBC Papers in Press, May 2, 2000, DOI 10.1074/jbc.M001436200

	ABBREVIATIONS

The abbreviations used are: HIV, human immunodeficiency virus; HIV-1 IN, human immunodeficiency virus type 1 integrase; TEMED, N,N,N',N'-tetramethylethylenediamine; MOPS, 4-morpholinepropanesulfonic acid; AZTMP, azidothymidine 5'-monophosphate.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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