FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2019949690> ?p ?o ?g. }

• W2019949690 endingPage "21467" @default.
• W2019949690 startingPage "21460" @default.
• W2019949690 abstract "The G-tetrad-forming oligonucleotides T30177 andT30695 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 andT30695 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. The G-tetrad-forming oligonucleotides T30177 andT30695 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 andT30695 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. human immunodeficiency virus human immunodeficiency virus type 1 integrase N,N,N′,N′-tetramethylethylenediamine 4-morpholinepropanesulfonic acid azidothymidine 5′-monophosphate 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.De Clercq E. J. Med. Chem. 1995; 38: 2491-2517Crossref PubMed Scopus (771) Google Scholar). 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.Chun T.W. Stuyver L. Mizell S.B. Enler L.A. Mican J.A.M. Baseler M. Lloyd A.L. Nowak M.A. Fauci A.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13193-13197Crossref PubMed Scopus (1556) Google Scholar, 3.Wong J.K. Hezareh M. Gunthard H.F. Havlir D.V. Ignacio C.C. Spina C.A. Richman D.D. Science. 1997; 278: 1921-1925Crossref PubMed Scopus (1785) Google Scholar, 4.Finizi D. Hermankova M. Pierson T. Carruth L.M. Buck C. Chaisson R.E. Quinn T.C. Chadwick K. Margolick J. Bookmeyer R. Gallant J. Markowitz M. Ho D.D. Richman D.D. Siliciano R. Science. 1998; 278: 1295-1300Crossref Scopus (2471) Google Scholar). The development of new agents against human immunodeficiency virus type 1 integrase (HIV-1 IN) (5.Pommier Y. Neamati N. Adv. Virus Res. 1999; 52: 427-458Crossref PubMed Scopus (104) Google Scholar), 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.Rando R.F. Ojwang J. Elbaggari A. Reyes G.R. Tinder R. McGrath M.S. Hogan M.E. J. Biol. Chem. 1995; 270: 1754-1760Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 7.Mazumder A. Neamati N. Ojwang J.O. Sunder S. Rando R.F. Pommier Y. Biochemistry. 1996; 35: 13762-13771Crossref PubMed Scopus (131) Google Scholar). 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.Rando R.F. Ojwang J. Elbaggari A. Reyes G.R. Tinder R. McGrath M.S. Hogan M.E. J. Biol. Chem. 1995; 270: 1754-1760Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). IC50(50% inhibitory concentration) values of T30177 for HIV-1 IN are in the nanomolar range in vitro (7.Mazumder A. Neamati N. Ojwang J.O. Sunder S. Rando R.F. Pommier Y. Biochemistry. 1996; 35: 13762-13771Crossref PubMed Scopus (131) Google Scholar). 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.Jing N. Rando R.F. Pommier Y. Hogan M.E. Biochemistry. 1997; 36: 12498-12505Crossref PubMed Scopus (137) Google Scholar). 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.Jing N. Rando R.F. Pommier Y. Hogan M.E. Biochemistry. 1997; 36: 12498-12505Crossref PubMed Scopus (137) Google Scholar, 9.Jing N. Gao X. Rando R.F. Hogan M.E. J. Biomol. Struct. Dyn. 1997; 15: 573-585Crossref PubMed Scopus (44) Google Scholar, 10.Jing N. Hogan M.E. J. Biol. Chem. 1998; 273: 34992-34999Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). 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.Jing N. Hogan M.E. J. Biol. Chem. 1998; 273: 34992-34999Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). 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. 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. Oligonucleotides at 5 μm strand equivalents (20 mmLi3PO4, 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.Jing N. Rando R.F. Pommier Y. Hogan M.E. Biochemistry. 1997; 36: 12498-12505Crossref PubMed Scopus (137) Google Scholar, 11.Jing N. De Clercq E. Rando R.F. Pallansh L. Lackman-Smith C. Lee S. Hogan M.E. J. Biol. Chem. 2000; 275: 3421-3430Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar):A(T) = (1 − α)A rc+ αA st, α = (0.5 + 0.5(K eq − 1)/((1 −K eq)2 + 4ςK eq)1/2), andK eq = exp((−ΔH 0 +TΔS 0)/RT), whereK eq is the constant for the random coils to folded oligonucleotide equilibrium, α is the fraction of folded strands, 1 − α is the fraction of random coils,A(T) is the absorbance at temperatureT, A rc is the absorbance when all strands are random coils, A st is the absorbance when all strands are folded, and ς is the cooperativity of the melting transition (referred to as the helix interruption constant) defined by ς = exp(ΔS i/R), where ΔS i is in units/mol of interruption. In our analysis study, the values of ς are in the range of 0.9–0.999, determined by an optimized fitting program. Values forT m and ΔG were obtained on the basis of the fitting procedure, which inputs the values of ΔH 0, ΔS 0,A rc, and A st, estimated from the experimental measurements, and then uses an optimized fitting program to search for the best fit. The G-rich oligonucleotides, T30695 and its derivatives, plus 10 mm KCl in 20 mmLi3PO4, 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. CD spectra of the G-quartet oligonucleotides were obtained in 15 μm strand concentration plus 10 mm KCl in 20 mmLi3PO4, 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·dmol−1). The pET-15b-IN-(1–288)/F185K/C280S plasmid was expressed in Escherichia coli strain BL21 as described previously (12.Jenkins T.M. Engelman A. Ghirlando R. Cragie R. J. Biol. Chem. 1996; 271: 7712-7718Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar) 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-β-d-thiogalactopyranoside. Cells were harvested and resuspended in lysis buffer containing 25 mm HEPES, pH 7.5, 1 m NaCl, 5 mmimidazole, 2 mm β-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 mmβ-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 mmEDTA, 10 mm dithiothreitol, 2 mmβ-mercaptoethanol, 100 mm imidazole, and 10% glycerol. The HIV-1 IN assays were performed as described previously (7.Mazumder A. Neamati N. Ojwang J.O. Sunder S. Rando R.F. Pommier Y. Biochemistry. 1996; 35: 13762-13771Crossref PubMed Scopus (131) Google Scholar) 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 mmMnCl2, 0.1 mg/ml bovine serum albumin, and 14.3 mm β-mercaptoethanol. Then, a 5 nmconcentration 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. The molecular structures of T40215 andT40216 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.Maignan S. Guilloteau J.-P. Qing Z.-L. Clement-Mella C. Mikol V. J. Mol. Biol. 1998; 282: 359-368Crossref PubMed Scopus (265) Google Scholar), was obtained from the Protein Data Bank. Next, we docked the NMR structure of T30695 (10.Jing N. Hogan M.E. J. Biol. Chem. 1998; 273: 34992-34999Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) into this x-ray structure using the GRAMM docking program with a high resolution matching mode (15.Katchalski-Katzri E. Shariv I. Eisenstein M. Friesem A.A. Aflalo C. Vakser I.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2195-2199Crossref PubMed Scopus (862) Google Scholar, 16.Vakser I.A. Protein Eng. 1996; 9: 37-41Crossref PubMed Scopus (63) Google Scholar, 17.Vakser I.A. Biopolymers. 1996; 39: 455-464Crossref PubMed Google Scholar, 18.Vakser I.A. Protein Eng. 1996; 9: 741-744Crossref PubMed Scopus (41) Google Scholar). 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. 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.Jing N. Hogan M.E. J. Biol. Chem. 1998; 273: 34992-34999Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). 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.Wang K.Y. McCurdy S. Shea S.G. Swaminathan S. Bolton P.H. Biochemistry. 1993; 32: 1899-1904Crossref PubMed Scopus (338) Google Scholar, 20.Schultze P. Macaya R.F. Feigon J. J. Mol. Biol. 1994; 235: 1532-1547Crossref PubMed Scopus (348) Google Scholar), were further studied by nondenaturing gel electrophoresis (11.Jing N. De Clercq E. Rando R.F. Pallansh L. Lackman-Smith C. Lee S. Hogan M.E. J. Biol. Chem. 2000; 275: 3421-3430Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). 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 G1and G15 positions are replaced by two phosphodiester linkages. We previously showed that T30923 has the same structure and physical properties as T30695 (11.Jing N. De Clercq E. Rando R.F. Pallansh L. Lackman-Smith C. Lee S. Hogan M.E. J. Biol. Chem. 2000; 275: 3421-3430Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). 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. Further evidence to support intramolecular G-quartet formation forT30923, T40215, and T40216 was obtained from nondenaturing gel electrophoresis (Fig. 3 A) and from melting and annealing measurements (Fig. 3 B and TableI). Fig. 3 A 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.Jing N. Hogan M.E. J. Biol. Chem. 1998; 273: 34992-34999Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). 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, andT40216 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.Table IMelting and annealing measurementsOligonucleotideHeatingT mHCoolingT mCΔT mΔT/T mH× 100%°C°C°C%T30923(5.0 μm)85.283.81.41.6T40215 (2.0 μm)72.772.700T40215 (5.0 μm)72.770.32.43.3T40215 (20.0 μm)72.767.94.86.6T40216 (5.0 μm)73.770.33.44.6Note that all T m values were measured in 5.0 mm KCl. Open table in a new tab Note that all T m values were measured in 5.0 mm KCl. 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 meltingT 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 ΔT 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 ΔT values. Interestingly, the ΔT values of T40215 at strand concentrations of 2 and 20 μm are 0 and 4.8 °C, respectively, which suggests that ΔT may also depend on oligonucleotide concentration and that higher concentrations induce greater ΔT. At 2 μm, there was no measurable temperature shift (ΔT = 0) between the melting and annealing curves ofT40215, 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 mdepends 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 higherT 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. 3 A. The ion binding stoichiometry of the G-quartet structures has been established previously (8.Jing N. Rando R.F. Pommier Y. Hogan M.E. Biochemistry. 1997; 36: 12498-12505Crossref PubMed Scopus (137) Google Scholar). First, the thermal denaturation curves ofT30923, 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 ΔH 0, ΔS 0,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 TableII, 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 ΔG 0 versus log[K+] (Fig.4). Based upon Δn = ΔΔG 0/2.3RTΔlog10[K+] and k slope = ΔΔG 0/Δlog10[K+], the Δn 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.Jing N. Gao X. Rando R.F. Hogan M.E. J. Biomol. Struct. Dyn. 1997; 15: 573-585Crossref PubMed Scopus (44) Google Scholar, 10.Jing N. Hogan M.E. J. Biol. Chem. 1998; 273: 34992-34999Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The Δn values obtained here indicate thatT30923 also coordinates three K+ ions, whereas T40215 andT40216 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.Table IIFitting coefficientsOligomerKClT mΔH 0ΔS 0ΔG 0 (T = 295)Fitting coefficientmm°Ckcal/molcal/K/molkcal/molT309230.163.6−109.55−325.49−13.530.996T309230.575.1−109.58−314.83−16.700.995T309231.080.6−112.73−318.8−18.680.993T309235.085.4−113.38−316.35−20.060.995T402150.147.7−46.47−144.90−3.730.994T402150.562.2−56.10−167.39−6.720.997T402151.068.4−74.86−219.27−10.170.997T402155.074.1−78.03−224.81−11.710.994T402160.149.8−47.62−147.5−4.100.994T402160.563.1−79.21−235.71−9.680.993T402161.069.2−88.33−258.10−12.190.993T402165.074.9−100.20288.00−15.230.978 Open table in a new tab Figure 5Molecular structures of , , and . The structure of T30695 was determined by NMR (10.Jing N. Hogan M.E. J. Biol. Chem. 1998; 273: 34992-34999Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). 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 T40216with two, three, and four G-quartets, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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.Mazumder A. Neamati N. Ojwang J.O. Sunder S. Rando R.F. Pommier Y. Biochemistry. 1996; 35: 13762-13771Crossref PubMed Scopus (131) Google Scholar). As shown in Fig.6 A, T30923, T40215, and T40216were 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. 6 A). The strand transfer products yield larger molecular species with slower migration compared with the 21-mer substrate. The inhibition of HIV-1 IN activity byT30923, T40215, and T40216 is shown in Fig. 6 B. 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.6 C). IC50 values for T30923, T40215, and T40216were 70, 100, and 80 nm in 3′-processing and 85, 90, and 60 nm in strand transfer, respectively (TableIII).Table IIIIC50 values for 3′-processing and strand transferOligomerSequence (5′ → 3′)IC503′-ProcessingStrand transfernmT30923GGGTGGGTGGGTGGGT7085T40215(GGGGT)410090T40216(GGGGGT)48060 Open table in a new tab 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.Jing N. Rando R.F. Pommier Y. Hogan M.E. Biochemistry. 1997; 36: 12498-12505Crossref PubMed Scopus (137) Google Scholar, 10.Jing N. Hogan M.E. J. Biol. Chem. 1998; 273: 34992-34999Abs" @default.
• W2019949690 created "2016-06-24" @default.
• W2019949690 creator A5006520998 @default.
• W2019949690 creator A5010282324 @default.
• W2019949690 creator A5034494201 @default.
• W2019949690 creator A5042114293 @default.
• W2019949690 creator A5085352453 @default.
• W2019949690 creator A5089734215 @default.
• W2019949690 date "2000-07-01" @default.
• W2019949690 modified "2023-10-14" @default.
• W2019949690 title "Mechanism of Inhibition of HIV-1 Integrase by G-tetrad-forming Oligonucleotides in Vitro" @default.
• W2019949690 cites W1601304840 @default.
• W2019949690 cites W1601518034 @default.
• W2019949690 cites W1602825981 @default.
• W2019949690 cites W1936017520 @default.
• W2019949690 cites W1965883561 @default.
• W2019949690 cites W1983644993 @default.
• W2019949690 cites W1986118467 @default.
• W2019949690 cites W2000383223 @default.
• W2019949690 cites W2001601356 @default.
• W2019949690 cites W2006572311 @default.
• W2019949690 cites W2013134024 @default.
• W2019949690 cites W2015559549 @default.
• W2019949690 cites W2020555083 @default.
• W2019949690 cites W2024913717 @default.
• W2019949690 cites W2026184248 @default.
• W2019949690 cites W2026268551 @default.
• W2019949690 cites W2031598812 @default.
• W2019949690 cites W2032891258 @default.
• W2019949690 cites W2037145841 @default.
• W2019949690 cites W2050228834 @default.
• W2019949690 cites W2055112989 @default.
• W2019949690 cites W2057042085 @default.
• W2019949690 cites W2059067652 @default.
• W2019949690 cites W2059305343 @default.
• W2019949690 cites W2060829964 @default.
• W2019949690 cites W2089417027 @default.
• W2019949690 cites W2090646713 @default.
• W2019949690 cites W2113202468 @default.
• W2019949690 cites W2113428012 @default.
• W2019949690 cites W2114817191 @default.
• W2019949690 cites W2124211547 @default.
• W2019949690 cites W2132289602 @default.
• W2019949690 cites W2139250164 @default.
• W2019949690 cites W2140854337 @default.
• W2019949690 cites W2165130939 @default.
• W2019949690 cites W2171044922 @default.
• W2019949690 cites W218277912 @default.
• W2019949690 cites W2413846099 @default.
• W2019949690 cites W3027678366 @default.
• W2019949690 doi "https://doi.org/10.1074/jbc.m001436200" @default.
• W2019949690 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10801812" @default.
• W2019949690 hasPublicationYear "2000" @default.
• W2019949690 type Work @default.
• W2019949690 sameAs 2019949690 @default.
• W2019949690 citedByCount "91" @default.
• W2019949690 countsByYear W20199496902012 @default.
• W2019949690 countsByYear W20199496902013 @default.
• W2019949690 countsByYear W20199496902014 @default.
• W2019949690 countsByYear W20199496902015 @default.
• W2019949690 countsByYear W20199496902016 @default.
• W2019949690 countsByYear W20199496902017 @default.
• W2019949690 countsByYear W20199496902018 @default.
• W2019949690 countsByYear W20199496902019 @default.
• W2019949690 countsByYear W20199496902020 @default.
• W2019949690 countsByYear W20199496902021 @default.
• W2019949690 countsByYear W20199496902022 @default.
• W2019949690 crossrefType "journal-article" @default.
• W2019949690 hasAuthorship W2019949690A5006520998 @default.
• W2019949690 hasAuthorship W2019949690A5010282324 @default.
• W2019949690 hasAuthorship W2019949690A5034494201 @default.
• W2019949690 hasAuthorship W2019949690A5042114293 @default.
• W2019949690 hasAuthorship W2019949690A5085352453 @default.
• W2019949690 hasAuthorship W2019949690A5089734215 @default.
• W2019949690 hasBestOaLocation W20199496901 @default.
• W2019949690 hasConcept C121332964 @default.
• W2019949690 hasConcept C129312508 @default.
• W2019949690 hasConcept C142462285 @default.
• W2019949690 hasConcept C14430832 @default.
• W2019949690 hasConcept C147727578 @default.
• W2019949690 hasConcept C159047783 @default.
• W2019949690 hasConcept C185592680 @default.
• W2019949690 hasConcept C202751555 @default.
• W2019949690 hasConcept C2777302000 @default.
• W2019949690 hasConcept C2993143319 @default.
• W2019949690 hasConcept C3013748606 @default.
• W2019949690 hasConcept C54355233 @default.
• W2019949690 hasConcept C552990157 @default.
• W2019949690 hasConcept C55493867 @default.
• W2019949690 hasConcept C62520636 @default.
• W2019949690 hasConcept C71240020 @default.
• W2019949690 hasConcept C86803240 @default.
• W2019949690 hasConcept C89611455 @default.
• W2019949690 hasConceptScore W2019949690C121332964 @default.
• W2019949690 hasConceptScore W2019949690C129312508 @default.
• W2019949690 hasConceptScore W2019949690C142462285 @default.
• W2019949690 hasConceptScore W2019949690C14430832 @default.
• W2019949690 hasConceptScore W2019949690C147727578 @default.

• next