MolPharm xPharm- The Comprehensive Pharmacology Reference

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Neamati, N.
Right arrow Articles by Pommier, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Neamati, N.
Right arrow Articles by Pommier, Y.

Vol. 54, Issue 2, 280-290, August 1998

Highly Potent Synthetic Polyamides, Bisdistamycins, and Lexitropsins as Inhibitors of Human Immunodeficiency Virus Type 1 Integrase

Nouri Neamati, Abhijit Mazumder, Sanjay Sunder, Joshua M. Owen, Manju Tandon, J. William Lown, and Yves Pommier

Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, Bethesda, Maryland 20892 (N.N., A.M., S.S., J.M.O., Y.P.), and Department of Chemistry, University of Alberta, Edmonton A8, T6G 2G2, Canada (M.T., J.W.L.)

    Summary
Top
Summary
Introduction
Materials & Methods
Results
Discussion
References

Alignment of the available human immunodeficiency virus type 1 (HIV-1) viral DNA termini [U5 and U3 long terminal repeats (LTRs)] shows a high degree of conservation and the presence of a stretch of five or six consecutive adenine and thymine (AT) sequences ~10 nucleotides away from each LTR end. A series of AT-selective minor-groove binders, including distamycin and bisdistamycins, bisnetropsins, novel lexitropsins, and the classic monomeric DNA binders Hoechst 33258, 4'-diamino-2-phenylindole, pentamidine, berenil, spermine, and spermidine, were tested for their inhibitory activities against HIV-1 integrase (IN). Although netropsin, distamycin, and all other monomeric DNA binders showed weak activities in the range of 50-200 µM, some of the polyamides, bisdistamycins, and lexitropsins were remarkably active at nanomolar concentrations. Bisdistamycins were 200 times less potent when the conserved AAAAT stretch present in the U5 LTR was replaced with GGGGG, consistent with the preferred binding of these drugs to AT sequences. DNase I footprinting of the U5 LTR further demonstrated the selectivity of these bisdistamycins for the conserved AT sequence. The tested compounds were more potent in Mg+2 than in Mn+2 and inhibited IN50-212 deletion mutant in disintegration assays and the formation of IN/DNA complexes. The lexitropsins also were active against HIV-2 IN. Some of the synthetic polyamides exhibited significant antiviral activity. Taken together, these data suggest that selective targeting of the U5 and U3 ends of the HIV-1 LTRs can inhibit IN function. Polyamides might represent new leads for the development of antiviral agents against acquired immune deficiency syndrome.

    Introduction
Top
Summary
Introduction
Materials & Methods
Results
Discussion
References

The rapid emergence of HIV strains resistant to available drugs (Arts and Wainberg, 1996; De Clercq, 1996; Erickson and Burt, 1996) implies that effective treatment modalities will require the use of a combination of drugs targeting different sites of the HIV life cycle (Schinazi, 1991; Johnson, 1994; De Clercq, 1995; Larder et al., 1995). As part of a program to develop novel antiviral agents, we sought to determine the role of specific DNA binding agents as possible inhibitors of HIV-1 IN. IN is an important target for intervention by chemotherapeutics, and to date several inhibitors of this enzyme have been reported (for recent reviews, see Neamati et al., 1997c; Pommier et al., 1997). IN is responsible for the insertion of the viral DNA into a host chromosome. This process is essential for effective viral replication and can be reproduced in vitro using recombinant IN and short oligonucleotides (Katz and Skalka, 1994; Rice et al., 1996). Integration takes place in two consecutive steps. Initially, IN processes the linear viral DNA by removing two nucleotides from each 3'-end, leaving the recessed 3'-OH termini. This reaction is followed by transesterification of phosphodiester bonds in which each processed viral 3' terminus becomes linked to a 5'-phosphate of host DNA strand. These two steps, known as 3'-processing and 3'-end joining (strand transfer), can be easily measured in an in vitro assay using purified recombinant HIV-1 IN and an oligonucleotide corresponding to the U5 region of HIV LTR sequence.

IN binds to the viral DNA sequences located at both extremities of the LTRs. Because these sequences are highly conserved in all HIV genomes (Fig. 1), they could provide potential targets for the selective inhibition of integration. In addition, both the U5 and U3 LTRs contain a conserved AT-rich sequence ~10 base pairs from the viral ends (Fig. 1). Netropsin and distamycin (Fig. 2) bind tightly to AT sequences of B-DNA with little affinity for single-stranded nucleic acids, double-stranded RNA, or DNA/RNA hybrids. These molecules have an intrinsic twist that favors insertion into the minor groove of B-DNA (Zimmer et al., 1977, 1983; Kopka et al., 1985; Kopka and Larsen, 1992).


View larger version (54K):
[in this window]
[in a new window]
  		Fig. 1.   Nucleotide sequence alignment of the LTR U3 and U5 regions of 43 HIV-1 strains. Top, HIV-1 provirus with the LTRs and the three genes gag, pol, and env. Dashes, identity. Periods, gaps in the nucleotide sequence. Shaded region, location of the conserved AT sequence. The sequences were obtained from the Los Alamos HIV Sequence Database Web site (http://hiv-web.lanl.gov/).


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 2.   Structures of minor-groove binders netropsin, distamycin, Hoechst 33258, 4'-diamino-2-phenylindole, pentamidine, and berenil.

In a search for selective DNA binders with modified properties, novel lexitropsins were designed and synthesized (Lown, 1988, 1994; Lown et al., 1989). Recent studies demonstrated that polyamides can be synthesized to achieve highly selective recognition of the four Watson-Crick base pairs in the DNA minor groove (Helene, 1998; White et al., 1998). In addition, polymethylene-linked lexitropsins were shown to exhibit antiviral activity (Lown et al., 1989; Wang and Lown, 1992). However, their antiviral mechanism is not well understood.

The current report stems from our continuing efforts to identify novel IN inhibitors from compounds that exhibit antiviral activity in cellular assays. Herein, we show that synthetic polyamides that interact selectively with the conserved AT stretch present in the HIV LTRs can inhibit integrations at submicromolar concentrations.

    Materials and Methods
Top
Summary
Introduction
Materials & Methods
Results
Discussion
References

Chemistry. All chemicals used were of reagent grade. The reactions were carried out in anhydrous tetrahydrofuran that was dried over sodium/benzophenone and distilled fresh at the time of reaction. Dimethylformamide and triethylamine were distilled and stored over molecular sieves (4 Å). The progress of the reaction was monitored by analytical TLC using silica gel (60F-254 mesh; Merck Research Labs, West Point, PA)-coated aluminum-backed plates. Preparative separations were performed by column chromatography on flash silica gel (70-230 mesh; Merck). Melting points were determined on an electrothermal melting point apparatus (Fisher-John) and are uncorrected. 1H NMR spectra were recorded on a Bruker AM-300 spectrometer, the samples were prepared in dimethylsulfoxide-d6 unless otherwise specified, and the chemical shifts were reported in delta  ppm with respect to tetramethylsilane as an internal standard. New products were characterized by elemental analysis, mass spectroscopic analysis, or both using ES on a Micromass Zabspec Hybrid Sector TOF.

Representative synthesis: N,N'-di{1-methyl-2-[1-methyl-2-(1-methyl-2-carboxamido(3-dimethylaminopropyl)-4-pyrrole)-carboxamido-4-pyrrole]carboxamido-4-pyrrolyl}-phenyl-1,4-dicarboxamide (26). Nitrodistamycin 7a (0.3 g, 0.6 mmol) was taken in DMF and methanol (10 ml, 1:1, v/v/v) and hydrogenated using Pd-C (10%, 150 mg) on a Parr shaker apparatus at 40 psi for 2 hr at 22°. The catalyst was removed by filtration and washed with methanol. The filtrate was evaporated, and the contents were dried under high vacuum to remove the traces of the solvent. The aminodistamycin 7b, so obtained, was redissolved in anhydrous DMF (4 ml), the solution cooled down to 0°, and 1,4-phenyl diacid dichloride (61 mg, 0.3 mmol), predissolved in anhydrous tetrahydrofuran (4 ml), was added to it followed by 0.5 ml of triethylamine. The reaction mixture was stirred for 2 hr at 22°, at which time the TLC showed complete consumption of 7b. The solvent was removed in vacuo, and the crude product was purified on a silica gel column using CH2Cl2/MeOH/NH4OH (7:3:0.1, v/v/v) as eluent to collect pure 26 (160 mg, 50%); melting point >300° [1H NMR delta  1.60 [p, J = 7.0 Hz, 4 H, 2 CH2·CH2·CH2·N(CH3)2], 2.16 [s, 12 H, 2 CH2·N(CH3)2], 2.28 [t, J = 7.0 Hz, 4 H, 2 CH2·CH2·CH2·N(CH3)2], 3.18 [dt, J = 6.0 Hz, J = 7.0 Hz, 2 CH2·CH2·CH2·N(CH3)2], 3.78, 3.86, and 3.90 (3 s, 18 H, each for 6 protons of pyrrolyl N---CH3), 6.84, 7.04, 7.14, 7.20, 7.25, and 7.38 (6 d, J = 2.0 Hz, 12 H, each for 2H of pyrrole ring), 8.08 (s, 4 H, phenyl protons), 8.09 (t, J = 6.0 Hz, two CONH·CH2, merged with phenylic protons), 9.9, 10.02, and 10.50 (3 s, 6 H, each s for two pyrrolyl NHCO); ES+ calc. for C54H66N16O8, 1067.18; found 1067.50 (M+, 100%)].

Other bisdistamycins were synthesized in a similar way, and the related data are described below.

N,N'-Di{1-methyl-2-[1-methyl-2-(1-methyl-2-carboxamido(3-dimethylaminopropyl)-4-pyrrole)-carboxamido-4-pyrrole]carboxamido-4-pyrrolyl}-phenyl-1,3-dicarboxamide (27). This product was isolated in same way as 26. Yield 225 mg (70%); melting point 295° [1H NMR delta  1.64 [p, J = 7.0 Hz, 4 H, two CH2·CH2·CH2·N(CH3)2], 2.16 [s, 12 H, 2 N(CH3)2], 2.30 (t, J = 7.0 Hz, 4 H, 2 CH2·CH2·CH2·N(CH3)2], 3.20 [dt, J = 6.0 Hz, J = 7.0 Hz, 2 CH2·CH2·CH2·N(CH3)2], 3.80, 3.88, and 3.94 (3 s, 18 H, each for 6 protons of pyrrolyl N-CH3), 6.86, 7.06, 7.14, 7.20, 7.30, and 7.40 (6 d, J = 2.0 Hz, 12 H, each for 2 H of pyrrole ring), 7.70 (t, J = 7.5 Hz, 1 H, phenylic H-5), 8.10 (m, 4 H, phenylic H-4 and H-5 merged with 2 CONH·CH2 protons), 8.50 (s, 1 H, phenyl H-2), 9.90, 10.02, and 10.54 (3 s, 6 H, each s for 2 pyrrolyl NHCO); ES+ calc. for C54H66N16O8, 1067.18; found 1067.60 (M+, 100%)].

2N-{1-Methyl-2-[1-methyl-2-(1-methyl-2-carboxamido(3-dimethylaminopropyl)-4-pyrrole)-carboxamido-4-pyrrole]carboxamido-4-pyrrolyl}-2-carboxy benzamide (28). The reaction of 7b (285 mg, 0.6 mmol) with 1,2-phenyl diacid chloride (50 mg, 0.3 mmol) in the presence of triethylamine (0.1 ml) using DMF as a solvent afforded this product at a higher than anticipated Rf value. Raising the temperature of the reaction mixture to 65° did not result in any change in the nature of the reaction, and some unconsumed 7b always existed in the reaction mixture (detected on TLC plate). The solvent was removed in vacuo, and the crude product was purified on a silica gel column using CH2Cl2/MeOH/NH4OH (7:3:0.1, v/v/v) as eluent to afford 100 mg (54%) of pure 28. melting point 173° [1H NMR delta  1.80 [p, J = 7.0 Hz, 4 H, 2 CH2·CH2·CH2·N(CH3)2], 2.48 [s, 6 H, N(CH3)2], 2.68 [t, J = 7.0 Hz, 2 H, CH2·CH2·CH2·N(CH3)2], 3.24 [dt, J = 6.0 Hz, J = 7.0 Hz, 2 H, CH2·CH2·CH2·N(CH3)2], 3.80, 3.85, and 3.90 (3 s, 9H, each for 3 pyrrolyl NCH3), 6.86, 6.98, 7.05, 7.20, 7.26, and 7.30 (6 d, J = 2.0 Hz, 6H, each for 2 H of pyrrole ring), 7.50 (m, 2 H, phenylic H-4 and H-5), 7.65 (dd, J = 7.0 Hz, J = 2.0 Hz, 1 H, phenylic H-6), 7.75 (dd, J = 7.0 Hz, J = 2.0 Hz, 1 H, phenylic H-3), 8.15 (t, J = 6.0 Hz, CONH·CH2), 9.90, and 10.00 (2 s, 2 H, each s for 2 pyrrolyl NHCO); 11.50 (broad s, 1 H, COOH); ES+ calc. for C31H36N8O6 616.65, found 617.20 (M+ 100%)].

N-{1-Methyl-2-[1-methyl-2-(1-methyl-2-carboxamido(3-dimethylaminopropyl)-4-pyrrole)-carboxamido-4-pyrrole]carboxamido-4-pyrrolyl}-phthalimide (29). Phthalic acid (50 mg, 0.3 mmol) and 1,1'-carbonyldiimidazole (100 mg, 0.6 mmol) were heated at 80° in anhydrous DMF and CH3CN (3:1, 3.0 ml, v/v/v) for 2 hr and cooled to 0°. Distamycin 7b (285 mg, 0.6 mmol), predissolved in anhydrous DMF (2.0 ml), was added to this reaction mixture, and the contents were stirred for 2 hr at 22°. A TLC examination at this time showed complete disappearance of aminodistamycin and formation of a new product. The solvent was evaporated in vacuo, and the crude product was purified on a silica gel column using CH2Cl2/MeOH/NH4OH (7:3:0.05) as eluent to give pure 29. Yield 80 mg (44%); melting point 220° [1H NMR delta  1.60 [p, J = 7.0 Hz, 2 H, CH2·CH2·CH2·N(CH3)2], 2.10 [s, 6 H, N(CH3)2], 2.25 (t, J = 7.0 Hz, 2 H, CH2·CH2·CH2·N(CH3)2], 3.20 [dt, J = 6.0 Hz, J = 7.0 Hz, 2 H, CH2·CH2·CH2·CH2·N(CH3)2], 3.80, 3.88, and 3.98 (3 s, 9 H, each for 3 pyrrolyl NCH3), 6.82, 7.08, 7.20, 7.30, 7.34, and 7.38 (6 d, J = 2.0 Hz, 6 H, each for one pyrrolyl CH), 7.88-7.95 (m, 4 H, aromatic), 8.10 (t, J = 6.0 Hz, CONH·CH2), 9.90 and 10.10 (2 s, 2 H, each s for 2 pyrrolyl NHCO); ES+ calc. for C31H34N8O5 598.63, found 599.10 (M+ 100%)].

N,N'-Di{1-methyl-2-[1-methyl-2-(1-methyl-2-carboxamido(3-dimethylaminopropyl)-4-pyrrole)-carboxamido-4-pyrrole]carboxamido-4-pyrrolyl}-pyridine-2,5-dicarboxamide (30). Reaction of 2,5-pyridine dicarboxy dichloride (61 mg, 0.3 mmol) with amino distamycin 7b (285 mg, 0.6 mmol) in the presence of triethylamine (0.5 ml), using anhydrous DMF as a solvent, gave the crude product. This material was purified the same way as described for 26. Yield 270 mg (85%); melting point >300° [1H NMR delta  1.60 [p, J = 7.0 Hz, 4 H, 2 CH2·CH2·CH2·N(CH3)2], 2.14 [s, 12 H, 2 N(CH3)2], 2.25 [t, J = 7.0 Hz, 4 H, 2 CH2·CH2·CH2·N(CH3)2], 3.20 [dt, J = 6.0 Hz, J = 7.0 Hz, 2 CH2·CH2·CH2·N(CH3)2], 3.78 and 3.85 (2 s, 12 H, each for 6 protons of pyrrolyl N-CH3), 3.90 and 3.92 (2 s, 6 H, each for 3 protons of pyrrolyl N-CH3), 6.84, 7.05, 7.12, 7.19, 7.26, 7.90, and 7.95 (7 d, J = 2.0 Hz, total 10 H of pyrrole ring) 8.10 (t, J = 6.0 Hz, 2 H of 2 CONH·CH2), 8.25 (d, J = 7.0 Hz, 1 H, pyridyl H-3), 8.50 (dd, J = 7.0 Hz, J = 2.0 Hz, 1 H, pyridyl H-4), 9.20 (d, J = 2.0 Hz, 1 H, pyridyl H-6), 9.90 and 10.00 (2 s, 4 H, each for 2 NHCO), 10.75 and 10.90 (2 s, 2 H, each for 1 H of NHCO); ES+ calc. for C53H65N17O8 1068.17, found 1068.50 (M+ 100%)].

N,N'-Di{1-methyl-2-[1-methyl-2-(1-methyl-2-carboxamido(3-dimethylaminopropyl)-4-pyrrole)-carboxamido-4-pyrrole]carboxamido-4-pyrrolyl}-pyridine-2,4-dicarboxamide (31). This product was obtained following the synthetic procedure as described for 26. The chromatographic purification, using CH2Cl2/MeOH/NH4OH (7:3:0.05, v/v) as eluent afforded 240 mg (75%) of pure 31 as yellow solid; melting point 200° [1H NMR delta  1.60 [p, J = 7.0 Hz, 4 H, 2 CH2·CH2·CH2·N(CH3)2], 2.18 [s, 12 H, 2 N(CH3)2], 2.26 [t, J = 7.0 Hz, 4 H, 2 CH2·CH2·CH2·N(CH3)2], 3.20 [dt, J = 6.0 Hz, J = 7.0 Hz, 2 CH2·CH2·CH2·N(CH3)2], 3.80, 3.86, 3.89, and 3.91 (4 s, 24 H, each for 6 protons of pyrrolyl N-CH3), 6.84, 7.06, 7.16, 7.18, 7.26, 7.42, and 7.44 (7 d, J = 2.0 Hz, total 12 H of 6 pyrrole rings) 8.10 (m, 3 H; 2 H of 2 CONH·CH2 and 1 H of pyridyl H-5), 8.62 (d, J = 1.5 Hz, 1 H, pyridyl H-3), 8.90 (d, J = 5.5 Hz, 1 H, pyridyl H-6), 9.90 and 10.02, 10.88, and 10.91 (4 s, 4 H, 1 H for each NHCO); ES+ calc. for C53H65N17O8 1068.17, found 1068.30 (M+ 100%)].

N,N'-Di{1-methyl-2-[1-methyl-2-(1-methyl-2-carboxamido(3-dimethylaminopropyl)-4-pyrrole)-carboxamido-4-pyrrole]carboxamido-4-pyrrolyl}-pyridine-2,6-dicarboxamide (32). The crude mixture was purified on a silica gel column using CH2Cl2:MeOH:NH4OH (7:3:0.05, v/v/v) to obtain 260 mg (81%); melting point 228-30° [1H NMR delta  1.65 [p, J = 7.0 Hz, 4 H, 2 CH2·CH2·CH2·N(CH3)2], 2.25 [s, 12 H, 2 N(CH3)2], 2.40 (t, J = 7.0 Hz, 4 H, 2 CH2·CH2·CH2·N(CH3)2], 3.20 [dt, J = 6.0 Hz, J = 7.0 Hz, 2 CH2·CH2·CH2·N(CH3)2], 3.80, 3.88 and 3.96 [3 s, 18H, each for 6 protons of pyrrolyl N-CH3), 6.86, 7.10, 7.18, 7.25, 7.30 and 7.45 (6 d, J = 2.0 Hz, 12 H, each for 2 H of pyrrole ring), 8.08 (t, J = 6.0 Hz, 2 CONH·CH2), 8.30 (dd, J = 6.5 Hz, 1 H, pyridyl H-4), 8.35 (dd, J = 6.5 Hz, J = 2.0 Hz, 2 H, pyridyl H-3 and H-5), 9.90, 10.10 and 11.10 (3 s, 6H, each s for 2 pyrrolyl NHCO); ES+ calc. for C53H65N17O8, 1068.17; found 1068.50 (M+, 100%)].

N,N'-Di{1-methyl-2-[1-methyl-2-(1-methyl-2-carboxamido(3-dimethylaminopropyl)-4-pyrrole)-carboxamido-4-pyrrole]carboxamido-4-pyrrolyl}-pyridine-3,5-dicarboxamide (33). Yield 241 mg (76%); melting point 210° [1H NMR delta  1.60 [p, J = 7.0 Hz, 4 H, 2 CH2·CH2·CH2·N(CH3)2], 2.20 [s, 12 H, 2 N(CH3)2], 2.25 [t, J = 7.0 Hz, 4 H, 2 CH2·CH2·CH2·N(CH3)2], 3.20 [dt, J = 6.0 Hz, J = 7.0 Hz, 2 CH2·CH2·CH2·N(CH3)2], 3.78, 3.84, and 3.90 (3 s, 18H, each for 6 protons of pyrrolyl N-CH3), 6.84, 7.06, 7.15 7.20, 7.28, and 7.40 (6 d, J = 2.0 Hz, 12 H, each for 2 H of pyrrole ring), 8.10 (t, J = 6.0 Hz, 2 CONH·CH2), 8.80 (t, J = 2.0 Hz, 1 H, pyridyl H-4), 9.26 (d, J = 2.0 Hz, 2 H, pyridyl H-2 and H-6), 9.90, 10.05, and 10.78 (3 s, 6 H, each s for 2 pyrrolyl NHCO); ES+ calc. for C53H65N17O8, 1068.17; found 1068.60 (M+, 100%)].

N-{1-Methyl-2-[1-methyl-2-(1-methyl-2-carboxamido(3-dimethylaminopropyl)-4-pyrrole)-carboxamido-4-pyrrole]carboxamido-4-pyrrolyl}-4-carboxy pyridine-3-carboxamide (34). This product was obtained following the procedure described for 28. Yield 95 mg (51%); melting point 192°; [1H NMR delta  1.78 [p, J = 7.0 Hz, 2 H, CH2·CH2·CH2·N(CH3)2], 2.48 [s, 6 H, N(CH3)2], 2.78 [t, J = 7.0 Hz, 2 H, CH2·CH2·CH2·N(CH3)2], 3.24 [dt, J = 6.0 Hz, J = 7.0 Hz, 2 H, CH2·CH2·CH2·N(CH3)2], 3.80, 3.85 and 3.90 (3 s, 9 H, each for 3 pyrrolyl NCH3), 6.90, 7.04,7.06, 7.20, 7.30, and 7.40 (6 d, J = 2.0 Hz, 6 H, each for 1 pyrrolyl CH), 7.46 (d, J = 5.0 Hz, 1 H, pyridyl H-5), 8.12 (t, J = 6.0 Hz, CONH·CH2), 8.56 (d, J = 5.0 Hz, 1 H, pyridyl H-6), 9.0 (s, 1 H, pyridyl H-2), 9.94 and 10.00 (2 s, 2 H, each for 1 H of NHCO), 13.50 (broad s, 1 H, COOH); ES+ calc. for C30H35N9O6 618.64, found 618.64 (M+ 100%)].

N-{1-Methyl-2-[1-methyl-2-(1-methyl-2-carboxamido(3-dimethylaminopropyl)-4-pyrrole)-carboxamido-4-pyrrole]-carboxamido-4-pyrrolyl}-3-carboxy pyridine-2-carboxamide (35). This product was obtained following the synthetic procedure as described for 28. Yield 100 mg (54%); melting point 187° [1H NMR delta  1.70 [p, J = 7.0 Hz, 2 H, CH2·CH2·CH2·N(CH3)2], 2.30 [s, 6 H, N(CH3)2], 2.50 (t, J = 7.0 Hz, 2 H, CH2·CH2·CH2·N(CH3)2], 3.22 [dt, J = 6.0 Hz, J = 7.0 Hz, 2 H, CH2·CH2·CH2·N(CH3)2], 3.80, 3.85, and 3.90 (3 s, 9 H, each for 3 pyrrolyl NCH3), 6.85, 7.06,7.08, 7.18, 7.25, and 7.32 (6 d, J = 2.0 Hz, 6 H, each for 1 pyrrolyl CH), 7.50 (dd, J = 5.0 Hz, J = 7.5 Hz, 1 H, pyridyl H-5), 8.02 (dd, J = 1.5 Hz, J = 7.5 Hz, 1 H, pyridyl H-4), 8.10 (t, J = 6.0 Hz, 1 H, CONH·CH2), 8.62 (dd, J = 5.0 Hz, J = 1.5 Hz, 1 H, pyridyl H-6), 9.90 and 10.00 (2 s, 2 H, each for 1 H of NHCO), 10.98 (broad s, 1 H, COOH); ES+ calc. for C30H35N9O6 618.64, found 618.10 (M+ 100%)].

Preparation of radiolabeled DNA substrates, IN proteins and assays, electrophoresis and quantification, and anti-HIV assays in cultured cell lines. These methods were performed essentially as described previously (Neamati et al., 1997b). The Mg+2-based assays were carried in the presence of 5% polyethylene glycol as described previously (Engelman and Craigie, 1995). The anti-HIV drug testing were performed at National Cancer Institute essentially as described by Weislow et al. (1989).

Schiff base formation and chemical trapping. IN was incubated with an oligonucleotide-containing an abasic site (see Fig. 7A) in reaction buffer as described above (Mazumder et al., 1996a) for 2 min at room temperature. A freshly prepared solution of sodium borohydride (0.1 M final concentration) was added, and reaction was continued for an additional 5 min. An equal volume (16 µl) of 2× SDS-polyacrylamide gel electrophoresis buffer (100 mM Tris, pH 6.8, 4% 2-mercaptoethanol, 4% SDS, 0.2% bromphenol blue, 20% glycerol) was added to each reaction, and the reaction was heated at 95° for 3 min before loading a 20-µl aliquot on a 12% SDS-polyacrylamide gel. The gel was run at 120 V for 1.5 hr, dried, and exposed in a PhosphorImager cassette. For inhibition of DNA binding experiments, IN (200 nM) was preincubated with the inhibitor (at the indicated concentration) for 30 min at 30° before the subsequent addition of the radiolabeled viral DNA substrate (20 nM) and borohydride. Gels were analyzed using a Molecular Dynamics PhosphorImager (Sunnyvale, CA).

Footprinting experiments. DNase I footprinting was performed in buffer containing 50 mM NaCl, 1 mM HEPES, pH 7.5, 50 µM EDTA, 50 µM dithiothreitol, 10% glycerol (w/v), 7.5 mM MgCl2, 0.1 mg/ml bovine serum albumin, 10 mM 2-mercaptoethanol, 25 mM MES, pH 7.2, and 10 nM oligonucleotide. Lexitropsins were added to the reaction mixture and incubated at room temperature for 5 min. Digestion was achieved by the addition of DNase I (3 units/ml) for 1 min. The reaction was quenched with EDTA and 2× SDS-polyacrylamide gel electrophoresis buffer (100 mM Tris, pH 6.8, 4% 2-mercaptoethanol, 4% SDS, 0.2% bromphenol blue, 20% glycerol), and an aliquot (5 µl) was electrophoresed on a denaturing 20% polyacrylamide gel as described above.

    Results
Top
Summary
Introduction
Materials & Methods
Results
Discussion
References

Synthesis of Dimethylaminopropylamido Analogs of Distamycin (Novel Lexitropsins)

Preparation of aryl acid chlorides. Except for commercially available diacid chlorides, these linkers were prepared fresh, before their coupling with distamycin, from diacids. The acids were taken in thionyl chloride (5-10 mol in excess) with 2 or 3 drops of anhydrous dimethylformamide, and the mixture was heated at 65° for 2-3 hr. Excess of thionyl chloride was removed in vacuo, the contents were coevaporated with anhydrous methylene chloride, and the diacid chloride formed was used as such in its coupling with aminodistamycin.

3-{1-Methyl-4-[1-methyl-4-(1-methyl-4-nitropyrrole-2-carboxamido)pyrrole-2-carboxamido)pyrrole-2-carboxamido]pyrrole-2-carboxamido}dimethylaminopropane (distamycin, 7a). Distamycin (7a) was synthesized according to a reported procedure (Nishiwaki et al., 1988). Distamycin (7a) was the key intermediate for the synthesis (Figs. 3 and 4) of new rigidly linked bislexitropsins (26-35).


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 3.   Catalytic reduction of nitrolexitropsin 7a to aminolexitropsin reagent 7b.


View larger version (6K):
[in this window]
[in a new window]
  		Fig. 4.   General synthetic scheme to linked bislexitropsins.

Targeting HIV-1 LTR as an Alternative Strategy to Inhibit IN Function

The viral DNA sequences that are required and sufficient for recognition by IN are the distal ends of the LTRs. To determine how conserved are the HIV-1 LTRs among all the reported HIV-1 strains, we aligned the sequences from the Los Alamos HIV sequence Database (http://hiv-web.lanl.gov/). The termini of the HIV-1 LTRs seemed highly conserved among 43 available HIV-1 strains (Fig. 1). It is noticeable that a conserved AT stretch was identified ~10 bases away from both ends of the U3 and U5 LTRs. Thus, we envisaged that lexitropsins, or information-reading molecules, with a predictable base and sequence recognition capability could target these sites and block integration.

Inhibition of IN Catalytic Activities by a Series of Polyamides

Netropsin and distamycin analogs. Thirty-five polyamides were tested for anti HIV-1 IN activity (Tables 1 and 2) using a dual assay measuring both 3'-processing and 3'-end joining (strand transfer or integration) (Fig. 5). The bisnetropsin (1-6) and bisdistamycin (8-19) derivatives composed of two netropsin or distamycin molecules separated by aliphatic or aromatic linkers with different size and geometry were examined (Table 1). The result presented in Table 1 attests to the remarkable relationship between the structure of various derivatives and their IC50 values against IN. For example, the striking difference in potency shown by distamycin 7 compared with the potency of bisdistamycin derivatives suggests the importance of the dimeric structure. The linkers separating the two distamycin moieties contribute to potency, and the geometry of the linker seems critical for effective inhibition. The 1,4 disubstituted para derivative 8 exhibited markedly higher potency than the 1,2-ortho and 1,3-disubstituted meta derivative 9 and 10, respectively. A similar observation was made when the pyridinyl derivative with para substitution (compound 11) was compared with the meta substituted derivative 12. In addition, the linear 1,2-trans substituted compound 13 was 30-50 times more potent than the more rigid cyclobutanyl and norbornyl derivatives 14 and 15, respectively. Compound 12 exhibited remarkable selectivity for the 3'-end joining (strand transfer) step (Fig. 5B). Moreover, the length of the linker also contributes to potency. The dimers with the short dimethylene linker (derivative 16) or a long aliphatic chain linker (derivative 19) exhibited a significantly reduced potency compared with compound 17 or 18 with hexamethylene or octamethylene linkers, respectively.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 1
Inhibition of HIV-1 IN catalytic activities and inhibition of HIV-1 replication in CEM cells by a series of polyamides

                              
View this table:
[in this window]
[in a new window]
  		TABLE 2
Inhibition of HIV-1 and HIV-2 IN catalytic activities and inhibition of HIV-1 replication in CEM cells by a series of novel lexitropsins


View larger version (64K):
[in this window]
[in a new window]
  		Fig. 5.   HIV-1 IN catalytic assays. A, A 21-mer blunt-end oligonucleotide corresponding to the U5 end of the HIV-1 proviral DNA, 5'-end-labeled with 32P, is reacted with purified HIV-1 IN. The initial step involves nucleolytic cleavage of two bases from the 3'-end, resulting in a 19-mer oligonucleotide. The second step, 3'-end joining or strand transfer, involves joining of this recessed 3'-end to the 5'-end of an IN-induced break in another identical oligonucleotide, which serves as the target DNA. B, Concentration-dependent inhibition of HIV-1 IN by compounds 8, 11, 13, and 12. Lane 1, DNA alone. Lanes 2, 13, and 24, DNA plus IN. Lanes 3-23, DNA and IN in the presence of indicated concentrations (µM) of drugs.

Novel lexitropsins. A series of novel polyamides also was examined (Table 2). As in the case of the bisdistamycins presented in Table 1, a common structural feature required for potency seems to be the para substitution. For example, the 1,4-disubstituted derivative 26 was markedly more potent than its corresponding 1,3-disubstituted analog, 27. In addition, the 1,4-disubstituted pyridinyl derivative 30 was more potent than its corresponding 1,3-disubstituted derivatives 31, 32, and 33. In accord with the results for the monosubstituted minor-groove binders presented in Table 1, the monosubstituted lexitropsins 28, 34, and 35 were practically inactive. We also found that the novel lexitropsins were active against HIV-2 IN (Table 2).

Classic minor-groove binders. Netropsin and distamycin (Fig. 2) are natural oligopeptide antibiotics with antitumor, antiviral, and antibacterial activities (Zimmer et al., 1977, 1983; Kopka et al., 1985; Kopka and Larsen, 1992). Both antibiotics are known to bind to AT-rich regions in the minor groove of B-DNA in nonintercalative fashion (Zimmer et al., 1977, 1983; Kopka et al., 1985; Kopka and Larsen, 1992). Netropsin, distamycin, Hoechst 33258, 4'-diamino-2-phenylindole, pentamidine, and berenil (Fig. 2), which possess a crescent shape, bind noncovalently in the DNA minor groove without insertion between the base pairs (Zimmer et al., 1977, 1983; Kopka et al., 1985; Kopka and Larsen, 1992). None of these monomeric groove binders (compounds 20-25) exhibited significant activity at 100 µM against IN (Table 1). Distamycin 7 was only weakly active with an IC50 value of ~50 µM. Thus, the ability of the polyamides examined to inhibit IN varied considerably. The difference in activity exceeded 2-3 orders of magnitude.

Probing the Mechanism of IN Inhibition

Effect of the polyamides on the HIV-1 IN core region. To examine the mechanism of inhibition of IN, we used an IN deletion mutant, IN50-212, which lacks the amino-terminal zinc-binding region and the carboxyl-terminal DNA-binding domain (Chow et al., 1992; Bushman et al., 1993). This mutant can catalyze an apparent reversal of the integration reaction known as disintegration (Chow et al., 1992) (Fig. 6A). In the disintegration assay, the lexitropsin 26 was markedly more potent than lexitropsin 34 (Fig. 6B). The bisdistamycins 16, 17, 18, and 19 exhibited IC50 values of 2.3, 0.009, 0.03, and 3.7 µM (data not shown). The activity of the novel lexitropsins 27-36 in the disintegration assay with IN50-212 are indicated in Table 2. These results demonstrate that polyamides can interfere with the activity of the IN core region and that their inhibitory activity does not require the presence of the zinc-binding and carboxyl-terminal domains of IN.


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 6.   HIV-1 IN disintegration assay using the core truncated mutant IN50-212. A, The substrate oligonucleotide mimics a strand transfer step product (i.e., a Y oligonucleotide containing a 15-mer oligonucleotide 5'-end-labeled with 32P). HIV-1 IN-mediated disintegration generates a 30-mer oligonucleotide. B, Concentrationdependent inhibition of HIV-1 IN-mediated disintegration by compounds 26 and 34.

Global nucleophilic inhibition. The 3'-processing reaction involves hydrolysis of a single phosphodiester bond 3' of the conserved CA-3' dinucleotide (Fig. 7A). However, in addition to this hydrolysis reaction, retroviral INs can use glycerol or the hydroxyl group of the viral DNA terminus as the nucleophile in the 3'-processing reaction, yielding a glycerol esterified to the 5'-phosphate, a circular dinucleotide or trinucleotide, respectively (Engelman et al., 1991; Vink et al., 1991; Mazumder et al., 1996b) (Fig. 7A). To examine the effect of synthetic polyamides on the choice of nucleophiles in the 3'-processing reaction, a substrate DNA labeled at the 3'-end with 32P-cordycepin was used (Mazumder et al., 1996b). Compounds 8, 11, 13, and 12 inhibited glycerolysis, hydrolysis, and circular nucleotide formation similarly (Fig. 7B). This result indicates that lexitropsins block indiscriminately all the IN nucleophilic reactions.


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 7.   Differential use of nucleophiles in the site-specific cleavage of 3'-end-labeled substrate (3'-processing) by HIV-1 IN. A, Schematic representation of nucleophilic substitution of water, glycerol, or the 3'-hydroxyl group of the viral DNA terminus yielding a linear trinucleotide with a 5'-phosphate (L), a linear trinucleotide with a glycerol esterified to the 5'-phosphate (G), and a cyclic trinucleotide (C). *, 32P- labeled cordycepin. B, Global inhibition of nucleophilic attack in the 3'-processing reaction by compounds 8 (), 11 (bullet ), 13 (black-down-triangle ), and 12 (triangle ).

Divalent ion effects. Although in vitro assays are generally more efficient with Mn+2 as a cofactor, it has been proposed that the physiological cation is Mg+2. We compared the extent of 3'-processing and strand transfer for two representative derivatives, 8 and 11, in the presence of Mg+2 and Mn+2. Both compounds were more potent with Mg+2 than with Mn+2 (Fig. 8). This suggests that in contrast to polyhydroxylated aromatics (Fesen et al., 1994; Hazuda et al., 1997b; Neamati et al., 1997a), polyamides do not interact selectively with the divalent metal of the IN catalytic site.


View larger version (23K):
[in this window]
[in a new window]
  		Fig. 8.   Concentration-dependent inhibition of HIV-1 IN by compounds 8 (, black-square) and 11 (open circle , bullet ) in the presence of Mn+2 (open circle , ) or Mg+2 (black-square, bullet ).

Inhibition of DNA IN binding by polyamides. A recently described DNA IN cross-linking assay (Mazumder et al., 1996a) was used. Briefly, a 21-mer oligonucleotide in which uracil was substituted for adenine in the conserved CA dinucleotide on the distal end of the U5 LTR was treated with uracil DNA glycosylase to generate an abasic site (Fig. 9A, U). Subsequent incubation with IN and stabilization with sodium borohydride of the Schiff base formed between free aldehyde on the DNA backbone and the amino group of lysines in the IN produced a cross-linked product running as a 39-kDa band after electrophoresis (Fig. 9B, lane 2). Compound 26 effectively inhibited the formation of IN/DNA complexes, and the lexitropsin 27 was considerably less potent (Fig. 9C). These results are in accord with the inhibition of DNA integration by these compounds (Table 2) and suggest that lexitropsins inhibit IN activity by blocking IN binding to its DNA substrate.


View larger version (41K):
[in this window]
[in a new window]
  		Fig. 9.   Inhibition of DNA binding of HIV-1 IN by lexitropsins 26 and 27. A, Depurinated (U) duplex U5 oligonucleotide substrate. B, PhosphorImager picture showing the inhibition of 39-kDa DNA IN cross-linked product in the presence of indicated concentrations of lexitropsins 26 and 27. C, Quantification of the gel in B.

DNA sequence selectivity of polyamides. Because minor-groove binders such as distamycin and netropsins preferentially bind to AT sequences, we mutated the U5 DNA substrate to replace the conserved AT sequence with GC (Fig. 10A). This replacement decreased the efficiency of 3'-processing by ~30%, whereas 3'-end joining became almost undetectable. The two most potent bisdistamycins, 17 and 18, were compared for their ability to inhibit 3'-processing using the two DNA substrates. For both polyamides, inhibitory activity was ~200-fold less with the GC-containing oligonucleotide than with the physiological 21-mer oligonucleotide containing the conserved AT stretch (Fig. 10B). These results are consistent with the importance of DNA binding for IN inhibition by polyamides.


View larger version (23K):
[in this window]
[in a new window]
  		Fig. 10.   Importance of the conserved AT sequence for HIV-1 IN inhibition by bisdistamycins. A, The 21-mer blunt-end oligonucleotides where the AT stretch was replaced with GC (underlined). B, Concentration-dependent inhibition of HIV-1 IN by compounds 18 (, black-square) and 17 (open circle , bullet ) using native 21-mer oligonucleotide (open circle , bullet ) or the GC-modified oligonucleotide (black-square, bullet ).

DNase I footprinting. A DNase I footprinting experiment using a labeled 21-mer was performed to examine further the binding of the highly potent bisdistamycins 17 and 18 to the U5 LTR (Fig. 11). Protection of the AT-rich domain was observed with both compounds, and this protection tended to extend toward the U5 end at higher concentrations with 17. No such protection was observed using the GC oligonucleotide (see Fig. 10A and data not shown), which is consistent with the selective binding of lexitropsins to AT stretches.


View larger version (63K):
[in this window]
[in a new window]
  		Fig. 11.   DNase I footprinting of the 21-mer oligonucleotide corresponding to the U5 end of the HIV-1 proviral DNA, 5' end-labeled with 32P in the presence of indicated concentrations of lexitropsins 17 and 18.

    Discussion
Top
Summary
Introduction
Materials & Methods
Results
Discussion
References

Earlier work showed that DNA intercalators can effectively inhibit HIV-1 integration (Carteau et al., 1993; Fesen et al., 1993; for review, see Neamati et al., 1997c). However, intercalators have limited sequence specificity because they primarily interact with two adjacent base pairs. Thus, they would be expected to bind to numerous DNA sites, to affect cellular metabolism relatively indiscriminately, and to be too toxic for anti-acquired immune deficiency syndrome therapy. In the current study, we examined the activity of synthetic polyamides that can recognize with high selectivity DNA sequences from the DNA minor groove and exhibit antiviral activity.

Selective minor-groove binding to the conserved AT sequences in the LTRs seems to be the primary mechanism of inhibition of IN by the polyamides studied. First, the polyamides inhibited the binding of IN to its DNA substrate in the Schiff base assay at concentrations that effectively inhibited integration. Second, DNase I footprinting demonstrated the binding of polyamides to the U5 LTR in the conserved AT stretch. Third, changing the AT stretch into a GC stretch reduced the inhibitory activity of the bisdistamycins by 2 orders of magnitude.

The difference in anti-integration potency among the polyamides tested exceeded 2-3 orders of magnitude. This provided a basis for structure-activity relationships. Monomers were relatively inactive even at micromolar concentrations, thus suggesting a requirement of a dimeric form (bislexitropsins) for potent anti-IN activity. Among the bisdistamycins and novel lexitropsins, isomers with para substituted aromatic linkers consistently exhibited higher potency than ortho or meta substituted derivatives (Tables 1 and 2), implying the importance of a linear versus a folded arrangement for activity. Actually, the bisdistamycins with rigid linkers that tended to fold the molecule into a hairpin configuration (compounds 9, 10, 14, and 15) were not more potent inhibitors than distamycin (compound 7). The greater activity of the linear bisdistamycins suggests that drug binding to an extended DNA segment is essential for inhibition of integration. Compounds with an aliphatic flexible linker composed of a hexamethylene or an octamethylene chain (compounds 17 and 18) also were remarkably active, implying the importance of the size of the aliphatic chain linker for optimum potency. Finally, the finding that lexitropsins with substitutions on the distamycin moiety (compare compounds in Tables 1 and 2) remained active inhibitors of IN suggests that further chemical modifications can be made to improve antiviral activity.

In contrast to other IN inhibitors, such as catechol-containing compounds (Hazuda et al., 1997a; Mazumder et al., 1997), the lexitropsins were more active in Mg2+-based assays than in the Mn2+-based assays commonly used in vitro. Thus, if Mg+2 is more physiologically relevant than Mn+2, it seems that polyamides are potential candidates to block integration in vivo.

Targeting of the conserved AT stretches of the LTRs (Bouziane et al., 1996) with lexitropsins (current study) represents a clear strategy for interfering with provirus integration and retrovirus replication. The remarkable conservation of the AT stretches in all the available HIV-1 LTRs (Fig. 1) suggests that synthetic polyamides might generally be active against a broad spectrum of HIV-1 viruses. It is noticeable that some of the synthetic polyamides tested exhibited significant antiviral activity (Table 1). Independent experiments demonstrated that such compounds inhibited reverse transcriptase only at high concentrations (150-200 µM for compounds 1, 2, 5, 6, 10, and 16) (Lown JW, unpublished observations). Such concentrations are much higher than those required to inhibit integration. Further studies are warranted to determine whether integration is a prime mechanism for the antiviral activity of synthetic polyamides and to investigate whether other sites of the HIV genome and other viral processes are targeted by lexitropsins.

    Acknowledgments

The assistance of the staff of the Drug Synthesis and Chemistry Branch, National Cancer Institute, is gratefully acknowledged. We also thank Drs. T. Jenkins and R. Craigie (Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD) for the mutant HIV-IN proteins and the expression system for wild-type HIV-IN.

    Footnotes

Received February 10, 1998; Accepted April 17, 1998

This work was supported by grants from the National Institutes of Health Intramural AIDS Targeted Antiviral Program (N.N., A.M., S.S., J.M.O., Y.P.) and the National Cancer Institute of Canada (M.T., J.W.L).

Send reprint requests to: Dr. Yves Pommier, Lab of Molecular Pharmacology, Division of Basic Sciences, NCI/NIH, Bldg. 37, Rm. 5D02, Bethesda, MD 20892-4255. E-mail: pommier{at}nih.gov

    Abbreviations

HIV-1, human immunodeficiency virus type-1; IN, integrase; LTR, long terminal repeat; ES, electrospray ionization; SDS, sodium dodecyl sulfate; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; TLC, thin layer chromatography, DMF, dimethylformamide; MES, 3-(N-morpholino)propanesulfonic acid.

    References
Top
Summary
Introduction
Materials & Methods
Results
Discussion
References

0026-895X/98/020280-11$3.00/0
MOLECULAR PHARMACOLOGY, 54:280-290 (1998).
Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
A. A. Johnson, J. M. Sayer, H. Yagi, G. P. Kalena, R. Amin, D. M. Jerina, and Y. Pommier
Position-specific Suppression and Enhancement of HIV-1 Integrase Reactions by Minor Groove Benzo[a]pyrene Diol Epoxide Deoxyguanine Adducts: IMPLICATIONS FOR MOLECULAR INTERACTIONS BETWEEN INTEGRASE AND DNA SUBSTRATES
J. Biol. Chem., February 27, 2004; 279(9): 7947 - 7955.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
C. A. Hawkins, C. Watson, Y. Yan, B. Gong, and D. E. Wemmer
Structural analysis of the binding modes of minor groove ligands comprised of disubstituted benzenes
Nucleic Acids Res., February 15, 2001; 29(4): 936 - 942.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager