Peptides derived from HIV-1 Rev inhibit HIV-1 integrase in a shiftide mechanism

The HIV-1 Integrase protein (IN) mediates the integration of the viral cDNA into the host genome. IN is an emerging target for anti-HIV drug design, and the first IN-inhibitor was recently approved by the FDA. We have developed a new approach for inhibiting IN by "shiftides": peptides derived from its cellular binding protein LEDGF/p75 that inhibit IN by shifting its oligomerization equilibrium from the active dimer to an inactive tetramer. In addition, we described two peptides derived from the HIV-1 Rev protein that interact with IN and inhibit its activity in vitro and in cells. In the current study, we show that the Rev-derived peptides also act as shiftides. Analytical gel filtration and cross-linking experiments showed that IN was dimeric when bound to the viral DNA, but tetrameric in the presence of the Rev-derived peptides. Fluorescence anisotropy studies revealed that the Rev-derived peptides inhibited the DNA binding of IN. The Rev-derived peptides inhibited IN catalytic activity in vitro in a concentration-dependent manner. Inhibition was much more significant when the peptides were added to free IN before it bound the viral DNA than when the peptides were added to a preformed IN-DNA complex. This confirms that the inhibition is due to the ability of the peptides to shift the oligomerization equilibrium of the free IN toward a tetramer that binds much weaker to the viral DNA. We conclude that protein-protein interactions of IN may serve as a general valuable source for shiftide design.     

Mechanism of inhibition of HIV-1 integrase by G-tetrad-forming oligonucleotides in Vitro

The G-tetrad-forming oligonucleotides and 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 and, composed of three and four G-quartets with stem lengths of 19 and 24 A, respectively. The fact that increasing the G-quartet stem length from 15 to 24 A 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 (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 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.     

HIV-1 integrase inhibition by modified oligonucleotides: optimization of the inhibitor structure

Integration of human immunodeficiency virus type 1 DNA into the infected cell genome is one of the key steps of the viral replication cycle. Therefore viral enzyme integrase, which realizes the integration, is of interest as a target for new antiviral drugs. Conjugates of 11-mer single stranded oligonucleotides with hydrophobic molecules are shown to be efficient integrase inhibitors since they induce dissociation of the integrase-viral DNA complex. The effect of the oligonucleotide length and structure as well as the structure of hydrophobic molecules on the conjugate inhibitory activity has been studied. Conjugates with eosin and oleic acid are shown to be the most active. Conjugates of these molecules with 2'-O-methyl-oligonucleotide inhibit integrase at 50-100 nM and have no influence on a number of other DNA-binding enzymes.     

HIV-1 integrase crosslinked oligomers are active in vitro

The oligomeric state of active human immunodeficiency virus type 1 (HIV-1) integrase (IN) has not been clearly elucidated. We analyzed the activity of the different purified oligomeric forms of recombinant IN obtained after stabilization by platinum crosslinking. The crosslinked tetramer isolated by gel chromatography was able to catalyze the full-site integration of the two viral LTR ends into a target DNA in vitro, whereas the isolated dimeric form of the enzyme was involved in the processing and integration of only one viral end. Accurate concerted integration by IN tetramers was confirmed by cloning and sequencing. Kinetic studies of DNA-integrase complexes led us to propose a model explaining the formation of an active complex. Our data suggest that the tetrameric IN bound to the viral DNA ends is the minimal complex involved in the concerted integration of both LTRs and should be the oligomeric form targeted by future inhibitors.     

Peptides that inhibit HIV-1 integrase by blocking its protein-protein interactions

HIV-1 integrase (IN) is one of the key enzymes in the viral replication cycle. It mediates the integration of viral cDNA into the host cell genome. IN activity requires interactions with several viral and cellular proteins, as well as IN oligomerization. Inhibition of IN is an important target for the development of anti-HIV therapies, but there is currently only one anti-HIV drug used in the clinic that targets IN. Several other small-molecule anti-IN drug leads are either undergoing clinical trials or in earlier stages of development. These molecules specifically inhibit one of the IN-mediated reactions necessary for successful integration. However, small-molecule inhibitors of protein-protein interactions are difficult to develop. In this review, we focus on peptides that inhibit IN. Peptides have advantages over small-molecule inhibitors of protein-protein interactions: they can mimic the structures of the binding domains within proteins, and are large enough to competitively inhibit protein-protein interactions. The development of peptides that bind IN and inhibit its protein-protein interactions will increase our understanding of the IN mode of action, and lead to the development of new drug leads, such as small molecules derived from these peptides, for better anti-HIV therapy.     

Development of tricyclic hydroxy-1H-pyrrolopyridine-trione containing HIV-1 integrase inhibitors

New tricyclic HIV-1 integrase (IN) inhibitors were prepared that combined structural features of bicyclic pyrimidinones with recently disclosed 4,5-dihydroxy-1H-isoindole-1,3(2H)-diones. This combination resulted in the introduction of a nitrogen into the aryl ring and the addition of a fused third ring to our previously described inhibitors. The resulting analogues showed low micromolar inhibitory potency in in vitro HIV-1 integrase assays, with good selectivity for strand transfer relative to 3′-processing.     

Locked nucleic acid containing antisense oligonucleotides enhance inhibition of HIV-1 genome dimerization and inhibit virus replication

We have evaluated antisense design and efficacy of locked nucleic acid (LNA) and DNA oligonucleotide (ON) mix-mers targeting the conserved HIV-1 dimerization initiation site (DIS). LNA is a high affinity nucleotide analog, nuclease resistant and elicits minimal toxicity. We show that inclusion of LNA bases in antisense ONs augments the interference of HIV-1 genome dimerization. We also demonstrate the concomitant RNase H activation by six consecutive DNA bases in an LNA/DNA mix-mer. We show ON uptake via receptor-mediated transfection of a human T-cell line in which the mix-mers subsequently inhibit replication of a clinical HIV-1 isolate. Thus, the technique of LNA/DNA mix-mer antisense ONs targeting the conserved HIV-1 DIS region may provide a strategy to prevent HIV-1 assembly in the clinic.     

Discovery of novel non-cytotoxic salicylhydrazide containing HIV-1 integrase inhibitors

The previously discovered salicylhydrazide class of compounds displayed potent HIV-1 integrase (IN) inhibitory activity. The development of this class of compounds as antiretroviral agents was halted due to cytotoxicity in the nanomolar to sub-micromolar range. We identified a novel class of non-cytotoxic hydrazide IN inhibitors utilizing the minimally required salicylhydrazide substructure as a template in a small-molecule database search. The novel hydrazides displayed low micromolar IN inhibitory activity and are several hundred-fold less cytotoxic than previously disclosed salicylhydrazide IN inhibitors.     

Inhibition of HIV-1 integrase by modified oligonucleotides: Optimization of the inhibitor structure

Integration of human immunodeficiency virus type 1 DNA into the infected cell genome is one of the key steps of the viral replication cycle. Therefore, viral integrase is of interest as a target for new antiviral drugs. Conjugates of 11-mer single-stranded oligonucleotides with hydrophobic molecules were shown to be efficient integrase inhibitors, inducing dissociation of the integrase-viral DNA complex. The dependence of the conjugate inhibitory activity on the oligonucleotide length and structure as well as on the structure of hydrophobic molecules was studied. Conjugates with eosin and oleic acid proved to be the most active. Conjugates of these molecules with 2′-O-methyl-oligonucleotide inhibited integrase at concentrations 50–100 nM but did not influence some other DNA-binding enzymes.     

Structure-activity of inhibition of HIV-1 integrase and virus replication by G-quartet oligonucleotides

As novel anti-HIV agents, the G-tetrad-forming oligonucleotides have been explored for their structure-activity relations with regard to inhibition of integrase (IN) (N. Jing, Expert Opin. Investig. Drugs (2000) 9, 1777-1785). We have now developed two families of G-quartet oligonucleotides: T40217-T40222, with potential formation of a tail-to-tail G-quartet dimer, and T40224-T40227, with phosphorothioate (PT) linkages in the guanine loops. The results obtained from biophysical measurements and the assays of the inhibition of HIV-1 IN and virus replication demonstrated that an increase in the length of the G-quartet structure from a monomer (15A) to a tail-to-tail dimer (47A) does not distinctly disrupt the inhibition of HIV-1 IN activity or the inhibition of HIV-1 replication in cell cultures. G-quartet oligonucleotides were observed to induce molecular aggregation of HIV-1 IN and interrupt the binding of viral DNA to HIV-1 IN. Also, PT substitutions did not confer any advantages compared with the regular phosphodiesters for the inhibition of HIV-1 replication by intramolecular G-quartets. The G-quartet motif is the primary requirement for the remarkable nuclease resistance and pronounced biological efficacy of these oligonucleotides.     

Mapping of HIV-1 integrase preferences for target site selection with various oligonucleotides

HIV integrase (IN) catalyzes the insertion of proviral DNA into the host cell chromosome. While IN has strict sequence requirements for the viral cDNA ends, the integration site preference has been shown to be very diverse. Here, we mapped the HIV IN strand transfer reaction requirements using various short oligonucleotides (ON) that mimic the target DNA. Most double stranded DNA dodecamers served as excellent IN targets with variable integration efficiency depending mostly on the ON sequences. The preferred integration was lost with any changes in the geometry of the DNA double helical structures. Various hairpin-loop-forming ONs also served as efficient integration targets. Similar integration preferences were also observed for ONs, in which the nucleotide hairpin loop was replaced with a flexible aliphatic linker. The integration biases with all target DNA structures tested were significantly influenced by changes in the resulting secondary ON structures.     

HIV-1 integrase complexes with DNA dissociate in the presence of short oligonucleotides conjugated to acridine

The human immunodeficiency virus type 1 (HIV-1) integrase is an essential enzyme in the life cycle of the virus and is therefore an attractive target for the development of new antiviral drugs. Among them, inhibitors which are capable of targeting the preassembled integrase/DNA complex are of particular interest, because they could suppress integrase activity in the context of the HIV-1 preintegration complex. Here, we study the mechanism of action of 11-mer oligonucleotides, which are efficient inhibitors of the catalytic activity of integrase, provided that they are conjugated to a hydrophobic compound, acridine. To understand the mechanism of the conjugate inhibitory action, we used a steady-state fluorescence anisotropy assay, which allowed us to study the stability of the integrase/DNA complex in various conditions. We found that oligonucleotide-acridine conjugates induced the efficient dissociation of preassembled integrase/DNA complexes. The simultaneous presence of both acridine and an oligonucleotidic moiety is required for the inhibitory activity of conjugates. However, the dissociation effect is not dependent on the oligonucleotide sequence. Finally, our results suggest that the conjugates bind directly to integrase within its complex with DNA at a site different from the viral DNA binding site.     

Modulation of HIV-1 integrase activity by single-stranded oligonucleotides and their conjugates with eosin

Integration of the DNA copy of the genomic RNA into an infected cell genome is one of the key steps of the replication cycle of all retroviruses. It is catalyzed by the viral enzyme, integrase. We have shown that conjugates of short single-stranded oligonucleotides with eosin efficiently inhibit the catalytic activity of the HIV-1 integrase. In this article, we have found that the dependence of the integrase catalytic activity on the concentration of oligonucleotides has a bell-shaped pattern. The modulation of HIV-1 integrase activity correlated with the oligonucleotide length and was not associated with specific sequences. Moreover, a similar mode of the oligonucleotide action was found for integrase from the prototype foamy virus. This dual effect of the oligonucleotide and their conjugates with eosin might be explained by their binding with retroviral integrase in two different sites; the oligodeoxynucleotide binding in the first site results in integrase activation, whereas interactions with another one lead to inhibition of the enzyme activity. Eosin coupling to oligonucleotides did not change the mode of their action but enhanced their affinity to both binding sites. The affinity increase was found to be much more important for the site responsible for the integrase inhibition, thus explaining the high inhibitory potency of oligonucleotide-eosin conjugates.     

Effect of acyclic nucleoside phosphonates on the HIV-1 integrase in vitro.

Integrase (IN) is an essential enzyme in the human immunodeficiency virus type-1 (HIV-1) replication cycle and, thus, a potential target for chemotherapeutic agents. Because various nucleotide analogues have been reported to inhibit IN in vitro, we investigated the effect of acyclic nucleoside phosphonates. Both unphosphorylated and diphosphorylated derivatives were inhibitory to IN at concentrations ranging between 60 and 800 microM, with diphosphorylated derivatives being 5- to 8-fold more potent than unphosphorylated counterparts.     

High affinity interaction of HIV-1 integrase with specific and non-specific single-stranded short oligonucleotides.

Retroviral integrase (IN) catalyzes the integration of double-stranded viral DNA into the host cell genome. The reaction can be divided in two steps: 3'-end processing and DNA strand transfer. Here we studied the effect of short oligonucleotides (ODNs) on human immunodeficiency virus type 1 (HIV-1) IN. ODNs were either specific, with sequences representing the extreme termini of the viral long terminal repeats, or nonspecific. All ODNs were found to competitively inhibit the processing reaction with Ki values in the nM range for the best inhibitors. Our studies on the interaction of IN with ODNs also showed that: (i) besides the 3'-terminal GT, the interaction of IN with the remaining nucleotides of the 21-mer specific sequence was also important for an effective interaction of the enzyme with the substrate; (ii) in the presence of specific ODNs the activity of the enzyme was enhanced, a result which suggests an ODN-induced conformational change of HIV-1 IN.     

Inhibition of HIV-1 integrase by modified oligonucleotides derived from U5' LTR.

Retroviral integrase catalyzes integration of double-stranded viral DNA into the host chromosome by a process that has become an attractive target for drug design. In the 3' processing reaction, two nucleotides are specifically cleaved from both 3' ends of viral DNA yielding a 5' phosphorylated dimer (pGT). The resulting recessed 3' hydroxy groups of adenosine provide the attachment sites to the host DNA in the strand transfer reaction. Here, we studied the effect of modified double-stranded oligonucleotides mimicking both the unprocessed (21-mer oligonucleotides) and 3' processed (19-mer oligonucleotides) U5 termini of proviral DNA on activities of HIV-1 integrase in vitro. The inhibitions of 3' processing and strand transfer reactions were studied using 21-mer oligonucleotides containing isopolar, nonisosteric, both conformationally flexible and restricted phosphonate internucleotide linkages between the conservative AG of the sequence CAGT, and using a 21-mer oligonucleotide containing 2'-fluoroarabinofuranosyladenine. All modified 21-mer oligonucleotides competitively inhibited both reactions mediated by HIV-1 integrase with nanomolar IC50 values. Our studies with 19-mer oligonucleotides showed that modifications of the 3' hydroxyl significantly reduced the strand transfer reaction. The inhibition of integrase with 19-mer oligonucleotides terminated by (S)-9-(3-hydroxy-2-phosphonomethoxypropyl)adenine, 9-(2-phosphonomethoxyethyl)adenine, and adenosine showed that proper orientation of the 3' OH group and the presence of the furanose ring of adenosine significantly influence the strand transfer reaction.     

Antisense oligonucleotides containing modified bases inhibit in vitro translation of Leishmania amazonensis mRNAs by invading the mini-exon hairpin

Complementary oligodeoxynucleotides (ODNs) that contain 2-aminoadenine and 2-thiothymine interact weakly with each other but form stable hybrids with unmodified complements. These selectively binding complementary (SBC) agents can invade duplex DNA and hybridize to each strand (Kutyavin, I. V., Rhinehart, R. L., Lukhtanov, E. A., Gorn, V. V., Meyer, R. B., and Gamper, H. B. (1996) Biochemistry 35, 11170-11176). Antisense ODNs with similar properties should be less encumbered by RNA secondary structure. Here we show that SBC ODNs strand invade a hairpin in the mini-exon RNA of Leishmania amazonensis and that the resulting heteroduplexes are substrates for Escherichia coli RNase H. SBC ODNs either with phosphodiester or phosphorothioate backbones form more stable hybrids with RNA than normal base (NB) ODNs. Optimal binding was observed when the entire hairpin sequence was targeted. Translation of L. amazonensis mRNA in a cell-free extract was more efficiently inhibited by SBC ODNs complementary to the mini-exon hairpin than by the corresponding NB ODNs. Nonspecific protein binding in the cell-free extract by phosphorothioate SBC ODNs rendered them ineffective as antisense agents in vitro. SBC phosphorothioate ODNs displayed a modest but significant improvement of leishmanicidal properties compared with NB phosphorothioate ODNs.     

Disruption of HIV-1 integrase-DNA complexes by short 6-oxocytosine-containing oligonucleotides

We recently found that oligonucleotides containing the 6-oxocytosine heterocyclic base are efficient inhibitors of the HIV-1 integrase in vitro [Brodin, P., et al. (2001) Nucleosides Nucleotides Nucleic Acids 20, 481-486]. In this report, we demonstrate that the inhibition arises from a noncompetitive mechanism in which the modified oligonucleotide attacks the integrase-DNA complex, leading to its active disruption. This conclusion is based on the following results. First, despite the fact that the respective affinities of a 6-oxocytosine-containing oligonucleotide and of its nonmodified counterpart for integrase were identical, only the modified compound inhibited the enzyme activities. Second, DNA binding and UV cross-linking assays indicated that the 6-oxocytosine-containing oligonucleotide prevented the formation of a stable integrase-DNA complex. Third, the kinetics of the dissociation of the integrase-DNA complex were dramatically accelerated in the presence of the modified ODN, whereas the nonmodified counterpart did not influence the dissociation. This mechanism was supported by the ability of the 6-oxocytosine-containing oligonucleotide to inhibit the strand transfer activity of HIV-1 preintegration complexes in vitro. Disruption of integrase-DNA complexes by 6-oxocytosine-containing oligonucleotides constitutes an original mechanism of integration inhibition, therefore suggesting a strategy for searching for inhibitors of the HIV-1 preintegration complexes.