Design and synthesis of specific inhibitors of the 3'-processing step of HIV-1 integrase

The novel dinucleotide 5'-phosphate, [(L,D)-pIsodApdC], discovered in our laboratory, is a strong inhibitor of HIV-1 integrase for both the 3'-processing and the strand transfer steps. The rationale used in this molecular design was that residues immediately upstream of the dinucleotide cleavage site in the 3'-processing step might provide critical recognition/binding sites on integrase. The rationale for the second type of inhibitors was based on the elimination products (linear and cyclic dinucleotides) of 3'-processing. However, while the linear dinucleotide 5'-phosphate (pdGpdT) was active, its cyclic counterpart was inactive against both wild-type and mutant HIV integrase.     

Integration requires a specific interaction of the donor DNA terminal 5'-cytosine with glutamine 148 of the HIV-1 integrase flexible loop

Integration is essential for retroviral replication and gene therapy using retroviral vectors. Human immunodeficiency virus, type 1 (HIV-1), integrase specifically recognizes the terminal sequences of each long terminal repeat (LTR) and cleaves the 3'-end terminal dinucleotide 5'-GT. The exposed 3'-hydroxyl is then positioned for nucleophilic attack and subsequent strand transfer into another DNA duplex (target or chromosomal DNA). We report that both the terminal cytosine at the protruding 5'-end of the long terminal repeats (5'-C) and the integrase residue Gln-148 are critical for strand transfer. Proximity of the 5'-C and Gln-148 was demonstrated by disulfide cross-linking. Cross-linking is inhibited by the inhibitor 5CITEP 1-(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl)-propenone. We propose that strand transfer requires a conformational change of the integrase-viral (donor) DNA complex with formation of an H-bond between the N-3 of the 5'-C and the amine group of Gln-148. These findings have implications for the molecular mechanisms coupling 3'-processing and strand transfer as well as for the molecular pharmacology of integrase inhibitors.     

Changes to the HIV long terminal repeat and to HIV integrase differentially impact HIV integrase assembly, activity, and the binding of strand transfer inhibitors

Human immunodeficiency virus (HIV) integrase enzyme is required for the integration of viral DNA into the host cell chromosome. Integrase complex assembly and subsequent strand transfer catalysis are mediated by specific interactions between integrase and bases at the end of the viral long terminal repeat (LTR). The strand transfer reaction can be blocked by the action of small molecule inhibitors, thought to bind in the vicinity of the viral LTR termini. This study examines the contributions of the terminal four bases of the nonprocessed strand (G(2)T(1)C(-1)A(-2)) of the HIV LTR on complex assembly, specific strand transfer activity, and inhibitor binding. Base substitutions and abasic replacements at the LTR terminus provided a means to probe the importance of each nucleotide on the different functions. An approach is described wherein the specific strand transfer activity for each integrase/LTR variant is derived by normalizing strand transfer activity to the concentration of active sites. The key findings of this study are as follows. 1) The G(2):C(2) base pair is necessary for efficient assembly of the complex and for maintenance of an active site architecture, which has high affinity for strand transfer inhibitors. 2) Inhibitor-resistant enzymes exhibit greatly increased sensitivity to LTR changes. 3) The strand transfer and inhibitor binding defects of a Q148R mutant are due to a decreased affinity of the complex for magnesium. 4) Gln(148) interacts with G(2), T(1), and C(-1) at the 5' end of the viral LTR, with these four determinants playing important and overlapping roles in assembly, strand transfer catalysis and high affinity inhibitor binding.     

HIV-1 integrase can process a 3'-end crosslinked substrate.

Integrase of the human immunodeficiency virus type-1 (HIV-1) recognizes specific sequences located in the U3 and U5 regions at the ends of viral DNA. We synthesized DNA duplexes mimicking the U5 region and containing either 2'-aminonucleosides or non-nucleoside 1,3-propanediol insertions at the third and terminal positions and studied their interactions with HIV-1 integrase. Both modifications introduced a local structural distortion in the DNA double helix. Replacement of the terminal nucleosides by corresponding 2'-aminonucleosides had no significant effect on integrase activity. We used an integrase substrate bearing terminal 2'-aminonucleosides in both strands to synthesize a duplex with cross-linked strands. This duplex was then used to determine whether terminal base pair disruption is an obligatory step of retroviral DNA 3'-processing. Processing of the cross-linked analog of the integrase substrate yielded a product of the same length as 3'-processing of the wild-type substrate but the reaction efficiency was lower. Replacement of the third adenosine in the processed strand by a corresponding 2'-aminonucleoside did not affect integrase activity, whereas, its replacement by 1,3-propanediol completely inhibited 3'-processing. Both modifications of the complementary thymidine in the nonprocessed strand increased the initial rate of 3'-processing. The same effect was observed when both nucleosides, at the third position, were replaced by corresponding 2'-aminonucleosides. This indicates that the local duplex distortion facilitated the cleavage of the phosphodiester bond. Thus, a localized destabilization of the third A-T base pair is necessary for efficient 3'-processing, whereas 3'-end-fraying is important but not absolutely required.     

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 substrates

The viral protein HIV-1 integrase is required for insertion of the viral genome into human chromosomes and for viral replication. Integration proceeds in two consecutive integrase-mediated reactions: 3'-processing and strand transfer. To investigate the DNA minor groove interactions of integrase relative to known sites of integrase action, we synthesized oligodeoxynucleotides containing single covalent adducts of known absolute configuration derived from trans-opening of benzo-[a]pyrene 7,8-diol 9,10-epoxide by the exocyclic 2-amino group of deoxyguanosine at specific positions in a duplex sequence corresponding to the terminus of the viral U5 DNA. Because the orientations of the hydrocarbon in the minor groove are known from NMR solution structures of duplex oligonucleotides containing these deoxyguanosine adducts, a detailed analysis of the relationship between the position of minor groove ligands and integrase interactions is possible. Adducts placed in the DNA minor groove two or three nucleotides from the 3'-processing site inhibited both 3'-processing and strand transfer. Inosine substitution showed that the guanine 2-amino group is required for efficient 3'-processing at one of these positions and for efficient strand transfer at the other. Mapping of the integration sites on both strands of the DNA substrates indicated that the adducts both inhibit strand transfer specifically at the minor groove bound sites and enhance integration at sites up to six nucleotides away from the adducts. These experiments demonstrate the importance of position-specific minor groove contacts for both the integrase-mediated 3'-processing and strand transfer reactions.     

Functional identification of nucleotides conferring substrate specificity to retroviral integrase reactions.

The long terminal repeats (LTRs) that flank the retroviral DNA genome play a distinct role in the integration process by acting as specific substrates for the integrase (IN). The role of LTR sequences in providing substrate recognition and specificity to integration reactions was investigated for INs from human immunodeficiency virus type 1 (HIV-1), Moloney murine leukemia virus (M-MuLV), human T-cell leukemia virus type 1 (HTLV-1), and human T-cell leukemia virus type 2 (HTLV-2). Overall, these INs required specific LTR sequences for optimal catalysis of 3'-processing reactions, as opposed to strand transfer and disintegration reactions. It is of particular note that in strand transfer reactions the sites of integration were similar among the four INs. In the 3'-processing reaction, sequence specificity for each IN was traced to the three nucleotides proximal to the conserved CA. Reactions catalyzed by M-MuLV IN were additionally influenced by upstream regions. The nucleotide requirements for optimal catalysis differed for each IN. HIV-1 IN showed a broad range of substrate specificities, while HTLV-1 IN and HTLV-2 IN had more defined sequence requirements. M-MuLV IN exhibited greater activity with the heterologous LTR substrates than with its own wild-type substrate. This finding was further substantiated by the high levels of activity catalyzed by the IN on modified M-MuLV LTRs. This work suggests that unlike the other INs examined, M-MuLV IN has evolved with an IN-LTR interaction that is suboptimal.     

Integrase of Mason-Pfizer monkey virus

The gene encoding an integrase of Mason-Pfizer monkey virus (M-PMV) is located at the 3'-end of the pol open reading frame. The M-PMV integrase has not been previously isolated and characterized. We have now cloned, expressed, isolated, and characterized M-PMV integrase and compared its activities and primary structure with those of HIV-1 and other retroviral integrases. M-PMV integrase prefers untranslated 3'-region-derived long-terminal repeat sequences in both the 3'-processing and the strand transfer activity assays. While the 3'-processing reaction catalyzed by M-PMV integrase was significantly increased in the presence of Mn(2+) and Co(2+) and was readily detectable in the presence of Mg(2+) and Ni(2+) cations, the strand transfer activity was strictly dependent only on Mn(2+). M-PMV integrase displays more relaxed substrate specificity than HIV-1 integrase, catalyzing the cleavage and the strand transfer of M-PMV and HIV-1 long-terminal repeat-derived substrates with similar efficiency. The structure-based sequence alignment of M-PMV, HIV-1, SIV, and ASV integrases predicted critical amino acids and motifs of M-PMV integrase for metal binding, interaction with nucleic acids, dimerization, protein structure maintenance and function, as well as for binding of human immunodeficiency virus type 1 and Rous avian sarcoma virus integrase inhibitors 5-CI-TEP, DHPTPB and Y-3.     

Methylphosphonodiester substitution near the conserved CA dinucleotide in the HIV LTR alters both extent of 3'-processing and choice of nucleophile by HIV-1 integrase.

We present evidence suggesting that the 3'-processing activity of HIV-1 integrase is dramatically affected by electrostatic and/or steric perturbations 3' to the conserved CA dinucleotide. When the phosphodiester bond 3' to the scissile phosphodiester is replaced by a methylphosphonodiester linkage, 3'-processing decreases by two orders of magnitude. This block of cleavage can be somewhat overcome by increasing the pH of the reaction. Labeling of the substrates at the 3'-end revealed blockage of water and glycerol, but stimulation of the viral DNA 3'-hydroxyl, acting as the nucleophile with the methylphosphonodiester substrate. Interestingly, a circular trinucleotide was formed using the phosphodiester and methylphosphonodiester substrates when the terminal nucleotide was 3'-deoxyadenosine but not 2'-deoxyadenosine. Mutagenesis of the enzyme active site has previously been shown to alter the choice of nucleophile in the 3'-processing reaction. Taken together, the results in this study suggest that 'mutagenesis' of the DNA backbone can also alter the choice of nucleophile.     

The strand transfer oligonucleotide inhibitors of HIV-integrase

Retroviral integrase participates in two catalytic reactions, which require interactions with the two ends of the viral DNA in the 3'processing reaction, and with a targeted host DNA in the strand transfer reaction. The 3'-hydroxyl group of 2'-deoxyadenosine resulting from the specific removing of GT dinucleotide from the viral DNA in the processing reaction provides the attachment site for the host DNA in a transesterification reaction. We synthesized oligonucleotides (ONs) of various lengths that mimic the processed HIV-1 U5 terminus of the proviral long terminal repeat (LTR) and are ended by 2'-deoxyadenosine containing a 3'-O-phosphonomethyl group. The duplex stability of phosphonomethyl ONs was increased by covalent linkage of the modified strand with its complementary strand by a triethylene glycol loop (TEG). Modified ONs containing up to 10 bases inhibited in vitro the strand transfer reaction catalyzed by HIV-1 integrase at nanomolar concentrations.     

Requirement of active human immunodeficiency virus type 1 integrase enzyme for productive infection of human T-lymphoid cells.

The human immunodeficiency virus type 1 (HIV-1) integrase enzyme exhibits significant amino acid sequence conservation with integrase proteins of other retroviruses. We introduced specific amino acid substitutions at a number of the conserved residue positions of recombinant HIV-1 integrase. Some of these substitutions resulted in proteins which were not able to be purified in the same manner as the wild-type enzyme, and these were not studied further. The remaining mutant enzymes were assessed for their abilities to perform functions characteristic of the integrase protein. These included specific removal of the terminal dinucleotides from oligonucleotide substrates representative of the viral U5-long terminal repeat, nonspecific cleavage of oligonucleotide substrates, and mediation of the strand transfer (integration) reaction. Substitution at position 43, within the protein's zinc finger motif region, resulted in an enzyme with reduced specificity for cleavage of the terminal dinucleotide. In addition, a double substitution of aspartic acid and glutamine for valine and glutamic acid, respectively, at positions 151 and 152 within the D,D(35)E motif region rendered the integrase protein inactive for all of its functions. The introduction of this double substitution into an infectious HIV-1 provirus yielded a mutant virus that was incapable of productively infecting human T-lymphoid cells in culture.     

Mutations in the U5 sequences adjacent to the primer binding site do not affect tRNA cleavage by rous sarcoma virus RNase H but do cause aberrant integrations in vivo

In most retroviruses, the first nucleotide added to the tRNA primer becomes the right end of the U5 region in the right long terminal repeat (LTR); the removal of this tRNA primer by RNase H defines the right end of the linear double-stranded DNA. Most retroviruses have two nucleotides between the 5' end of the primer binding site (PBS) and the CA dinucleotide that will become the end of the integrated provirus. However, human immunodeficiency virus type 1 (HIV-1) has only one nucleotide at this position, and HIV-2 has three nucleotides. We changed the two nucleotides (TT) between the PBS and the CA dinucleotide of the Rous sarcoma virus (RSV)-derived vector RSVP(A)Z to match the HIV-1 sequence (G) and the HIV-2 sequence (GGT), and we changed the CA dinucleotide to TC. In all three mutants, RNase H removes the entire tRNA primer. Sequence analysis of RSVP(HIV2) proviruses suggests that RSV integrase can remove three nucleotides from the U5 LTR terminus of the linear viral DNA during integration, although this mutation significantly reduced virus titer, suggesting that removing three nucleotides is inefficient. However, the results obtained with RSVP(HIV1) and RSVP(CATC) show that RSV integrase can process and integrate the normal U3 LTR terminus of a linear DNA independently of an aberrant U5 LTR terminus. The aberrant end can then be joined to the host DNA by unusual processes that do not involve the conserved CA dinucleotide. These unusual events generate either large duplications or, less frequently, deletions in the host genomic DNA instead of the normal 5- to 6-base duplications.     

Homogeneous high-throughput screening assays for HIV-1 integrase 3beta-processing and strand transfer activities

HIV-1 integrase (HIV-IN) is a well-validated antiviral drug target catalyzing a multistep reaction to incorporate the HIV-1 provirus into the genome of the host cell. Small molecule inhibitors of HIV-1 integrase that specifically target the strand transfer step have demonstrated efficacy in the suppression of virus propagation. However, only few specific strand transfer inhibitors have been identified to date, and the need to screen for novel compound scaffolds persists. Here, the authors describe 2 homogeneous time-resolved fluorescent resonance energy transfer-based assays for the measurement of HIV-1 integrase 3'-processing and strand transfer activities. Both assays were optimized for high-throughput screening formats, and a diverse library containing more than 1 million compounds was screened in 1536-well plates for HIV-IN strand transfer inhibitors. As a result, compounds were found that selectively affect the enzymatic strand transfer reaction over 3beta processing. Moreover, several bioactive molecules were identified that inhibited HIV-1 reporter virus infection in cellular model systems. In conclusion, the assays presented herein have proven their utility for the identification of mechanistically interesting and biologically active inhibitors of HIV-1 integrase that hold potential for further development into potent antiviral drugs.     

Interactions of HIV-1 DNA heterocyclic bases with viral DNA

Human immunodeficiency virus type 1 integrase is one of three viral enzymes, and it realizes a key process of the viral replication cycle, i.e. viral DNA integration into infected cell genome. Integrase recognizes nucleotide sequences located at the ends of the viral DNA U3 and U5 LTRs and catalyzes 3'-processing and strand transfer reactions. To study the interactions between integrase and viral DNA at present work, we used modified integrase substrates mimicking the terminal U5 LTR sequence and containing non-nucleoside insertions in one or/and both strands. It is shown that the substrate modifications have no influence on the integrase binding rate, while the heterocyclic bases removal in the 5th and 6th substrate positions and in the 3rd position of the substrate processed strand distinctly inhibits the integrase catalytic activity. This fact demonstrates these bases significance for the active enzyme/substrate complex formation. On the contrary, modification of the 3rd position within substrate non-processed strand stimulates 3'-processing. Since heterocyclic base elimination results in disruption of the DNA complementary and staking interactions, this result shows that DNA double helix destabilization close to the cleaved bond promotes the 3'-processing.     

The strand transfer oligonucleotide inhibitors of HIV-integrase

Retroviral integrase participates in two catalytic reactions, which require interactions with the two ends of the viral DNA in the 3′processing reaction, and with a targeted host DNA in the strand transfer reaction. The 3′-hydroxyl group of 2′-deoxyadenosine resulting from the specific removing of GT dinucleotide from the viral DNA in the processing reaction provides the attachment site for the host DNA in a transesterification reaction. We synthesized oligonucleotides (ONs) of various lengths that mimic the processed HIV-1 U5 terminus of the proviral long terminal repeat (LTR) and are ended by 2′-deoxyadenosine containing a 3′-O-phosphonomethyl group. The duplex stability of phosphonomethyl ONs was increased by covalent linkage of the modified strand with its complementary strand by a triethylene glycol loop (TEG). Modified ONs containing up to 10 bases inhibited in vitro the strand transfer reaction catalyzed by HIV-1 integrase at nanomolar concentrations.     

Influence of substrate structure on disintegration activity of Moloney murine leukemia virus integrase.

The disintegration activity of Moloney murine leukemia virus (M-MuLV) integrase (IN) was investigated through structural and sequence modifications of a Y substrate that resembles an integration intermediate. The Y substrates, constructed from individual oligonucleotides, contain a single viral long terminal repeat (LTR) joined to a nicked target DNA. Truncation of the double-stranded LTR sequences distal to the conserved 5'-CA-3' dinucleotide progressively diminished disintegration activity. M-MuLV IN was also able to catalyze disintegration of a heterologous double-stranded LTR sequence. Significantly, the activity of M-MuLV IN on single-stranded LTR Y substrates was more dependent on the sequence and length of the LTR strand than that reported for human immunodeficiency virus type 1 (HIV-1) IN. Modifications introduced at the Y-substrate junction demonstrated that the 3'-hydroxyl group at the terminus of the target strand was necessary for efficient joining of the target DNA strands. The presence of a 2'-hydroxyl group at the 3' end of the target strand, as well as a single-nucleotide gap at the LTR-target junction, reduced disintegration activity. The absence of hydroxyl groups on the terminal nucleotide abolished joining of the target strands. The results presented here suggest that M-MuLV IN disintegration activity is dependent on substantially different LTR sequence requirements than those reported for HIV-1 IN and may be mediated primarily through a structural recognition event.     

Enzymatic capability of HIS-tagged HIV-1 integrase using oligonucleotide disintegration substrates

Disintegration, wherein a half-site integration substrate is resolved into separate viral and host DNA components via DNA strand transfer, is one of three well-established in vitro activities of HIV-1 integrase. The role of disintegration in the HIV-1 replicative cycle, however, remains a mystery. In this report, we describe the expression inEscherichia coli and purification of HIV-1 integrase as a fusion protein containing a 6×His tag at its amino terminus. Integrase resolved dumbbell and Y-substrates optimally at pH 6.8–7.2 in the presence of 2 mM MnCl₂. Substrate requirements for intramolecular disintegration included a 10 base pair viral U5 LTR arm and a CA dinucleotide located at the 3 end of the LTR. Disintegration was not sensitive to changes in the host DNA portion of the substrate. A dumbbell substrate with a 5 oligo-dA tail also underwent disintegration. The released LTR arm with an oligo-dA tail was utilized as a template primer by several DNA polymerases indicating that disintegration occurred via nucleophilic attack on the phosphodiester bond located immediately adjacent to the CA dinucleotide at the 3 end of the LTR. Coupled disintegration-DNA polymerase reactions provided a highly efficient and sensitive means of detecting disintegration activity. Integrase also catalyzed an apparently concerted disintegration-5-end joining reaction in which an LTR arm was transferred from one dumbbell substrate molecule to another.     

HIV-1 integrase preassembled on donor DNA is refractory to activity stimulation by LEDGF/p75

LEDGF/p75 is known to enhance the integrase strand transfer activity in vitro, but the underlying mechanism is unclear. Using an integrase assay with a chemiluminescent readout adapted to a 96-well plate format, the effect of LEDGF/p75 on both the 3'-processing and strand transfer steps was analyzed. Integrase inhibitors of the strand transfer reaction remained active in the presence of LEDGF/p75, but displayed 3- to 7-fold higher IC50 values. Our analyses indicate that, in the presence of 150 nM LEDGF/p75, active integrase/donor DNA complexes were increased by 5.3-fold during the 3'-processing step. In addition, these integrase/donor DNA complexes showed a 4.5-fold greater affinity for the target DNA during the subsequent strand transfer step. We also observed a 3.7-fold increase in the rate constant of catalysis of the strand transfer step when 150 nM LEDGF/p75 was present during the 3'-processing step. In contrast, when LEDGF/p75 was added at the beginning of the strand transfer step, no increase in either the concentration of active integrase/donor DNA complex or its rate constant of strand transfer catalysis was observed. This observation suggested that the integrase/donor DNA formed in the absence of LEDGF/p75 became refractory to the stimulatory effect of LEDGF/p75. Instead, this LEDGF/p75 added at the start of the strand transfer step was able to promote the formation of a new cohort of active integrase/donor DNA complexes which became functional with a delay of 45 min after LEDGF/p75 addition. We propose a model whereby LEDGF/p75 can only bind integrase before the latter binds donor DNA whereas donor DNA can engage either free or LEDGF/p75-bound integrase.     

Structural determinants for HIV-1 integrase inhibition by beta-diketo acids.

Among all the HIV-1 integrase inhibitors, the beta-diketo acids (DKAs) represent a major lead in anti-HIV-1 integrase drug design. These derivatives inhibit the integration reaction in vitro with a strong specificity for the 3'-end joining step. They are also antiviral and inhibit integration in vivo. The aim of the present study has been to investigate the molecular interactions between DKAs and HIV-1 integrase. We have compared 5CITEP with one of the most potent DKAs reported by the Merck group (L-708,906) and found that 5CITEP inhibits 3'-processing at concentrations where L-708,906 is only active on strand transfer. We also report a novel bifunctional DKA derivative that inhibits 3'-processing even more effectively than 5CITEP. The interactions of these inhibitors with the viral DNA donor ends have been studied by performing experiments with oligonucleotides containing defined modifications. We propose that the bifunctional DKA derivative binds to both the acceptor and donor sites of HIV-1 integrase, whereas the monofunctional L-708,906 derivative binds selectively to the acceptor site.     

Major and minor groove contacts in retroviral integrase-LTR interactions

The 3'-processing activities of HIV-1, HTLV-2, and M-MuLV integrases (INs) with their corresponding U5 end of the viral DNA molecule were examined to define functional group determinants of U5 terminus recognition and catalysis. Nucleotide analogues were incorporated into the U5 terminus to produce conservative modifications in the surface of the major and/or minor grooves to map the hydrogen-bonding contacts required for LTR-IN interaction. Specifically, the phylogenetically conserved CA (positions 4 and 3, respectively) and the 5'-proximal nucleotide (position 5) were replaced with base analogues in plus and/or minus strands. For each integrase, similar major and minor groove contacts were identified in the guanine and adenine of the conserved CA/GT. Overall, perturbances in the minor groove resulted in a greater decrease in 3'-processing activity than the major groove substitutions. Additionally for HIV-1 and HTLV-2 INs, we observed an increase in the 3'-processing activity with an O4-MeThy substitution at position 3 of the minus strand. O4-MeThy may act to destabilize Watson-Crick base pairing and in doing so provide these INs with a more favorable interaction with the adjacent scissile bond. At position 5, a substantial divergence among the three INs was noted in the functional groups required for 3'-processing activity, thereby supporting the role of this position in providing some level of substrate specificity.     

Single amino acid substitution in HIV-1 integrase catalytic core causes a dramatic shift in inhibitor selectivity

HIV-1 integrase (IN) mediates the insertion of viral cDNA into the cell genome, a vital process for replication. This step is catalyzed by two separate DNA reaction events, termed 3'-processing and strand transfer. Here, we show that six inhibitors from five structurally different classes of compounds display a selectivity shift towards preferential strand transfer inhibition over the 3'-processing activity of IN when a single serine is substituted at position C130. Even though IN utilizes the same active site for both reactions, this finding suggests a distinct conformational dissimilarity in the mechanistic details of each IN catalytic event.