This work provides evidence to support the use of Tn5 Tnp as a surrogate model for HIV-1 IN inhibitor development. Six compounds were identified that inhibit the activities of both Tn5 Tnp and HIV-1 IN, yet they do not inhibit the restriction enzyme BsmAI. In addition, these compounds were not identified as hits in any other screen used at the facility, including other FP assays, and it should be noted that for compounds tested, inhibition of Tnp synapsis reactions were not affected by the addition of an excess of unlabeled plasmid to chase away any potential inhibition due to nonspecific DNA-compound interactions (data not shown). Thus, the most likely conclusion is that these compounds are not interacting with the DNA but with a region along the protein conserved between Tnp and IN and not BsmAI.
These compounds were originally identified as inhibitors of Tnp complex assembly and are likely inhibiting IN-DNA interactions as well, providing some of the first evidence that both coumarins and cinnamoyl IN inhibitors target this step of the integration mechanism. It has been suggested that IN inhibitors, which target complex assembly, are undesirable because several compounds found to inhibit IN-DNA interactions were shown to be ineffective at inhibiting viral preintegration complexes (20
). However, we identified three compounds that inhibit IN in vitro which also inhibit Tnp assembly and HIV-1 infection in cells at a point in the viral life cycle consistent with inhibition of integration (compounds 10, 10-B, and 10-F).
These compounds contain a cinnamoyl moiety attached to a central pyrrole. This cinnamoyl lacks the pharmacophoric aromatic ring hydroxlyations previously described for cinnamoyl inhibitors (1
). These previously reported nonhydroxylated cinnamoyls inhibit IN with IC50
s in the low micromolar range, which approaches the concentration of Tnp and IN in our in vitro assays, 0.8 and 1.0 μM, respectively. Thus, although these IC50
values appear modest, even a very potent inhibitor would not inhibit at a much lower concentration in our assays, since inhibition at stoichiometric levels would occur in the low μM range. This is best illustrated by the fact that cold unlabeled DNA, which serves as a competitive inhibitor, exhibits an IC50
of 5.1 μM in our gel shift assay.
Disruption of the cinnamoyl moiety through the removal of the ethylene group (Table , compound 10-A) increases cytotoxicity and impedes its efficacy as an IN inhibitor, suggesting that this moiety is important for inhibition. Interestingly, two reactive groups, a ketone and an adjacent enol, form a diketo-like motif within the central pyrrole. This is reminiscent of the diketo moiety found in diketo acids, another extensively described class of IN inhibitors (24
). This diketo-like motif is also found in 5ClTEP. In fact, the 5ClTEP IN cocrystal structure revealed that this moiety forms a hydrogen bond with E152 of the IN DDE motif (21
). Thus, the activity we observe could stem, in part, from this diketo-like moiety.
However, it is likely that neither the cinnamoyl nor the central pyrrole is the exclusive pharmacophore for these compounds because two additional groups attached to the central pyrrole also have an impact on inhibition (Table ). Furthermore, it is worth noting that compound 10-F partially inhibits BsmAI activity (data not shown), suggesting that although this compound appears to inhibit HIV transduction, it may lack the desired specificity, a phenomenon previously reported for some cinnamoyl derivatives (35
). Future studies should therefore involve the identification of related structures that enhance inhibition and specificity without increasing toxicity.
Additionally, we measured compound efficacy against in vivo Tn5 transposition and determined that none of these compounds had a significant inhibitory effect on transposition in vivo (data not shown). This may stem from poor uptake of these compounds in a bacterial cell culture, and further analysis of inhibition of in vivo transposition should aim at improving the delivery of these compounds into bacteria.
In addition, we found several compounds that appear to represent novel inhibitor classes for this superfamily. Five thiazol derivatives (compounds 5, 11, 14, 18, and 19) were identified as Tnp inhibitors, two of which inhibit both Tn5 Tnp and HIV-1 IN (compounds 14 and 18). It is noteworthy that the keto group, in combination with the benzene ring and linker, forms a cinnamoyl variant in four of the five thiazol derivatives (compound 5) (Fig. ). For three of these compounds (compounds 5, 11, and 14) the cinnamoyl is trapped in a configuration not previously reported for IN inhibitors, so it remains unclear whether this moiety is the functioning pharmacophore in these compounds. In addition, several benzoic acids were identified as Tn5 Tnp inhibitors (compounds 8, 15, 16, 17, and 20). The similarity between the aromatic diketo acid IN inhibitors and these benzoic acid Tn5 Tnp inhibitors suggests that this moiety is potentially a pharmacophore in this group.
Several coumarin dimers, one of the most extensively described classes of IN inhibitors, were also identified in this screen. The IC50
values we report are, in fact, surprisingly close to those previously reported for similar coumarin derivatives (29
). Our results provide the first evidence that coumarins inhibit Tn5
Tnp, indicating that coumarins are interacting with a conserved region of these proteins. Both this work and previous reports find that potent IN inhibition is limited to coumarins containing large moieties attached to the phenyl linker (43
). However, all five coumarins identified in this screen inhibit Tn5
Tnp at the same approximate concentration, suggesting that the coumarin dimer interacts with a region conserved in both proteins and that the moieties attached to the phenyl linker may interact with a nonconserved region of HIV-1 IN. Modeling coumarin binding using this information could further aid in deciphering the nature of these interactions and in developing more effective coumarin derivatives that specifically inhibit a diverse range of related drug targets.
Our work thus illustrates that Tn5
is suited as a model for the development of therapeutic agents against HIV-1. Approaches have been reported for both HIV-1 reverse transcription and virus-induced translational frameshifting utilizing components of HIV-1 in alternate systems (23
). To identify novel HIV-1 reverse transcription inhibitors, a hybrid Ty1/HIV-1 element was generated, replacing the Ty1 reverse transcription region with that of HIV-1 for use in an inhibitor screen, whereas to study HIV-1-induced translational frameshifting, the HIV-1 −1 frameshift signal was introduced as a transgene in Saccharomyces cerevisiae
. Both assays serve to aid in identifying and characterizing inhibitors by targeting pieces of HIV-1 in alternate systems, and it is our hope that our approach utilizing a surrogate protein will serve to harness the power of the Tn5
structural data for the development of improved therapeutics for an alternate step in the viral life cycle.
In conclusion, the success of using Tn5
transposase as a surrogate for finding HIV-1 inhibitors suggests that similar surrogates can be used for other protein superfamilies. This would facilitate the use of simpler screens and the use of the best available structural data for inhibitor screening and development. This also alerts one to the possibility of undesirable cross activity with other members of the same superfamily of proteins. In this case, such cross-reactivity could occur with the RAG proteins. These proteins are involved in DNA cleavage during immunoglobulin gene formation and have a catalytic mechanism and, presumably, structure similar to both transposase and retroviral integrases (39