Despite the two models for the

Despite the two models for the HIV-1 IN-aptamer complexes appeared to be comparable, disintegration assays revealed that 93-del inhibited C– or N-terminally truncated enzyme at the same extent, [18] whereas, T30695 required N-terminal domain (NTD) of the enzyme to exert its integrase inhibitory activity [19]. These observations suggest that the two aptamers differently interact with the NTD, and the proposed molecular model of the (HIV-1 IN)4/T30695 complex needs to be further refined. To gain additional information on the mechanism of action of T30695 and its analogues we have extended structure−activity relationships (SARs) studies through the development of new derivatives of T30695. In particular, for the reasons given above, modifications at the loop residues were targeted as they could influence the binding ability of T30695 GQ to HIV-1 IN. We synthesized T30695 variants, in which the R or S acyclic nucleoside 1, named glycerol T, (Fig. 2) singly replaced the topotecan sale residues at the T30695 loops. Because of its impact on both phosphate backbone and/or spatial orientation of nucleobase, R and S glycerol T are good candidates as T analogues to modify T30695 GQ loops. The rational basis of this approach lays in a previous study on the anticoagulant activities of some GQs variants of the Thrombin Binding Aptamer (TBA). In those variants a R or S glycerol-nucleobase derivative replaced, one at time, a specific T in T3T4 or T12T13 or T7G8T9 loops [20]. Although the replacement of T3 (or T12) with the R or S glycerol-nucleobase derivative, gave rise to GQs having similar thermal stabilities, nevertheless the same was not valid comparing the biological activities. Indeed, while the R glycerol TBA derivatives were more active than TBA, those variants containing the S glycerol derivative were unable to inhibit thrombin activity. Molecular modeling results indicated that, as a consequence of the orientation of the modified nucleobase at T3 or T12, only the TBA-GQs containing R glycerol-derivative could easily adopt the thrombin binding conformation of the TT loops [20]. These results prompted us to examine the influence of T loop replacement with either R or S glycerol T on thermal stability and on biological activity of T30695-GQ. Circular dicroism (CD) melting, electrophoretic mobility shift assay (EMSA), and high resolution mass spectrometry (HRMS) experiments provided information about the thermal stability and the stoichiometry of T30695-GQ variants, whereas CD and 1H NMR studies were performed to evaluate the effect of the modification on T30695-GQ typology. Furthermore, Lens Epithelium Derived Growth Factor p75 (LEDGF/p75) dependent and independent integration assay were carried out to evaluate the impact of T loop modification on T30695-GQ biological activities. LEDGF/p75 is a chromatin-associated protein that interacts and preferentially associates with actively transcribed chromatin [21]. Importantly, LEDGF/p75 is a key cellular cofactor of HIV integrase that is responsible for the selective integration of HIV into active transcription units of transcriptionally active genes [22].
Materials and methods Chemicals and anhydrous solvents were purchased from Fluka-Sigma-Aldrich. TLCs were run on Merck silica gel 60 F254 plates. Silica gel chromatography was performed using Merck silica gel 60 (0.063–0.200 mm). Reagents and phosphoramidites for DNA syntheses were purchased from Sigma-Aldrich. ON syntheses were performed on a PerSeptive Biosystem Expedite DNA synthesizer. HPLC purifications and analyses were carried out using a JASCO PU-2089 Plus HPLC pump equipped with a JASCO BS-997-01 UV detector. CD experiments were performed on a JASCO 715 spectropolarimeter equipped with a PTC-348 temperature controller. The data were processed using the Varian VNMR and the NMR (http://www.inmr.net) software packages. Mass spectrometry data were collected on a hybrid linear ion trap LTQ Orbitrap XL™ Fourier Transform MS (FTMS) equipped with an ESI ION MAX™ source (Thermo-Fisher; San Josè, CA, USA).