Development and in Vitro Evaluation of a Microbicide Gel Formulation for a Novel Non-Nucleoside Reverse Transcriptase Inhibitor Belonging to the N-Dihydroalkyloxybenzyloxopyrimidines (N-DABOs) Family

17openPreventing HIV transmission by the use of a vaginal microbicide is a topic of considerable interest in the fight against AIDS. Both a potent anti-HIV agent and an efficient formulation are required to develop a successful microbicide. In this regard, molecules able to inhibit the HIV replication before the integration of the viral DNA into the genetic material of the host cells, such as entry inhibitors or reverse transcriptase inhibitors (RTIs), are ideal candidates for prevention purpose. Among RTIs, S- and N-dihydroalkyloxybenzyloxopyrimidines (S-DABOs and N-DABOs) are interesting compounds active at nanomolar concentration against wild type of RT and with a very interesting activity against RT mutations. Herein, novel N-DABOs were synthesized and tested as anti-HIV agents. Furthermore, their mode of binding was studied by molecular modeling. At the same time, a vaginal microbicide gel formulation was developed and tested for one of the most promising candidates.openTintori, Cristina; Brai, Annalaura; DASSO LANG, MARIA CHIARA; Deodato, Davide; Greco, Antonia Michela; Bizzarri, Bruno Mattia; Cascone, Lorena; Casian, Alexandru; Zamperini, Claudio; Dreassi, Elena; Crespan, Emmanuele; Maga, Giovanni; Vanham, Guido; Ceresola, Elisa; Canducci, Filippo; Ariën, Kevin K.; Botta, MaurizioTintori, Cristina; Brai, Annalaura; DASSO LANG, MARIA CHIARA; Deodato, Davide; Greco, Antonia Michela; Bizzarri, Bruno Mattia; Cascone, Lorena; Casian, Alexandru; Zamperini, Claudio; Dreassi, Elena; Crespan, Emmanuele; Maga, Giovanni; Vanham, Guido; Ceresola, Elisa; Canducci, Filippo; Ariën, Kevin K.; Botta, Maurizi

Pol λ appears to preferentially generate G:T over C:T mismatches and its misincorporation ability is suppressed by RP-A

<p><b>Copyright information:</b></p><p>Taken from "Human replication protein A can suppress the intrinsic mutator phenotype of human DNA polymerase λ"</p><p>Nucleic Acids Research 2006;34(5):1405-1415.</p><p>Published online 6 Mar 2006</p><p>PMCID:PMC1390690.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () Misincorporation of dGTP (lanes 1–3) or dCTP (lanes 4–6) was monitored on the 19/40merT template in the presence of 0.1 mM Mn and increasing concentrations of pol λ (25, 100 and 250, corresponding to 0.25, 1 and 2.5 pmol, respectively). Lane 7: control reaction in the presence of 25 nM pol λ and all four dNTPs at 5 µM each. The positions of the differently migrating dGMP- and dCMP-terminated +1 products are shown on the left side of the panel. () Misincorporation of increasing concentrations of dGTP (lanes 1–6) or dCTP (lanes 7–12) were monitored on the 19/40merT template in the presence of 0.1 mM Mn and 25 nM (0.25 pmol) pol λ. The positions of the differently migrating dGMP- and dCMP-terminated +1 products are shown on the left side of the panel. () Incorporation of dTTP or dATP (5 µM each) was measured in the presence of 50 nM (0.5 pmol) polλ, 5′-labelled d(T)/d(A) primer/template, 1 mM Mg and in the absence (lanes 1 and 9) or in the presence of different concentrations of either PCNA (lanes 2, 3, 10 and 11) or RP-A (lanes 4–6, 12–14), or an equimolar combination of both proteins (lanes 7, 8, 15 and 16). () Incorporation of 5 µM dCTP (lanes 1–8) or 10 µM dGTP (lanes 9–16) was monitored on the 5′-labelled 20/40merG primer/template, in the presence 150 nM (1.5 pmol) pol λ, 0.1 mM Mn, and in the absence (lanes 1 and 9) or in the presence of 0.5 (lanes 2, 10) and 2 pmol (lanes 3, 11) of PCNA, or 0.2 (lanes 4, 12), 0.5 (lanes 5, 13) and 2 pmol (lanes 6, 14) of RP-A, or a combination of 0.2 (lanes 7, 15) or 2 pmol (lanes 15, 16) of PCNA and RP-A in equimolar ratios. The positions of the differently migrating dCMP- and dGMP-terminated primers are indicated on the left side of the panel with an asterisc

The specific insertion of human DDX3 is important for nucleic-acid stimulation of ATPase activity.

<p>Reactions were performed as described in Material and Methods. <b>A</b>. Product analysis for the ATPase reaction catalyzed by the DDX3wt (lanes 2–6) and the DDX3ΔINS (lanes 7–11) mutant proteins in the absence (lanes 6, 11) or in the presence of increasing concentrations of ssDNA. Lane 1, control reaction without enzyme. Reactions with the full length DDX1 protein (lanes 12–14) in the absence (lane 12) or in the presence of a fixed amount of DNA (lane 13) or RNA (lane 14), were included for comparison. <b>B</b>. Variation of the increase in the ATPase reaction rate (Δv) as a function of the ssDNA concentration in the presence of DDX3wt (circles) or DDX3ΔINS (triangles). The Δv values were derived as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019810#s4" target="_blank">Materials and Methods</a>. Data were fitted to Eq.(2) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019810#s4" target="_blank">Materials and Methods</a>). Values are the means of three independent determinations. Error bars are ±SD. <b>C</b>. Progress curves for the product formation during the ATPase reaction catalyzed by 0.2 µM of DDX3wt as a function of time, in the absence (circles) or in the presence of 10 µM ssRNA (triangles) or 10 µM ssDNA (squares). Data were fitted to Eq. (3) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019810#s4" target="_blank">Materials and Methods</a>). Values are the means of three independent determinations. Error bars are ±SD. <b>D</b>. As in panel C, but in the presence of 0.2 µM of the DDX3ΔINS mutant. <b>E</b>. Increasing amounts of DDX3wt (second row) or DDX3ΔINS mutant (third row), were incubated with a fixed concentration of (6-FAM)-5′-labelled ssDNA oligonucleotide. Nitrocellulose filter bound protein-DNA complexes were revealed by laser scanning. First row, control in the absence of proteins. <b>F</b>. Binding of DDX3 to ssDNA, as revealed by filter-binding assays. Data were fitted to Eq.(5) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019810#s4" target="_blank">Materials and Methods</a>). Values are the means of three independent determinations. Error bars are ±SD. <b>G</b>. Increasing amounts of DDX3wt (lanes 2–5) or DDX3ΔINS (lanes 6–9) were incubated in the presence of a (6-FAM)-5′-labelled ss RNA oligonucleotide. Enzyme-RNA ([E:RNA]) complexes were resolved by non denaturing PAGE and visualized by laser scanning. Lane 1, oligonucleotide alone.</p

The specific insertion of human DDX3 is important for RNA unwinding.

<p>Reactions were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019810#s4" target="_blank">Materials and Methods</a>. <b>A</b>. Representative RNA unwinding assay for DDX3wt, or the DDX3ΔINS mutant. Lanes 8, 16, boiled controls. <b>B</b>. As in A, but with the addition of a 50- fold molar excess of a r(A)₄₀mer RNA trap ss oligonucleotide. <b>C</b>. Quantitative analysis of the RNA unwinding reaction catalyzed by DDX3wt and the DDX3ΔINS mutant. Values are the means of three independent determinations. Error bars are ±SD.</p

Kinetics of RNA unwinding by DDX3wt and the DDX3ΔINS mutant.

a<p>The <i>k_uw</i> values were was estimated by Eq.(4) as described in Material and Methods. Values are the means of three independent replicates ± SD.</p

Kinetics of the ATP-dependent reaction of DDX3wt and mutants with nucleic acids.

a<p>The A_burst, <i>k</i>_burst and <i>k</i>_ss parameters were determined by Eq.(3) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019810#s4" target="_blank">Materials and Methods</a>. Values are the means of three independent replicates ± SD.</p>b<p>RNA was oligo(rU)₂₀; DNA was oligo(dT)₂₀.</p

The DDX3 insert is functionally implicated in the HIV-1 cofactor activity of DDX3.

<p><b>A</b>. HIV genomic RNA pull down experiments with purified DDX3wt and DDX3ΔINS proteins. Bound HIV RNA was detected by RT-PCR with <i>gag</i>-specific primers in three independent experiments (lanes 1 and 2; lanes 3 and 4; lanes 8 and 9). Lanes 6 and 7 correspond to RT-PCR fragments of input viral RNA and PCR of the HIV-1 pLai plasmid, respectively; lane 5 is the negative PCR control. <b>B</b>. Peptide DDX3-INS1 selected with the DDX3 insertion by phage display shows homology to XPO1. The crystal structure of XPO1 (DOI:10.2210/pdb3gb8/pdb) is shown and amino acids identical to the peptide sequence are represented as spheres. The corresponding positions are indicated on the right side of the panel, along with the conservative substitution V408L. <b>C</b>. Antiviral activity of the selected peptide DDX3-INS1 fused to a protein transduction domain. Peptides DDX3-INS1 and a control peptide were transduced into HelaP4 cells after infection with HIV-1Lai and virus in supernatants was quantified on Tzm-bl cells by luminometry after 44 hours. Bars represent the mean of 3 independent experiments and error bars indicate SD. For the control peptide, one representative experiment is shown.</p

ATPase reaction efficiency of DDX3wt and mutants and DDX1 in the absence or presence of nucleic acids.

a<p>The kinetic parameters K_D, K_m, <i>k</i>_cat, were determined by Eq.(1) and Eq. (5), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019810#s4" target="_blank">Materials and Methods</a>. Values are the means of three independent replicates ± SD. K_m = K_D+k_cat/k_on.</p>b<p>RNA was oligo(rU)₂₀; DNA was oligo(dT)₂₀.</p

Human DDX3 can act as a DNA helicase.

<p>Experiments were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019810#s4" target="_blank">Materials and Methods</a>. The 18-mer oligonucleotide was fluorescently labelled at its 5′-end in all the experiments. The position of the unreacted substrate (18/38-mer) and of the released strand 18-mer are indicated on the left side of each panel. <b>A</b>. Strand-displacement reactions catalyzed by increasing amounts of DDX3wt in the presence of the different partially dsDNA substrates: 18/[5′-3′]-38 mer (lanes 1–5); 18/[3′]-38 mer (lanes 6–10); 18/[5′]-38 mer (lanes 11–15). Lanes 1, 6, 11: control reactions incubated in the absence of enzyme. Lanes 2, 7, 12: reaction mix with no enzyme loaded on gel without incubation. Lane 16: boiled substrate. <b>B</b>. Strand displacement activity of increasing amounts of the DDX1 (lanes 3–5) or DDX3ΔINS (lanes 7–9) proteins on the 18/[5′-3′]-38 mer substrate. Lanes 2, 6, control reactions incubated in the absence of enzyme; lane 1, boiled substrate. <b>C</b>. Sequence alignment of human DDX3, <i>S. cerevisiae</i> Dpb9, human DDX1, <i>D. melanogaster</i> Vasa and human Dpb5, showing the structural motifs Ia, Ib and IV. Residues known to mediate specific 2′-OH ribose contacts in the Vasa crystal structure are highlighted. The corresponding helicase substrates for each enzyme are indicated on the right side of the panel.</p

Characterization of the ATPase activity of recombinant human DDX3.

<p>Reactions were performed as described in Material and Methods. <b>A</b>. Product analysis of a representative experiment for the ATPase reaction catalyzed by 0.1 µM full length DDX3 in the presence of 10 µM ss RNA. <b>B</b>. As in panel A, but in the presence of 10 µM ss DNA. <b>C</b>. As in panel A, but in the absence of nucleic acids. <b>D</b>. Variation of the initial velocities of the reaction as a function of ATP concentrations, in the absence (triangles) or in the presence of 10 µM ss DNA (circles) or 10 µM ss RNA (squares). Data were fitted to Eq.(1) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019810#s4" target="_blank">Materials and Methods</a>). Values are the means of three independent determinations. Error bars are ±SD. <b>E</b>. Product analysis of the ATPase reaction catalyzed by increasing amounts of the DDX3ΔINS mutant in the absence (lanes 2–5) or in the presence of of 25 µM ss DNA (lanes 7–10) or ssRNA (lanes 12–15). Lanes 1, 6 and 11, control reactions in the absence of enzyme. <b>F</b>. [α-³³P] ATP was UV crosslinked to increasing amounts of DDX3wt (upper panel) or ΔINS mutant (lower panel). Radioactive proteins were resolved on SDS-PAGE and revealed by phosphoImaging. <b>G</b>. Binding of DDX3 to ATP, as revealed by UV-crosslinking. Data were fitted to Eq.(5) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019810#s4" target="_blank">Materials and Methods</a>). Values are the means of three independent determinations. Error bars are ±SD. <b>H</b>. Comparison of the catalytic efficiencies (k_cat/K_m) for ATP hydrolysis of DDX3wt (aa 1–662), and the N-DDX3 (aa 1–427), DDX3ΔINS and I-DDX3 (aa 160–427) mutants, in the absence (white bars) or in the presence of ssDNA (grey bars) or ssRNA (black bars). Determination of the kinetic constants k_cat and K_m was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019810#s4" target="_blank">Materials and Methods</a>. Values represent the means between two independent estimates of the k_cat/K_m values from two sets of experiments. Error bars represent ±SD.</p