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

Unconventional Plasticity of HIV‑1 Reverse Transcriptase: How Inhibitors Could Open a Connection “Gate” between Allosteric and Catalytic Sites

Targeted molecular dynamics (TMD) simulations allowed for identifying the chemical/structural features of the nucleotide-competitive HIV-1 inhibitor DAVP-1, which is responsible for the disruption of the T-shape motif between Try183 and Trp229 of the reverse transcriptase (RT). DAVP-1 promoted the opening of a connection “gate” between allosteric and catalytic sites of HIV-1 RT, thus explaining its peculiar mechanism of action and providing useful insights to develop novel nucleotide-competitive RT inhibitors

Novel Macrocyclic Amidinoureas: Potent Non-Azole Antifungals Active against Wild-Type and Resistant Candida Species

Novel macrocyclic amidinourea derivatives <b>11</b>, <b>18</b>, and <b>25</b> were synthesized and evaluated as antifungal agents against wild-type and fluconazole resistant Candida species. Macrocyclic compounds <b>11</b> and <b>18</b> were synthesized through a convergent approach using as a key step a ring-closing metathesis macrocyclization reaction, whereas compounds <b>25</b> were obtained by our previously reported synthetic pathway. All the macrocyclic amidinoureas showed antifungal activity toward different Candida species higher or comparable to fluconazole and resulted highly active against fluconazole resistant Candida strains showing in many cases minimum inhibitory concentration values lower than voriconazole

Free Energy Profile and Kinetics Studies of Paclitaxel Internalization from the Outer to the Inner Wall of Microtubules

Several pieces of experimental evidence led us to hypothesize that the mechanism of action of paclitaxel (Taxol) could involve a two-steps binding process, with paclitaxel first binding within the outer wall of microtubules and then moving into the inner binding site. In this work, we first used multiply targeted molecular dynamics (MTMD) for steering paclitaxel from the outer toward the inner binding site. This rough trajectory was then submitted to a refinement procedure in the path collective variables space. Paclitaxel binding energy was monitored along the refined pathway, highlighting the relevance of residues belonging to the H6–H7 and the M- loops. Computational results were supported by kinetics studies performed on fluorescent paclitaxel derivatives

Pharmacophore models.

<p>(A), Left panel, Structure-based pharmacophore generated from the Mg⁺⁺ loaded ERK8/ADP complex (coordinates were taken from the refined ERK8 structure) by using the Ligandscout software. Right panel, Structure-based pharmacophore generated by the GRID-based pharmacophore modeling approach, starting from the ligand-bound refined structure of ERK8. Features code: HYD = hydrophobic; HBA = H-bond acceptor; HBD = H-bond donor; AROM = aromatic ring; grey spheres are excluded volumes. (B), The two ligand-based pharmacophores generated with the training set of 18 different inhibitors active towards ERK8 (from Bain J, et al., 2007). Features code same as above.</p

ERK8 kinase domain model.

<p>(A), Multiple sequence alignment between ERK8 and the selected templates FUS3 and ERK2. Numbering is referred to human ERK8 cDNA sequence as defined in Uniprot accession number Q8TD08. Consensus code: “yellow” indicates positions which have a single, fully conserved residue; “green” indicates conservation between groups of strongly similar properties; “blue” indicates conservation between groups of weakly similar properties. Gatekeeper residue is in bold and indicated by a full black circle. The TEY activation motif is in red (activation loop spans from the DFG motif to the APE motif, residues 155–187). The region in square brackets has been substituted (starting from the position indicated with the dashed red line) with the alignment highlighted in the bottom square that includes p38α. (B), Model of the ERK8 kinase domain (residues 12–345 of the full-length 1–544 protein) obtained by means of homology modeling protocol. Conserved kinase domain features are indicated, β-sheets colored in yellow, α-helices colored in red, loops colored in green, TEY activation motif colored in blue. (C), Superimposition of the same ERK8 model (grey) with the ERK2 template (purple). (D), Evolution of ERK8 structure with the MD refinement. Superimposition of the ERK8 model (grey), used as MD input, with the representative final structure (the refined ERK8 structure) (cyan) obtained after the simulation. (E), Superimposition of the refined ERK8 model (cyan) with the ERK2 template (purple).</p

<i>In vitro</i> characterization.

<p>(A), Dose/response curves for ITT53 and ITT57 on GST-ERK8_Bac. Results are reported as residual MBP phosphorylation levels compared with the control (DMSO). The average results of two independent experiments done in triplicate ± SD are plotted with the curve-fitting PRISM software (GraphPad). The concentration of drug that inhibited activity by 50% (IC₅₀) is shown. (B), ITT53, ITT57 and Ro-318220 ATP competition assay on GST-ERK8_Bac. Inhibition values are reported as percentage of residual MBP phosphorylation levels (i.e., residual kinase activity) compared with the control (DMSO). Results for the two indicated concentrations of ITT53, ITT57 and Ro-318220 (top, middle, bottom panel, respectively) at four different ATP doses were plotted. The average results of two independent experiments done in triplicate ± SD are plotted.</p

Flowchart of the <i>in silico</i> protocol.

<p>Computational steps applied to select all the hit compounds to be tested <i>in vitro</i>. In each set the percentage of success rate refers to the ratio between the number of active molecules and the number of tested molecules in the following experimental screening: purified GST-ERK8 protein (50 ng/sample) was used in kinase assays. Candidate compounds were dissolved in dimethyl sulfoxide (DMSO) and tested at fixed concentration of 50 µM (an equal volume of DMSO was added to control samples). Reactions were resolved by SDS-PAGE and ³²P incorporation on MBP was estimated by densitometry. Molecules were classified as active when the residual kinase activity was less than 50% in comparison to control samples.</p

Effect of selected molecular scaffolds on bacterial and eukaryotic GST-ERK8.

<p>(A), Molecular structure of selected compounds. (B), Binding mode of each compound as obtained after the molecular docking step. The ITT molecules are showed as sticks and colored by atom type. ERK8 protein structure is represented by secondary structure cyan elements. (C), Samples of GST-ERK8 from <i>E. coli</i> with the indicated concentration of inhibitors were subjected to kinase assay. Reactions were resolved by SDS-PAGE and ³²P incorporation on MBP was estimated by densitometry (upper panel). Coomassie staining verified that equal amounts of substrate were loaded (lower panel). (D), The average results of three independent experiments done in duplicate ± SD are plotted. (E), Samples of GST-ERK8_Bac with the indicated concentration of inhibitors were subjected to kinase assay. Reactions were resolved by SDS-PAGE and ³²P incorporation on MBP was estimated by densitometry (upper panel). Coomassie staining verified that equal amounts of substrate were loaded (lower panel). (F), The average results of three independent experiments done in duplicate ± SD are plotted.</p

Gatekeeper mutants.

<p>(A), Multiple sequence alignment of gatekeeper region among different members of the MAPK and CDK families of kinases. The position corresponding to the gatekeeper residue is highlighted. (B), Superimposition of the refined ERK8 structure (cyan) and CDK2 (magenta) X-ray structure. (C), Western Blot control of GST-fusion proteins from <i>E. coli</i>. Each lane was loaded with 100 ng of purified protein. ERK8_KD sample (lane 6) is a point mutant on the conserved lysine (Lys, K) in position 42 to arginine (Arg, R). (D), Representative kinase assay blot of gatekeeper mutants (200 ng/sample of purified protein) (upper panel). Reactions were resolved by SDS-PAGE and ³²P incorporation on MBP was estimated by densitometry. Coomassie staining verified that equal amounts of substrate were loaded (lower panel). Quantification of kinase activity in comparison to WT, as scored by MBP phosphorylation, from three independent experiments is reported in the lower panel.</p