Adamantyl Derivative As a Potent Inhibitor of <i>Plasmodium</i> FK506 Binding Protein 35

FKBP35, FK506 binding protein family member, in <i>Plasmodium</i> species displays a canonical peptidyl-prolyl isomerase (PPIase) activity and is intricately involved in the protein folding process. Inhibition of <i>Pf</i>FKBP35 by FK506 or its analogues were shown to interfere with the in vitro growth of <i>Plasmodium falciparum</i>. In this study, we have synthesized adamantyl derivatives, Supradamal (SRA/4a) and its analogues SRA1/4b and SRA2/4c, which demonstrate submicromolar inhibition of <i>Plasmodium falciparum</i> FK506 binding domain 35 (FKBD35) PPIase activity. SRA and its analogues not only inhibit the in vitro growth of <i>Plasmodium falciparum</i> 3D7 strain but also show stage specific activity by inhibiting the trophozoite stage of the parasite. SRA/4a also inhibits the <i>Plasmodium vivax</i> FKBD35 PPIase activity and our crystal structure of <i>Pv</i>FKBD35 in complex with the SRA provides structural insights in achieving selective inhibition against <i>Plasmodium</i> FKBPs

Direct interaction of luteolin with VRK1.

<p>(A), Pull-down assay using luteolin-conjugated sepharose 4B beads or control sepharose 4B beads with recombinant VRK1 protein. Purified GST and GST-VRK1 proteins are incubated with indicated beads, and then pull down assay was performed. Each proteins were detected by immunoblotting with GST antibody. (B), Pull-down assay using luteolin conjugated sepharose 4B beads with SH-SY5Y cell lysate. Cell lysate are incubated with indicated beads, and then pull-down was performed. The proteins were detected by immunoblotting with VRK1 antibody. (C), SPR detection for the interaction of luteolin with VRK1. The data were obtained by kinetic titration method with sequentially injection of analytes without regeneration steps. Data for eupatilin and wogonin were obtained by classical methods.</p

Luteolin exhibits selective cytotoxicity towards tumorigenic cells and induces apoptosis.

<p>(A), The effects of luteolin on cell viability of tumorigenic (HeLa and U2OS) and non-tumorigenic (BEAS-2B and HEK293A) cell lines. Cells were treated with indicated concentration of luteolin for 24 hours. Afterward, cell viability was analyzed by the MTT assay. (B), Time-dependent effects of luteolin on HeLa cell viability. HeLa cells were treated with indicated luteolin 10 µM for indicated times. Cell viability was assessed by the MTT assay. Error bars represents means ±SEM (n = 10). Symbols (***) represent p value <0.0001. (C), The effects of flavonoids on A549 cell viability were compared. Error bars represent means ±SEM (n = 10). Symbols (***) represent p value <0.0001. The P-values in (A), (B) and (C) were calculated using Student's t-test. (D), Proapoptotic effect of luteolin on HeLa cells. HeLa cells treated with indicated concentrations of luteolin were double stained with PI/Annexin-V allophycocyanin. Apoptosis analysis was performed by flow cytometry.</p

NMR titration assay and in silico modeling for interaction of luteolin with VRK1.

<p>(A), NMR titration experiments were performed. Spectrum of chemical shift perturbations versus amino acid residues of the VRK1 protein after binding of luteolin. (B), Mapping of chemical shift perturbations on the VRK1 protein. Most of the perturbed residues (shown in red) are located close to the catalytic domain of VRK1. (C), <i>in silico</i> modeling of the binding mode of luteolin to the VRK1 protein. Luteolin is predicted to fit in the vicinity of the G-loop, catalytic site, and α-C lobe.</p

Luteolin-induced cell cycle arrest and nuclear envelope disassembly defects.

<p>(A), Cell cycle analysis was carried out by flow cytometry. HeLa cells transfected with GFP or GFP-VRK1 were treated with luteolin for 24 hours, and 10,000 cells were gated for analysis. Quantitative data are below the histogram plots. (B) and (C), HeLa cells treated with vehicle or luteolin were stained with lamin B antibody to visualize nuclear envelope and with Hoechst 33342 to visualized DNA. Alexa 488 dye-conjugated antibody was used as secondary antibody. The slides were visualized by fluorescence microscopy (B), or confocal laser scanning microscopy (C). (D), Fluorescent staining of VRK1, BAF, and DNA for analysis of the phosphorylation-mediated re-localization of BAF. Cells co-transfected with GFP or GFP-BAF with RFP or RFP-VRK1 were treated with DMSO or 10 µM luteolin for 24 hours.</p

Kinetic parameters of the binding of luteolin to VRK1.

<p>Kinetic parameters of the binding of luteolin to VRK1.</p

SRF cytotoxicity involves JNK kinases and proceeds via caspase-3 activation and mitochondrial membrane potential loss.

<p>(A) SRF (10 µM) activates JNK kinase but not ERK and p38. Phosphorylation status of the JNK, ERK and p38 was probed by immunoblotting using phospho-specific antibodies against the kinases. SRF selectively induces JNK phosphorylation [Panel (i)] without altering protein levels [Panel (ii)]. (B and C) Pre-treatment of cells with JNK-specific inhibitor prior to SRF (10 µM) exposure abrogates Bcl-2 and Bad phosphorylation. No phosphorylation of Bcl-2 (Panel B, Lane 4) or Bad (Panel C, Lane 3) were seen when cells were pre-treated with JNK-specific inhibitor, SP600125. Similar results were not observed with highly selective non-competitive ERK1/2 inhibitor, PD98059 (Panel B, Lane 6) or p38 inhibitor, SB203580 (Panel B, Lane 8). (D) Inhibition of JNK-kinase protects cells against SRF-induced toxicity. Cell viability was determined by MTT assay and reported as percentage control. SP600125 was able to retain viability in approximately 70% cells. Data are shown as means ± SEM. **<i>P</i><0.01 versus control. (E) Cells pre-treated with SP600125 were able to overcome SRF-induced G₂/M phase cell cycle blockage. Percentage cells in the different stages of cell cycle were determined by flow cytometric analysis. Data are shown as means ± SEM. *<i>P</i><0.05; **<i>P</i><0.01 versus control. (F) SP600125 treated cells retain cellular microtubule network. Fluorescence micrographs of cells treated with SRF in the presence or absence of SP600125. Microtubules (green) and nucleus (blue) were stained with FITC-conjugated anti-tubulin antibody and DAPI, respectively. Scale bar = 10 µM. (G) SRF induces loss of mitochondrial membrane potential as shown by flow-cytometric analysis of cells stained with JC-1. Events were counted in the green channel. SP600125 pre-treatment prevented cells from undergoing apoptosis as percentage of cells having fluorescence in the green channel decreased from 84.2% in SRF treated cells to 47.8% for cells that were pre-treated with SP600125 prior to SRF (10 µM) exposure. (H) Apoptotic death mediated by SRF proceeds through caspase-3 activation. A cleaved band corresponding to activated caspase-3 is present in SRF-treated lysates but absent from SP600125 pre-treated lysates.</p

SRF - a novel tubulin-binding agent that depolymerizes microtubules.

<p>(A) Chemical structure of SRF. (B) Effect of SRF on microtubule polymerization <i>in vitro</i>. Purified tubulin was incubated at 37°C in the absence (DMSO) or presence of drugs like taxol (3 µM), nocodazole (10 µM), SRF and absorbance was measured at 420 nm every 1 min over a 60 min period. SRF inhibited microtubule polymerization in a concentration-dependent manner as was indicated by a decrease in absorbance with time. (C) Confocal micrographs of HeLa cells exposed to SRF, taxol and nocodazole. Cells were labeled with FITC-conjugated anti-tubulin antibody. SRF treatment completely destroys the intricate microtubule network. Scale bar = 10 µM.</p

Inhibitory effects of luteolin on VRK1 kinase activity.

<p>(A) and (B), <i>in vitro</i> kinase activity measurements for VRK1 with BAF (A) or VRK1 with histone H3 (B) were performed by increasing concentrations of luteolin (0.0, 1.0, 10, 50, 100, and 250 µM), and then VRK1 and substrate proteins were stained with silver nitrate. (C), Chemical structures and molecular weights of luteolin, eupatilin, and wogonin. (D) and (E), <i>in vitro</i> kinase assay for VRK1 with BAF were performed by increasing concentrations (0.0, 1.0, 10, 50, 100, and 250 µM) of eupatilin (D) or wogonin (E), and then VRK1 and BAF proteins were stained with silver nitrate. (F) and (G), Quantification of VRK1 auto-phosphorylation (F) or BAF phosphorylation (G) described in (B), (D) and (E). Data in (F) and (G) represent means of three independent experiments ±SEMs.</p

Cartoon representing plausible mechanism of SRF mediated toxicity in cancer cells.

<p>SRF can bypass P-gp mediated drug efflux via mechanism(s) that are currently unknown. Inside the cell, SRF binds and inhibits microtubule polymerization resulting in cell cycle arrest at the G₂/M phase. These events, in turn, activate JNK-mediated stress-response signaling cascade leading to the phosphorylation and inactivation of anti-apoptotic proteins like Bcl-2 and Bad. Consequently, there is loss of mitochondrial membrane potential and integrity, release of cytochrome-c, activation of caspase-3 and eventual cell death by apoptosis. Thus, SRF-mediated cell death proceeds via the intrinsic/mitochondrial apoptotic pathway.</p