MOESM1 of Fabrication of device with poly(N-isopropylacrylamide)-b-ssDNA copolymer brush for resistivity study

Additional file 1: Figure S1. Thickness of (a) PN4, PN8, PN12, PN16 and PN20, and (b) PN4D, PN8D, PN12D, PN16D and PN20D plotted as a function of temperature

Moiety-Linkage Map Reveals Selective Nonbisphosphonate Inhibitors of Human Geranylgeranyl Diphosphate Synthase

Bisphosphonates are potent inhibitors of farnesyl pyrophosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS). Current bisphosphonate drugs (e.g., Fosamax and Zometa) are highly efficacious in the treatment of bone diseases such as osteoporosis, Paget’s disease, and tumor-induced osteolysis, but they are often less potent in blood and soft-tissue due to their phosphate moieties. The discovery of nonbisphosphonate inhibitors of FPPS and/or GGPPS for the treatment of bone diseases and cancers is, therefore, a current goal. Here, we propose a moiety-linkage-based method, combining a site-moiety map with chemical structure rules (CSRs), to discover nonbisphosphonate inhibitors from thousands of commercially available compounds and known crystal structures. Our moiety-linkage map reveals the binding mechanisms and inhibitory efficacies of 51 human GGPPS (hGGPPS) inhibitors. To the best of our knowledge, we are the first team to discover two novel selective nonbisphosphonate inhibitors, which bind to the inhibitory site of hGGPPS, using CSRs and site-moiety maps. These two compounds can be considered as a novel lead for the potent inhibitors of hGGPPS for the treatment of cancers and mevalonate-pathway diseases. Moreover, based on our moiety-linkage map, we identified two key residues of hGGPPS, K202, and K212, which play an important role for the inhibitory effect of zoledronate (IC₅₀ = 3.4 μM and 2.4 μM, respectively). This result suggests that our method can discover specific hGGPPS inhibitors across multiple prenyltransferases. These results show that the compounds that highly fit our moiety-linkage map often inhibit hGGPPS activity and induce tumor cell apoptosis. We believe that our method is useful for discovering potential inhibitors and binding mechanisms for pharmaceutical targets

Identification, Synthesis, and Evaluation of New Neuraminidase Inhibitors

High-throughput screening was performed on ∼6800 compounds to identify <b>KR-72039</b> as an inhibitor of H1N1 and H5N1 neuraminidases (NAs). Structure–activity relationship studies led to <b>3e</b>, which inhibited H5N1 NA with an IC₅₀ of 2.8 μM and blocked viral replication. Docking analysis shows that compounds bind to loop-430 around the NA active site. Compound <b>3l</b> additionally inhibited H7N9 NA, making it a dual inhibitor of N1- and N2-type NAs

Roles of Amino Acids in the <i>Escherichia coli</i> Octaprenyl Diphosphate Synthase Active Site Probed by Structure-Guided Site-Directed Mutagenesis

Octaprenyl diphosphate synthase (OPPS) catalyzes consecutive condensation reactions of farnesyl diphosphate (FPP) with five molecules of isopentenyl diphosphates (IPP) to generate C₄₀ octaprenyl diphosphate, which constitutes the side chain of ubiquinone or menaquinone. To understand the roles of active site amino acids in substrate binding and catalysis, we conducted site-directed mutagenesis studies with <i>Escherichia coli</i> OPPS. In conclusion, D85 is the most important residue in the first DDXXD motif for both FPP and IPP binding through an H-bond network involving R93 and R94, respectively, whereas R94, K45, R48, and H77 are responsible for IPP binding by providing H-bonds and ionic interactions. K170 and T171 may stabilize the farnesyl carbocation intermediate to facilitate the reaction, whereas R93 and K225 may stabilize the catalytic base (MgPP_i) for H_R proton abstraction after IPP condensation. K225 and K235 in a flexible loop may interact with FPP when the enzyme becomes a closed conformation, which is therefore crucial for catalysis. Q208 is near the hydrophobic part of IPP and is important for IPP binding and catalysis

MMPs, especially MMP-9, were responsible for the invadopodia and degrading ability of A431-III cells.

<p>A, A431-III cells were plated on gelatin or Oregon Green® 488-conjugated gelatin and treated with DMSO or 25 µM GM6001 for 5 h to observe the formation of invadopodia and the matrix degrading ability. Tks5, invadopodia component protein, was used as a marker. B, Quantification of cells associated with matrix degradation (left panel). Quantification of degradation area normalized against cell number (right panel). C, Effect of GM6001 on MMPs’ activities and TIMPs’ expression were measured by zymography and western blot. D, The cells were treated with 40 nM MMP-9 siRNA or control siRNA. Knockdown efficiency was measured by qPCR (left) or gelatin zymography (right). E, A431-III cells (expressing control or MMP-9 knockdown siRNA) were plated on gelatin or Oregon Green® 488-conjugated gelatin to investigate the formation of invadopodia and the matrix degrading ability. F, Quantification of cells associated with matrix degradation (left panel) and degradation area normalized against cell number (right panel).*<i>p</i><0.05. Error bars present the standard error of the mean. Scale bar are 22 µm.</p

Effects of SU6656 on invadopodia formation and functioning.

<p>A, A431-III cells were plated on gelatin or Oregon Green® 488-conjugated gelatin and treated with DMSO or 5 µM SU6656 for 5 h to investigate the formation of invadopodia and matrix degradation. B, Quantification of cells associated with matrix degradation (upper panel). Quantification of the degradation area normalized against cell number (lower panel). C, Total cell lysates were prepared for immunoblotting analysis. Active Src and downstream target cortactin (Y421) were analyzed. D, Invasion assays were performed. *<i>p</i><0.05. P values are compared with control A431-III. Error bars present the standard error of the mean. Scale bar are 22 µm.</p

Src kinase activity and phosphorylation of cortactin were responsible for invadopodia formation in A431-III cells.

<p>A, Expression of invadopodia regulators, core components and MMPs/TIMPs in A431-P and A431-III were analyzed by microarray. B, Expression of invadopodia regulators, components and MMPs/TIMPs were validated by qPCR. C, Total cell lysates were subjected for immunoblotting analysis. The active status of Src kinase and the phosphorylation of cortactin were determined.</p

A431-III forms invadopodia and exhibits higher ability to degrade gelatin than A431-P.

<p>A, Upper panel: A431-P and A431-III cells were stained with cortactin (red), F-actin (green), and DAPI (blue). Arrowheads, examples of invadopodia that are identified as cortactin and actin-positive dots. Representative images taken of both cells. Lower panel: Both cells were plated on Oregon Green® 488-conjugated gelatin. Degraded ECM was identified as a dark area on the gelatin. B, Upper panel: Quantification of cells associated with matrix degradation. Lower panel: Quantification of the degradation area normalized against cell number. C, Invasion assays were performed. *<i>p</i><0.05. Error bars present the standard error of the mean. Scale bar are 22 µm. P (A431-P); III (A431-III).</p

Conjugation of Benzylvanillin and Benzimidazole Structure Improves DNA Binding with Enhanced Antileukemic Properties

<div><p>Benzyl-o-vanillin and benzimidazole nucleus serve as important pharmacophore in drug discovery. The benzyl vanillin (2-(benzyloxy)-3-methoxybenzaldehyde) compound shows anti-proliferative activity in HL60 leukemia cancer cells and can effect cell cycle progression at G2/M phase. Its apoptosis activity was due to disruption of mitochondrial functioning. In this study, we have studied a series of compounds consisting of benzyl vanillin and benzimidazole structures. We hypothesize that by fusing these two structures we can produce compounds that have better anticancer activity with improved specificity particularly towards the leukemia cell line. Here we explored the anticancer activity of three compounds namely 2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2MP, N-1-(2-benzyloxy-3-methoxybenzyl)-2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2XP, and (R) and (S)-1-(2-benzyloxy-3-methoxyphenyl)-2, 2, 2-trichloroethyl benzenesulfonate, 3BS and compared their activity to 2-benzyloxy-3-methoxybenzaldehyde, (Bn1), the parent compound. 2XP and 3BS induces cell death of U937 leukemic cell line through DNA fragmentation that lead to the intrinsic caspase 9 activation. DNA binding study primarily by the equilibrium binding titration assay followed by the Viscosity study reveal the DNA binding through groove region with intrinsic binding constant 7.39 µM/bp and 6.86 µM/bp for 3BS and 2XP respectively. 2XP and 3BS showed strong DNA binding activity by the UV titration method with the computational drug modeling showed that both 2XP and 3BS failed to form any electrostatic linkages except via hydrophobic interaction through the minor groove region of the nucleic acid. The benzylvanillin alone (Bn1) has weak anticancer activity even after it was combined with the benzimidazole (2MP), but after addition of another benzylvanillin structure (2XP), stronger activity was observed. Also, the combination of benzylvanillin with benzenesulfonate (3BS) significantly improved the anticancer activity of Bn1. The present study provides a new insight of benzyl vanillin derivatives as potential anti-leukemic agent.</p> </div

The activity on caspase activation enzymes after 2XP, 3BS and betulinic acid exposure.

<p>(i) The fluorescence intensity of caspase enzymes ratio on U937 cells after exposure to 2XP, 3BS and betulinic acid (as a positive control) at different time intervals. caspase 3&7 with significant difference between the treatments and the control group at 5hrs, 7hrs and 12hrs incubation period. p<0.01, (ii) comparison caspase 8 & 9 during the maximum peak period with significant difference between the caspases, p<0.0001 and within the treatment, p=0.047, (iii) caspase 8 with significant difference between the treatments and the control (DMSO) at 3hrs and 5hrs of incubation period. p<0.01, (iv) caspase 9 with significant difference among the treatments at 3hrs, 5hrs, 7hrs and 9hrs of incubation period. p<0.01. Note: The results for 3BS and betulinic acid are not displayed for 7hrs and 9 hrs as their respective caspase 3 activation occurs at 5 hours.</p