Medical Big Data and Artificial Intelligence for Healthcare

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Shape-Dependent Biomimetic Inhibition of Enzyme by Nanoparticles and Their Antibacterial Activity

Enzyme inhibitors are ubiquitous in all living systems, and their biological inhibitory activity is strongly dependent on their molecular shape. Here, we show that small zinc oxide nanoparticles (ZnO NPs)pyramids, plates, and spherespossess the ability to inhibit activity of a typical enzyme β-galactosidase (GAL) in a biomimetic fashion. Enzyme inhibition by ZnO NPs is reversible and follows classical Michaelis–Menten kinetics with parameters strongly dependent on their geometry. Diverse spectroscopic, biochemical, and computational experimental data indicate that association of GAL with specific ZnO NP geometries interferes with conformational reorganization of the enzyme necessary for its catalytic activity. The strongest inhibition was observed for ZnO nanopyramids and compares favorably to that of the best natural GAL inhibitors while being resistant to proteases. Besides the fundamental significance of this biomimetic function of anisotropic NPs, their capacity to serve as degradation-resistant enzyme inhibitors is technologically attractive and is substantiated by strong shape-specific antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), endemic for most hospitals in the world

Biochemical Effect of Resistance Mutations against Synergistic Inhibitors of RSV RNA Polymerase

<div><p>ALS-8112 is the parent molecule of ALS-8176, a first-in-class nucleoside analog prodrug effective in the clinic against respiratory syncytial virus (RSV) infection. The antiviral activity of ALS-8112 is mediated by its 5'-triphosphate metabolite (ALS-8112-TP, or 2'F-4'ClCH₂-cytidine triphosphate) inhibiting the RNA polymerase activity of the RSV L-P protein complex through RNA chain termination. Four amino acid mutations in the RNA-dependent RNA polymerase (RdRp) domain of L (QUAD: M628L, A789V, L795I, and I796V) confer in vitro resistance to ALS-8112-TP by increasing its discrimination relative to natural CTP. In this study, we show that the QUAD mutations specifically recognize the ClCH₂ group of ALS-8112-TP. Among the four mutations, A789V conferred the greatest resistance phenotype, which was consistent with its putative position in the active site of the RdRp domain. AZ-27, a non-nucleoside inhibitor of RSV, also inhibited the RdRp activity, with decreased inhibition potency in the presence of the Y1631H mutation. The QUAD mutations had no effect on the antiviral activity of AZ-27, and the Y1631H mutation did not significantly increase the discrimination of ALS-8112-TP. Combining ALS-8112 with AZ-27 in vitro resulted in significant synergistic inhibition of RSV replication. Overall, this is the first mechanistic study showing a lack of cross-resistance between mutations selected by different classes of RSV polymerase inhibitors acting in synergy, opening the door to future potential combination therapies targeting different regions of the L protein.</p></div

Loss of inhibition of AZ-27 with RSV polymerase Y1631H mutant.

<p><b>(A)</b> RSV L-P pull-down assay. The enzyme-inhibitor complex (DMSO in Lane 1, and AZ-27 Lane 2) was pre-incubated for 30 minutes, after which nickel beads were used to pull down the histidine-tagged P subunit. The bound protein was washed on the beads and visualized by SDS PAGE. (<b>B</b>) Dose-response inhibition of RSV L-P RdRp in the biotin-primer extension assay. Reduction in the enzymatic activity of WT, QUAD, and Y1631H mutant was measured in the presence of increasing concentrations of AZ-27. (<b>C</b>) K_m determination for CTP and ALS-8112-TP by RSV polymerase containing the Y1631H mutation. K_{m CTP} = 0.042±0.009 μM (n = 4), and K_{m ALS-8112-TP} = 0.75±0.46 μM (n = 2).</p

Inhibition of primer extension by AZ-27.

<p><b>(A)</b> Standard WT RSV L-P primer extension assay with in Lane 1, *GTP; in Lane 2, *GTP+ATP; in Lane 3, *GTP+ATP+CTP; in Lane 4, *GTP+ATP+CTP+AZ-27. (<b>B</b>) Each individual primer extension RNA product (from +1 to +7) was quantified for Lane 4 (AZ-27), and compared with the corresponding amount of RNA in Lane 3 (no inhibitor). (<b>C</b>) The same experiment was repeated, using increasing concentrations of ATP+CTP from 1 to 100 μM (n = 2). The amount of +1 and +4–7 RNA products were measured in presence of AZ-27 at 10 μM, and compared with the corresponding amount of RNA without inhibitor (n = 2).</p

Effect of the QUAD mutations on the discrimination of 2'F-4'N₃-CTP.

<p>(<b>A</b>) Principle of the standard primer extension reaction for single CTP incorporation. The L subunit of RSV polymerase binds to the primer/template duplex (green) and carries the RdRp activity. The radiolabeled α³³P-GTP (*G or *GTP) is incorporated first at position +1, after which ATP and CTP are required to fully extend the primer to the +7 position. (<b>B</b>) The RSV L-P proteins (WT and QUAD) were incubated in the presence of *GTP + ATP and increasing concentrations of CTP. Lane 1, no CTP; lane 2, 0.0021 μM; lane 3, 0.0062 μM; lane 4, 0.019 μM; lane 5, 0.056 μM; lane 6, 0.17 μM; and lane 7, 0.5 μM CTP. Product formation was quantified and expressed as percentage primer extension from the +3 position. WT K_{m CTP} = 0.056±0.010 μM (n = 4), and QUAD K_{m CTP} = 0.028±0.005 μM (n = 2) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154097#pone.0154097.ref008" target="_blank">8</a>]. (<b>C</b>) The same experiment was repeated for each enzyme with 2'F-4'N₃-CTP, resulting in a WT K_{m 2'F-4'N3-CTP} = 0.62±0.042 μM (n = 2), and QUAD K_{m 2'F-4'N3 CTP} = 0.28±0.07 μM (n = 2). (<b>D</b>) For each enzyme, the level of discrimination was calculated as K_{m CTP analog} / K_{m CTP}, and resistance as QUAD_{discrimination} / WT_{discrimination}.</p

Individual contribution of each of the QUAD mutations.

<p><b>(A)</b> SDS PAGE analysis of RSV L-P proteins containing one or four (QUAD) amino acid changes in the L subunit. (<b>B</b>) Relative enzyme activity of each protein variant compared to WT RSV L-P, as measured by the total formation of RNA products in the standard primer extension assay. (C) Chemical structure of 4'ClCH₂-CTP. (<b>D)</b> Efficiency of incorporation of 4'ClCH₂-CTP by WT and mutated RSV L-P. K_m values were determined by incubating each enzyme with increasing concentrations of 4'ClCH₂-CTP, in the presence of *GTP + ATP as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154097#pone.0154097.g001" target="_blank">Fig 1</a>. For 4'ClCH₂-CTP, WT K_m = 19.7±6.7 μM (n = 2), M628L WT K_m = 23.1±0.07 μM (n = 2), QUAD K_m = 235±77 μM (n = 4), A789 K_m = 61.0±26.9 μM (n = 2), L795I K_m = 16.5±6.4 μM (n = 2), and I796V K_m = 22.0±9.9 μM (n = 2). (E) A homology-based three dimensional structure of the RSV polymerase L protein was generated using RaptorX [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154097#pone.0154097.ref010" target="_blank">10</a>], using the VSV L protein (cyan, PDB = 5a22) as reference. The molecular graphics of the resultant structure (blue) were generated using the UCSF Chimera package [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154097#pone.0154097.ref035" target="_blank">35</a>]. The proposed positioning of residues 628, 789, 795, and 796 from the RSV polymerase model is shown in red, as indicated by arrows. The asterisk (orange) shows the position of the GDNQ catalytic motif.</p

Synergistic antiviral effect of ALS-8112 and AZ-27 on RSV replication.

<p>(<b>A</b>) Isobologram analysis of ALS-8112 and AZ-27 interaction in HEp-2 cells against RSV replication. AZ-27 and ALS-8112 concentrations were on the x- and y- axis respectively. The three lines intersecting both axes represent additivity for EC₅₀ (red), EC₇₅ (green) and EC₉₀ (blue). The calculated CI for EC₅₀, EC₇₅, and EC₉₀ all fall below their respective additivity lines, indicating a synergy (0.3–0.7). (<b>B</b>) Prichard’s model for 3-D drug interaction dose-responses. The synergy volume was above the 0% plane and falls within 50–100 range, indicating a significant synergy (n = 5).</p

Discrimination of purine analogs by the QUAD mutant.

<p>(<b>A</b>) Incorporation of 2'F-4'ClCH_2-ATP by WT L-P and chain termination effect. Lane 1: *GTP only (+1 product), Lane 2: *GTP+ATP (+3 product), Lane 3: *GTP+2'F-4'ClCH₂-ATP (+2 product). (<b>B</b>) The RSV L-P proteins (WT and QUAD) were incubated in the presence of *GTP and increasing concentrations of either ATP or 2'F-4'ClCH₂-ATP. (<b>C</b>) Incorporation of 2'F-4'ClCH₂-GTP by WT L-P and chain termination effect. The sequence of the standard template was modified to enable single G incorporation at position +4. Lane 1: *CTP+ATP, Lane 2: *CTP+ATP+GTP, Lane 3: *CTP+ATP+2'F-4'ClCH₂-GTP. (<b>D</b>) The RSV L-P proteins (WT and QUAD) were incubated in the presence of *CTP+ATP and increasing concentrations of either GTP or 2'F-4'ClCH₂-GTP.</p

Effect of the QUAD mutations on the discrimination of 2'F-CTP analogs.

<p>(<b>A</b>) Measurement of resistance level of QUAD RSV L-P against a series of 2'F-CTP analogs with the following substitution at the 4'position: chloromethyl (ClCH₂), fluoromethyl (FCH₂), bromomethyl (BrCH₂), 1-fluoroethyl (FCH₃CH₂), azido (N₃-), cyclopropyl (CH₂CH₂CH₂), and methyl-thio-methyl (CH₃SCH₂). For each CTP analog, the level of resistance was calculated as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154097#pone.0154097.g001" target="_blank">Fig 1</a> (n = 2). (<b>B</b>) In vitro inhibition potency of 2'F-4'N₃-cytidine against the RSV minigenome luciferase-based reporter assay. HEp-2 cells were co-transfected to transiently express the RSV N, P, M2-1 and L proteins containing either the WT or the QUAD mutated L sequence (n = 4). (<b>C</b>) Inhibition of luciferase-based WT and QUAD RSV minigenome activity by 2'F-4'BrCH₂-cytidine (n = 4).</p