AChE Enzyme Reactivation Assays

Assays are used to test how effectively the oximes reactivate the enzyme acetylcholinesterase after it has been deactivated by paraoxon. The frozen enzyme is first thawed. The AChE is then vortexed with one-part enzyme, 499 parts pH 8 PBS. The amount of enzyme needed varies depending on how many oximes will be tested. We diluted POX with a serial dilution to test it at different concentrations. A serial dilution like this can be used to create a calibration curve of POX concentration vs enzymatic reactivity. In pH 8 PBS, we dilute the organophosphate anywhere from 20 µM to 0.156 µM. In the following order, solutions are added to the 96 well plates: pH 8 PBS (50, 70 or 80 µL), AChE (100 µL), and POX (10 µL). The plates are then incubated for 30 minutes to let the POX deactivate the enzyme AChE. Afterwards, 20 µL of the different oxime conjugates are added to each row (except for the control row), ranging anywhere from 0.01mM to 10mM. Our oxime molecules were synthesized in the lab apart from 2-PAM and Obidoxime chloride, which were purchased through Fisher Scientific. Although the synthesis of attaching the oxime molecules to the PAMAM dendrimers is not discussed here, Bharathi’s (2014) research and design on oxime-conjugated PAMAM dendrimers was modeled. (Bharathi, p. 1068–1078). We used Obidoxime and 2-PAM as controls, which are already marketed oxime products. A fresh 1:1 mixture of acetylthiocholine chloride, ATCh, and 5,5’-dithio-bis (2-nitrobenzoic acid), DTB, are weighed out and dissolved into DMSO and PBS 8.0. This 1:1 mixture of ATCh (5.0 mM, 3.0 mL) and DTNB (5 mM, 3.0 mL) is labeled as “AT/DT,” and it shall be used as a substrate to measure the enzymatic reactivity of acetylcholinesterase. 20 µL of the substrate is pipetted swiftly into each well with a multi-channel pipette. Prior to adding the substrate, the plate reader is set to an absorbance of 412nm and to read enzymatic activity every 2 minutes for 20 minutes. Enzymatic activity is measured by detecting absorbance via color change from clear to yellow upon addition of the AT/DT mixture. The higher the reactivity of the enzyme acetylcholinesterase, the darker the yellow would appear in the wells after the reaction has reached completion. Figure 2 shows an example of a 96 well plate setup that was tested:

Figure 2:

Figure 2: A typical well plate set up to screen for oxime conjugates. Some of the oxime small molecules that we narrowed our focus on were 693–3a, 693–3b, 693–8a, 693–8b, 693–19a, 693–19b, 693–70a, 693–70b, 693–29a, 693–29b, 693–29c, 693–29d, 693–29d, 693–77a, 693–77b, 693–77c, 693–77d, 693–77e, and 693–77f.

Plate 706–41C series (each well 200 μL)

• Buffer, PBS pH 8.0: variable volume (80, 70 or 50 μL)

• AChE: 100 μL each fixed

• POX = 10 μL each fixed volume

  • Row A: variable concentration

  • Row B to H: 10 µM

• Test oxime solution: 20 μL each, variable concentration (0.01–10 mM) as noted in the upper row

• AT/DT: 20 μL each fixed

POX decontamination in porcine skin tissue

We test how long it takes for the POX to be removed from the skin using Franz cells, which clamp the skin tissue in place. Pig skin is used for this experiment because it is most similar to human skin. In this experiment, we recreated a makeshift human-synapsis with a pH buffer of 7.4 absorbing the internal side of our pig skin, since the human body has a natural pH of around 7.4. We add a buffer of pH 10.5 to the donor side, the exterior side of the skin, allowing it to equilibrate. We then remove the pH 10.5 buffer and add the paraoxon (a type of organophosphate) to the outer surface of the skin. We incubate the OP on the skin for 2 hours to allow for full deactivation of AChE. After the incubation, we add 300 microliters of the dendrimers to the top of the Franz cell. Once the dendrimers are added, we remove 50 microliter samples at indicated time points and add them to formic acid to quench the reaction. The usual time points used are at t=0 minutes, 6 minutes, 15, 30, 60, and 120 minutes. Ultra-Pressure Liquid Chromatography (UPLC) is used to separate small compounds into complex mixtures. Using UPLC analysis, we want to see the levels of activated POX decreasing over time. We expect almost 100% of POX at 0 minutes and deactivation of nearly all POX at 120 minutes. One way we measure this is by doing AUC, or area under the curve. The smaller the peak’s area that shows up on the UPLC, the less POX remains. We also took samples of the pH 7.4 buffer on the receptor side of the skin to see how much of the POX went through the skin. This is also measured by UPLC. Next, a skin extraction is performed: we soak the decontaminated skin in ethanol overnight. The following day, a sample of the ethanol was removed and tested by UPLC to see how much POX and dendrimer remained in the epidermal and basal layers of the skin.

Enzyme reactivation

We found that the higher the enzyme percent reactivity, the more effective the oxime is at cleaving the organophosphate from the active site. We expect to see higher reactivity at lower concentrations for the use of potential small molecule drug products.

In the graphs, we strive to see high enzyme reactivity, meaning that the dendrimer compound would effectively and efficiently deactivate the POX and reactive the enzyme. We see high enzyme reactivation in compounds 693–29f, 693–3b, 693–8b, 693–77c, and 693–77b (Figure 3). Not only were these molecules most effective but they showed the most consistency when the data was repeated. After addition of substrate, our pre-set Bioassay Plate Reader takes 25 absorbance readings in a span of 20 minutes. The kinetic rate of the enzymatic reaction is interpreted on a logarithmic scale. In Microsoft Excel, the initial slope is calculated for each oxime compound at various concentrations. Three data points are taken at each indicated oxime concentration; the average and standard deviation of the three absorbance values are calculated. Each 96-well setup contains a control row where no oxime is added (Figure 2). The first three columns of the control row contain no POX (wells A1, A2 and A3), and the last three columns contain the highest concentration of POX (wells A10, A11, and A12). Two averages of two slopes are found using these absorbance values: one is labeled “active” and the other “inactive.” Setting k=slope, percent enzyme (re)activity is calculated using the equation: Enzyme % Activity=100*[k-k(inactive)]/[k(active)-k(inactive)]. The averages and variance values are then calculated for each small oxime molecule at indicated concentrations. These averages are then plotted with concentration of the oxime conjugate on the x-axis and enzyme percent activity on the y-axis (Figure 3).

Figure 3: Results of 21 different oxime conjugates, two of which (Obidoxime and 2-PAM) are marketed products of OP poisoning. The data in these graphs come from plate assays using 96 well plates. On the x-axis, we have the concentration [M] of the oxime being added to the wells. On the y-axis, we have the percent reactivation of the enzyme calculated by the equation 100*[k-k(inactive)]/[k(active)-k(inactive)]. ‘k’ is the mean reactivity of the enzyme or each conjugate, ‘inactive’ is a constant for when the enzyme is least reactive, and ‘active’ is a constant for when the enzyme is most active. Enzyme % activity is relative to the control.

POX decontamination in porcine skin

The graphs shown below are results of UPLC (ultra-performance liquid chromatography) analysis for POX decontamination in the donor compartment of the Franz cell. In the skin decontamination experiment, our goal is to find a conjugate that will allow the least POX penetration through human skin.

Below is data collected from a UPLC of how long it took for POX to be fully inactivated on the surface of the porcine skin (Figure 4). The faster the dendrimer latches onto and deactivates the POX, the less likely the POX will penetrate through the skin to get into the bloodstream, meaning a lower toxicity level. The absorption unit (proportional to the POX remaining) was measured at 215nm and 275nm. The dendrimers eluted from the UPLC at a retention time of about 10.25 minutes, represented by the x-axis in Figure 4. An internal standard was used to calibrate the absorption unit of each dendrimer at the various time points (Absorption Unit is represented by the y-axis). The Area Under the Curve (AUC) was measured for each sample of each dendrimer pipetted from the Franz cells at various time points ranging from 0 to 120 minutes. Data taken by the UPLC are then transferred to an Excel spreadsheet where the results can be better displayed. As seen in Figure 4, the readings taken at 275 nanometers appear to show more sensitive results than those taken at 215 nanometers. The peaks on the graphs represent the remaining volume of POX on the apical side of the Franz cell. By adding the dendrimers, it is expected that the active form of the organophosphate will dissipate within 2 hours. The more ideal results would show a faster dissipation of the active form of POX. One example of an ideal candidate can be seen in the results 693–40A because there is no remaining POX after 1 hour of incubation with the dendrimer, whereas 706–8A is an example of a bad candidate because there continues to be traces of POX after 2 hours of incubation (Figure 4).

Figure 4:

Figure 4: Ultra-Pressure Liquid Chromatography data taken at 215nm and 275nm for five different dendrimers. Time (t) is measured in minutes. Wavelengths 215 and 275 nanometers were used for the dendrimers because these wavelengths showed the best sensitivity of our analytes. 275 nanometers tended to show clearer “peaks” for most of our dendrimers compared to at 215 nanometers.