P7C3 neuroprotective chemicals function by activating the rate-limiting enzyme in NAD salvage

Abstract

The P7C3 class of aminopropyl carbazole chemicals fosters the survival of neurons in a variety of rodent models of neurodegeneration or nerve cell injury. To uncover its mechanism of action, an active derivative of P7C3 was modified to contain both a benzophenone for photocrosslinking and an alkyne for CLICK chemistry. This derivative was found to bind nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme involved in the conversion of nicotinamide into nicotinamide adenine dinucleotide (NAD). Administration of active P7C3 chemicals to cells treated with doxorubicin, which induces NAD depletion, led to a rebound in intracellular levels of NAD and concomitant protection from doxorubicin-mediated toxicity. Active P7C3 variants likewise enhanced the activity of the purified NAMPT enzyme, providing further evidence that they act by increasing NAD levels through its NAMPT-mediated salvage.

Copyright © 2014 Elsevier Inc. All rights reserved.

Figures

Figure 1. Protection of cultured U2OS cells from doxorubicin-mediated toxicity by active derivatives of P7C3

(A) U2OS cells were treated with 5μM P7C3-A20 for 2 h prior to incubation with the indicated concentrations of doxorubicin for 72h. (B) Comparison of the P7C3-S243 enantiomers in protection of cells from doxorubicin revealed that (−)-P7C3-S243 was more active than (+)-P7C3-S243, consistent with their respective neuroprotective activities in vivo (see text). In all cell survival graphs, data are expressed as mean ± standard deviation (SD) of experimental duplicates. See also Figure S1, S2 and S3, and Table S1.

Figure 2. Identification of the P7C3 binding protein p70 using the P7C3-S326 photo-crosslinking probe

(A) Chemical structure of P7C3-S326. (B) Full gel image of photo-crosslinking in H2122 cells with P7C3-S326 followed by click chemistry with Alexa 532 dye. The active analog P7C3-A20 competed away the UV-dependent binding of p70 to P7C3-S326. See also Figure S4. (C) Formulas used for calculating doxorubicin toxicity (dox:tox) protection activity. Protection activity is expressed by (Sinf-S0)/AC50 in the twelve-point dose response curve (DRC) corresponding to each compound, wherein both efficacy and potency of compounds were considered. Sinf represents the expected maximal protection. S0 is the baseline. AC50 is the concentration of a compound where 50% of its maximal protection effect was observed. See also Figure S3 and Table S2. (D) Scatter plot of 168 derivatives of P7C3 revealed a significant correlation between protective activity of P7C3 analogs from doxorubicin-mediated toxicity and their ability to compete for photo-crosslinking of P7C3-S326 to p70. In the photo-crosslinking assays, the p70 band intensity was quantified by Image J, and compared the sample of P7C3-S326 alone with that of P7C3-S326 plus competitor. Pearson correlation coefficient (r) and two tailed P value were determined by GraphPad Prism 6 and Spearman Rank Correlation (v1.0.1) software. See also Table S1.

Figure 3. Identification of p70 and p55 targets of P7C3 by two-dimensional gel electrophoresis and mass spectrometry

Lysates from cells exposed to 0.3 μM P7C3-S326 were CLICKed with a green dye, Alexa 532, and those from cells co-exposed to 0.3 μM P7C3-S326 and 5 μM of the active competitor P7C3-A20 were CLICKed with a red dye Cy5. CLICK reacted lysates were combined, subjected to two-dimensional gel electrophoresis, and scanned in the green channel (A) and the red channel (B). Images from both channels were merged to reveal green-only spots (C). After spot picking, total proteins were visualized by Sypro Ruby staining (D). White circles in (D) indicate spots excised for shotgun mass spectrometry analysis. See also Table S3.

Figure 4. Conversion of p70 into p55 in the presence of urea

(A) CLICK reacted lysates from H2122 cells treated with P7C3-S326 were separated on a standard 8% SDS-PAGE gel (left panel) or one supplemented with 7M urea (right panel), and visualized on a Typhoon scanner. Asterisk indicates the crosslinked protein that migrates as 70KDa in a standard SDS-PAGE gel and 55KDa in the SDS-PAGE gel supplemented with 7M urea. (B) The same lysates were resolved on a horizontal urea gradient gel co-loaded with crosslinked p70 and the same 75kD and 50kD size standard proteins displayed in panel A. The horizontal urea gradient proceeds from zero denaturant on the left to 7M denaturant on the right.

Figure 5. Active variants of P7C3 compensate NAD exhaustion induced by doxorubicin

(A) U2OS cells were treated with the indicated concentration of P7C3-A20. Cells were harvested and NAD metabolites were measured by LC-MS/MS. Abundance of NAD was normalized relative to total metabolites (Experimental Procedures). The data are represented as the mean ± SD of experimental duplicates. (B) Active derivatives P7C3-A20 and P7C3-S243, but not inactive derivatives P7C3-S6, or P7C3-S117, facilitate replenishment of NAD levels in doxorubicin-treated cells. Cells were grown in 96-well plates and treated with the indicated concentrations of P7C3-A20, P7C3-S243, P7C3-S6 or P7C3-S117 together with 0.5μM doxorubicin for 45h. Cellular NAD abundance was determined by NAD/NADH Glo assay kit. See also Figure S5 and Table S2. (C) Scatter plots revealed a strong correlation between dox:tox protective activities of 159 compounds and their relative abilities to replenish NAD levels. The activities in both assays are represented by (Sinf-S0)/AC50 in dose response curves of test compounds. See also Table S1.

Figure 6. P7C3-A20 enhances the flux of nicotinamide through the salvage pathway

(A) Cells were pre-treated with 0.5μM doxorubicin for 48 hours, followed by 6 hours treatment with 14C-nicotinamide in the presence of the indicated amount of P7C3-A20. Metabolites were extracted and analyzed by thin layer chromatography (a representative result from three experiments is shown here). Free 14C-nicotinamide was loaded (the first lane) as a standard. (B) Quantification of the relative intensities of NAD and NMN from the thin layer chromatogram.

Figure 7. Active variants of P7C3 enhance the activity of purified NAMPT enzyme

(A) P7C3-A20 was incubated at indicated concentrations in a reaction coupled with three enzymes; NAMPT, NMNAT, and ADH (Experimental Procedures). NAMPT activity was recorded for the indicated period of time at OD340nm by measuring NADH appearance, leading to the indicated concentration-time plot. Each assay was repeated three independent times with similar results. See also Figure S6. (B) Analysis of the activities of thirty P7C3 analogs was performed to assess their effects on NAMPT enzymatic activity. The reaction rate was calculated as the slope of the concentration-time curve. Relative reaction rate was normalized by the control reaction run prior to compound addition. Data represents the mean of experimental duplicates. Scatter graphs were plotted to compare the ability of compounds to activate NAMPT relative to their ability to compete away P7C3-S326 crosslinking (top scatter plot), ability to protect cells from doxorubicin-mediated toxicity (middle scatter plot), or ability to facilitate NAD restoration in doxorubicin-treated cells (bottom scatter plot). Significant correlations were observed from all three sets of data. See also Table S1. (C) Comparison of P7C3-S243 enantiomers showed that (−)-P7C3-S243 was superior to (+)-P7C3-S243 in activating the purified NAMPT enzyme. Data are expressed as mean ± SD of duplicate independent assays. See also Figure S7.