Chemical Modification of the Multitarget Neuroprotective Compound Fisetin

Many factors are implicated in age-related central nervous system (CNS) disorders, making it unlikely that modulating only a single factor will provide effective treatment. Perhaps a better approach is to identify small molecules that have multiple biological activities relevant to the maintenance of brain function. Recently, we identified an orally active, neuroprotective, and cognition-enhancing molecule, the flavonoid fisetin, that is effective in several animal models of CNS disorders. Fisetin has direct antioxidant activity and can also increase the intracellular levels of glutathione (GSH), the major endogenous antioxidant. In addition, fisetin has both neurotrophic and anti-inflammatory activity. However, its relatively high EC₅₀ in cell based assays, low lipophilicity, high topological polar surface area (tPSA), and poor bioavailability suggest that there is room for medicinal chemical improvement. Here we describe a multitiered approach to screening that has allowed us to identify fisetin derivatives with significantly enhanced activity in an in vitro neuroprotection model while at the same time maintaining other key activities

Structure activity relationship of a few CNB-001 derivatives.

<p>The structure of CNB-001 is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027865#pone-0027865-g001" target="_blank">Figure 1A</a>. A series of CNB-001 derivatives were synthesized with the goal of removing the hydroxyl group and maintaining and/or improving biological activity in the glutamate based oxytosis assay and extracellular amyloid toxicity. Et (ethyl), Me (Methyl).</p

J147 Increases the Levels of BDNF and BDNF Responsive Protein and Protein Phosphorylation.

<p>(A) BDNF expression in the hippocampus of normal adult rats following two weeks treatment of J147 in food at 200 ppm. J147 treatment increased BDNF levels in the normal rat. Two-tailed t test. *<i>P</i><0.05. (B) BDNF expression in hippocampus of huAPP/PS1 transgenic mice fed J147 for 7 months before sacrifice at 10 months. BDNF levels are decreased in AD transgenic mice and treatment with J147 restores levels to beyond control levels. (C) Homer-1 expression in huAPP/PS1 mice. J147 treatment increases Homer-1 levels. (D) Phosphorylation of PSD95 at Tyr 236, 240. J147 treatment increases phosphorylation of PSD95 in AD transgenic mice. Data are presented as ratio of phospho PSD95 to total PSD95. *<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001. All data shown are means ± SEM. n = 5–6 per group. C = control; C+ = control + J147; A = AD transgenic; A+ = AD transgenic fed J147.</p

Preparation of J147.

<p>J147 was prepared as described in the chemistry section of the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027865#s4" target="_blank">materials and methods</a>. The reagents and conditions for each step are as follows: (a) EtOH, rt, 1 h, 90%; (b) (CF₃CO)₂O, Et₃N, 30 min, 10°C, 77%.</p

J147 Reduces Heat Shock Protein Stress Response and Increases Markers for Synaptic Function.

<p>Cell lysates from hippocampal tissue of line 85 mice fed J147 for 7 months were analyzed by Western blotting. C = control; A = AD transgenic; A+ = AD transgenic fed J147. (A) HSP90 levels are decreased in AD transgenic mice fed J147. (B) HOP levels are decreased in AD transgenic mice fed J147. (C) HSP70 levels are increased in AD transgenic mice and these levels are decreased with treatment of J147. (D) Drebrin levels are decreased in AD transgenic mice and J147 treatment restores levels to above control levels. (E) Synapsin-1 levels are decreased in AD transgenic mice and are restored significantly beyond control by J147. (F) Synaptophysin levels are decreased in AD transgenic mice and levels are restored beyond control levels with J147. *<i>P</i><0.05, **<i>P</i><0.01), and ***<i>P</i><0.001. All data shown are means ± SEM, n = 5 per group.</p

J147 Facilitates the Induction of LTP in Schaffer Collateral CA1 Pyramidal Cell Synapses in Rat Hippocampal Slices.

<p>(A) Effect of J147 (1 µM) on basal synaptic transmission. Hippocampal slices were exposed to J147 during the time indicated by the black bar. The fEPSP slope is expressed as the percentage of the value immediately before the addition of J147. J147 does not affect basal synaptic transmission. (B) J147 facilitates the induction of LTP after a weak tetanic stimulation (15 pulses at 100 Hz) which alone does not induce LTP in control slices. The effect of J147 is concentration-dependent. Time course of changes in the fEPSP slope. The hippocampal slices were untreated (o, n = 9) or exposed to J147 (▾, 0.01 µM, n = 6; ▴, 0.1 µM, n = 5; •, 1 µM, n = 7) for the time indicated by the black bar and weak tetanic stimulation was applied at time 0. The fEPSP slope is expressed as the percentage of the value immediately before the application of weak tetanic stimulation. (C) Concentration-dependency. To compare the data among the groups, the averages of the fEPSP slopes 30–60 min after tetanic stimulation were calculated as an index of LTP magnitude. J147 demonstrated a concentration dependent effect with 1000 nM having the greatest effect on the fEPSP slope (n = 7 slices per rat: one-way ANOVA F(3,23) = 4.4, **<i>P</i> = 0.01). (D) Negative Control. −187–88, the alkene form of J147 in which nitrogens are replaced by carbons (o, n = 9; ▴, 0.1 µM, n = 5; •, 1 µM, n = 5) showed no effect. All data shown are means ± SEM.</p

Selection of J147.

<p>CNB-001 (A) is a broadly neuroprotective and neurotrophic derivative of curcumin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027865#pone.0027865-Liu1" target="_blank">[8]</a>. CNB-023 (B) is a derivative of CNB-001 lacking hydroxyl groups but with similar activity that was identified by SAR analysis of CNB-001. The boxed area shows the hypothesized biologically active fragment of CNB-023. A large collection of molecules around this chemical space was generated by the reaction of m-anisaldehyde with 2.4 dimethylphenyl hydrazine (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027865#s4" target="_blank">Materials and Methods</a>) and J147 (C) was selected from the reaction products on the basis of its activity in trophic factor withdrawal (TFW), oxidative stress (oxytosis) and Aβ toxicity assays (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027865#pone-0027865-g002" target="_blank">Figure 2</a>). (D) Final HPLC run of reaction mixture that generated J147. The structure of the most active product (fraction 10) was determined by NMR. J147 was then synthesized, and its crystal structure (insert) determined, confirming its biological activity and structural identity. The following are de-risking and target identification screens, with no significant reproducible hits. J147 was used at 10 micromolar unless indicated. (1) LeadProfiling+P450 screen. Over 60 CNS receptors and transporters (work done by MDS Pharma). (2) hERG (work done by MDS and Absorption Systems). (3) Acute toxicity in rats. Negative at 2 grams/kilogram (work done by Absorption systems). (4) CeeTox “Safe” up to 90 micromolar plasma concentration (work done by CEETOX, INC). (5) 352 protein kinases (done by Ambit). (6) MDRI-MDCK brain penetration classification “High” (work done by Absorption Systems). (7) Enzyme Assays. Cox 1; Cox 2; Lox 5,12,15; Sirt 1,2,3; LT4 hydrolase; LTC4 synthase; cathepsin B; matrix metalloprotease 1; phosphodiesterases 10A1, 11A1, 1A, 2A, 3A, 4A1A, 4B1, 5A, 6, 7A, 7B, 8A1, 9A2; proteasome (done by MDS). (8) Enzyme assays. PPARα, γ; deubiquitinases, BAP1, UCH-L1, L3, USP5; acetylcholine esterase; MAOA; MAOB; phosphatases (19 of them), proteases and caspases (8) (done by Caliper).</p

Biological Activities of J147.

<p>J147 is active with EC_{50 s} between 10 and 200 nM in six different assays for neurotrophic activity and neurotoxicity. o-o, J147; x-x, CNB-001; Δ-Δ, curcumin. (A) <u>Trophic Factor Withdrawal</u>. Primary cortical neurons were prepared from 18-day-old rat embryos and cultured at low cell density with or without the three compounds. Cell viability was assayed 2 days later. (B) <u>BDNF-like Activity</u>. HT22 cells expressing the TrkB (BDNF) (open circles, J147) receptor or no TrkB (black circles, J147) were placed in serum-free medium in the presence of 50 ng/ml BDNF or the indicated amounts of compounds. Cell viability was determined 2 days later. Curcumin had no activity in this assay up to one micromolar. BDNF was used at 50 ng/ml and active only in cells expressing TrkB (open bar), not in its absence (black bar). (C) <u>Oxidative Stress</u>. E18 rat cortical neurons were treated with 5 mM glutamate and different concentrations of compounds one day after plating when no ionotropic glutamate receptors are expressed. Cell viability was measured 24 hr later. (D) <u>Glucose Starvation</u>. PC12 cells were starved for glucose plus or minus 20 nM J147, 0.2 µM CNB-001 or 10 µM curcumin and cell viability determined 48 hr later. J147 and NGF increase cell viability in the absence of glucose, *<i>P</i><0.001 vs. control. CNB-001 and curcumin are inactive at 0.2 and 10 µM respectively (curcumin not shown). (E) <u>Chemical ischemia</u>. HT22 cells were treated with 20 µM iodoacetic acid for 2 hr alone or in the presence of varying concentrations of J147, CNB-001 or curcumin. Percent survival was measured after 24 hr. (F) <u>Amyloid toxicity</u>. Primary hippocampal cells were exposed to 5 µM Aβ_1–42 in the presence of increasing amounts of compounds and cell viability determined 48 hr later. All data shown are mean ± SEM, n = 3 or 4. The curcumin and CNB-001 data which were included for comparison with J147 have been presented, in part, previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027865#pone.0027865-Liu1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027865#pone.0027865-Lapchak1" target="_blank">[84]</a>.</p

Immunohistochemical analysis of condensed Aβ deposits.

<p>Immunohistochemical analysis was done using brain coronal sections of 13-month old AD mice on control diet (A) or J147 diet (B) using antibody 6E10. In comparison to AD control mice (A), condensed Aβ deposits were significantly reduced in the hippocampus region of AD mice on J147 diet (B). 30 micron thick sections of similar regions from each mouse, (n = 6) were examined and plaque counts in the hippocampus were quantified. All images were quantified using Image J Software. (C) The average of plaque counts for each mouse group is expressed as a number of plaques ± the SD (n = 6 per group: unpaired t-test, *P<0.05). (D) Reduced Aβ plaque load in AD mice on J147 diet. The Aβ plaque load was determined by measuring the area occupied by Aβ-immunoreactive condensed deposits, (n = 6 per group: unpaired t-test, *P<0.05). (E) The plaque size did not change between AD mice on control and J147 diets (n = 6 per group: unpaired t-test, *P>0.05).</p