Back to the future with phenotypic screening

Abstract

There are no disease-modifying drugs for any old age associated neurodegenerative disease or stroke. This is at least in part due to the failure of drug developers to recognize that the vast majority of neurodegenerative diseases arise from a confluence of multiple toxic insults that accumulate during normal aging and interact with genetic and environmental risk factors. Thus, it is unlikely that the current single target approach based upon rare dominant mutations or even a few preselected targets is going to yield useful drugs for these conditions. Therefore, the identification of drug candidates for neurodegeneration should be based upon their efficacy in phenotypic screening assays that reflect the biology of the aging brain, not a single, preselected target. It is argued here that this approach to drug discovery is the most likely to produce safe and effective drugs for neurodegenerative diseases.

Figure 1

Time course of major metabolic events associated with oxytosis, a form of programmed cell death initiated by glutathione (GSH) depletion that occurs in conditions of oxidative stress and even normal aging.

Figure 2

Structure activity relationship analysis results in the identification of potent small molecule J147.

Figure 3

Biological activities of J147. J147 is active with EC₅₀’s between 10 and 200 nM in six different assays for neurotrophic activity and neurotoxicity. Blue circles, J147; green cubes, CNB-001; red triangles, curcumin. (A) Trophic factor withdrawal: 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) BDNF-like activity: HT22 cells expressing the TrkB (BDNF) (yellow circles, J147) receptor or no TrkB (blue 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 1 μM. BDNF was used at 50 ng/mL and active only in cells expressing TrkB (open bar), not in its absence (black bar). (C) Oxytosis: E18 rat cortical neurons were treated with 5 mM glutamate and different concentrations of compounds 1 day after plating when no ionotropic glutamate receptors are expressed. Cell viability was measured 24 h later. (D) Glucose starvation: PC12 cells were starved for glucose plus or minus 20 nM J147, 0.2 mM CNB-001, or 10 μM curcumin, and cell viability determined 48 h later. J147 and NGF increase cell viability in the absence of glucose, *P, 0.001 vs control. CNB-001 and curcumin are inactive at 0.2 and 10 μM, respectively (curcumin not shown). (E) Chemical ischemia: HT22 cells were treated with 20 μM iodoacetic acid for 2 h alone or in the presence of varying concentrations of J147, CNB-001, or curcumin. Percent survival was measured after 24 h. (F) Amyloid toxicity: Primary hippocampal cells were exposed to 5 μM Aβ_1–42 in the presence of increasing amounts of compounds, and cell viability determined 48 h later. All data shown are mean ± SEM, n = 3 or 4. The curcumin and CNB-001 data were included for comparison with J147.

Figure 4

Improvement of fisetin in terms of potency (in vitro ischemia and oxytosis), medicinal chemistry properties (cLogP, MW, tPSA), and transport through CaCO-2 gut-epithelial cells.