P7C3 and an unbiased approach to drug discovery for neurodegenerative diseases

Abstract

A novel neuroprotective small molecule was discovered using a target-agnostic in vivo screen in living mice. This aminopropyl carbazole, named P7C3, is orally bioavailable, crosses the blood-brain barrier, and is non-toxic at doses several fold higher than the efficacious dose. The potency and drug-like properties of P7C3 were optimized through a medicinal chemistry campaign, providing analogues for detailed examination. Improved versions, such as (-)-P7C3-S243 and P7C3-A20, displayed neuroprotective properties in rodent models of Parkinson's disease, amyotrophic lateral sclerosis, traumatic brain injury and age-related cognitive decline. Derivatives appended with immobilizing moieties may reveal the protein targets of the P7C3 class of neuroprotective compounds. Our results indicate that unbiased, in vivo screens might provide starting points for the development of treatments for neurodegenerative diseases as well as tools to study the biology underlying these disorders.

Fig. 1

Unbiased screen for neuroprotective small molecules. (A) Schematic of hippocampal neurogenesis illustrating the incorporation of BrdU into new born cells, the month-long period of maturation and roles of pro-proliferative and neuroprotective agents. Modified from ref. J. M. Encinas, A. Vaahtokari, G. Enikolopov, Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 8233. Copyright (2006) National Academy of Sciences, U. S. A. (B) Outline of the in vivo screen. Drugs were infused directly into the brain of live mice over 7 days. Subsequent sectioning and immunohistochemical staining revealed newly formed neurons (black cells). (C) Chemical structure of active component of pool 7. (D) Increase in number of BrdU + cells following oral dosing with P7C3.

Fig. 2

Chemical optimization of P7C3. Derivatives of P7C3 can be accessed rapidly using epoxides as lynchpins. Fluorination of the secondary alcohol provides additional analogues. Compounds are grouped as those that were less active than P7C3, equivalent to P7C3 or more active than P7C3 in the in vivo hippocampal neurogenesis assay via ICV administration. Changes from P7C3 are highlighted in red.

Fig. 3

Efficacy of P7C3 derivatives in an in vivo mouse model of neuro- genesis. Compounds were administered as indicated for 7 days along with daily injection of BrdU. Data are expressed as mean ± SEM.

Fig. 4

Neuroprotection in the MPTP model of Parkinson’s disease. (A) Mice were administered MPTP (30 mg k⁻¹ d⁻¹) for 5 days. On the 6th day, treatment with drug was initiated at the indicated dose (IP, BID, 21 d). Bars indicate number of tyrosine hydroxylase positive cells detected by immunohisotochemical staining of the substantial nigra. Error bars indicate SEM. Asterisks indicate p < 0.01 (*), p < 0.001 (**) or p < 0.0001 (***) relative to Veh + MPTP group. Numbers of mice in each group are shown within the bars. (B) Correlation between efficacy in the MPTP model of PD and the in vivo neurogenesis assay for analogs of P7C3. (C) Representative immunohistochemical pictures of TH staining in the SNc are shown for 5 mg kg⁻¹ d⁻¹ treatment groups. Bar = 300 mM.

Fig. 5

Efficacy of P7C3-A20 in the SOD1 mouse model of ALS. (A) P7C3- A20 blocks motor neuron death in the spinal cord when administered (20 mg kg⁻¹ d⁻¹, ip, bid) starting at the time of disease onset. N = 5 for each timepoint. (B) P7C3-A20 preserves performance in the accelerating rotarod test when administered (20 mg kg⁻¹ d⁻¹, ip, bid) starting at the time of disease onset. N = 20 until week 16 when n = 13. For A and B, asterisks indicate p < 2 × 10⁻² (*), p < 2 × 10⁻⁴ (**) or p < 2 × 10⁻⁶ (***) relative to vehicle. (C) Representative images following immunohisoto- chemical staining with antibodies for choline acetyltransferase