Combining the rapid MTT formazan exocytosis assay and the MC65 protection assay led to the discovery of carbazole analogs as small molecule inhibitors of Abeta oligomer-induced cytotoxicity.
The discovery of small molecule inhibitors of cytotoxicity induced by amyloid-beta (Abeta) oligomers, either applied extracellularly or accumulated intraneuronally, is an important goal of drug development for Alzheimer's disease (AD), but has been limited by the lack of efficient screening methods. Here we describe our approach using two cell-based methods. The first method takes advantage of the unique ability of extracellularly applied Abeta oligomers to rapidly induce the exocytosis of formazan formed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). We employed a short protocol to quantify this toxicity, and quickly identified two novel inhibitors, code-named CP2 and A5, from two compound libraries. A second independent screen of the same libraries using our previously published MC65 protection assay, which identifies inhibitors of toxicity related to intracellular Abeta oligomers, also selected the same two leads, suggesting that both assays select for the same anti-Abeta oligomer properties displayed by these compounds. We further demonstrated that A5 attenuated the progressive aggregation of existing Abeta oligomers, reduced the level of intracellular Abeta oligomers, and prevented the Abeta oligomer-induced death of primary cortical neurons, effects similar to those demonstrated by CP2. Our results suggest that, when combined, the two methods would generate fewer false results and give a high likelihood of identifying leads that show promises in ameliorating Abeta oligomer-induced toxicities within both intraneuronal and extracellular sites. Both assays are simple, suitable for rapid screening of a large number of medicinal libraries, and amenable for automation.
Hong
Hyun-Seok
HS
M.I.N.D. Institute and Department of Pathology, UC Davis Cancer Center, University of California Davis, Sacramento, CA 95817, USA.
Maezawa
Izumi
I
Yao
Nianhuan
N
Xu
Bailing
B
Diaz-Avalos
Ruben
R
Rana
Sandeep
S
Hua
Duy H
DH
Cheng
R Holland
RH
Lam
Kit S
KS
Jin
Lee-Way
LW
D016229:Amyloid beta-Peptides / Q000008:administration & dosage / Q000633:toxicity*; D000818:Animals; D001681:Biological Assay / Q000379:methods; D002227:Carbazoles / Q000494:pharmacology*; D002460:Cell Line; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology; D005562:Formazans / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009419:Nerve Tissue Proteins / Q000187:drug effects / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D018696:Neuroprotective Agents / Q000494:pharmacology; D013778:Tetrazolium Salts / Q000378:metabolism*
D016229:Amyloid beta-Peptides; D002227:Carbazoles; D005562:Formazans; D009419:Nerve Tissue Proteins; D018696:Neuroprotective Agents; D013778:Tetrazolium Salts; C079782:MTT formazan