Recently, gamma-secretase modulators (GSM) have been shown to interact directly with the amyloid precursor protein (APP) and simultaneously inhibit the activity of the Presenilin domain of gamma-secretase. A clear understanding of the molecular recognition pathways by which GSM can target both gamma-secretase and All precursor protein can lead to the development of more effective inhibitors. To examine whether this direct interaction with APP affects the downstream A beta fibril formation, we chose to investigate three different molecules in this study: Sulindac sulfide, Semagacestat and E2012 from the class of generation I GSMs, gamma-secretase inhibitors (GSI), and generation II GSM molecules, respectively. Firstly, through NMR based ligand titration, we identified that Sulindac sulfide and Semagacestat interact strongly with A beta 40 monomers, whereas E2012 does not. Secondly, using saturation transfer difference (STD) NMR experiments, we found that all three molecules bind equally well with A beta 40 fibrils. To determine if these interactions with the monomer/fibril lead to a viable inhibition of the fibrillation process, we designed an NMR based time-dependent assay and accurately distinguished the inhibitors from the non-inhibitors within a short period of 12 h. Based on this pre-seeded fibril assay, we conclude that none of these molecules inhibit the ongoing fibrillation, rather ligands such as Semagacestat and E2012 accelerated the rate of aggregation. (C) 2014 Elsevier Inc. All rights reserved.