Surprisingly, only two of these compounds lowered tau levels in our HEK293T tauopathy cell model: carbocyanine and anthraquinone (Figure 2A,B). tau-lowering efficacy in cells and slices. Moreover, other Hsp70 inhibitor scaffolds with weaker tau-lowering activity in cells inhibited tau aggregation cysteine oxidation.29,30 Because tau has two naturally occurring cysteine residues located in the microtubule binding domain name, it can form intermolecular disulfide bonds with neighboring tau molecules leading to aggregate formation.31C33 MB creates disulfide bonds within the same tau molecule disrupting fibrillization.29 Since MB has been shown to reduce tau levels in multiple tauopathy models,34C36 which has precipitated clinical trials of related derivatives for AD and FTD, it is difficult to know which activity, Hsp70 inhibition or aggregation inhibition, is most responsible for its ability to facilitate tau clearance.34,37,38 In this regard, several other studies have identified tau aggregation inhibitors, but the ability of Glucocorticoid receptor agonist these compounds to promote tau clearance has not been presented for most of those. For example, the olive oil phenols, aminothienopyridazine (ATPZ), rhodanines, and anthraquinones all prevent tau aggregation activity alone was a strong predictor of tau-lowering in cells. Rather, only those molecules possessing potent activity against both Hsp70 ATPase function and tau aggregation facilitated tau clearance impartial of toxicity. Here, we describe the implications of these findings for tau-based drug discovery efforts, and how this information could be used to improve the success rate for translation of prospects recognized from assays into preclinical and clinical studies. RESULT AND Conversation On the basis of our previous reports that methylene blue and the compound YM-01, a derivative of MKT-077 from your rhodacyanine scaffold, both inhibited Hsp70 activity and lowered tau levels in a cell tauopathy model,8,14 we hypothesized Glucocorticoid receptor agonist that it was in fact the Hsp70 ATPase inhibition that was the best predictor of tau-lowering activity in cells. To investigate this, we examined the tau-lowering capability of several other published Hsp70 inhibitors, outlined in Table 1. Compounds for each scaffold were assessed for tau-lowering efficacy. Human embryonic kidney (HEK293T) cells, transiently overexpressing WT4R0N tau were treated with increasing concentrations of each compound for 24 h. Interestingly, vast differences in tau-lowering activity were found among the molecules. Compounds from your piperidine-3-carboxamide and the adenosine analog scaffolds surprisingly increased tau levels. In contrast, the rhodacyanine and phenothiazine compounds still potently reduced tau levels at all concentrations. However, the dihydropyrimidine, phenoxy-N-arylacetamide, sulfonamide, and flavonol scaffolds only lowered tau levels at the highest concentration tested, 30 M (Physique 1A,B). Comparable trends were observed for these compounds in a Rabbit polyclonal to A2LD1 stably transfected HEK P301L tau cell collection (Supporting Information Physique 1). These data show that allosteric Hsp70 inhibitors might be more likely to possess tau-lowering activity than those that directly target the ATP binding site. Perhaps more importantly, because all of these compounds target the same mechanism of action, we concluded that Hsp70 inhibition alone was insufficient to predict tau lowering activity by greater than ~60%. Open in a separate window Physique 1 Diverse Hsp70 inhibitor scaffolds having differing effects on tau levels. (A) Representative Western blot analysis of HEK293T cells transiently transfected WT4R0N tau and treated with each Hsp70 inhibitor at indicated concentrations for 24 h. (B) Quantification of tau levels in panel A as a percentage of vehicle treated standard error of the mean (SEM), = 3. By linear regression analyses, *** indicates 0.001, and ** indicates 0.01. Glucocorticoid receptor agonist Table 1 Summary of Published Hsp70 Glucocorticoid receptor agonist Inhibitor Scaffolds tau aggregation, we then speculated that tau lowering efficacy could be better predicted by anti-tau aggregation activity. To test this, we evaluated the tau lowering activity of several commercially available tau aggregation inhibitor scaffolds including carbocyanine, aminothienopyridazine (ATPZ), polyphenols, anthraquinone, and rhodanine (Table 2). Surprisingly, only two of these compounds lowered tau levels in our HEK293T tauopathy cell model: carbocyanine and anthraquinone (Physique.