Cellular Models of Aggregation-dependent Template-directed Proteolysis to Characterize Tau Aggregation Inhibitors for Treatment of Alzheimer Disease.

Alzheimer disease (AD) is a degenerative tauopathy characterized by aggregation of Tau protein through the repeat domain to form intraneuronal paired helical filaments (PHFs). We report two cell models in which we control the inherent toxicity of the core Tau fragment. These models demonstrate the properties of prion-like recruitment of full-length Tau into an aggregation pathway in which template-directed, endogenous truncation propagates aggregation through the core Tau binding domain. We use these in combination with dissolution of native PHFs to quantify the activity of Tau aggregation inhibitors (TAIs). We report the synthesis of novel stable crystalline leucomethylthioninium salts (LMTX®), which overcome the pharmacokinetic limitations of methylthioninium chloride. LMTX®, as either a dihydromesylate or a dihydrobromide salt, retains TAI activity in vitro and disrupts PHFs isolated from AD brain tissues at 0.16 µM. The Ki value for intracellular TAI activity, which we have been able to determine for the first time, is 0.12 µM. These values are close to the steady state trough brain concentration of methylthioninium ion (0.18 µM) that is required to arrest progression of AD on clinical and imaging end points and the minimum brain concentration (0.13 µM) required to reverse behavioral deficits and pathology in Tau transgenic mice.

Multigram-scale synthesis of short peptides via a simplified repetitive solution-phase procedure.

A rapid repetitive solution-phase synthesis of peptides is described. The procedure involves coupling of amino acids and peptide acids, instead of the usual amino esters and peptide esters, to slight excesses of pentafluorophenyl active esters in a THF/water solvent mixture. Due to their poor solubility, peptide acid intermediates are easily isolated in high purity by acidification under controlled conditions and removal of excess active esters by selective extraction. Contrary to modern repetitive solution-phase peptide synthesis procedures, our approach does not require time-consuming neutralization reactions and reduces significantly the number of operation units that are necessary to obtain peptide intermediates. Efficiency of the method was demonstrated by the rapid synthesis of short hydrophobic and hydrophilic peptides, the antimalarial cycloheptapeptide mahafacyclin B, and a protected form of the hydrophilic pentapeptide GRGDS.