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Transthyretin (TTR) tetramer dissociation is rate limiting for aggregation and subunit exchange. Slowing of TTR tetramer dissociation via kinetic stabiliser binding slows cardiomyopathy progression. Quadruplicate subunit exchange comparisons of the drug candidate AG10, and the drugs tolcapone, diflunisal, and tafamidis were carried out at 1, 5, 10, 20 and 30 mM concentrations in 4 distinct pooled wild type TTR (TTRwt) human plasma samples. These experiments reveal that the concentration dependence of the efficacy of each compound at inhibiting TTR dissociation was primarily determined by the ratio between the stabiliser's dissociation constants from TTR and albumin, which competes with TTR to bind kinetic stabilisers. The best stabilisers, tafamidis (80 mg QD), AG10 (800 mg BID), and tolcapone (3 x 100 mg over 12 h), exhibit very similar kinetic stabilisation at the plasma concentrations resulting from these doses. At a 10 mM plasma concentration, AG10 is slightly more potent as a kinetic stabiliser vs. tolcapone and tafamidis (which are similar), which are substantially more potent than diflunisal. Dissociation of TTR can be limited to 10% of its normal rate at concentrations of 5.7 mM AG10, 10.3 mM tolcapone, 12.0 mM tafamidis, and 188 mM diflunisal. The potency similarities revealed by our study suggest that differences in safety, adsorption and metabolism, pharmacokinetics, and tissue distribution become important for kinetic stabiliser clinical use decisions.

Autophagy activation has the potential to ameliorate neurodegenerative disease phenotypes, including protein aggregation, lipid level perturbations and axonal trafficking defects. We performed a high content imaging-based screen assessing 940,000 small molecules to identify those that accelerate lipid droplet clearance. Hits were validated in diverse cell lines and by counter-screening. Of the 77 structurally diverse validated hits, 24 increase autophagy flux. Herein, we highlight CCT020312 as a mammalian target of rapamycin (mTOR) inhibitor-independent autophagy activator, which should function without compromising human immune function. CCT020312 dose-dependently reduces cytotoxic axonal mutant prion protein aggregate levels within endosomes of primary murine hippocampal neurons and normalizes axonal trafficking deficiencies. Moreover, CCT020312 robustly clears phosphorylated insoluble tau, while reducing tau-mediated neuronal stress vulnerability in patient-derived neuronal models. CCT020312 also restores lysosomal function in neurons differentiated from sporadic Alzheimer's patients' fibroblasts bearing epigenetic marks of aging. Taken together, we describe a promising strategy to uncover novel pharmacological agents that normalize cellular neurodegenerative disease pathology.

No abstract

The aggregation and amyloid deposition of soluble transthyretin (TTR) has been linked to the development sporadic and familial forms of cardiac amyloidosis. TTR associated cardiomyopathies are often diagnosed when the disease state has reached advanced stages (if at all), precluding effective clinical intervention. This diagnostic problem can largely be attributed to a lack of fundamental understanding of the mechanism of disease pathogenesis and the clinical similarities of TTR cardiomyopathies to other heart failure related conditions. In the human prion diseases and tau protein related neurodegenerative diseases, the amyloidogenic protein conformations have the ability to “seed” and transform non‐amyloidogenic states into pathogenic amyloid‐like states. Therefore, we hypothesized that TTR related cardiomyopathies may undergo similar seed based aggregation mechanisms. To test this hypothesis, we synthesized C‐terminal fragments of Transthyretin that are fluorescently labeled and upon assembly, the fluorescent signal is quenched and provides a convenient method to measure aggregation. We specifically chose the C‐terminal peptide fragment (TTR50–127) because at autopsy it has been shown that the C‐terminal region is preferentially deposited in heart tissue compared to the full length sequence. First we used these fluorescent probes to analyze the kinetics involved in the transition from monomeric TTR to the stable polymeric form of TTR and thus demonstrated proof‐of‐principle. As C‐terminally cleaved TTR is primarily found in biopsies of cardiomyopathy patients, introduction of rhodamine labeled TTR50–127 to cardiomyopathy and polyneuropathy patient plasma revealed distinct aggregation profiles in Native PAGE gels. While preliminary in nature, these results suggest that plasma of symptomatic patients may accelerate the aggregation of Transthyretin.Support or Funding InformationSpecial thanks to staff of the Kelly Lab at TSRI as well as mentors James Smart Ph.D. and Jeff Duerr Ph.D.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.