Amyloid fibrils are nanoparticles that target lysosomes.

The amyloidoses are a group of debilitating disorders which include neurodegenerative diseases such as Alzheimer’s disease and systemic diseases such as dialysis-related amyloidosis (DRA). Amyloidoses are associated with the aggregation of proteins into amyloid fibrils with a highly organised cross-β structure. Amyloid fibrils are formed by a variety of proteins and peptides despite differences in sequence and native structure. Amyloid formation occurs by a nucleated growth mechanism and proceeds via oligomeric intermediates into mature fibrils. Despite intense research, the molecular mechanisms involved in disease pathogenesis remain unclear. This thesis discusses the mechanism by which amyloid fibrils cause cellular disruption. Chapter 3 describes work performed to validate the use of β2-microglobulin (β2m), the protein that self-associates into amyloid fibrils found in DRA deposits, as a model to study amyloidosis. Fragmentation of mature β2m fibrils, increased their internalisation and access to intracellular compartments, and were therefore used to investigate mechanisms of cellular disruption. Building on previous work in the laboratory showing trafficking of β2m fibrils to the lysosome, chapter 4 examined the effect of fragmented fibrils on lysosomal function and demonstrates that fragmented fibrils impair lysosome-mediated degradation of endocytosed proteins. Following on from this, chapter 5 discusses the effect of fragmented fibrils on membrane trafficking. Fragmented fibrils perturbed the trafficking of lysosomal membrane proteins and also reduced the trafficking of endocytosed cargo to lysosomes. This may rationalise the impairment in degradation of endocytosed proteins. The molecular chaperone, heat shock protein 70 (Hsp70) has been shown to be protective in amyloid disease. The role of Hsp70 in fibril-mediated cell disruption was investigated in chapter 6. Hsp70 protected fibril-treated cells from impairment in degradation of endocytosed protein but not from membrane trafficking defects. This work demonstrates that fragmented fibrils are nanoparticles which target lysosomes and implicates the lysosome in the pathogenesis of amyloidosis

Development of a small molecule that corrects misfolding and increases secretion of Z α1 -antitrypsin.

Severe α1 -antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α1 -antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA-encoded chemical library to undertake a high-throughput screen to identify small molecules that bind to, and stabilise Z α1 -antitrypsin. The lead compound blocks Z α1 -antitrypsin polymerisation in vitro, reduces intracellular polymerisation and increases the secretion of Z α1 -antitrypsin threefold in an iPSC model of disease. Crystallographic and biophysical analyses demonstrate that GSK716 and related molecules bind to a cryptic binding pocket, negate the local effects of the Z mutation and stabilise the bound state against progression along the polymerisation pathway. Oral dosing of transgenic mice at 100 mg/kg three times a day for 20 days increased the secretion of Z α1 -antitrypsin into the plasma by sevenfold. There was no observable clearance of hepatic inclusions with respect to controls over the same time period. This study provides proof of principle that "mutation ameliorating" small molecules can block the aberrant polymerisation that underlies Z α1 -antitrypsin deficiency

Development of a small molecule that corrects misfolding and increases secretion of Z α1‐antitrypsin

Abstract Severe α1‐antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α1‐antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA‐encoded chemical library to undertake a high‐throughput screen to identify small molecules that bind to, and stabilise Z α1‐antitrypsin. The lead compound blocks Z α1‐antitrypsin polymerisation in vitro, reduces intracellular polymerisation and increases the secretion of Z α1‐antitrypsin threefold in an iPSC model of disease. Crystallographic and biophysical analyses demonstrate that GSK716 and related molecules bind to a cryptic binding pocket, negate the local effects of the Z mutation and stabilise the bound state against progression along the polymerisation pathway. Oral dosing of transgenic mice at 100 mg/kg three times a day for 20 days increased the secretion of Z α1‐antitrypsin into the plasma by sevenfold. There was no observable clearance of hepatic inclusions with respect to controls over the same time period. This study provides proof of principle that “mutation ameliorating” small molecules can block the aberrant polymerisation that underlies Z α1‐antitrypsin deficiency