The discovery of methods suitable for the conversion in vitro of native
proteins into
amyloid fibrils has shed light on the molecular basis of
amyloidosis and has provided fundamental tools for drug discovery. We have studied the capacity of a small library of
tetracycline analogues to modulate the formation or destructuration of β2-microglobulin fibrils. The inhibition of fibrillogenesis of the wild type
protein was first established in the presence of 20%
trifluoroethanol and confirmed under a more physiologic environment including
heparin and
collagen. The latter conditions were also used to study the highly amyloidogenic variant, P32G. The NMR analysis showed that
doxycycline inhibits β2-microglobulin self-association and stabilizes the native-like species through fast exchange interactions involving specific regions of the
protein. Cell viability assays demonstrated that the drug abolishes the natural cytotoxic activity of soluble β2-microglobulin, further strengthening a possible in vivo therapeutic exploitation of this drug.
Doxycycline can disassemble preformed fibrils, but the IC(50) is 5-fold higher than that necessary for the inhibition of fibrillogenesis. Fibril destructuration is a dynamic and time-dependent process characterized by the early formation of cytotoxic
protein aggregates that, in a few hours, convert into non-toxic insoluble material. The efficacy of
doxycycline as a drug against dialysis-related
amyloidosis would benefit from the ability of the drug to accumulate just in the skeletal system where
amyloid is formed. In these tissues, the
doxycycline concentration reaches values several folds higher than those resulting in inhibition of amyloidogenesis and
amyloid destructuration in vitro.