Effect of tetracyclines on the dynamics of formation and destructuration of beta2-microglobulin amyloid fibrils.
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.
Department of Biochemistry, University of Pavia, via Taramelli 3b, 27100 Pavia, Italy.
Sofia
S
Giorgetti
Sara
S
Raimondi
Katiuscia
K
Pagano
Annalisa
A
Relini
Monica
M
Bucciantini
Alessandra
A
Corazza
Federico
F
Fogolari
Luca
L
Codutti
Mario
M
Salmona
Palma
P
Mangione
Lino
L
Colombo
Ada
A
De Luigi
Riccardo
R
Porcari
Alessandra
A
Gliozzi
Massimo
M
Stefani
Gennaro
G
Esposito
Vittorio
V
Bellotti
Monica
M
Stoppini
D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism; D000686:Amyloidosis / Q000188:drug therapy / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000627:therapeutic use; D045744:Cell Line, Tumor; D004318:Doxycycline / Q000737:chemistry* / Q000627:therapeutic use; D004353:Drug Evaluation, Preclinical; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D014270:Trifluoroethanol / Q000737:chemistry; D001613:beta 2-Microglobulin / Q000737:chemistry* / Q000378:metabolism
D000682:Amyloid; D000900:Anti-Bacterial Agents; D001613:beta 2-Microglobulin; D014270:Trifluoroethanol; D004318:Doxycycline