First author Jie Li and colleagues tested the antibiotic in an in vitro α-synuclein assay, finding that 100-micromolar rifampicin completely abolished aggregation of the protein, while lower doses (10-micromolar) reduced aggregation by over 60 percent. To elucidate what effect rifampicin might have on the aggregation process, the researchers used circular dichroism spectroscopy, which can give valuable information about three-dimensional protein structure, to measure conformational changes accompanying fibril formation. They found that the drug dramatically delayed the formation of α-synuclein β-sheet structures in solution, which, the authors posited, is because it prevents soluble oligomeric species from forming.

This suggestion was supported by subsequent chromatographic experiments. When Li and colleagues incubated α-synuclein alone, they found that it rampantly formed soluble oligomers, while in the presence of the antibiotic, monomers were predominant. In addition, when the authors examined the protein by electron microscopy, they found that rifampicin prevented the formation of long, needle-like fibrils that formed in its absence. And remarkably, when Li added the drug to α-synuclein fibrils that had already formed, it led, within three days, to almost complete disaggregation of the fibrils.

So how does rifampicin prevent fibrillization of α-synuclein? A clue may come from studies of "aged" rifampicin, which proved to be best at preventing fibrils. Could the drug be undergoing a chemical modification? Well, rifampicin in solution is relatively unstable and oxidizes easily. When Li and colleagues tested the drug under anaerobic conditions, or in the presence of the antioxidants glutathione or ascorbic acid, they found it was much less effective. The authors state that “it is likely that an oxidation product is the major species responsible for inhibition of fibrillation,” and they also suggest that a quinone, formed when the drug oxidizes, may be the business end of the molecule. If so, then the mechanism of action may be similar to that proposed for dopamine's blocking of α-synuclein fibrillization—it covalently modifies the protein (see ARF related news story). Indeed, the authors did find that rifampicin (a known chromophore), or another species that absorbs visible light, gets incorporated into monomers and oligomers of the protein.

In an accompanying Chemistry & Biology Preview, Aphrodite Kapurniotu, from the University Hospital of the RWTH Aachen, Germany, raises some interesting questions, perhaps the most fundamental being “are they [the covalently modified synucleins] neurotoxic oligomers or are they nontoxic oligomers?” If the latter, suggests Kapurniotu, then rifampicin and related compounds may offer a reasonable start for the development of drugs to combat PD.—Tom Fagan.

References:
Li J, Zhu M, Rajamani S, Uversky VN, Fink AL. Rifampicin inhibits alpha-synuclein fibrillation and disaggregates fibrils. Chemistry & Biology. 2004 November 29;11:1513-1521. Abstract

Kapurniotu A. Targeting alpha-synuclein in Parkinson’s disease. Chemistry & Biology. 2004 November 29;11:1476-1478. Abstract