Recognition of Proteins with Aminopyrazoles

Protein-folding diseases are characterized by the formation of insoluble protein plaques in the brains' nerve cells. The mechanism of these misfolding events is often unclear. In BSE/CJD an infectious protein particle could be identified, leading to the famous prion hypothesis [22]. It is called prion protein scrapie (PrPSc). After refolding of the native protein newly formed ¡-sheet domains aggregate with those in other PrPSc molecules and lead to precipitation of the protein from physiological solution [23]. A similar process might be operating in the Alzheimer's protein, which also forms amyloid plaques in the brain consisting of very regular, stacked twisted ¡-sheets. Our concept intervenes at the beginning of the aggregation process - we aim at preventing dimerization of the pathological protein domains, by capping the solvent-exposed ¡-sheet region with our oligomeric aminopyrazole

Fig. 2.4.11. Ultracentrifugation experiments with PrP alone (10 |xm) and after incubation with AmpOx 9 (1 mM) under harsh aggregation conditions (37 °C, 1 day);

S = solution, P = pellet. Left: typical prion rods (white bar ~ 100 nm) [25]. Right: schematic representation of the concept: capping the j-sheets [26].

Fig. 2.4.11. Ultracentrifugation experiments with PrP alone (10 |xm) and after incubation with AmpOx 9 (1 mM) under harsh aggregation conditions (37 °C, 1 day);

S = solution, P = pellet. Left: typical prion rods (white bar ~ 100 nm) [25]. Right: schematic representation of the concept: capping the j-sheets [26].

Fig. 2.4.12. FCS measurements of the aggregates formed from soluble AJ (10 mM). Ten different 30 s runs are superimposed. Left: without additive; right: with dimeric aminopyrazole 9 (1 mM).
Fig. 2.4.13. Left: typical amyloid fibrils are twisted j-sheets [27]. Right: differential ultracentrifugation experiment with Aj (1-42, 33 and 16 |im) in the absence (right) or presence (left) of dimeric aminopyrazole 9 (Ampox, 1 mM). S = solution, P = pellet.

ligands. Because the back face of these ligands contains no hydrogen-bond donors, the aggregation process should be completely stopped or even be reversed.

The biophysical experiments of our cooperation partners focus mainly on two different techniques - a rough estimate of aggregation prevention is found by differential ultracentrifugation (UC), but more detailed information about the kinetics and aggregate size comes from fluorescence correlation spectroscopy (FCS) [24].

The dimeric j-sheet-binders were first tested on the prion-protein by the Riesner group. An initial precipitation experiment showed that 9 was able to keep about one third of PrPC in solution (Figure 2.4.11 - the detergent sodium dodecylsulfate was diluted out, a procedure which always leads to complete aggregation of the protein.). This was the starting point of our tests; monomeric aminopyrazole derivatives had no influence on the aggregation behavior of PrP. That one of the dimeric aminopyrazoles of the second generation was a promising candidate for a new therapeutic approach to protein folding diseases encouraged us to develop higher generations and generally test all new candidates in bioassays.

The dimeric aminopyrazole ligands were also tested to determine their effect on the aggregation behavior of the Alzheimer's protein. To this end we used the model compound Aj (1-42), which was fluorescence-labeled with Oregon green. The FCS spectra demonstrate beautifully how Aj spontaneously aggregates in sodium phosphate buffer containing 5% DMSO after incubation for 12-72 h. After preincubation of the same samples with selected aminopyrazole ligands, e.g. 9 and 20, however, the aggregation could be completely suppressed (Figure 2.4.12).

The results of the respective differential ultracentrifugations correlate very well with the FCS measurements. Without addition of our ligands Aj (1-42) almost completely precipitates and is found exclusively in the pellet (P). Addition of 9 or 20 before the start of the aggregation process keeps most of the protein in solution (S, Figure 2.4.13), however.

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