1. Aly MOUSSA Agence Nationale de Sécurité Sanitaire de l’Alimentation, de l’Environnement et du Travail (Anses) - Laboratoire de Lyon, France The biological activity of streptomycin and related molecules are associated with the presence of functional guanidine groups

2. In 1986 an epidemic of mad cow disease was developed in great Brittan followed by the new variant of Creuztfeldt-Jacob disease in man. Later new cases of BSE and vCJD appeared world wide and give an incitation to this study.

3. Protein misfolding diseases (Amyloidosis) Amyloid proteins are insoluble and inappropriately folded versions of proteins or polypeptides present naturally in the body. The change from normal forms to amyloid one is associated with conformational changes in the peptide back-bond from a higher degree of alpha- helical structure to a conformer in which a much higher degree of beta-sheet structure are present. In humans there are about 22 diseases associated with the presence of the amyloid proteins i.e. Alzheimer's disease (Aβ), Parkinson's disease (Alphasynuclein), Creuztfeldt-Jacob disease (PrPsc), Diabetes mellitus type 2 (Amylin), Medullary carcinoma of the thyroid (ACal), Cardiac arrhythmias (AANF), Rheumatoid arthritis (AA), Dialysis related amyloidosis (Beta 2 microglobulin ) …etc.

4. The Prion, the prion diseases and the prion proteins

5. The pathogenic prion is an infectious amyloid protein which can alone induce diseases. These are called Transmissible Spongiform Encephalitis (TSE) and are sub-acute fatal infections characterized by the presence of vacuoles in neurons Exp: Scrapie in sheep& gaots, BSE in cattle, chronic wasting disease in deer and Creutzfeld-Jakob Disease in humans (CJD).

6. The cellular Prion protein PrPc is coded by the prnp Gene situated on the chromosome 20 in humans, 13 in bovine and 2 in mice. This gene was found in all vertebrates and invertebrates and is expressed, transported and anchored on the outer membrane of cells in CNS and the reticular-endothelial system. The physiological function of PrPc:- * Protect cells against apoptosis and oxidative stress, *cellular uptake by binding of copper ions, *transmembrane signal transduction, *formation and maintenance of synapses,

7. The pathogenic Prion protein PrPsc (scrapie) is produced after conformational transformation of the PrPc induced either by gene mutation or after infection with the pathogenic PrPsc.

8. Tridimensional structure of the PrPc rich in  helices (left) and the PrPsc rich in  sheets PrPc PrPsc PrPsc Sensitive to Proteinase K resistant proteinase k detergant Insoluble

9. The Prion protein PrP sc is formed of 3 bands representing the isoforms bi-, mono and non glycosylated forme PrPres : Forme di-diglycoe Forme mono-glycosylée Forme non - glycosylée

10. Streptomycin and related molecules: * Story Streptomycin was shown in the year 1940 to interact with globulin and albumin during dialysis studies. By accident we also observed the interaction of streptomycin with the prion proteins and later with the Alzheimer peptide P53.

11. sc PrP sc treated with streptomycin sulfate lanes 1 and 2 respectively, and with penicillin G sodium salt, polymyxin B sulfate, geneticin (G418), ampicillin and amphotericin B, lanes 3–7.

12. Electrophoresis and western blot were used for detection the intermolecular interaction of prion protein and streptomycin. Addition of variable concentration of streptomycin to constant amount of PrPsc leads to a graduel increase of the molecular weight mass of the PrPsc bands. 1 2 3 4 5 6 7 8 9 10 11 12 0mM 3,75mM 7,5mM 15mM 30mM 120mM Streptomycine Concentrations

13. Precipitation of PrP res by streptomycin:- to 100 µl PrPsc was added 0, 5, 10 or 20 µl of streptomycin at 0.7 M incubation 1 hour at 37° Addition of streptomycin Centrifugation: 10000 RPM for 10 min. Collection of the supernatants and suspension of the pellets Electrophoresis & western blot µl of 0,7 M streptomycin 0 5 10 20 SupernatantPrecipitate

14. Streptomycin Dihydrostreptomycin sesquisulfate reduction of one aldehyde group

15. Precipitation of PrPres extracted from 2 mg of brain tissue (lanes 1– 7 supernatants and 9–15 pellets) Lanes 1 and 9 without additive, lanes 2–4 and 10–12 with 2.5, 5 or 10 ul streptomycin 0,7M, lanes 5–7 and 13–15 with 2.5, 5 or 10 ul Dihydrostreptomycin 0,7M. Molecular weight marker lane 8.

16. Mode of action: The interaction between the prion protein and streptomycin is through a hydrogen bond transfer between the guanidine groups on streptomycin and the negativly charged amino acids on the prion protein and is often described as a strong electrostatic dipole-dipole interaction (a schiff base reaction). The binding energy produced through this intramolecular hydrogen bonding can cause a conformational change in the prion protein.

17. Guanidine Hcl spermine triethylenetetramine Bis(3-aminopropyl)amine

18. Adding 0, 30, 60, 120, 180 and 240 mM triethylenetetramine (tri) or 0, 1, 2, 4, 6 and 8 µl of 0.5 M bis-3-aminopropylamine (Bis) to a constant quantity of the PrP sc showed an increase of the molecular weight of the prion proteins proportional to the concentration of the added molecule. The PrPsc three bands tend to migrate altogether in a smeared distribution of aggregated proteins.

19. Guanidine hydrochloride increased the apparent molecular mass of the 3 prion protein bands proportional to its concentration added.

20. An increase of the molecular weight of the 3 PrPsc peptides proportional to the spermine quantity added and in a similar way as the other tested aggregating molecules.

21. The Interaction of the prion protein with either one of the two antibiotics or 4 chemicals having in common the presence of guanidine groups leads to the following conclusions:- 1-Increases of the apparent molecular weight of the prion protein was proportional to the added molecule. 2-Formation of multimolecular prion protein aggregates can be recovered by a low speed centrifugation step. 3-The interaction with the guanidine groups induce conformational changes which leads to alteration of the surface electrostatic charges on the PrPsc. 4-Electrophoresis and western blot proved useful for the detection of the intermolecular interaction of proteins with molecules possessing guanidine groups.