1Harnessing Chaperones to GenerateSmall-Molecule Inhibitors ofAmyloid β AggregationJason E. Gestwicki, Gerald R. Crabtree,Isabella A. GraefScience 29 October 2004 306: 865-869Amyloid• Extracellular filamentous protein aggregates, usuallyprimarily beta-sheet character• Associated with a large number of diseases andsyndromes, local or systemic• Often involves misfolding of protein subunits, probablymultiple steps (Example: lysozyme)• Cause or consequence?2Examples of amyloid diseases• Disease Major component of amyloid• Alzheimer’s disease A-beta peptide• primary systemic amyloidosis antibody light chain and fragments• secondary systemic amyloidosis fragment of amyloid A protein• senile systemic amyloidosis transerythrin and fragments• hereditary cerebral amyloid angiopathy cystatin-C fragments• hemodialysis-related amyloidosis beta-2 microglobulin• familial amyloidotic polyneuropathy II apolipoprotein A-I fragments• Finnish hereditary amyloidosis gelsolin fragment• type II diabetes islet-associated polypeptide fragment• medullary carcinoma of the thyroid calcitonin fragments• atrial amyloidosis atrial natriuretic factor• lysozyme amyloidosis lysozyme variants• insuline-related amyloid insulin• fibrinogen alpha-chain amyloidosis fibrinogen alpha-chain variantsGeneration of Aβ peptide• Mutations in presinilins (PS complex) isassociated with familial early-onset Alzheimer’s• Addition of Aβ amyloid to cultured neurons iscytotoxic3Hypothesis:• Formation of beta-amyloid causes neuronaldamage in Alzheimer’s disease• Inhibition of Aβ aggregation would slow orstop progression of disease• Aggregation may be inhibited by molecules thatbind aggregation intermediates• Efficiency of inhibition might be increased byincreasing the size of the inhibitory molecule(e.g. recruiting a protein binding partner)Design (F1)• Congo Red binds amyloid, used in histological stains• High concentrations of Congo Red inhibit aggregation• FK506 immunosuppressant, same pathway as cyclosporin• FK506 binds and inhibits FKBP, cis-trans prolyl isomerases withmultiple family members• Downstream inhibition of calcineurin prevents T cell activationand causes immunosuppressive effects• FKBP/FK506 are beloved of “chemical biologists”4Does it bind? (S1)• Note: No difference in affinity for SLF-CR vs. SLF-CR + FKBP,suggests FKBP is not interacting with the amyloid fibrilDoes it inhibit aggregation? (F1)Assay 1: light scattering25 µM Ab, 1 µM FKBP, 10 µM drugDose-response5Does it inhibit aggregation? (F1)Assay 2: thioflavin T bindingThioflavin T is also used in histological staining for amyloidDoes it inhibit fibril formation? (F2)EMAFM6Does it reduce cytotoxicity? (F3, S2)Assay 1: MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)Does it reduce cytotoxicity? (F3, S2)Assay 2: TUNEL (terminal deoxynucleotidyl transferase-mediateddeoxyuridine triphosphate nickend labeling)7Can chemical modificationsmake it better? (F4)Can chemical modificationsmake it better? (F4, S3)8Can chemical modificationsmake it better? (F4, S3)How does it work? (S2)• Prolyl isomerase activity of FKBP is not involved– Equimolar FKBP does not work alone– Addition of FK506 has no effect– ??Is SLF an inhibitor of FKBP??• Slight difference in aggregation intermediate pattern between CRand SLF-CR (what about the drugs alone with no FKBP?9Conclusions• Recruiting a binding protein improves the efficacy of smallmolecule inhibitors of protein aggregation in a variety of invitro and tissue culture assays• This is probably a more viable strategy than increasing thesize of the small molecule inhibitors themselves(accessibility)• The identity of the recruited protein probably doesn’t matter• Open questions:– Will this work in a live animal or person?– How will the drug gain access to the brain?– Will the binding and recruitment occur in the correct cellularcompartment?– Will inhibition of protein aggregation have any effect onAlzheimer’s disease
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