Investigating the influence of Creactive protein CRP on heart attacks and strokes, and finding ways

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Investigating the influence of C-reactive protein
(CRP) on heart attacks and strokes, and finding
ways to inhibit this protein

• Jochen Aernoudt
• Protein Technology
• Project work

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Other measurements

• Lipid level
• Cholesterol level
• Blood viscosity
• Body Mass Index (BMI)
• CRP atherosclerose stimulates immune system

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Introduction

• Acute phase proteins
• Atherosclerotic lesions and CRP
• Molecular structure of CRP
• Known inhibitors
• This protject

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Acute phase proteins

• Inflammation/infection gt cytokines gt
  stimulation of immune system

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Why CRP?

• Remarkable sensitivity
• Speed
• Half life (19h)
• No drugs ? CRP gt few adjustments
• Only regulated by expression severity

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Atherosclerotic lesions and CRP

• No CRP in healthy arteries
• Lipid accumulation hand in hand with inflammation
• CRP C-polysaccharide teichoic acid (S.
  pneumoniae)
• Oxidised/enzyme-modified LDL present in lesions
• Ligand bound stimulation of complement
  (classical/alternative)

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Molecular structure of CRP

• Pentraxin family ? five subunits, 206 AA,
• 4 H-bonds, 3 salt bridges
• Pdb-entries for free and complexed CRP
• Site-directed mutations gt imortant residues
  known

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Known inhibitorsThis project

• Statins (cholesterol)
• Angiotensin-converting enzyme inhibitors
• ?not directly inhibiting CRP (not well known)
• Aim inhibitor acting immediately on CRP
• No side effects

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Experimental plan

• Choice of the expression vector
• Cell cultures, expression, purification
• Design of inhibitor
• Assay of the CRP-inhibitor complex
• Design of drugs

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Choice of expression vector

• Human/animal cells?
• No glycosylation at all
• Rat and mouse
• Chinese hamster ovary cells (Agrawal et al.,
  2002) for stable expression of native CRP.
• Vector known p91023

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Cell cultures, expression, purificationDesign
of inhibitor

• Follow protocol (Agrawal et al., 2002).
• Prestwick Chemical Library
• Search for substrate analogs
• First filter in silico docking (computational
  way of calculating enzyme-substrate interactions)
• Best inhibitor for C1q

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Assay of the CRP-inhibitor complexDesign of
drugs

• CRP-inh complex injected in rat/mouse
• gt measure complement proteins
• Binding column chromatography add inhibitors
  normal substrate
• Orally/injected
• Intake easy to standardise

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