Chip-based protein-protein interaction studied by atomic force microscopy.

Area of Science:

• Biophysics
• Biochemistry
• Pharmacology

Background:

• Accurate measurement of protein-to-protein interactions is crucial for drug discovery.
• Existing methods like Surface Plasmon Resonance (SPR) have limitations in quantitative analysis.
• Atomic Force Microscopy (AFM) offers a potential high-resolution technique for probing molecular interactions.

Purpose of the Study:

• To develop and validate a direct measurement technique for protein-to-protein interactions using AFM.
• To assess the effect of a drug candidate on these interactions.
• To compare the efficacy of AFM with existing methods like SPR for drug screening.

Main Methods:

• Immobilization of T-cell/CD28 on-chip and B-cell/CD80 on an AFM tip.
• Direct measurement of unbinding forces between T-cell/CD28 and B-cell/CD80 before and after drug introduction.
• Determination of loading rates, maximum probability of bindings, and average unbinding forces.
• Application of Echinacea purpurea derived cynarin as a known immunosuppressant drug candidate.

Main Results:

• Cynarin introduction significantly reduced binding events from 61 ± 5% to 47 ± 6% at an AFM loading rate of 1.44 × 10(4) pN/s.
• Maximum probability of bindings decreased from 70% to 35%, indicating a ≈ 35% blocking effect.
• Average unbinding forces were reduced from 61.4 to 38.9 pN, showing a ≈ 37% blocking effect, superior to SPR's ≈ 9%.

Conclusions:

• AFM provides accurate quantitative measurements for assessing drug candidate effects on protein-protein interactions.
• The developed AFM method is effective for drug screening and demonstrates significant blocking effects of cynarin.
• This technique holds promise for broader drug candidate screening with advancements in bio-chip technology.