IUVSTA 15th International Vacuum Congress (IVC-15), AVS 48th International Symposium (AVS-48), 11th International Conference on Solid Surfaces (ICSS-11)
    Biomaterials Wednesday Sessions
       Session BI+NS-WeA

Paper BI+NS-WeA2
The Direct Measurement of Drug-enzyme Interactions by Atomic Force Microscopy

Wednesday, October 31, 2001, 2:20 pm, Room 103

Session: Nanobiology
Presenter: S.J.B.T. Tendler, University of Nottingham, UK
Authors: S.M. Rigby-Singleton, University of Nottingham, UK
S.J.B.T. Tendler, University of Nottingham, UK
S. Allen, University of Nottingham, UK
M.C. Davies, University of Nottingham, UK
C.J. Roberts, University of Nottingham, UK
P.M. Williams, University of Nottingham, UK
Correspondent: Click to Email
AFM has been employed to directly probe the rupture forces upon the mechanical dissociation of the drug-enzyme complex formed between the anticancer compound methotrexate and the protein dihydrofolate reductase (DHFR). AFM probes were functionalized with methotrexate immobilized beads and rupture forces recorded between the probe and a DHFR monolayer attached via Lys residues. Three variables were studied, AFM retraction rates, the presence of the enzyme cofactor NADPH and the protonation of the key enzyme Asp26 residue. Rupture forces of 91 pN were recorded at a retract velocity of 1 micrometer per second, a ten fold decrease in velocity resulted in an observed decrease in rupture force. The influence of the enzyme cofactor was negligible suggesting little effect on the dissociation pathway, this is in marked contrast to literature fluorescent binding assays. Notably a decrease in rupture force of approximately 25pN was observed when the pH was decreased below the pKa of the key Asp26 residue which is situated deep within the methotrexate binding site. These studies indicate that the AFM will be a valuable tool in the drug discovery process.