A single‐molecule force spectroscopy nanosensor for the identification of new antibiotics and antimalarials

Abstract

An important goal of nanotechnology is the application of individual molecule handling techniques to the discovery of potential new therapeutic agents. Of particular interest is the search for new inhibitors of metabolic routes exclusive of human pathogens, such as the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway essential for the viability of most human pathogenic bacteria and of the malaria parasite. Using atomic force microscopy single-molecule force spectroscopy (SMFS), we have probed at the single-molecule level the interaction of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), which catalyzes the first step of the MEP pathway, with its two substrates, pyruvate and glyceraldehyde-3-phosphate. The data obtained in this pioneering SMFS analysis of a bisubstrate enzymatic reaction illustrate the substrate sequentiality in DXS activity and allow for the calculation of catalytic parameters with single-molecule resolution. The DXS inhibitor fluoropyruvate has been detected in our SMFS competition experiments at a concentration of 10 μM, improving by 2 orders of magnitude the sensitivity of conventional enzyme activity assays. The binding of DXS to pyruvate is a 2-step process with dissociation constants of k(off) = 6.1 × 10(-4) ± 7.5 × 10(-3) and 1.3 × 10(-2) ± 1.0 × 10(-2) s(-1), and reaction lengths of x(β) = 3.98 ± 0.33 and 0.52 ± 0.23 Å. These results constitute the first quantitative report on the use of nanotechnology for the biodiscovery of new antimalarial enzyme inhibitors and open the field for the identification of compounds represented only by a few dozens of molecules in the sensor chamber.

1-Deoxy-d-xylulose 5-Phosphate Synthase Catalyzes a Novel Random Sequential Mechanism

“…While this work was being prepared, a study describing single-molecule force spectroscopy for the identification of novel DXP synthase inhibitors was reported (14). The method allows measurement of adhesion forces between immobilized substrates and enzyme.…”

“…The authors suggest a possible dissociation of substrate recognition properties and substrate transformation properties that could permit the use of this catalytically less active nanosensor as a tool for identifying high affinity ligands without the need for substrate turnover. Interestingly, Sisquella et al (14) reported binding of GAP tethered through the phosphoryl moiety and enhancement of its affinity for the enzyme in the presence of soluble pyruvate. This is particularly interesting, in light of our finding that removal of the phosphoryl moiety inverts the binding mode of the acceptor aldehyde.…”

“…2A). A more recent study reported a single-molecule force spectroscopy nanosensor to measure E. coli DXP synthase substrate binding and application of single-molecule force spectroscopy for identifying ␤-fluoropyruvate as an inhibitor of the enzyme (14). Using immobilized enzyme and substrate, the authors determined substrate binding constants and suggested that the observed 1.7-fold enhancement in binding of D-GAP to DXP synthase in the presence of soluble pyruvate provides further support for an ordered mechanism.…”

Using Substrate Analogues To Probe the Kinetic Mechanism and Active Site of Escherichia coli MenD

“…Subsequent work by Sisquella et al with E. coli DXPS using single-molecule force spectroscopy also supported an ordered mechanism in which pyruvate binds tightly, and GAP binds before the reaction occurs. 19 This precedent within the enzyme family demonstrates that experimental evidence is required to establish the kinetic mechanism of MenD.…”

Nanomaterials—Tools, Technology and Methodology of Nanotechnology Based Biomedical Systems for Diagnostics and Therapy

“…Another direct application of the AFM principle for the search for bio-active compounds is the single-molecule force spectroscopy (Figure 4) [28]; the authors elected to screen for new inhibitors of the 2- C -methyl- d -erythritol-4-phosphate (MEP) pathway designed to specifically affect members of the kingdom bacteria, including human pathogens, such as malaria parasites, but not members of the kingdom animalia (see refs. [29,30], and below for details).…”