by Keyword: pyruvate

Yeste, J, Azagra, M, Ortega, MA, Portela, A, Matajsz, G, Herrero-Gómez, A, Kim, Y, Sriram, R, Kurhanewicz, J, Vigneron, DB, Marco-Rius, I, (2023). Parallel detection of chemical reactions in a microfluidic platform using hyperpolarized nuclear magnetic resonance Lab On A Chip 23, 4950-4958

The sensitivity of NMR may be enhanced by more than four orders of magnitude via dissolution dynamic nuclear polarization (dDNP), potentially allowing real-time, in situ analysis of chemical reactions. However, there has been no widespread use of the technique for this application and the major limitation has been the low experimental throughput caused by the time-consuming polarization build-up process at cryogenic temperatures and fast decay of the hyper-intense signal post dissolution. To overcome this limitation, we have developed a microfluidic device compatible with dDNP-MR spectroscopic imaging methods for detection of reactants and products in chemical reactions in which up to 8 reactions can be measured simultaneously using a single dDNP sample. Multiple MR spectroscopic data sets can be generated under the same exact conditions of hyperpolarized solute polarization, concentration, pH, and temperature. A proof-of-concept for the technology is demonstrated by identifying the reactants in the decarboxylation of pyruvate via hydrogen peroxide (e.g. 2-hydroperoxy-2-hydroxypropanoate, peroxymonocarbonate and CO2). dDNP-MR allows tracing of fast chemical reactions that would be barely detectable at thermal equilibrium by MR. We envisage that dDNP-MR spectroscopic imaging combined with microfluidics will provide a new high-throughput method for dDNP enhanced MR analysis of multiple components in chemical reactions and for non-destructive in situ metabolic analysis of hyperpolarized substrates in biological samples for laboratory and preclinical research.

JTD Keywords: injections, nmr, pyruvate, Polarization

Sisquella, X., de Pourcq, K., Alguacil, J., Robles, J., Sanz, F., Anselmetti, D., Imperial, S., Fernàndez-Busquets, X., (2010). A single-molecule force spectroscopy nanosensor for the identification of new antibiotics and antimalarials FASEB Journal , 24, (11), 4203-4217

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 mu M, 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 x 10(-4) +/- 7.5 x 10(-3) and 1.3 x 10(-2) +/- 1.0 x 10(-2) s(-1), and reaction lengths of x(beta) = 3.98 +/- 0.33 and 0.52 +/- 0.23 angstrom. 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.

JTD Keywords: Malaria, 2-C-methyl-D-erythritol-4-phosphate pathway, 1-deoxy-D-xylulose 5-phosphate synthase, Pyruvate, Glyceraldehyde-3-phosphate, Drug discovery