Measurement of enzyme kinetics and inhibitor constants using enthalpy arrays

Recht, Michael I.; Torres, Frank E.; Bruyker, Dirk De; Bell, Alan G.; Klumpp, Martin; Bruce, Richard H.

doi:10.1016/j.ab.2009.02.028

Cited by 31 publications

(30 citation statements)

References 31 publications

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“…Each enthalpy array detector contains a sample region and a reference region, which are designated based on the material deposited on the region (see Fig. 1 of Recht et al 2009; 16 ). The sample region materials consisted of a drop of PDE10A (30 µM) and a drop of substrate solution (4 mM 3′,5′-cGMP).…”

Section: Methodsmentioning

confidence: 99%

Identification and Optimization of PDE10A Inhibitors Using Fragment-Based Screening by Nanocalorimetry and X-ray Crystallography

et al. 2014

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Fragment-based lead discovery (FBLD) is a technique in which, small, low-complexity chemical fragments of 6 to 15 heavy atoms are screened for binding to or inhibiting activity of the target. Hits are then linked and/ or elaborated into tightly binding ligands, ideally yielding early lead compounds for drug discovery. Calorimetry provides a label-free method to assay binding and enzymatic activity that is unaffected by the spectroscopic properties of the sample. Conventional microcalorimetry is hampered by requiring large quantities of reagents and long measurement times. Nanocalorimeters can overcome these limitations of conventional isothermal titration calorimetry. Here we use enthalpy arrays, which are arrays of nanocalorimeters, to perform an enzyme activity-based fragment screen for competitive inhibitors of phosphodiesterase 10A (PDE10A). Two dozen fragments with KI <2 mM were identified and moved to crystal soaking trials. All soak experiments yielded high resolution diffraction with two-thirds of the fragments yielding high-resolution co-crystal structures with PDE10A. The structural information was used to elaborate fragment hits, yielding leads with KI <1 µM. This study shows how array calorimetry can be used as a prescreening method for fragment-based lead discovery with enzyme targets and paired successfully with an x-ray crystallography secondary screen.

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Section: Methodsmentioning

confidence: 99%

Identification and Optimization of PDE10A Inhibitors Using Fragment-Based Screening by Nanocalorimetry and X-ray Crystallography

et al. 2014

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“…Each merged drop pair results in a single thermodynamic experiment (e.g. enzyme activity, protein-ligand binding) [22]. …”

Section: Rapid Drop Mixing On Enthalpy Arraysmentioning

confidence: 99%

Rapid mixing of sub-microlitre drops by magnetic micro-stirring

et al. 2011

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We demonstrate rapid mixing of sub-microliter droplets (250nl) using miniaturized magnetic stir bars (400 μm by 200 μm by 15 μm). The stir bars are fabricated using laser micromachining and placed on the substrate on which the drops are manipulated. They are activated by an externally applied magnetic field and used in combination with on-demand drop merging in enthalpy arrays. This technique results in a 10-fold increase in mixing rate, and a mixing time constant of about 2 seconds. Drop mixing times are measured by Förster resonance energy transfer (FRET) and verified by thermodynamic measurements of binding and enzymatic reactions.

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“…The miniaturized DSC unit will also allow us to integrate multiple DSC on one silicon wafer or polymer substrate. There were already some attempts to develop such calorimeter array [18,24,25].…”

Section: Introductionmentioning

confidence: 99%

Review of MEMS differential scanning calorimetry for biomolecular study

Wang

et al. 2017

Front. Mech. Eng.

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Differential scanning calorimetry (DSC) is one of the few techniques that allow direct determination of enthalpy values for binding reactions and conformational transitions in biomolecules. It provides the thermodynamics information of the biomolecules which consists of Gibbs free energy, enthalpy and entropy in a straightforward manner that enables deep understanding of the structure function relationship in biomolecules such as the folding/unfolding of protein and DNA, and ligand bindings. This review provides an up to date overview of the applications of DSC in biomolecular study such as the bovine serum albumin denaturation study, the relationship between the melting point of lysozyme and the scanning rate. We also introduce the recent advances of the development of micro-electro-mechanic-system (MEMS) based DSCs.

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Measurement of enzyme kinetics and inhibitor constants using enthalpy arrays

Cited by 31 publications

References 31 publications

Identification and Optimization of PDE10A Inhibitors Using Fragment-Based Screening by Nanocalorimetry and X-ray Crystallography

Identification and Optimization of PDE10A Inhibitors Using Fragment-Based Screening by Nanocalorimetry and X-ray Crystallography

Rapid mixing of sub-microlitre drops by magnetic micro-stirring

Review of MEMS differential scanning calorimetry for biomolecular study

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