Enthalpy Arrays for Compound Screening

Assay Technology

  • Fig. 1: (a) Each site on an enthalpy array consists of a sample region and a reference region. (b) The drops are merged with an electrostatic mechanism, initiating the reaction. (c) The detector consists of two identical sensing elements arranged in a Wheatstone bridge configuration. The voltage difference between the sample region (S) and reference region (R) is recorded. (d) The voltage data is converted to rate of heat generation vs. time. Fig. 1: (a) Each site on an enthalpy array consists of a sample region and a reference region. (b) The drops are merged with an electrostatic mechanism, initiating the reaction. (c) The detector consists of two identical sensing elements arranged in a Wheatstone bridge configuration. The voltage difference between the sample region (S) and reference region (R) is recorded. (d) The voltage data is converted to rate of heat generation vs. time.
  • Fig. 1: (a) Each site on an enthalpy array consists of a sample region and a reference region. (b) The drops are merged with an electrostatic mechanism, initiating the reaction. (c) The detector consists of two identical sensing elements arranged in a Wheatstone bridge configuration. The voltage difference between the sample region (S) and reference region (R) is recorded. (d) The voltage data is converted to rate of heat generation vs. time.
  • Fig. 2: Competitive inhibitors of enzymatic reactions. (a) Trypsin hydrolysis of BAEE. Rate vs. remaining substrate concentration for individual reactions in the absence of inhibitor (circles) and in the presence of 1 mM benzamidine (triangles) or 10 µM leupeptin (squares). The solid curves are the fit of the data to a modified form of the Michaelis-Menten equation [3]. (b) cAMP dependent kinase reactions. Rate of heat generation vs. time for reaction in absence (black) and presence (red) of 4 µM staurosporine. Both reactions contained 2% DMSO. Inlay: Rate vs. remaining limiting substrate concentration for reaction in absence of inhibitor, and fit to modified Michaelis-Menten equation (solid curve) [3].
  • Dr. Michael I. Recht, Palo Alto Research Center

Enthalpy arrays enable label-free, solution-based calorimetric detection of molecular interactions in an array format [1-3]. The combination of the small size of the detectors and ability to perform measurements in parallel results in a significant reduction of sample volume and measurement time compared with conventional calorimeters. The technology can be applied to characterizing binding and enzymatic reactions and determining inhibition constants and inhibition mode of low molecular weight compounds and peptides [3].

Array Layout and Experimental Design


Each site in the enthalpy array consists of two identical detector regions, providing a differential temperature measurement between a sample and reference specimen. Two 250 nl drops are deposited on each of the two regions (fig. 1a). The array is placed in a measurement chamber and a polymer cap is applied over the samples to provide evaporation control. Each region has its own electrodes for electrostatically-driven isothermal merging of the two drops (fig. 1b). To provide good mixing of the drops, magnetic mixing is employed by using PEGylated stir bars in one of the two drops in each region and a rotating magnet below the array. After merging, the detector measures temperature changes of the sample relative to a simultaneous merging of similar but non-reacting materials in the reference region (fig. 1c). This relative measurement subtracts out common-mode changes in temperature, thereby improving sensitivity. The differential temperature data is converted to a rate of heat generation versus time (fig. 1d). This data can be integrated to obtain the total heat of the reaction [2] or further analyzed to obtain enzyme kinetic parameters [3].

Compound Screening with Enthalpy Arrays

Like other calorimetric techniques [4-6], the measurements are label-free and solution based, enabling measurement of both binding [2] and enzymatic reactions [3]. This provides advantages in compound screening, as no immobilization of the target or tagging of a competitor ligand or enzyme substrate is required, leading to a significant reduction in assay development time compared to fluorescence or surface plasmon resonance techniques.

Enzymatic reactions are performed in a continuous assay, allowing one to obtain both the kcat and KM in a single measurement that takes only a few minutes to perform. In addition, the KI for competitive inhibitors can be determined from a single measurement down to 130 nM (fig. 2a) [3].
The signal for an enzymatic reaction is proportional to ΔHapp*kcat*[E]0. For reactions with a low turnover number (kcat), the concentration of enzyme can be increased to improve the signal, but this places limits on the quantitation of K, for potent inhibitors (fig. 2b) [3]. Producing a detectable signal in an enzymatic reaction is dependent on the thermal dissipation time constant of the technology, placing the lower limit for detection of an enzymatic reaction at a kcat > 0.5 s-1 [2]. Conventional microcalorimeters, with larger sample volumes (0.2 to 1.4 ml) and longer time constants, allow enzymatic reactions with lower turnover number to be measured [4-6], but at significantly lower throughput than the enthalpy array, making it impractical to screen a large number of compounds with that technology.
Enthalpy arrays provide a label-free, solution based method to measure enzyme activity in an array format, providing an opportunity to perform activity screening of compounds in new ways. Provided the reaction has sufficient enthalpy and turnover number to produce a measurable signal, the kinetic parameters and inhibition constants for competitive inhibitors can be obtained from single samples.
Since compound libraries are routinely stored in DMSO, it is important that the technology be compatible with performing measurements in the presence of DMSO. The mixing of two solutions of differing DMSO concentration will produce a heat signal, which poses a challenge for calorimetric measurements. With an extended enzymatic reaction, the matching of DMSO is important, but the mixing of samples of variable DMSO produces a transient signal which can be excluded from the data analysis if the signal from the enzymatic reaction is significantly longer than the characteristic time for thermal dissipation (fig. 2b).

Summary

Enthalpy arrays are a label-free, solution based technology for measuring the thermodynamics of molecular interactions and kinetics of enzymatic reactions, allowing quantification of inhibitor constants over a large range in KI. Enthalpy arrays should prove useful in drug discovery for hit validation and lead characterization of enzyme inhibitors.

References
[1] Torres F. et al.:Proc. Natl. Acad. Sci. USA 101, 9517-9522 (2004)
[2] Recht M.I. et al.: Anal. Biochem. 377, 33-39 (2008)
[3] Recht M.I. et al.: Anal. Biochem. 388, 204-212 (2009)
[4] Todd M. J. and Gomez J.: Anal. Biochem. 296, 179-187 (2001)
[5] Bianconi M.L. et al.: Biophys. Chem. 126, 59-64 (2007)
[6] Olsen S.N.: Thermochim. Acta 448, 12-18 (2006)

 

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