Calorimetry is used to measure enthalpic changes, including enthalpic changes arising from reactions, phase changes, changes in molecular conformation, temperature variations, and other variations of interest that may occur for a particular specimen. By measuring enthalpic changes over a series of conditions, other thermodynamic variables may be deduced. For example, measurements of enthalpy as a function of temperature reveal the heat capacity of a specimen, and titrations of reacting components can be used to deduce the equilibrium constant and effective stoichiometry for a reaction. Calorimetry measurements are useful in a broad variety of applications, including, for example, pharmaceuticals (drug discovery, decomposition reactions, crystallization measurements), biology (cell metabolism, drug interactions, fermentation, photosynthesis), catalysts (biological, organic, or inorganic), electrochemical reactions (such as in batteries or fuel cells), and polymer synthesis and characterization, to name a few. In general, calorimetry measurements can be useful in the discovery and development of new chemicals and materials of many types, as well as in the monitoring of chemical processes. It is understood that an enthalpy array may be considered an array of calorimeters (e.g., nanocalorimeters).
Calorimeters and enthalpy arrays can, therefore, be used to screen for substrates, cofactors, activators, and inhibitors of enzymes, including at the proteome level, and can also be used to quantify the enzymatic kinetics. Calorimeters/enthalpy arrays detect the amount of heat evolved from an enzymatic reaction. The heat evolved depends on the enthalpy of the reaction, enzyme concentration, substrate concentrations, the presence of inhibitors, activators, or cofactors, values for the kinetic parameters for the reaction of interest, buffer conditions, as well as various other factors and parameters. In particular, the concentrations of the enzyme, one or more substrates, and/or regulators (e.g. agonists, inhibitors, and inverse agonists) are often a limiting factor in analyzing enzymatic reactions by detecting the enthalpy of reaction. Specifically, it is often necessary to use low concentrations of enzyme and/or substrates when examining enzymatic reactions, yet at the low concentrations of interest, the amount of heat evolved is low.
One benefit of low concentration studies is that the use of smaller concentrations provides for more selective reactions. Consider, for example, the study of a binding reaction with a dissociation constant Kd:
            A      +      B        ->    C              K      d        =                            [          A          ]                ⁡                  [          B          ]                            [        C        ]            In this reaction, A and B bind to form the complex C, and the dissociation constant is written in terms of concentrations denoted by square brackets. This equation assumes ideal solution behavior, but it is sufficient for the purposes herein. In testing for binding, it is often desired to obtain an indication of the magnitude of Kd. In many biochemical studies, including drug screening and development studies and proteome-wide investigations of protein-protein interactions, among others, Kd values of interest are typically <1-10 μM, and values from 1-1000 nM—and especially <100 nM—are not uncommon and often of particular interest. In order to measure Kd, the reaction must be studied at concentrations that are not too distant from the value of Kd. At the upper end of this range, titrations may be performed at concentrations of 10 to 100 times Kd, but titrations at concentrations near the value of Kd are preferred when possible. Thus, there is a benefit to performing studies at as low a concentration as possible. In particular, there is a benefit to being able to perform studies at concentrations as low as 10−6 to 10−7 M. Likewise, it is a benefit to be able to measure kinetics of enzymatic reactions at low concentrations, including enzymatic reactions with slow turnover rates.
It is also useful to work with substrates at low concentrations. In analyzing enzymatic reactions, benefits arise from determining the Michaelis constant, Km, which provides an indication of the substrate concentration at which an enzyme is most effective at increasing the rate of reaction. If the concentration of the substrate needs to be increased beyond the Km for that substrate, it then becomes difficult or impossible to accurately determine Km using calorimetry. Measuring reactions at lower concentrations allows one to differentiate, e.g. strong binding from weaker binding, or larger Km in the case of enzymatic reactions from smaller Km, etc.
Likewise, in drug screening studies, reaction information at lower concentrations is often necessary to determine binding constants, for example, or to differentiate strongly binding hits in drug screening campaigns from hits that do not bind as strongly. The concentrations used in the experiments will then set the lower limits of reactions that are being distinguished. It is therefore advantageous to work at low concentrations in a variety of areas, including but not limited to drug screening and biochemical research.
In addition to the needs for using low concentrations, it is also important in many studies to use as small a volume of materials as possible, since the combination of small volume and low concentration means small amounts of the materials of interest. The amount of sample, e.g. purified enzyme or other protein, is often limited, and making the samples is often labor intensive and costly. Additionally, the quantity of desired experiments may require the use of low concentrations and small volumes. In drug screening or proteome scale experiments, for example, researchers may be running anywhere from 1,000 to 100,000 or more different experiments. Also, one may desire to study a natural extract or synthesized compound for biological interactions, but in some cases the available amount of material at concentrations large enough for calorimetry might be no more than a few milliliters. The use of enzymes and/or substrates in such studies can only be kept within a tolerable range if sample concentrations and volumes are kept as low as possible. In particular, performing such studies is not feasible using sample sizes of about 1 ml, as required for measurement using commercially available microcalorimeters such as products sold, for example, by MicroCal® Inc. (model VP-ITC) or Calorimetry Sciences Corporation® (model CSC4500).
In certain situations, the enzymatic reactions of interest, at the low concentrations and small volumes required by many studies, may not generate a sufficient amount of heat to be reliably detected by calorimetric methods. Thus, using small quantities of materials may not yield a signal and prevents researchers from monitoring the reactions. It would, therefore, be advantageous to provide a method for monitoring enzymatic reactions using calorimetric measurements and/or enthalpy arrays that allows researchers to operate and perform experiments within the boundaries in which they are required to work, including limitations in concentration and sample volume. In that regard, it would be a further advantage to provide a method for monitoring enzymatic reaction via, calorimetry and/or enthalpy arrays using low concentrations and small volumes of enzymes, substrates, activators, regulators, inhibitors and/or co-factors.
Binding of ligands, such as agonists, inverse agonists, or inhibitors, to GPCRs (G-protein coupled receptors) is an example of a reaction that may not produce a sufficient amount of heat to be reliably detected using a desired calorimetric method. It may be desirable in drug screening applications to test for binding of library compounds to GPCRs by high throughput calorimetry, using arrays of calorimeters such as those described in, U.S. application Ser. No. 10/114,611, filed Apr. 1, 2002, titled “Apparatus and Method for a Nanocalorimeter for Detecting Chemical Reactions”; U.S. Pat. Nos. 6,380,605 B1 and 6,545,334 B2, each entitled, “Device and a Method for Thermal Sensing”, to Verhaegen; U.S. Pat. No. 6,193,413, entitled, “System and Method for an Improved Calorimeter for Determining Thermodynamic Properties of Chemical and Biological Reactions”, to Lieberman; Johannessen, et al., Applied Physics Letters, vol 80(11):2029-2031; Johannessen, et al., Analytical Chemistry A 2002 May 1; 74(9):2190-7; K. Verhaegen, et al., Sensors and Actuators 82(2000):186-190; and F. Hellman, NSF Award Abstract—#9513629 AWSFL008-DS3: “Development of Instrumentation for Microcalorimetry of Biological Systems”. However, preparing samples with a high enough GPCR concentration to yield a detectable signal in such systems can be difficult or even impractical, in part because the cost of expressing GPCRs in the desired amounts is large and in part because GPCRs are membrane proteins. Membrane proteins can be provided in suspensions of cell membranes, membrane fragments, vesicles, or micelles, but they are only a part of the membranes, fragments, vesicles, or micelles, which makes increasing the concentration more challenging than for soluble proteins.
GPCRs are one of the most important classes of protein targets for the pharmaceutical industry. GPCR's are membrane proteins involved in signaling cells based on extracellular signaling molecules, and approximately 50% of the top 200 drugs currently on the market target GPCRs. Today, GPCRs with known ligands are screened by measuring how test compounds affect the behavior of the GPCR, using assays that include the known ligand. Accordingly, traditional drug screening for GPCR targets has relied on the identification of small molecules that interfere with binding of a know ligand to the GPCR, as described in “The Use of Constitutively Active GPCRS in Drug Discovery and Functional Genomics”, Derek T. Chalmers & Dominic P. Behan, Nature Reviews Drug Discovery 1, 599-608 (2002). The situation for orphan GPCRs, which do not have known ligands, is more complicated. In an article on assays for ligand regulation of orphan GPCRs (Drug Discovery Today vol. 8, No. 13, July 2003, pp. 579-585) by G. Milligan, the author states that efforts to identify ligands that interact with orphan GPCRs have shifted from ligand-binding assays to functional assays because it is currently impossible to perform binding assays in the absence of a known ligand. Methods for screening for ligands of orphan GPCRs use cellular assays that involve fluorescent tagging, hypotheses about cellular behavior of the GPCRs, and possibly modifying the GPCR itself, e.g. to make it constitutively active. Examples include AequoScreen™ technology from Euroscreen, which is based on using fluorescent reporters of Ca+2 activity in cellular assays, Ca+2 changes being a common result of GPCR activity, and CART Constitutively Activating Receptor Technology (CART), described in an article in, “Nature Reviews Drug Discovery 1, 599-608 (2002); “The Use of Constitutively Active GPCRS in Drug Discovery and Functional Genomics”, by Chalmer and Behan, which is based on modifying GPCRs to make them constitutively active and using these GPCRs in cellular assays. These methods do not directly detect ligand binding to a GPCR or the associated G-protein activity. A method that directly probes ligand binding to a GPCR without requiring an assay based on a previously known ligand would be very useful. In particular, a calorimetric method would be very useful, especially if it could be used with arrays of calorimeters that enable parallel measurements at appropriate concentrations and with small amounts of material for each measurement.
Similarly, calorimetric studies of kinases, proteases, ion channels, phosphatases, metabolic enzymes, nucleic acid binding and modifying enzymes, transcription/translation factors, signaling enzymes, protein modifying enzymes, transport/trafficking enzymes, and orphan enzymes are all of potential benefit in biological research and drug discovery activities, especially if they could be performed with arrays of calorimeters that enable parallel measurements at appropriate concentrations and with small amounts of material for each measurement.