As our knowledge of the genetic and molecular basis of disease continues to grow, drug development programs focus increasingly on those proteins associated with disease states. Successfully identifying and developing therapeutic molecules to target these proteins and their activities requires a detailed understanding of the underlying biological pathways by which they are created and in which they are involved.
Examination of the biomolecular interactions that occur in both normal and disease-compromised individuals, and those interactions that result from the action of potential therapeutic molecules, requires sensitive analytical techniques. These must be coupled with appropriate and effective experimental methodologies. Isothermal titration calorimetry (ITC) delivers comprehensive information on a wide range of biomolecular interactions, and has become an essential tool in drug discovery and in the study and regulation of protein interactions with both small and large therapeutic molecules. This article explores how ITC supports the elucidation of biological interactions and the mechanisms associated with different diseases, and its central role in associated drug discovery processes.
Focus on proteins in drug discovery
Gaining an essential understanding of the biological pathways that determine a cell’s activities, and how and where defects in these pathways can result in certain types of disease, is a critical early step in targeted drug development. The pivotal roles of proteins may be summed up in the statement: “Almost all approved drugs on the market today are directed towards protein targets.”1 While many proteins involved in cellular processes will become potential drug targets, other proteins and their variants are themselves promising therapeutic candidates.
In both fundamental research and drug development, investigators employ a range of biochemical and biophysical techniques to characterize the interactions of proteins with one another and also with potential inhibitors. Among these, ITC is regarded as the “gold standard” method for understanding binding processes and is also widely used in studying enzyme kinetics.
Background to ITC
Isothermal titration calorimetry sensitively and reproducibly measures the heat evolved or absorbed when complexes are formed between molecules, providing detailed information on the binding affinity and thermodynamics of biomolecular interactions. It determines the thermodynamic properties that explain why interactions occur and provides data that reveal the forces driving the formation of complexes, helping describe function and mechanisms at the molecular level. When combined with structural information, ITC data provide deeper insights into structure–function relationships and the mechanisms of binding.
ITC is the only technique that can simultaneously determine all binding parameters in a single experiment. Furthermore, since it needs no modification of binding partners, either with fluorescent tags or through immobilization, ITC measures the affinity of binding partners in their native states. The data generated enable the accurate determination of binding constants (KD), reaction stoichiometry (n), enthalpy (ΔH), and entropy (ΔS), providing a complete thermodynamic profile of the molecular interaction.
When binding occurs, heat is either absorbed or released, and this is measured by the sensitive calorimeter during progressive titration of the ligand into the sample cell containing the biomolecule of interest. ITC instrumentation, general measurement principles, and data analysis processes are described in detail elsewhere,2 and methodology descriptions in this article are confined to those that are specific to the application examples outlined.
ITC in action
1. Applying ITC to the study of epigenetic interactions
According to Professor Andreas Ladurner, Chair of the Department of Physiological Chemistry at Ludwig-Maximilians-Universitӓt in Munich, and a scientist responsible for groundbreaking studies on bromodomains, ITC was a crucial tool for pioneers in the field of epigenetics and continues to be the workhorse of research laboratories today.3 Epigenetics refers to the study of heritable changes in gene expression that are caused by nongenetic mechanisms, with no alteration in either gene structure or DNA sequence. Each cell contains essentially the same genetic code, but epigenetic mechanisms permit the specialization of cell function. Thus, epigenetic modifications may simply manifest as the normal cellular differentiation that produces, for example, skin cells, liver cells, or brain cells. On the other hand, the role of epigenetic mechanisms in integrating environmental responses at the cellular level means they may also be important in disease development. Consequently, the proteins and processes that create these heritable changes are being actively characterized and are the focus of much drug discovery effort.
While epigenetic regulation of gene activity is not completely understood, it is known to involve the modification of chromatin, the protein–DNA–RNA complex that exists in eukaryotic cells. In simple terms, histones (alkaline proteins) package and order DNA to form nucleosomes; multiple nucleosomes then pack together to form chromatin. Epigenetic events may involve covalent modification of histones, DNA and RNA, chromatin remodeling, micro-RNA mediation, and other changes to chromatin structure.
The N-terminal tails of histones contain site-specific post-translational modifications (PTMs) known as marks. These include methylation, acetylation, and phosphorylation of particular amino acid residues. The state of chromatin and access to the genetic code are largely regulated by these specific modifications to the histone proteins and the subsequent recognition of these marks by other proteins and protein complexes. PTMs on the histone tails form patterns and are added, read, and removed by specific enzymes, resulting in more marks. The presence or absence of marks on a histone tail creates a unique docking module for the binding or release of downstream effector proteins and thus influences a range of functions, including transcription regulation, cell cycle control, differentiation, and apoptosis. In addition to histone tail modifications, another common epigenetic mark, which may have a role in gene silencing, is DNA methylation at the 5 position of the cytosine base.
Mediating these modifications are families of proteins that comprise “writers” to carry out chemical modifications, “readers” that dock to marks on the histone tail, and “erasers” whose role is to remove the marks. For each possible mark, there is a corresponding family of writer, reader, and eraser protein. Epigenetics has evolved to focus on multiple-domain multiple-mark interactions, and has revealed that there are hundreds of proteins involved in epigenetic regulation that are potential targets for drug discovery. For example, the enzymes that catalyze histone PTMs are considered druggable targets, and studies are also underway on drug molecules that bind reader proteins such as bromodomains.
The analysis challenge
Chromatin binding and modification activities are often found in multidomain and multiprotein complexes that have multiple biological and catalytic functions. Gaining a complete understanding of function, activity, and specificity requires the definition of each interaction using a series of biochemical and biophysical assays. While enzymatic assays are useful for measuring the activity of many writer and eraser proteins, the absence of enzyme activity in readers, for example, makes it necessary to examine binding interactions for a variety of molecules using other means.
Benefits of ITC
To completely characterize the structure, function, and activity of epigenetic proteins, it is necessary to establish a clear interaction between an isolated specific module or domain and its mark. It is also important to understand the interactions between the different protein modules of the multidomain, multiprotein complexes. The strength of the binding interaction is determined from precise measurements of the dissociation constant, KD, of the reaction— this is a concentration measurement, and the smaller the value, the tighter the binding affinity. KD is a critical parameter for characterizing and comparing different binding modules and marks, and there is a requirement for measurement from the nanomolar range applicable for drug interactions, through the millimolar range for weak binders.
While various techniques are used in epigenetics for high-throughput screening of binding reactions, reproducible and quantitative methods are required for confirmation and further work. Biophysical assays tend to be the most reliably quantitative. Of these, ITC is the most widely used in both primary and confirmatory testing and is the gold standard assay for determining KD. Applications in epigenetics range from characterizing protein–cofactor, protein–protein, and protein–nucleic acid interactions, for example, through the development of small-molecule inhibitors for drug discovery and development. In combination with structural information and in vivo studies, ITC data play a vital role in understanding complex epigenetic interactions, and in subsequent rational drug design to create specific inhibitors. See Refs. 5 and 6 for examples of current research and citations.
2. Applying ITC in enzyme kinetics and peptide substrate identification
Peptidases involved in metabolic processes are another increasingly important group of drug targets. With a mission to study peptidases from a clinical perspective, researchers at the University of Antwerp’s Laboratory of Medical Biochemistry are involved in the investigation of the roles of these enzymes in health and disease, evaluating them as drug targets and disease markers. Their work focuses particularly on proline-specific peptidases and basic carboxypeptidases.
The analysis challenge
Prolyl carboxypeptidase (PRCP) is a lysosomal enzyme that cleaves off a C-terminal amino acid residue when proline or alanine are in the penultimate position. It has a role in body weight control due to its ability to cleave an anorexigenic peptide (α-MSH-1-13) into its inactive form, and as such there is considerable interest in discovering novel PRCP substrates. However, the search for novel peptide substrates for proline-specific peptidases has often been held back by a lack of suitable enzyme assays. Existing technologies may be too expensive, too labor-intensive, or require derivatization and separation of the reaction products. In addition, conventional approaches to measuring enzyme kinetics may fail to reflect activity under native conditions, may be nonquantitative, lengthy to perform, require high levels of expertise, or exert a heavy demand in terms of quantity for enzymes and peptides.
A recent presentation7 describes work undertaken at the Laboratory of Medical Biochemistry to validate the use of ITC in studying enzyme kinetics, by comparing results with the more conventional methods of MALDI-TOF mass spectrometry and reversed-phase HPLC (RPHPLC).
Benefits of ITC
Underpinning the use of ITC for the study of enzyme kinetics is the relationship between reaction rate and heat generated as a function of time. In order to generate the required data, two sets of experiments were carried out, the first to determine the reaction enthalpy, and the second to determine the rate of heat exchange at different substrate concentrations.
Ahead of the ITC experiments, the ideal concentrations of enzyme and peptide were determined using MATLAB simulation software, drawing on published kinetic parameters for PRCP. Spectroscopic methods were used to accurately quantify the molar concentration of both the enzyme and substrates, and reactants were made up in sodium acetate buffer (pH 5.0) to reflect the lysosomal nature of the enzyme and its acidic pH optimum. The substrates tested with PRCP were angiotensin III and (Pyr)-apelin-13. Multiple injection measurements were performed and proprietary software was used for Michaelis-Menten fitting to give kinetic parameters.
The ITC results for the hydrolysis of angiotensin III compared well with those from RPHPLC, indicating the utility of ITC in studying enzyme kinetics in this system. Furthermore, the kinetic parameters of (Pyr)-apelin-13 hydrolysis were comparable with those of angiotensin III, a known substrate for PRCP, identifying (Pyr)-apelin-13 as a novel substrate for the enzyme. The team concluded that ITC is a very useful tool to study enzyme kinetics in a system of interest and has advantages over other techniques in terms of being easy to use, quicker, and being able to make measurements in a label-free environment.
3. Applying ITC in the development of therapeutic enzymes
ITC is being used by the team at Swedish Orphan Biovitrum (Sobi), based at the Karolinska Institutet in Stockholm, to support the development of therapeutic enzymes for the treatment of lysosomal storage diseases (LSDs).7 ITC methods developed at Sobi have been used to characterize enzyme batches and to establish structure–activity relationships supporting the development of the enzyme as a therapeutic molecule.
LSDs are monogenic inherited diseases and are caused by a protein deficiency that halts the processing of a specific metabolite, leading to its storage and resulting in cell death, tissue atrophy, and severe pathology in affected organs. Different tissues are affected by different LSDs. Enzyme replacement therapies, whereby a recombinant functional enzyme is administered through infusion, are proving promising. The enzyme is taken up by cells and transported to the lysosome, where function is restored. For such therapies, enzyme activity is the cardinal quality attribute in the release specification of the drug, but assay development is extremely challenging because of the complexity of the endogenous enzyme substrates. A surrogate substrate is normally required and must be able to provide the same information as the endogenous substrate about the quality of the batch of drug produced. Lysosomal enzymes that degrade heparan sulfate provide a good example of the work at Sobi.
Heparan sulfate is a linear polysaccharide found in all animal tissues. It occurs as a proteoglycan in which two or three heparan sulfate chains are attached in close proximity to the cell surface or extracellular matrix proteins, and is involved in regulating a wide variety of biological activities. Heparan sulfate degradation in the lysosome is sequential, with different enzymes acting at various points. If any enzymes are missing, are present only in low amounts, or have low activity, then storage of metabolite material will result. The different steps affected are related to different LSDs.
The aim of the Sobi team was several-fold: to develop assays orthogonal to conventional methods, to increase their ability to examine endogenous substrates, to perform full characterization of enzyme kinetics, and to develop methods applicable to more than one enzyme in the pathway. Potential substrates investigated included surrogates containing a fluorophore, well-defined small endogenous heparan sulfate fragments, and complex mixtures of heparan sulfate.
Benefits of ITC
While conventional (non-ITC) assay development has generally involved multiple sequential and time-consuming steps, assay development with ITC proved to be straightforward. Using a simple endogenous tetrasaccharide substrate, for example, setup was completed in just one day. The sequence of setup experiments first checked the quality of the substrate by injecting the substrate in buffer alone and looking at the thermal power change. This was followed by determining the enthalpy change (ΔH) by injecting enzyme into substrate, and finally determining the reaction rate as a function of the substrate concentration.
Testing the quality of the substrate was a critical step. Using the tetrasaccharide, results showed that there was rapid thermal equilibration, no slow dissociation of aggregates, no disintegration of substrate, and no contaminating enzyme activities, thus confirming the integrity of the substrate (Figure 1).
Figure 1 – Testing the quality of the tetrasaccharide substrate.The second experiment measured the thermal power shift when substrate was injected into a relatively high concentration of enzyme. The high concentration of the enzyme reflected the desire to have complete conversion of the substrate to the product in an acceptable timeframe. The area under the curve in Figure 2 corresponds with the total heat generated during the experiment, from which ΔH is determined. Thermal power is then related to the conversion of substrate to product. Multiple injection experiments at this stage were shown to aid more accurate enthalpy determination as well as helping to gain a rough estimate of the kinetic parameters and give an idea of the amount of product inhibition takin place during the reaction.
Figure 2 – Determining ΔH using the tetrasaccharide substrate.The final experiment, which provided the key information being sought, determined reaction rate as a function of substrate concentration. Here it was necessary to find an enzyme concentration that enabled determination of a steady state rate at several consecutive injections of substrate. This is possible either using simulations with data from the previous experiments, or simply by testing different enzyme concentrations.
Testing of two different enzyme concentrations for the tetrasaccharide substrate generated the data shown in Figure 3. Close examination of the traces led to the conclusion that an enzyme concentration of 8 nM produced steady-state conditions throughout the measurement period and that this was a sufficiently high concentration to use.
Figure 3 – Two concentrations of enzyme were tested in multiple injections for the tetrasaccharide substrate.Based on these measurements, the reaction rates were extracted and the substrate concentrations after each injection calculated, resulting in the curve shown in Figure 4, which corresponds well to the Michaelis-Menten curve.
Figure 4 – Kinetic profiles using a tetrasaccharide substrate.These experiments demonstrated to the researchers that they could generate a complete kinetic profile using the endogenous substrate in a single experiment, with an assay time of 15 minutes and a cycle time of less than 40 minutes per sample.
Similar experiments were conducted with surrogate substrates and more complex substrates, including a polydisperse high-molecular-weight fragment of heparin. While both these categories of substrate presented initial challenges, further experimentation resulted in the development of acceptable protocols. For example, ITC measurements with the surrogate substrate revealed a lag phase with no activity identified for the first six injections. Adhesion of the surrogate substrate to the sample chamber was confirmed when the addition of detergent resolved the problem. In contrast, the heparin substrate showed a large heat effect and slow processing upon injection into buffer, probably due to the polydispersity of the substrate, with entanglement of the polymers at high concentration and subsequent slow disentanglement upon dilution. Reducing the concentration of heparin in the syringe produced an acceptable profile for substrate into buffer.
Overall conclusions from this initial ITC work were that the activity of enzyme preparations could be assessed using several different relevant substrates and that the results correlated well with those from other assay formats already in use.
ITC was seen to have the major benefit over traditional assays of being label-free, removing the need to factor in the effects of labeling when performing assay development. The ability to use endogenous substrates is important in the work described, as is the ability to use complex substrates. Assay development proved to be straightforward with simple changes to parameters enabling real-time development. The ability to create a full kinetic profile in a single experiment was especially valuable.
Summary
ITC is a long-established technique that has many applications in drug discovery and development, and increasingly in understanding the biological pathways that allow identification of drug targets. The discussions of current research presented in this article highlight the central role that ITC plays in epigenetics, where it supports the identification and characterization of druggable targets, and in other areas of drug discovery that demand reliable techniques for sensitively and quantitatively profiling enzyme-substrate reactions. In the enzyme systems described, results compared favorably with other techniques. As a direct in-solution method that requires no immobilization or labeling and that offers straightforward method development, ITC delivers multiple advantages as a frontline technology or a truly orthogonal methodology.
References
- http://www.proteinatlas.org/humanproteome/druggable
- Ghai, R.; Falconer, R.J. et al. J. Mol. Recog. 2012, 25, 32–52; doi:10.1002/jmr.1167. Velazques-Campoy, A.; Leavitt, S.A. et al. Meth. Mol. Biol. 2015, 1278, 183–204; doi: 10.1007/978-1-4939-2425-7_11.
- https://www.youtube.com/watch?v=UTRBX75OQkQ
- Pande, V. J. Med. Chem. 2016, 59, 1299–1307; doi: 10.1021/acs.jmedchem.5b01507.
- https://www.malvern.com/en/support/resource-center/Whitepapers/WP160505-Epigenetics-ITC.html
- http://www.malvern.com/en/support/events-andtraining/ webinars/W160426DrugDiscoveryITC.aspx
- http://www.malvern.com/en/support/events-andtraining/webinars/W161026ITCEnzymes.aspx
Verna Frasca is field applications manager—Bioscience, Malvern Panalytical, Enigma Business Park, Grovewood Rd., Malvern, Worcestershire, WR14 1XZ, U.K.; tel.: +44 (0) 1684 892456; fax: +44 (0) 1684 892789; e-mail: [email protected]; www.malvern.com