Book Excerpt: Calorimetric Evaluation of Molecular Interactions Using Isothermal Titration Calorimeters (ITC)

Experimental Medicine extra edition “Perfect Interaction Analysis for Drug Discovery” (Yodosha, a specialized publisher in life sciences and medicine) Chapter 1 Standard of Interaction Analysis in Drug Discovery – I Low to Mid Molecular Drug Discovery – 5 (By Akira Nagatoishi and Kohei Tsumoto), we introduce an article related to ITC. This article is reprinted with the permission of Yodosha.

Calorimetric Evaluation of Molecular Interactions Using Isothermal Titration Calorimeters (ITC)

Akira Nagatoishi, Kohei Tsumoto

Purpose and Key Points of the Experiment

The isothermal titration calorimeter (ITC) detects heat changes involved in molecular interactions. By evaluating the heat changes involved in molecular interactions, one can directly gain insight into the thermodynamic quality of non-covalent bonding related to such interactions. For example, it can provide information on binding patterns and specificity of seed and hit compounds selected by screening. Further, in structure optimization, ITC can also precisely analyze whether designed interactions are occurring thermodynamically, allowing strategies to be reviewed and subsequent designs to be developed effectively by integrating structure information.

Introduction

The specific interaction of small molecules binding to proteins in small molecule drug development is crucial. Therefore, gathering specific insights at every stage, including exploration, validation, and optimization, is important. Non-covalent bonding formation between molecules, protein’s three-dimensional structure or conformation changes, and hydration state changes due to solution presence contribute to exothermic and endothermic reactions. Thus, thermal measurement is a crucial indicator in target molecular interaction analysis. The isothermal titration calorimeter (ITC) is the only analytical method that allows observation of the heat changes involved in desired interactions, enabling precise thermodynamic analysis. Refer to Chapter 1-13 for basic ITC use.

Optimization: A hit is confirmed by validating the specificity for target proteins, leading from the hit compound to enhancing binding affinity and functional activity by adding/modifying the skeleton and functional groups. These structural developments are called optimizations.

In optimizing small molecule drugs, we introduce important retrospective studies on the utility of thermodynamic insights. The thermodynamic parameters for HIV protease inhibitors, which are AIDS treatment drugs, targeting proteins are summarized in Fig. 1A^1,2. In this figure, inhibitors developed within ten years from the initially approved IDV in 1996 are listed in approved order. With new inhibitors being approved, trends show higher binding affinity with target proteins (ΔG becoming more negatively significant). Noteworthy is their thermodynamic profile (Fig. 1B).

Initial drugs (like IDV) were entropy-driven with unfavorable binding enthalpy (ΔH) and favorable binding entropy (ΔS). However, as drugs are optimized, the balance of thermodynamic parameters shifts from entropy-driven to enthalpy-driven (Fig. 1B). In initial inhibitors, the contribution of entropy is significant, while newly approved inhibitors rely more on enthalpy. It is also seen that these inhibitors have a ΔH−ΔS compensation correlation. Similar trends are observed in hypercholesterolemia drugs such as statins and osteoporosis treatment bisphosphonate compounds^3,4. These results suggest that high specificity with target proteins, i.e., contributions from both enthalpy-driven and entropy-driven factors, are crucial factors in drug optimization. In structural optimization to enhance the potency, decoding the thermodynamic contribution of functional groups is very useful in understanding the quality of molecular targeted drugs. While it is not easy to derive generalities about which functional groups contribute to enthalpy and which to entropy, verifying thermodynamic parameters and rationally designing drugs provides key information for creating specificity in small molecule designs for target proteins.

Preparation

For basic preparation methods, refer to Chapter 1-13. Extreme care must be taken to ensure the concentration is consistent when using compounds, often containing DMSO.

Protocol

For basic operations and experimental procedures, refer to Chapter 1-13. Generally, it’s recommended to set protein on the cell side and compound on the syringe side.

Troubleshooting

Be cautious of heat changes due to DMSO
When using compounds, the assay system often contains DMSO, and we observe unexpectedly large reaction or dilution heat. Similar to SPR, DMSO affects interaction-related reaction heat in ITC. Separate sample preparations for the cell side and syringe side are recommended to avoid DMSO concentration shifts. Minor DMSO concentration errors may manifest as dilution heat, hiding desired interaction heat. It’s crucial to conduct a dilution heat measurement of the compound only and prepare samples to minimize heat changes from DMSO.

Example Experiment 1: Exploration Using Competitive Assays with ITC Measurements^5）

A comprehensive chemical space exploration of protein surfaces is an efficient and effective screening method in discovering small molecule compounds that specifically bind to drug discovery proteins. Highlighted here is a drug discovery strategy using a fragment library with an average molecular weight smaller than general compound libraries, below 300 Da. However, since fragment compounds generally have simple chemical structures, their binding affinity to proteins is low, with dissociation constants sometimes in the mM range. Therefore, ITC becomes an effective evaluation method due to its high sensitivity and physicochemical analysis approach. As with the mentioned example of HIV protease inhibitors, specific binding often shows significant exothermic reactions and convergence of the reaction. Hydrogen bond formation is expected in such profiles, while non-specific interactions may not converge, and hydrophobic interactions may show endothermic reactions. Hence, selecting compounds showing exothermic reactions as strong candidates within hit compounds is a desirable strategy. When a binding site is clear on the target protein, and substrates or low molecular ligands binding to that site are known, competitive assays are an effective strategy. By using ITC in competitive assays, site-specific and exothermic hit compounds can be selected (Fig. 2). Below, we demonstrate examples practiced by the authors.

1. Target Protein

The target was keto-steroid isomerase (KSI), as well as 3-oxo- Δ5 keto-steroid isomerase, an enzyme that catalyzes isomerizing 3-oxo- Δ5 keto-steroids to hormonally active conjugated isomers.

2. Small Molecule Exploration

Compounds were selected from a fragment library via SPR screening for KSI.

3. ITC Competitive Assay (SITE)

In the competitive assay via ITC of hit candidate compounds obtained from screening, we conducted effective hit validation. This analysis technique adds known ligand molecules in solution with the target protein and screening compounds together (Fig. 2A). When a fragment compound interacts with the designated binding site on KSI, a higher affinity known ligand (deoxycholate: DOC) used as a positive control compound is added to displace the fragment compound and stabilize the site. When fragment compounds cause interactions with exothermic reactions, the displacement by DOC manifests as an endothermic reaction.

To increase throughput, we devised the SITE method (single-injection thermal extinction), allowing evaluation of this heat change competitive assay in a single injection. Using this SITE method, we validated fragment compound binding hits for KSI, resulting in acquiring an enthalpy-driven hit compound that competes with KSI substrate DOC and shows significant exothermic reactions (Fig. 2B). Additionally, for protein kinase ERK2, involved in the intracellular MAPK signaling cascade pathway, we similarly succeeded in capturing compounds from fragment screening.

Thus, it is clear that ITC shows its power not only in drug optimization but also in the exploration phase by evaluating heat reactions. General exothermic reactions are considered to create specificity through non-covalent bonds like hydrogen bonds. However, rationally designing hydrogen bonds is relatively challenging. In such cases, discovering low molecular compounds with hydrogen bond formation at the early stages of drug design and synthetically developing them can be a useful method leading to high-quality lead compounds.

Example Experiment 2: ITC Analysis in Small Molecule Optimization^6）

1. Target Protein

The target was the protein DJ-1, noted for its deep involvement in Parkinson’s disease and cancer (Fig. 3A). DJ-1 is known as an enzyme exhibiting glyoxal activity, yet many unclear points remain about its link between this activity and disease. Our research aimed to clarify the relationship between DJ-1 enzymatic activity and disease by capturing compounds showing inhibition activity against DJ-1, expected to bridge towards new treatments.

2. Small Molecule Exploration

Several hit candidate compounds were selected via SPR screening. Among these, an Isatin scaffold-containing compound was selected as a promising hit candidate (Fig. 3B).

3. Hit Validation (ITC)

Following ITC measurements on hit candidate compounds, Isatin showed an enthalpy-driven binding mode with DJ-1, demonstrating an exothermic reaction (ΔH = -11.6 kcal/mol, –TΔS = 4.1 kcal/mol, K_D = 3.2 μM) (Fig. 3C). –TΔS is calculated based on ΔG = ΔH – TΔS, and K_D is derived from K_A = 1/K_D. Additionally, DJ-1’s thermal stability analysis via differential scanning fluorimetry (DSF) showed that Isatin enhanced DJ-1’s thermal stability.

4. Structural Analysis

Unique complex structures were obtained showing Isatin forming covalent and non-covalent bonds with several amino acid side chains to interact with DJ-1 (Fig. 4A, B). Based on this structural information, mutational analysis suggested that Isatin similarly interacted at the same binding site in solution as in the crystal structures (Fig. 4C).

5. Compound Optimization

Based on this complex structural information, affinity enhancement trials were pursued. This led to Compound #15 demonstrating nM-order binding affinity (Fig. 5). Compound #15 contributed to entropy (ΔH = -11.5 kcal/mol, –TΔS = 2.0 kcal/mol, K_D = 0.1 μM). This compound also improved stability for DJ-1. Isatin and Compound #15 were tested for inhibition activity in cells, with both revealing inhibition of intracellular glyoxal activity. Interestingly, both these compounds were shown to significantly enhance DJ-1’s thermal stability in cells using cellular thermal shift assay (CETSA). These results demonstrate ITC’s ability to select enthalpy-driven compounds creating specificity, and show structural optimization’s contribution to structural complementarity, with entropy contributing. Such ITC approaches are effective for selecting compounds that act specifically on targets against intracellular target proteins.

Conclusion

This section introduces research examples related to the development of small molecule pharmaceuticals utilizing thermal measurement. In small molecule inhibitor exploration, high biological activity, inhibition activity, and high binding activity are generally used as selection indicators. However, advancing while unclear about the interaction scheme between target molecules and small molecule drugs frequently occurs, sometimes resulting in no lead design or bad structure-activity correlations in structural optimization. Furthermore, recent trends show that target molecules are not limited to enzymes and receptors but also include membrane proteins and intrinsically disordered proteins, which are biochemically difficult to handle, increasing difficulties in handling target molecules. When target molecules become hard to handle, existing drug exploration approaches and assay systems may not apply. Additionally, low molecular inhibitors may need to act on protein-protein interaction (PPI) interfaces or large protein complex interfaces, further increasing special requirements. To select compounds that bind specifically to target proteins under these circumstances, detection technologies capable of high-sensitivity and quantitative analysis of binding behavior are essential. In this environment of many challenges in molecular-targeted drug discovery, the most demanded aspect is creating high binding affinity and specificity against target molecules. High precision exploration and analysis technology is therefore necessary, and one example of the utility of ITC has been explained here. Though not covered in this section, other practical examples of ITC usage in small molecule inhibitors’ exploration include compounds selected via small molecule inhibitor screening for malaria infection and cancer target serine metabolism enzyme (SHMT), highlighted in recent years, revealing two types of hit compounds being enthalpy-driven and entropy-driven respectively7). ITC’s thermal profiles could be a useful method in understanding hit compound specificity and rational molecular design in structural optimization.

◆ References
1) Ohtaka H & Freire E：Prog Biophys Mol Biol, 88：193-208, 2005
2) Muzammil S, et al：J Virol, 81：5144-5154, 2007
3) Carbonell T & Freire E：Biochemistry, 44：11741-11748, 2005
4) Kawasaki Y, et al：Chem Pharm Bull (Tokyo), 62：77-83, 2014
5) Kobe A, et al：J Med Chem, 56：2155-2159, 2013
6) Tashiro S, et al：ACS Chem Biol, 13：2783-2793, 2018
7) Nonaka H, et al：Nat Commun, 10：876, 2019

Malvern Panalytical’s Isothermal Titration Calorimeter (ITC)
Click here for the MicroCal ITC series