Applying Microcalorimetry for Drug Discovery Process in Modern Pharmaceutical Industry

Drug discovery process is integral to today’s pharmaceutical sector, and thus a better understanding of the interactions between a drug and a target protein forms an essential part of this process.

In order to define the target protein as well as its interactions with inhibitors, complete and fully integrated biophysical and biochemical techniques have been developed.

With the help of these methods, various factors such as physical instability like surface denaturation, chemical instability such as deamination and oxidation, precipitation, soluble aggregation, chemical or proteolytic degradation, and post-translational modification can be detected easily.

A more improved characterization of the target protein provides a better understanding regarding the development of superior formulations, because the way the formulation components affect the stability of the target protein can be determined effectively.

The biophysical method is capable of studying the thermodynamic properties of a protein through DSC or ITC techniques, which are built on well-proven basic principles.

The ITC technique offers thermodynamic information that is not only utilized to validate the binding model, but also used to measure entropy (TΔS), binding enthalpy (ΔH), binding association constant Ka, and free energy (ΔG).

Widely used to interpret the folding and unfolding process of proteins, the DSC technique helps in determining the heat differences related to the protein’s thermal denaturation.

Compound binding renders the extent of stabilization, which is typically related to the affinity of the interaction. The thermodynamics related to unfolding, i.e., change of ΔH, ΔCp, and Tm provide critical data regarding the compound binding’s properties.

However, since the instrument has to be operated manually, large amounts of protein as well as prolonged operator time are required for both set of measurements. This means, these measurements cannot be applied at a wider level.

In order to overcome this issue, MicroCal developed two innovative instruments, namely the AutoDSC and Auto ITC which provide a number of benefits such as robotic automation, better sensitivity, reduced consumption of sample, and easy-to-use software packages.

These packages streamline the process of data analysis and experimental set-up. This article presents some instances of disciplines with respect to the discovery of small molecule drugs, and shows how the new instrument enhances the efficiency of different processes.

Recombinant protein construct design and expression for drug discovery at Exelixis

Today, DNA manipulation has become easier and is not a restricting factor, owing to significant advances in genome science. Recombinant protein construct design for expression is still not a predictable process.

Thus, in order to ensure that the target proteins are effectively expressed without involving many optimization rounds, a high throughput, parallel method is adopted in which 20 to 30 constructs are prepared concurrently for a specified protein.

While this is likely to provide a successful outcome, many experiments would need to be performed to ensure that the expression of all constructs is optimized, which further complicates the problem. The following section discusses a number of techniques that can be used to prioritize these constructs.

Prior to formulating the expression constructs, it would be helpful to look for literature standards, especially from the database of protein structure. Given that structural biology often needs protein in large amounts and of high quality, this approach serves as a good indicator for the viability of expression.

In the case of targets that are new or not studied properly, analysis of domains is carried out depending on the data acquired from structural modeling and in-house expression database, especially in the context of insertion sequences and boundary analysis.

The initial design panel also contains several arbitrary combinations of C-terminal and N-terminal boundaries, which are derived from either ortholog or homolog proteins. Significant changes can occur in protein stability, or protein expression yield, or a combination of both when even slight alterations take place at the termini.

Cleavable fusion tags are often integrated in the construct design so as to enable better detection and purification of the protein of interest. The desired affinity tag is the short poly-His tag. Using metal chelating chromatography for purification is not only less costly but also scalable. A number of techniques can be used for proteins having solubility issues.

MBP and GST are large protein tags that should be utilized carefully, because the tag helps in dragging a protein, which is partly folded, into the solution. When a protein ligand is required by the protein to form a stable complex, co-expression provides a suitable option. During the production of proteins, a tiny molecule ligand should be included in certain types of proteins so as to sustain the stability of proteins.

Based on orthologous protein modeling, residues with hydrophobic surface can be mutated. By designing all the constructs, it is possible to remove the affinity tags by subjecting them to highly specific proteases. The host cell would find the target protein expression highly toxic and this would lead to cell death or reduced yield.

For structural reasons and biophysical measurements, the toxic effects on the host cell can be effectively reduced by introducing activity attenuating mutations. Extreme care was taken when choosing these mutations so that they are far away from the inhibitor binding site. These mutations in kinases can result in 5 to 10 times yield improvement. Integral to intracellular protein expression are BEVS and E. coli expression systems.

Screening and optimizing recombinant protein

As soon as numerous constructs for the protein of interest have been produced, it is important to prioritize the constructs depending on their major biophysical characteristics. As such, three criteria have been devised and implemented to prioritize the constructs for both scale-up and optimization. Following the initial round of expression optimization, soluble expression yield is the first criterion.

Various factors like growth conditions, medium composition, harvest time, induction conditions such as inducer temperature and concentration, etc. can impact the protein expression yield in E. coli. While in the case of the BEVS system, the expression yield can be considerably affected by a number of factors like infection time, multiplicity of infection (MOI), agitation, harvest time, and sparging rate.

At the expression testing stage in small scale, the critical expression factors are separately validated so as to ensure result comparability. Eventually, the costs of production and purification resources needed for scale-up are governed by the protein yield. Therefore, it is important to perform a precise evaluation of yield and interpret crucial expression parameters for the specified target.

Testing the solubility of protein at high concentration is the next criterion to prioritize the constructs. Here, more than 3 g/ml is needed to optimize the crystallization. A wide range of buffers are used to concentrate the purified protein and the same is assessed by quantifying soluble precipitation and aggregation at different levels of concentrations. Static light scattering and dynamic light scattering techniques are used to assess the soluble fraction homogeneity.

A major governing factor for effective crystallization is monodispersity of the protein solution. Thermal stability of the construct is the last criterion, as determined by the DSC technique. Although the stability of proteins cannot be always predicted by protein yield, thermal stability and stability against aggregation in solution are good predictors of an effective crystallization.

Affinity tag effects on protein stability

In recombinant protein expression, affinity tags that are joined to the target protein are utilized extensively. These tags simplify the purification process, besides enabling the ease of identification of proteins by western immunoblots and ELISA methods.

The original purification process depends on the nature of the tag and not on the independent protein properties. This process has been redefined by the affinity tags to enable high throughput, parallel, and automatable small-scale processing of many samples.

However, it should be noted that the tag’s effect on the protein is not benign all the time. In fact, in a large number of cases, it has been shown that even when a small 6xHis tag is introduced, the stability of the protein reduces significantly.

At times, the effect of the tag on the protein can be quite striking, thus moving the Tm by 10°C or more. Using the DSC technique, the stability variation between tag-free and tagged protein can be determined easily.

When MBP or GST tags are utilized, the DSC technique can identify the protein domain’s thermal transitional peak. Both MBP and GST tags exhibit distinct high Tm, which is relatively higher than Tm of the protein of interest. The presence of independently folded protein domains is indicated by identifying a second Tm from the bound target protein.

It was observed that for a large number of protein domains, which did not solubly express with a poly-His tag, soluble expression may be facilitated by using larger protein tags. Conversely, upon closer analysis, it was observed that the target protein is usually non-functional or not properly folded. Upon tag cleavage, this protein instantly precipitates. Therefore, care must be taken when these tags are used.

Protein integrity assessment

An evenly folded protein is suitably indicated by a clear thermal transition, reflecting a population which reacts to high temperature and gradually unfolds using the same pathway. Nonetheless, a conclusion cannot be drawn that the folded protein is functional, instead of implementing a kinetically favored conformation or confining it in a local energy minimum.

The ITC and DSC techniques are used to validate the function of the protein by determining the way it interacts with ligands, natural substrates, or inhibitors. The inhibitors that were utilized may be either generic for specific types of enzymes, or highly specific compounds that are commercially available on the market or developed in-house. When the protein is fused with high affinity inhibitors or substrates, it is stabilized to thermal denaturation, followed by increasing the Tm in DSC analysis.

The DSC assay can be organized easily and is both robust and reliable. No major problems are associated with compound solubility. If required, ITC can be used to acquire the quantitative binding energy.

This aspect helps in measuring the binding stoichiometry, thereby substantiating the purity of the protein preparation process. The product and substrate’s relative binding affinities also offer useful data for designing activity assays. This provides a suitable method for identifying the possibility for product inhibition of the enzyme-catalyzed reaction.

Protein formulation optimization

An important aspect of protein chemistry efforts is formulating proteins to improve both their stability and solubility factors. Using the DSC technique, the effect of buffer component variations on the stability of proteins can be studied directly.

It is important to gain a better insight into the thermal sensitivity, co-solvent effects, and the intrinsic properties of the protein folding process, so that large-scale process can be designed in a better way, crystallization trials and assay development can be accelerated, and storage of products can be improved to a large extent.

For instance, samples of protein are allowed to undergo numerous freezing and thaw cycles and then subsequently evaluated through the DSC technique. This is done to improve the effects of solvents on the freezing-thaw stability of proteins. DSC can be used to validate protein integrity at different points of time of an assay condition simulation. This integrity of proteins calls for increased temperatures and prolonged assay times.

Free energy is acquired from the Gibbs-Helmholtz equation and ITC analysis, and the same is plotted as a function of temperature to act as a reference point for defining an optimum range of working temperature. The DSC method not only helps in identifying good additives, but also aids in eliminating co-solvents that are redundant. This forms a major step for streamlining the original sample provided for crystallization trials.

In one analysis, a wide array of co-solvents was required to sustain the protein stability, as specified by literature conditions. It was observed that these co-solvents prevent crystal formation during the trials. Here, the DSC technique was used to analyze the needs for individual additive, allowing detection of dominant additives. When nonessential components were removed, it led to a robust crystallization process.

DSC applications in protein purification and characterization

Recombinant proteins are capable of binding to host cell ligands such as macromolecules or small molecules that are sufficiently strong and remain fused during the entire purification process. Proteins having high pI binding to host cells’ nucleic acids is a well-known phenomenon.

It is possible to identify such processes by merely determining the ratio of UV280 and UV260. It was also seen that host proteins and the proteins of interest can be strongly complexed together. The former can be easily detected through a wide range of traditional techniques, namely mass spectrometry, multi-angle light scattering, SDS-PAGE, gel filtration, and so on.

However, these methods cannot easily detect these changes in recombinant proteins that are reversibly and tightly fused with tiny molecules. Nevertheless, the tiny molecules are tightly bound together that can considerably improve the protein’s thermal stability.

The DSC instrument is highly sensitive and can be used for detecting these energetic variations. In one of the experiments, it was discovered that the preparation of protein was found to be heterogeneous, even after purifying the protein thoroughly.

This was detected through a high resolution ion exchange separation technique. Based on electrospray mass spectrometry and SDS-PAGE, all three peaks were found to be similar. Individual peaks were studied through the DSC technique, which showed higher Tm in two peaks. These peaks were re-chromatographed, which revealed slow transition to the lowest form of Tm.

The peaks with higher Tm exhibited tiny molecule ligands from the host cells. Mass spectrometry was used to detect these tightly bound molecules following extraction from the basic preparation of proteins. Leveraging this information, a new method was developed to remove the small molecule ligands, which resulted in a better recovery of a uniform protein preparation required for optimum crystallization.

Microcalorimetry provides orthogonal information for understanding drug binding

As a rule, activity-based assays were used to derive data about the inhibition of compounds. Such types of assays often give a Ki or IC50. However, a number of experimental artifacts can affect the quality of these measurements.

If the target compound contains reactive impurities, the concentration of active protein will reduce considerably. In pharmaceutical small molecule screening, this is a common enough event, but data quality can be restored by applying careful analysis with modified mathematical models.

In other scenarios where enzymes have slow turn-over rates or stability problems or enzymes have extremely high Km, it can result in incorrect affinity measurements. The equations that are applied to determine Ki will not be applicable when the concentration of the enzyme required to acquire an experimental readout is relatively greater than the Ki for compound inhibition.

Another option is biophysical analysis, which can help in establishing the binding affinity that is not compromised by artifacts. Furthermore, crucial thermodynamic data regarding the binding mechanism can be obtained through isothermal calorimetry, which helps in determining ΔS, ΔH and Ka and even offers ΔCp, which is a parameter associated with the degree of conformational change in proteins.

In a large number of instances, a two-step binding event is required in pharmaceutical compounds that bind slowly. This includes fast binding followed by a slow event combined with a conformation change in enzymes. The former event is often diffusion restricted. The ITC technique can be used to view and analyze these components.

It proves handy in those situations where the inhibitor adheres to the enzyme’s inactive form. For instance, the inactive form of the cABL kinase is prevented by GLEEVEC®. When active cABL is used to determine the Ki in an assay, it does not give the exact value of the small molecule’s affinity for the inactive form.

Using the DSC technique, the compounds’ relative binding affinity can be roughly estimated by determining the Tm shift induced by compound binding. Racemic inhibitor mixtures having differential binding affinity are detected through the DSC technique. This binding affinity is indicated as two different peaks of melting temperature.

Moreover, the DSC technique can profile binding affinities to both forms of active and in-active enzymes in SAR analysis, and is capable of differentiating between non-competitive and competitive binding modes in mechanistic investigation when studying inhibitor, protein, and ligand mixtures.

According to Kroe RR et al. [4], a good linear association exists between Log Ki and Tm and this association was validated by data. Nevertheless, in rare instances, the correlation is significantly reduced where the binding ΔCp is extraordinarily huge.

Conclusion

The article has shown how calorimetry measurements can be effectively used to improve the analyses of interactions between compounds and proteins, and also to improve the efficiency of investigational optimization in protein biochemistry.

The wide range of applications described above is likely to grow as more and more laboratories implement advanced automated equipment to integrate standard calorimetry measurements in their routine workflow processes.

Acknowledgements

Produced from materials authored by Hangjun Zhan and Douglas Buckley from Malvern Panalytical Limited, Malvern Panalytical, UK.

References

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  2. Brandts JF and Lin LN. (1990) Study of strong to ultratight protein interactions using differential scanning calorimetry. Biochemistry. (29):6927-40.
  3. Kuzmic P, Hill C, Kirtley MP, Janc JW. (2003) Kinetic determination of tight binding impurities in enzyme inhibitors. Anal Biochem. 319(2):272-9. 4.
  4. Kroe RR, Regan J, Proto A, Peet GW, Roy T, Landro LD, Fuschetto NG, Pargellis CA, Ingraham RH. (2003) Thermal denaturation: a method to rank slow binding, high-affinity P38alpha MAP kinase inhibitors. J Med Chem.

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Last updated: Mar 22, 2019 at 7:03 AM

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