Application of microcalorimetry in drug discovery at Exelixis

Introduction

Understanding the molecular details of drug-target protein interactions is a critical component of the drug discovery process in the modern pharmaceutical industry. We have put in place a comprehensive set of highly integrated biochemical and biophysical methods to better characterize the target protein and its interactions with inhibitors. These techniques enable us to identify chemical instability (oxidation, deamination etc.), proteolytic or chemical degradation, post-translational modification, and physical instability such as surface denaturation, soluble aggregation, and precipitation. More complete routine characterization of the protein of interest informs the development of better formulations, as we are able to quantitate the effects of formulation components on protein stability. A critical component of our biophysical arsenal is the ability to investigating a protein’s thermodynamic properties by ITC and DSC. Both techniques are based in well established fundamental principles. ITC provides thermodynamic data used to confirm the binding model, as well as quantitation of binding enthalpy (ΔH), entropy (TΔS), free energy (ΔG) and binding association constant K_a. DSC measures the heat changes associated with thermal denaturation of the target protein and has been extensively used in understanding protein folding and unfolding. The degree of stabilization conferred by compound binding is often related to the affinity of the interaction. The thermodynamics associated with unfolding (change of T_m, ΔCp, and ΔH,) reveal information about the properties of compound binding.^1,2 Classically, both measurements required a significant amount of protein and commitment of significant operator time, as the instrumentation required manual operation. These factors limited wide application of these measurements. Recently, MicroCal LLC. introduced two new instruments, the AutoITC and AutoDSC, with significantly improved sensitivity, greatly reduced sample consumption, robotic automation and user friendly software packages that greatly simplify experimental set-up and data analysis. In this paper, we will present examples of disciplines in small molecule drug discovery where this new instrumentation has allowed us to improve the efficiency of our processes.

Recombinant protein construct design and expression for drug discovery at Exelixis

Protein construct design for expression is not yet a predictable process. Thanks to the rapid development of genome science, DNA manipulation is no longer a limiting factor. To ensure successful expression of our target proteins without multiple rounds of optimization, we take a highly parallel approach, preparing 20-30 constructs for a given protein simultaneously. This insures a high probability of success, but adds the additional complication of requiring unwieldy numbers of experiments to optimize expression of all constructs. Approaches to prioritizing these constructs will be discussed in the next section. The first step in designing expression constructs is to extensively search for literature precedents, particularly from the protein structural database (www.pdb.org). This provides a good indicator for expression feasibility, as structural biology usually requires both high quality and large quantities of protein. For novel or not well-studied targets, domain analysis is performed based on information from our in-house expression database and structural modeling (particularly in the areas of boundary analysis and insertion sequences). A number of random combinations of N-terminal and C-terminal boundaries derived from homolog or ortholog proteins are also included in the initial design panel. In our experience, even minor changes at the termini can result in dramatic differences in protein expression yield and/or protein stability. To facilitate identification and purification of the target protein, cleavable fusion tags are usually incorporated in the construct design. The poly-His tag is our preferred affinity tag. The tag is short and purification by metal chelating chromatography is inexpensive and scalable. For proteins with solubility issues, several approaches may be taken. Large protein tags such as GST and MBP are used with great caution, as frequently the tag helps drag partially folded protein into solution. Co-expression is another alternative when the protein requires a protein ligand to form a stable complex. For certain classes of proteins, it is necessary to include a small molecule ligand during protein production to maintain protein stability. Finally, hydrophobic surface residues can be mutated based on modeling from orthologous proteins. All of the constructs are engineered so that the affinity tags can be subsequently removed by treatment with highly specific proteases. Target protein expression can be toxic to the host cell, resulting in reduced yield or cell death. For biophysical measurements and structural purposes, activity attenuating mutations can be introduced to reduce the toxic effects on the host cell. These mutations are carefully chosen to be distant from the site of inhibitor binding. In the case of kinases, these mutations can often increase the yield 5-10 fold. E. coli and BEVS expression systems are our workhorses for intracellular protein expression.

Screening and optimizing recombinant protein

After generating multiple constructs for a target protein, prioritizing constructs based on their key biophysical properties is essential. We have developed and implemented three criteria for prioritizing constructs for optimization and scaleup. The first criterion is soluble expression yield after the first round of expression optimization. Protein expression yield in E.coli can be affected by multiple factors such as medium composition, growth conditions, induction conditions (inducer concentration and temperature), harvest time, etc. In the BEVS system, factors such as multiplicity of infection (MOI), infection time, agitation, sparging rate, and harvest time can significantly impact the expression yield. At the small scale expression testing stage, the key expression parameters are tested in parallel to insure comparability of the results. Protein yield ultimately dictates the production costs and purification resources required for scale-up. Therefore, accurate assessment of yield and understanding key expression parameters for the target of interest are crucial. The next criterion for construct prioritization is protein solubility testing at high concentration (typically >3 mg/ml is required for crystallization optimization). The purified protein is concentrated in a variety of buffers and evaluated by measuring soluble aggregation and precipitation at different concentrations. The homogeneity of the soluble fraction is assessed by both dynamic light scattering and static light scattering. Monodispersity of the protein solution is one of key determining factors for successful crystallization. The final criterion is thermal stability (high Tm) of the construct as measured by DSC. While protein yield is not always a good predictor of protein stability, both stability against aggregation in solution (mono-dispersity) and thermal stability are excellent predictors of successful crystallization

Affinity tag effects on protein stability

Affinity tags fused to the protein of interest are widely used in recombinant protein expression. In addition to the ease of protein identification by ELISA and western immunoblots, the tag greatly facilitates the purification process. The initial purification no longer relies on the individual protein properties, but rather on the nature of the tag. Affinity tags have transformed the purification process to allow parallel, high throughput, automatable small scale processing of multiple samples. Nevertheless, the effect of the tag on the protein is not always benign. In many cases, we have observed that even the addition of a small 6xHis tag can reduce protein stability. Sometimes, the effect can be dramatic, shifting the Tm by 10°C or more. DSC can readily be used to measure the stability difference between tagged and tag-free protein (after tag removal). DSC can also detect the thermal transitional peak of the expressed protein domain when large tags such as GST or MBP are used. Both GST and MBP have discrete high T_m, which frequently is much higher than T_m of our target protein. The detection of a second T_m from the fused target protein provides a good indication of the presence of independently folded domains. It is important to mention that we have found that for the majority protein domains that failed to be solubly expressed with a poly-His tag (insoluble aggregation, protein found in the pellet after lysis), the use of larger protein tags may appear to promote soluble expression. However, with closer study, the protein of interest is often found not to be functional or correctly folded and immediately precipitates upon tag cleavage. Therefore, one should be cautious when using these tags.

Protein integrity assessment

A distinct thermal transition is a good indication of uniformly folded protein. It reflects a population that responds to increased temperature and unfolds by the same pathway. However, one cannot conclude that this protein is folded to be functional, as opposed to having adopted a conformation that is kinetically favored or is trapped in a local energy minimum. We use DSC and ITC to confirm protein function by measuring its ability to interact with inhibitors, natural substrates, or ligands. The inhibitors used may be generic for certain class of enzymes, or highly specific compounds made in-house or available commercially. When high affinity substrates or inhibitors are bound to the protein, the protein is stabilized to thermal denaturation and the T_m is increased in DSC analysis. The DSC assay is robust, very consistent and easy to assemble. Compound solubility generally does not present a significant issue. The quantitative binding energy can also be obtained by ITC if desired. This has the added benefit of determining the stoichiometry of binding which in turn confirms the purity of the protein preparation. Furthermore, the relative binding affinities of substrate and product provide useful information for development of activity assays. It is a great way to identify the potential for product inhibition of the reaction catalyzed by the enzyme. >> Download the full Application Note as PDF

Malvern provides the materials and biophysical characterization technology and expertise that enables scientists and engineers to investigate, understand and control the properties of dispersed systems. These systems range from proteins and polymers in solution, particle and nanoparticle suspensions and emulsions, through to sprays and aerosols, industrial bulk powders and high concentration slurries. Used at all stages of research, development and manufacturing, Malvern’s instruments provide critical information that helps accelerate research and product development, enhance and maintain product quality and optimize process efficiency. Our products reflect Malvern’s drive to exploit the latest technological innovations. They are used by both industry and academia, in sectors ranging from pharmaceuticals and biopharmaceuticals to bulk chemicals, cement, plastics and polymers, energy and the environment. Malvern systems are used to measure particle size, particle shape, zeta potential, protein charge, molecular weight, mass, size and conformation, rheological properties and for chemical identification, advancing the understanding of dispersed systems across many different industries and applications. www.malvern.com Material relationships http://www.malvern.com/en/ [email protected]

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