Michael I. Recht

Quantitative analysis of protein-RNA interactions by gel mobility shift.

The gel mobility shift assay is routinely used to visualize protein-RNA interactions. Its power resides in the ability to resolve free from bound RNA with high resolution in a gel matrix. We review the quantitative application of this approach to elucidate thermodynamic properties of protein-RNA complexes. Assay designs for titration, competition, and stoichiometry experiments are presented for two unrelated model complexes.

Structural and Biophysical Characterization of the EphB4·EphrinB2 Protein-Protein Interaction and Receptor Specificity

Increasing evidence implicates the interaction of the EphB4 receptor with its preferred ligand, ephrinB2, in pathological forms of angiogenesis and in tumorigenesis. To identify the molecular determinants of the unique specificity of EphB4 for ephrinB2, we determined the crystal structure of the ligand binding domain of EphB4 in complex with the extracellular domain of ephrinB2. This structural analysis suggested that one amino acid, Leu-95, plays a particularly important role in defining the structural features that confer the ligand selectivity of EphB4. Indeed, all other Eph receptors, which promiscuously bind many ephrins, have a conserved arginine at the position corresponding to Leu-95 of EphB4. We have also found that amino acid changes in the EphB4 ligand binding cavity, designed based on comparison with the crystal structure of the more promiscuous EphB2 receptor, yield EphB4 variants with altered binding affinity for ephrinB2 and an antagonistic peptide. Isothermal titration calorimetry experiments with an EphB4 Leu-95 to arginine mutant confirmed the importance of this amino acid in conferring high affinity binding to both ephrinB2 and the antagonistic peptide ligand. Isothermal titration calorimetry measurements also revealed an interesting thermodynamic discrepancy between ephrinB2 binding, which is an entropically driven process, and peptide binding, which is an enthalpically driven process. These results provide critical information on the EphB4·ephrinB2 protein interfaces and their mode of interaction, which will facilitate development of small molecule compounds inhibiting the EphB4·ephrinB2 interaction as novel cancer therapeutics.

Higher Throughput Calorimetry: Opportunities, Approaches and Challenges

Higher throughput thermodynamic measurements can provide value in structure-based drug discovery during fragment screening, hit validation, and lead optimization. Enthalpy can be used to detect and characterize ligand binding, and changes that affect the interaction of protein and ligand can sometimes be detected more readily from changes in the enthalpy of binding than from the corresponding free-energy changes or from protein-ligand structures. Newer, higher throughput calorimeters are being incorporated into the drug discovery process. Improvements in titration calorimeters come from extensions of a mature technology and face limitations in scaling. Conversely, array calorimetry, an emerging technology, shows promise for substantial improvements in throughput and material utilization, but improved sensitivity is needed.

Enthalpy array analysis of enzymatic and binding reactions

Enthalpy arrays enable label-free, solution-based calorimetric detection of molecular interactions in a 96-detector array format. The combination of the small size of the detectors and the ability to perform measurements in parallel results in a significant reduction of sample volume and measurement time compared with conventional calorimetry. We have made significant improvements in the technology by reducing the temperature noise of the detectors and improving the fabrication materials and methods. In combination with an automated measurement system, the advances in device performance and data analysis have allowed us to develop basic enzyme assays for substrate specificity and inhibitor activity. We have also performed a full titration of 18-crown-6 with barium chloride. These results point to future applications for enthalpy array technology, including fragment-based screening, secondary assays, and thermodynamic characterization of leads in drug discovery.

Three-dimensional structure of the EphB2 receptor in complex with an antagonistic peptide reveals a novel mode of inhibition.

The Eph family of receptor tyrosine kinases has been implicated in tumorigenesis as well as pathological forms of angiogenesis. Understanding how to modulate the interaction of Eph receptors with their ephrin ligands is therefore of critical interest for the development of therapeutics to treat cancer. Previous work identified a set of 12-mer peptides that displayed moderate binding affinity but high selectivity for the EphB2 receptor. The SNEW antagonistic peptide inhibited the interaction of EphB2 with ephrinB2, with an IC50 of ∼15 μm. To gain a better molecular understanding of how to inhibit Eph/ephrin binding, we determined the crystal structure of the EphB2 receptor in complex with the SNEW peptide to 2.3-Å resolution. The peptide binds in the hydrophobic ligand-binding cleft of the EphB2 receptor, thus competing with the ephrin ligand for receptor binding. However, the binding interactions of the SNEW peptide are markedly different from those described for the TNYL-RAW peptide, which binds to the ligand-binding cleft of EphB4, indicating a novel mode of antagonism. Nevertheless, we identified a conserved structural motif present in all known receptor/ligand interfaces, which may serve as a scaffold for the development of therapeutic leads. The EphB2-SNEW complex crystallized as a homodimer, and the residues involved in the dimerization interface are similar to those implicated in mediating tetramerization of EphB2-ephrinB2 complexes. The structure of EphB2 in complex with the SNEW peptide reveals novel binding determinants that could serve as starting points in the development of compounds that modulate Eph receptor/ephrin interactions and biological activities.

Measurement of enzyme kinetics and inhibitor constants using enthalpy arrays.

Enthalpy arrays enable label-free, solution-based calorimetric detection of molecular interactions in a 96-detector array format. Compared with conventional calorimetry, enthalpy arrays achieve a significant reduction of sample volume and measurement time through the combination of the small size of the detectors and ability to perform measurements in parallel. The current capabilities of the technology for studying enzyme-catalyzed reactions are demonstrated by determining the kinetic parameters for reactions with three model enzymes. In addition, the technology has been used with two classes of enzymes to determine accurate inhibitor constants for competitive inhibitors from measurements at a single inhibitor concentration.

Rapid mixing of sub-microlitre drops by magnetic micro-stirring

We demonstrate rapid mixing of sub-microlitre droplets (250 nl) using miniaturized magnetic stir bars (400 μm × 200 μm × 15 μm). The stir bars are fabricated using laser micromachining and placed on the substrate on which the drops are manipulated. They are activated by an externally applied magnetic field and used in combination with on-demand drop merging in enthalpy arrays. This technique results in a 10-fold increase in mixing rate, and a mixing time constant of about 2 s. Drop mixing times are measured by Förster resonance energy transfer (FRET) and verified by thermodynamic measurements of binding and enzymatic reactions.

Fragment-Based Screening for Inhibitors of PDE4A Using Enthalpy Arrays and X-ray Crystallography

Fragment-based screening has typically relied on X-ray or nuclear magnetic resonance methods to identify low-affinity ligands that bind to therapeutic targets. These techniques are expensive in terms of material and time, so it useful to have a higher throughput method to reliably prescreen a fragment library to identify a subset of compounds for structural analysis. Calorimetry provides a label-free method to assay binding and enzymatic activity that is unaffected by the spectroscopic properties of the sample. Conventional microcalorimetry is hampered by requiring large quantities of reagents and long measurement times. Nanocalorimeters can overcome these limitations of conventional isothermal titration calorimetry. Here we have used enthalpy arrays, which are arrays of nanocalorimeters, to perform an enzyme activity-based fragment screen for competitive inhibitors of phosphodiesterase 4A (PDE4A). Several inhibitors with K I <2 mM were identified and moved to X-ray crystallization trials. Although the co-crystals did not yield high-resolution data, evidence of binding was observed, and the chemical structures of the hits were consistent with motifs of known PDE4 inhibitors. This study shows how array calorimetry can be used as a prescreening method for fragment-based lead discovery with enzyme targets and provides a list of candidate fragments for inhibition of PDE4A.

Identification and Optimization of PDE10A Inhibitors Using Fragment-Based Screening by Nanocalorimetry and X-ray Crystallography.

Fragment-based lead discovery (FBLD) is a technique in which small, low-complexity chemical fragments of 6 to 15 heavy atoms are screened for binding to or inhibiting activity of the target. Hits are then linked and/or elaborated into tightly binding ligands, ideally yielding early lead compounds for drug discovery. Calorimetry provides a label-free method to assay binding and enzymatic activity that is unaffected by the spectroscopic properties of the sample. Conventional microcalorimetry is hampered by requiring large quantities of reagents and long measurement times. Nanocalorimeters can overcome these limitations of conventional isothermal titration calorimetry. Here we use enthalpy arrays, which are arrays of nanocalorimeters, to perform an enzyme activity-based fragment screen for competitive inhibitors of phosphodiesterase 10A (PDE10A). Two dozen fragments with KI <2 mM were identified and moved to crystal soaking trials. All soak experiments yielded high-resolution diffraction, with two-thirds of the fragments yielding high-resolution co-crystal structures with PDE10A. The structural information was used to elaborate fragment hits, yielding leads with KI <1 µM. This study shows how array calorimetry can be used as a prescreening method for fragment-based lead discovery with enzyme targets and paired successfully with an X-ray crystallography secondary screen.

Monitoring Assembly of Ribonucleoprotein Complexes by Isothermal Titration Calorimetry

Isothermal titration calorimetry (ITC) is a useful technique to study RNA-protein interactions as it provides the only method by which the thermodynamic parameters of free energy, enthalpy, and entropy can be directly determined. This chapter presents a general procedure for studying RNA-protein interactions using ITC and gives specific examples for monitoring the binding of Caenorhabditis elegans GLD-1 STAR domain to TGE RNA and the binding of Aquifex aeolicus S6:S18 ribosomal protein heterodimer to an S15-ribosomal RNA complex.