Geoffrey A. Holdgate

Epidermal growth factor receptor tyrosine kinase: Investigation of catalytic mechanism, structure-based searching and discovery of a potent inhibitor

Inhibition of tyrosine kinases is a possible approach for the treatment of cancer. We have investigated the catalytic mechanism of the epidermal growth factor receptor tyrosine kinase (EGF-RTK) in order to obtain information for use in structure-based searching for inhibitors. Initial rate studies imply that EGF-RTK forms a ternary complex together with ATP and peptide substrate. Investigation of pH and temperature dependence suggests that the kinase reaction requires the ionised form of a carboxylate (pK = 6.3) and the protonated form of another group (pK = 9.1). These characteristics are consistent with a mechanism where the carboxylate of Asp813(pK = 6.3) facilitates deprotonation of the tyrosyl hydroxyl of the peptide substrate, activating it as a nucleophile to attack the gamma-phosphorus of ATP which interacts with a protonated enzyme side-chain (pK = 9.1), possibly the guanidinium group of Arg817. This proposed catalytic mechanism was used to define a query when searching for inhibitors in a database of predicted three-dimensional structures. The procedure involved searching for compounds that mimic the ATP gamma-phosphate, tyrosyl hydroxyl and the tyrosyl aromatic ring, all of which seem to interact strongly with the enzyme during catalysis. This search allowed identification of inhibitors of EGF-RTK which were used to define queries for two-dimensional searching of a larger database, leading to the discovery of 4-(3-chloroanilino)quinazoline (CAQ) which is a potent inhibitor (Ki = 16 nM) of the enzyme. The compound is believed to be the first representative from a new structural class of anilinoquinazoline tyrosine kinase inhibitors. It follows competitive kinetics with respect to ATP and noncompetitive kinetics when the peptide is varied, implying that it functions as an analogue of ATP. CAQ is a novel and potent lead in the search for tyrosine kinase inhibitors as potential agents for the treatment of cancer.

The entropic penalty of ordered water accounts for weaker binding of the antibiotic novobiocin to a resistant mutant of DNA gyrase: a thermodynamic and crystallographic study.

Novobiocin is an antibiotic which binds to a 24 kDa fragment from the B subunit of DNA gyrase. Naturally occurring resistance arises from mutation of Arg-136 which hydrogen bonds to the coumarin ring of novobiocin. We have applied calorimetry to characterize the binding of novobiocin to wild-type and R136H mutant 24 kDa fragments. Upon mutation, the Kd increases from 32 to 1200 nM at 300 K. The enthalpy of binding is more favorable for the mutant (DeltaH degrees shifts from -12.1 to -17.5 kcal/mol), and the entropy of binding is much less favorable (TDeltaS degrees changes from -1.8 to -9.4 kcal/mol). Both of these changes are in the direction opposite to that expected if the loss of the Arg residue reduces hydrogen bonding. The change in heat capacity at constant pressure upon binding (DeltaCp) shifts from -295 to -454 cal mol-1 K-1. We also report the crystal structure, at 2.3 A resolution, of a complex between the R136H 24 kDa fragment and novobiocin. Although the change in DeltaCp often would be interpreted as reflecting increased burial of hydrophobic surface on binding, this structure reveals a small decrease. Furthermore, an ordered water molecule is sequestered into the volume vacated by removal of the guanidinium group. There are large discrepancies when the measured thermodynamic parameters are compared to those estimated from the structural data using empirical relationships. These differences seem to arise from the effects of sequestering ordered water molecules upon complexation. The water-mediated hydrogen bonds linking novobiocin to the mutant protein make a favorable enthalpic contribution, whereas the immobilization of the water leads to an entropic cost and a reduction in the heat capacity of the system. Such a negative contribution to DeltaCp, DeltaH degrees , and TDeltaS degrees appears to be a general property of water molecules that are sequestered when ligands bind to proteins.

Measurements of binding thermodynamics in drug discovery

Thermodynamics governs the process of biomolecular recognition. The steps of characterizing, understanding and exploiting binding thermodynamics have the potential to contribute to an improved rational drug design process that is more robust and reliable. It is only relatively recently that instrumentation capable of direct and full thermodynamic characterization has been improved, giving impetus to the application of thermodynamic measurements in drug discovery. This review highlights current instruments and methods that can be employed to measure binding thermodynamics and their use in studies of biomolecular recognition and drug discovery.

Preclinical anticancer activity of the potent, oral Src inhibitor AZD0530

AZD0530, an orally available Src inhibitor, demonstrated potent antimigratory and anti‐invasive effects in vitro, and inhibited metastasis in a murine model of bladder cancer. Antiproliferative activity of AZD0530 in vitro varied between cell lines (IC50 0.2 –>10μM). AZD0530 inhibited tumor growth in 4/10 xenograft models tested and dynamically inhibited in vivo phosphorylation of Src substrates paxillin and FAK in both growth‐inhibition‐resistant and ‐sensitive xenografts. The activity of AZD0530 in NBT‐II bladder cancer cells in vitro was consistent with inhibition of cell migration and stabilization of cell–cell adhesion. These data suggest a dominant anti‐invasive pharmacology for AZD0530 that may limit tumor progression in a range of cancers. AZD0530 is currently in Phase II clinical trials.

Design and Synthesis of Novel Lactate Dehydrogenase A Inhibitors by Fragment-Based Lead Generation

Lactate dehydrogenase A (LDHA) catalyzes the conversion of pyruvate to lactate, utilizing NADH as a cofactor. It has been identified as a potential therapeutic target in the area of cancer metabolism. In this manuscript we report our progress using fragment-based lead generation (FBLG), assisted by X-ray crystallography to develop small molecule LDHA inhibitors. Fragment hits were identified through NMR and SPR screening and optimized into lead compounds with nanomolar binding affinities via fragment linking. Also reported is their modification into cellular active compounds suitable for target validation work.

Isothermal titration calorimetry in drug discovery.

Isothermal titration calorimetry (ITC) follows the heat change when a test compound binds to a target protein. It allows precise measurement of affinity. The method is direct, making interpretation facile, because there is no requirement for competing molecules. Titration in the presence of other ligands rapidly provides information on the mechanism of action of the test compound, identifying the intermolecular complexes that are relevant for structure-based design. Calorimetry allows measurement of stoichiometry and so evaluation of the proportion of the sample that is functional. ITC can characterize protein fragments and catalytically inactive mutant enzymes. It is the only technique which directly measures the enthalpy of binding (delta H degree). Interpretation of delta H degree and its temperature dependence (delta Cp) is usually qualitative, not quantitative. This is because of complicated contributions from linked equilibria and a single change in structure giving modification of several physicochemical properties. Measured delta H degree values allow characterization of proton movement linked to the association of protein and ligand, giving information on the ionization of groups involved in binding. Biochemical systems characteristically exhibit enthalpy-entropy compensation where increased bonding is offset by an entropic penalty, reducing the magnitude of change in affinity. This also causes a lack of correlation between the free energy of binding (delta G degree) and delta H degree. When characterizing structure-activity relationships (SAR), most groups involved in binding can be detected as contributing to delta H degree, but not to affinity. Large enthalpy changes may reflect a modified binding mode, or protein conformation changes. Thus, delta H degree values may highlight a potential discontinuity in SAR, so that experimental structural data are likely to be particularly valuable in molecular design.

Kinetic efficiency: the missing metric for enhancing compound quality?

The kinetics of ligand-target interactions have been recognised as being instrumental in dictating the efficacy of drug action. Increased focus on kinetic signatures in drug discovery has concincided with improvements in label free methodology for measuring kinetic parameters. Simultaneously, focus has also been applied to increasing the quality of compounds, in terms of their physicochemical properties. To facilitate this drive towards higher compound quality, metrics such as ligand efficiency and enthalpic efficiency have been employed. We propose another metric, kinetic efficiency, that may be used pragmatically to help identify those compounds displaying differentiated kinetic behaviour. The combination of these metrics has the potential to improve decision-making in drug discovery leading to higher quality compounds and series.

Enzyme kinetics and binding studies on inhibitors of MEK protein kinase.

Inhibition of the protein kinase, MEK1, is a potential approach for the treatment of cancer. Inhibitors may act by prevention of activation (PoA), which involves interfering with phosphorylation of nonactivated MEK1 by the upstream kinase, B-RAF. Modulation also may occur by inhibition of catalysis (IoC) during phosphorylation of the downstream substrate, ERK2, by activated MEK1. Here, five MEK inhibitors are characterized in terms of binding affinity, PoA, and IoC. The compounds are a butadiene (U-0126), an N-alkoxy amide (CI-1040), two CI-1040 analogues (an anthranilic acid and an N-alkyl amide), and a cyanoquinoline. Some compounds give different mechanisms of inhibition (ATP-competitive, noncompetitive, or uncompetitive) in PoA compared to IoC or show a change in potency between the assays. The inhibitors also exhibit different shifts in potency when either PoA or IoC is compared with binding to nonactivated MEK. The inhibitor potency ranking, therefore, is dependent upon the assay format. When the ATP concentration equals K m, IoC IC 50 increases in the order CI-1040 approximately cyanoquinoline < anthranilic acid approximately U-0126 < alkyl amide. Conversely, the K d from nonactivated MEK1 for four of the compounds varies between more than 6-fold lower and over 18-fold higher than this IC 50, with U-0126 having the lowest K d and CI-1040 having the highest. In PoA when the ATP concentration equals K m, U-0126 has the lowest IC 50, becoming more potent than CI-1040, the cyanoquinoline, and the anthranilic acid. These observations have implications for understanding structure-activity relationships of MEK inhibitors and illustrate how assays can be designed to favor different compounds.

Biophysical Methods in Drug Discovery from Small Molecule to Pharmaceutical

Biophysical methods have become established in many areas of drug discovery. Application of these methods was once restricted to a relatively small number of scientists using specialized, low throughput technologies and methods. Now, automated high-throughput instruments are to be found in a growing number of laboratories. Many biophysical methods are capable of measuring the equilibrium binding constants between pairs of molecules crucial for molecular recognition processes, encompassing protein-protein, protein-small molecule, and protein-nucleic acid interactions, and several can be used to measure the kinetic or thermodynamic components controlling these biological processes. For a full characterization of a binding process, determinations of stoichiometry, binding mode, and any conformational changes associated with such interactions are also required. The suite of biophysical methods that are now available represents a powerful toolbox of techniques which can effectively deliver this full characterization.The aim of this chapter is to provide the reader with an overview of the drug discovery process and how biophysical methods, such as surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), nuclear magnetic resonance, mass spectrometry (MS), and thermal unfolding methods can answer specific questions in order to influence project progression and outcomes. The selection of these examples is based upon the experiences of the authors at AstraZeneca, and relevant approaches are highlighted where they have utility in a particular drug discovery scenario.

FGFR1 Kinase Inhibitors: Close Regioisomers Adopt Divergent Binding Modes and Display Distinct Biophysical Signatures.

The binding of a ligand to its target protein is often accompanied by conformational changes of both the protein and the ligand. This is of particular interest, since structural rearrangements of the macromolecular target and the ligand influence the free energy change upon complex formation. In this study, we use X-ray crystallography, isothermal titration calorimetry, and surface-plasmon resonance biosensor analysis to investigate the binding of pyrazolylaminopyrimidine inhibitors to FGFR1 tyrosine kinase, an important anticancer target. Our results highlight that structurally close analogs of this inhibitor series interact with FGFR1 with different binding modes, which are a consequence of conformational changes in both the protein and the ligand as well as the bound water network. Together with the collected kinetic and thermodynamic data, we use the protein-ligand crystal structure information to rationalize the observed inhibitory potencies on a molecular level.