Tami Marrone

Characterizing the Effects of the Juxtamembrane Domain on Vascular Endothelial Growth Factor Receptor-2 Enzymatic Activity, Autophosphorylation, and Inhibition by Axitinib

The catalytic domains of protein kinases are commonly treated as independent modular units with distinct biological functions. Here, the interactions between the catalytic and juxtamembrane domains of VEGFR2 are studied. Highly purified preparations of the receptor tyrosine kinase VEGFR2 catalytic domain without (VEGFR2-CD) and with (VEGFR2-CD/JM) the juxtamembrane (JM) domain were characterized by kinetic, biophysical, and structural methods. Although the catalytic parameters for both constructs were similar, the autophosphorylation rate of VEGFR2-CD/JM was substantially faster than VEGFR2-CD. The first event in the autophosphorylation reaction was phosphorylation of JM residue Y801 followed by phosphorylation of activation loop residues in the CD. The rates of activation loop autophosphorylation for the two constructs were determined to be similar. The autophosphorylation rate of Y801 was invariant on enzyme concentration, which is consistent with an intramolecular reaction. In addition, the first biochemical characterization of the advanced clinical compound axitinib is reported. Axitinib was found to have 40-fold enhanced biochemical potency toward VEGFR2-CD/JM (K(i) = 28 pM) compared to VEGFR2-CD, which correlates better with cellular potency. Calorimetric studies, including a novel ITC compound displacement method, confirmed the potency and provided insight into the thermodynamic origin of the potency differences. A structural model for the VEGFR2-CD/JM is proposed based on the experimental findings reported here and on the JM position in c-Kit, FLT3, and CSF1/cFMS. The described studies identify potential functions of the VEGFR2 JM domain with implications to both receptor biology and inhibitor design.

Towards understanding the mechanisms of molecular recognition by computer simulations of ligand-protein interactions.

The thermodynamic and kinetic aspects of molecular recognition for the methotrexate (MTX)–dihydrofolate reductase (DHFR) ligand–protein system are investigated by the binding energy landscape approach. The impact of ‘hot’ and ‘cold’ errors in ligand mutations on the thermodynamic stability of the native MTX–DHFR complex is analyzed, and relationships between the molecular recognition mechanism and the degree of ligand optimization are discussed. The nature and relative stability of intermediates and thermodynamic phases on the ligand–protein association pathway are studied, providing new insights into connections between protein folding and molecular recognition mechanisms, and cooperativity of ligand–protein binding. The results of kinetic docking simulations are rationalized based on the thermodynamic properties determined from equilibrium simulations and the shape of the underlying binding energy landscape. We show how evolutionary ligand selection for a receptor active site can produce well‐optimized ligand–protein systems such as MTX–DHFR complex with the thermodynamically stable native structure and a direct transition mechanism of binding from unbound conformations to the unique native structure. Copyright

Parallel simulated tempering dynamics of ligand-protein binding with ensembles of protein conformations

Abstract Simulated tempering dynamics with the simplified energy model and the ensemble of protein conformations have been performed for the SB203386 inhibitor binding with HIV-1 protease. Equilibrium simulations with multiple protein conformations implicitly incorporate protein flexibility and rank HIV-1 protease conformations according to the average ligand–protein interaction energies. Subsequent energy refinement with a molecular mechanics force field accurately quantifies the energetics of the low-energy ligand binding modes. The results suggest that the mobility of the SB203386 inhibitor is effectively restricted to two symmetry-related binding modes and this may prevent the inhibitor from adapting to distorted binding sites in mutant conformations.

Discovering high-affinity ligands from the computationally predicted structures and affinities of small molecules bound to a target: A virtual screening approach

We describe a ‘virtual NMR screening’ method to assist in the design of inhibitors that occupy different sites within a target. We dock small molecules into the active site of an enzyme and score them. Keeping the tightest-binding lead fixed in space, we dock and score other small molecules in its presence. Using this approach, linker groups are used to join the compounds together to form a high-affinity inhibitor. We present validation of our computational approach by reproducing experimental results for FKBP and stromelysin. Docking simulations are not subject to experimental problems such as proteolysis, protein or compound insolubility, or enzyme size. Because docking is fast and our scoring method can distinguish between high- and low-affinity inhibitors, this docking procedure shows promise as integral part of a drug-design strategy.

Hierarchy of simulation models in predicting molecular recognition mechanisms from the binding energy landscapes: Structural analysis of the peptide complexes with SH2 domains

Computer simulations using the simplified energy function and simulated tempering dynamics have accurately determined the native structure of the pYVPML, SVLpYTAVQPNE, and SPGEpYVNIEF peptides in the complexes with SH2 domains. Structural and equilibrium aspects of the peptide binding with SH2 domains have been studied by generating temperature‐dependent binding free energy landscapes. Once some native peptide–SH2 domain contacts are constrained, the underlying binding free energy profile has the funnel‐like shape that leads to a rapid and consistent acquisition of the native structure. The dominant native topology of the peptide–SH2 domain complexes represents an extended peptide conformation with strong specific interactions in the phosphotyrosine pocket and hydrophobic interactions of the peptide residues C‐terminal to the pTyr group. The topological features of the peptide–protein interface are primarily determined by the thermodynamically stable phosphotyrosyl group. A diversity of structurally different binding orientations has been observed for the amino‐terminal residues to the phosphotyrosine. The dominant native topology for the peptide residues carboxy‐terminal to the phosphotyrosine is tolerant to flexibility in this region of the peptide–SH2 domain interface observed in equilibrium simulations. The energy landscape analysis has revealed a broad, entropically favorable topology of the native binding mode for the bound peptides, which is robust to structural perturbations. This could provide an additional positive mechanism underlying tolerance of the SH2 domains to hydrophobic conservative substitutions in the peptide specificity region. Proteins 2001;45:456–470.

Navigating ligand–protein binding free energy landscapes: universality and diversity of protein folding and molecular recognition mechanisms

Abstract Thermodynamic and kinetic aspects of ligand–protein binding are studied for the methotrexate–dihydrofolate reductase system from the binding free energy profile constructed as a function of the order parameter. Thermodynamic stability of the native complex and a cooperative transition to the unique native structure suggest the nucleation kinetic mechanism at the equilibrium transition temperature. Structural properties of the transition state ensemble and the ensemble of nucleation conformations are determined by kinetic simulations of the transmission coefficient and ligand–protein association pathways. Structural analysis of the transition states and the nucleation conformations reconciles different views on the nucleation mechanism in protein folding.

Design and Synthesis of Novel and Selective Phosphodiesterase 2 (PDE2a) Inhibitors for the Treatment of Memory Disorders

A series of potent and selective [1,2,4]triazolo[1,5-a]pyrimidine PDE2a inhibitors is reported. The design and improvement of the binding properties of this series was achieved using X-ray crystal structures in conjunction with careful analysis of electronic and structural requirements for the PDE2a enzyme. One of the lead compounds, compound 27 (DNS-8254), was identified as a potent and highly selective PDE2a enzyme inhibitor with favorable rat pharmacokinetic properties. Interestingly, the increased potency of compound 27 was facilitated by the formation of a halogen bond with the oxygen of Tyr827 present in the PDE2a active site. In vivo, compound 27 demonstrated significant memory enhancing effects in a rat model of novel object recognition. Taken together, these data suggest that compound 27 may be a useful tool to explore the pharmacology of selective PDE2a inhibition.

Mathematical and Structural Characterization of Strong Non-additive SAR Caused by Protein Conformational Changes.

In medicinal chemistry, additivity-based SAR analysis rests on three assumptions: (1) con-sistent binding pose of the central scaffold, (2) no interaction between the substitutions, and (3) a relatively rigid binding pocket in which the two substitutions act independently. Previously, non-additive SAR have been documented in systems that deviate from the first two assump-tions. Interestingly, protein structural change upon ligand binding, through induced fit or con-formational selection, although a well-known phenomenon that invalidates the third assump-tion, has not been linked to non-additive SAR conclusively. Here, for the first time, we show clear structural evidence that the formation of a hydrophobic pocket upon ligand binding in PDE2 catalytic site reduces the size of another distinct sub-pocket, and contribute to strong non-additive SAR between two otherwise distant R groups.

Abstract 2327: Structural and kinetic characterization of crizotinib with wild-type and mutant anaplastic lymphoma kinase

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Dysregulation of Anaplastic Lymphoma Kinase (ALK), primarily through gene translocations, has been shown to be involved in a variety of cancers. Crizotinib, an orally available small molecule inhibitor of the ALK tyrosine kinase has demonstrated marked efficacy in clinical trials of NSCLC patients harboring the EML4-ALK oncogenic gene rearrangement. Mutation of some residues within the ALK kinase domain have been reported to confer acquired or de novo resistance to crizotinib. To understand the binding of crizotinib to ALK and the mechanism of resistance of specific mutations we generated and kinetically characterized wild-type (WT) and mutant ALK kinase domains (KD). Additionally, ALK KD crystal structures were determined of the WT nonphosphorylated apoenzyme and complexes with crizotinib bound to WT and a L1196M gatekeeper mutation. No large protein conformational changes are necessary for crizotinib to bind to unliganded ALK. The interactions which crizotinib makes with ALK are similar to its binding to c-Met with the exception of notable differences in the position of the activation loop between ALK and c-Met. Mutation of the L1196 gatekeeper residue to methionine results in a ∼8-fold increase in catalytic efficiency of phosphorylation of an activation loop peptide and also more rapid enzyme auto-phosphorylation. In addition, inhibition of L1196M ALK by crizotinib was reduced ∼9-fold, compared to wild-type enzyme, from Ki determinations. The crystal structures show that L1196 or M1196 make direct contact with crizotinib. The diminished activity of crizotinib against L1196M ALK is therefore likely due to both higher intrinsic kinase activity and a subtle change in the ALK-crizotinib binding interactions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2327. doi:10.1158/1538-7445.AM2011-2327