Publications (12) View all
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Article: Flexibility of the P-loop of Pim-1 kinase: observation of a novel conformation induced by interaction with an inhibitor.
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ABSTRACT: The serine/threonine kinase Pim-1 is emerging as a promising target for cancer therapeutics. Much attention has recently been focused on identifying potential Pim-1 inhibitor candidates for the treatment of haematopoietic malignancies. The outcome of a rational drug-design project has recently been reported [Nakano et al. (2012), J. Med. Chem. 55, 5151-5156]. The report described the process of optimization of the structure-activity relationship and detailed from a medicinal chemistry perspective the development of a low-potency and nonselective compound initially identified from in silico screening into a potent, selective and metabolically stable Pim-1 inhibitor. Here, the structures of the initial in silico hits are reported and the noteworthy features of the Pim-1 complex structures are described. A particular focus was placed on the rearrangement of the glycine-rich P-loop region that was observed for one of the initial compounds, (Z)-7-(azepan-1-ylmethyl)-2-[(1H-indol-3-yl)methylidene]-6-hydroxy-1-benzofuran-3(2H)-one (compound 1), and was also found in all further derivatives. This novel P-loop conformation, which appears to be stabilized by an additional interaction with the β3 strand located above the binding site, is not usually observed in Pim-1 structures.Acta Crystallographica Section F Structural Biology and Crystallization Communications 08/2012; 68(Pt 8):860-6. · 0.51 Impact Factor -
Article: Structural approaches to probing metal interaction with proteins.
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ABSTRACT: In this mini-review we focus on metal interactions with proteins with a particular emphasis on the evident synergism between different biophysical approaches toward understanding metallobiology. We highlight three recent examples from our own laboratory. Firstly, we describe metallodrug interactions with glutathione S-transferases, an enzyme family known to attack commonly used anti-cancer drugs. We then describe a protein target for memory enhancing drugs called insulin-regulated aminopeptidase in which zinc plays a role in catalysis and regulation. Finally we describe our studies on a protein, amyloid precursor protein, that appears to play a central role in Alzheimer's disease. Copper ions have been implicated in playing both beneficial and detrimental roles in the disease by binding to different regions of this protein.Journal of inorganic biochemistry 03/2012; 115:138-47. · 3.25 Impact Factor -
Article: Studies of glutathione transferase P1-1 bound to a platinum(IV)-based anticancer compound reveal the molecular basis of its activation.
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ABSTRACT: Platinum-based cancer drugs, such as cisplatin, are highly effective chemotherapeutic agents used extensively for the treatment of solid tumors. However, their effectiveness is limited by drug resistance, which, in some cancers, has been associated with an overexpression of pi class glutathione S-transferase (GST P1-1), an important enzyme in the mercapturic acid detoxification pathway. Ethacraplatin (EA-CPT), a trans-Pt(IV) carboxylate complex containing ethacrynate ligands, was designed as a platinum cancer metallodrug that could also target cytosolic GST enzymes. We previously reported that EA-CPT was an excellent inhibitor of GST activity in live mammalian cells compared to either cisplatin or ethacrynic acid. In order to understand the nature of the drug-protein interactions between EA-CPT and GST P1-1, and to obtain mechanistic insights at a molecular level, structural and biochemical investigations were carried out, supported by molecular modeling analysis using quantum mechanical/molecular mechanical methods. The results suggest that EA-CPT preferentially docks at the dimer interface at GST P1-1 and subsequent interaction with the enzyme resulted in docking of the ethacrynate ligands at both active sites (in the H-sites), with the Pt moiety remaining bound at the dimer interface. The activation of the inhibitor by its target enzyme and covalent binding accounts for the strong and irreversible inhibition of enzymatic activity by the platinum complex.Chemistry 07/2011; 17(28):7806-16. · 5.93 Impact Factor -
Article: Influence of the H‐site residue 108 on human glutathione transferase P1‐1 ligand binding: Structure‐thermodynamic relationships and thermal stability
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ABSTRACT: The effect of the Y108V mutation of human glutathione S-transferase P1-1 (hGST P1-1) on the binding of the diuretic drug ethacrynic acid (EA) and its glutathione conjugate (EASG) was investigated by calorimetric, spectrofluorimetric, and crystallographic studies. The mutation Tyr 108 → Val resulted in a 3D-structure very similar to the wild type (wt) enzyme, where both the hydrophobic ligand binding site (H-site) and glutathione binding site (G-site) are unchanged except for the mutation itself. However, due to a slight increase in the hydrophobicity of the H-site, as a consequence of the mutation, an increase in the entropy was observed. The Y108V mutation does not affect the affinity of EASG for the enzyme, which has a higher affinity (Kd ∼ 0.5 μM) when compared with those of the parent compounds, K ∼ 13 μM, K ∼ 25 μM. The EA moiety of the conjugate binds in the H-site of Y108V mutant in a fashion completely different to those observed in the crystal structures of the EA or EASG wt complex structures. We further demonstrate that the ΔCp values of binding can also be correlated with the potential stacking interactions between ligand and residues located in the binding sites as predicted from crystal structures. Moreover, the mutation does not significantly affect the global stability of the enzyme. Our results demonstrate that calorimetric measurements maybe useful in determining the preference of binding (the binding mode) for a drug to a specific site of the enzyme, even in the absence of structural information.Protein Science 09/2009; 18(12):2454 - 2470. · 2.80 Impact Factor -
Article: Structural basis for the altered drug sensitivities of non-small cell lung cancer-associated mutants of human epidermal growth factor receptor.
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ABSTRACT: The epidermal growth factor receptor (EGFR) has an essential role in multiple signaling pathways, including cell proliferation and migration, through extracellular ligand binding and subsequent activation of its intracellular tyrosine kinase (TK) domain. The non-small cell lung cancer (NSCLC)-associated EGFR mutants, L858R and G719S, are constitutively active and oncogenic. They display sensitivity to TK inhibitors, including gefitinib and erlotinib. In contrast, the secondary mutation of the gatekeeper residue, T790M, reportedly confers inhibitor resistance on the oncogenic EGFR mutants. In this study, our biochemical analyses revealed that the introduction of the T790M mutation confers gefitinib resistance on the G719S mutant. The G719S/T790M double mutant has enhanced activity and retains high gefitinib-binding affinity. The T790M mutation increases the ATP affinity of the G719S mutant, explaining the acquired drug resistance of the double mutant. Structural analyses of the G719S/T790M double mutant, as well as the wild type and the G719S and L858R mutants, revealed that the T790M mutation stabilizes the hydrophobic spine of the active EGFR-TK conformation. The Met790 side chain of the G719S/T790M double mutant, in the apo form and gefitinib- and AMPPNP-bound forms, adopts different conformations that explain the accommodation of these ligands. In the L858R mutant structure, the active-site cleft is expanded by the repositioning of Phe723 within the P-loop. Notably, the introduction of the F723A mutation greatly enhanced the gefitinib sensitivity of the wild-type EGFR in vivo, supporting our hypothesis that the expansion of the active-site cleft results in enhanced gefitinib sensitivity. Taken together, our results provide a structural basis for the altered drug sensitivities caused by distinct NSCLC-associated EGFR mutations.Oncogene advance online publication 20 February 2012; doi:10.1038/onc.2012.21.Oncogene 02/2012; · 6.37 Impact Factor
