Structurally diverse MDM2-p53 antagonists act as modulators of MDR-1 function in neuroblastoma.

BACKGROUND: A frequent mechanism of acquired multidrug resistance in human cancers is overexpression of ATP-binding cassette transporters such as the Multi-Drug Resistance Protein 1 (MDR-1). Nutlin-3, an MDM2-p53 antagonist, has previously been reported to be a competitive MDR-1 inhibitor. METHODS: This study assessed whether the structurally diverse MDM2-p53 antagonists, MI-63, NDD0005, and RG7388 are also able to modulate MDR-1 function, particularly in p53 mutant neuroblastoma cells, using XTT-based cell viability assays, western blotting, and liquid chromatography-mass spectrometry analysis. RESULTS: Verapamil and the MDM2-p53 antagonists potentiated vincristine-mediated growth inhibition in a concentration-dependent manner when used in combination with high MDR-1-expressing p53 mutant neuroblastoma cell lines at concentrations that did not affect the viability of cells when given alone. Liquid chromatography-mass spectrometry analyses showed that verapamil, Nutlin-3, MI-63 and NDD0005, but not RG7388, led to increased intracellular levels of vincristine in high MDR-1-expressing cell lines. CONCLUSIONS: These results show that in addition to Nutlin-3, other structurally unrelated MDM2-p53 antagonists can also act as MDR-1 inhibitors and reverse MDR-1-mediated multidrug resistance in neuroblastoma cell lines in a p53-independent manner. These findings are important for future clinical trial design with MDM2-p53 antagonists when used in combination with agents that are MDR-1 substrates.

New epoetin molecules and novel therapeutic approaches.

Erythropoietin (EPO) is a 34 kDa protein that is the primary regulator of red blood cell production. EPO facilitates its effect by binding to the cell surface EPO receptor which initiates the JAK-STAT signal transduction cascade. The search for small mimetic molecules of EPO has led to the discovery of a family of peptides that demonstrate EPO mimetic activity. A member of this peptide family, EMP1 (EPO mimetic peptide 1), was used to solve the crystal structure of the soluble EPO receptor in complex with this peptide. The structure revealed a 2:2 stoichiometry of receptor to peptide, with each peptide contacting both receptor molecules in a symmetrical fashion. The potency of the EMPs could be improved through the covalent dimerization of two peptide molecules. Further investigations of EMP EPO receptor complex structures revealed the formation of a non-productive receptor dimer using an inactive peptide. An alternative approach towards the identification of an EPO-like mimetic is to target an intracellular signalling molecule such as haematopoietic cell phosphatase (HCP), also known as SHP1. Inhibiting HCP causes responsive cells to be hypersensitive to EPO. The cloned HCP protein has been utilized in screening assays to identify small molecule inhibitors of HCP.

Crystallographic evidence for preformed dimers of erythropoietin receptor before ligand activation.

Erythropoietin receptor (EPOR) is thought to be activated by ligand-induced homodimerization. However, structures of agonist and antagonist peptide complexes of EPOR, as well as an EPO-EPOR complex, have shown that the actual dimer configuration is critical for the biological response and signal efficiency. The crystal structure of the extracellular domain of EPOR in its unliganded form at 2.4 angstrom resolution has revealed a dimer in which the individual membrane-spanning and intracellular domains would be too far apart to permit phosphorylation by JAK2. This unliganded EPOR dimer is formed from self-association of the same key binding site residues that interact with EPO-mimetic peptide and EPO ligands. This model for a preformed dimer on the cell surface provides insights into the organization, activation, and plasticity of recognition of hematopoietic cell surface receptors.

Functional mimicry of a protein hormone by a peptide agonist: the EPO receptor complex at 2.8 A.

The functional mimicry of a protein by an unrelated small molecule has been a formidable challenge. Now, however, the biological activity of a 166-residue hematopoietic growth hormone, erythropoietin (EPO), with its class 1 cytokine receptor has been mimicked by a 20-residue cyclic peptide unrelated in sequence to the natural ligand. The crystal structure at 2.8 A resolution of a complex of this agonist peptide with the extracellular domain of EPO receptor reveals that a peptide dimer induces an almost perfect twofold dimerization of the receptor. The dimer assembly differs from that of the human growth hormone (hGH) receptor complex and suggests that more than one mode of dimerization may be able to induce signal transduction and cell proliferation. The EPO receptor binding site, defined by peptide interaction, corresponds to the smaller functional epitope identified for hGH receptor. Similarly, the EPO mimetic peptide ligand can be considered as a minimal hormone, and suggests the design of nonpeptidic small molecule mimetics for EPO and other cytokines may indeed be achievable.

Refolding, purification, and characterization of human erythropoietin binding protein produced in Escherichia coli.

The extracellular domain of the human erythropoietin receptor (EPO binding protein (EBP)) has been expressed and overproduced in Escherichia coli. Regardless of the presence ofpelB or ompT signal sequences the recombinant protein produced in this fashion appears, as with many other recombinant eukaryotic proteins produced in E. coli as an insoluble product in laboratory scale fermentations. The induction product of the pelB protein expression system appears as two protein forms with slightly different molecular weights. Based on N-terminal sequence analysis of recovered protein, these forms represent two variants, one with the signal sequence properly processed to yield the expected "native" amino terminus and another which retains the signal sequence. Both forms appear as insoluble fermentation products. Control of oxygen levels and pH during high density fermentation allows the production of only the protein variant with the native amino terminus. Methods reported here permit the efficient recovery of purified EBP which quantitatively binds EPO in solution as determined by high performance size exclusion chromatography. A long-lived refolding intermediate was observed which penultimately collapses into an active conformation. The active purified protein competes with membrane associated EPO receptor for binding [125I]EPO and neutralizes EPO-dependent stimulation in a cell based proliferation assay. Further, the radioligand equilibrium binding constant for this interaction has been determined by immobilizing EBP on agarose gel via a free cysteine. The production of EBP by these methods should facilitate the structural determination of the protein by NMR or crystallography and may serve as a guide for the refolding of other hematopoietic receptors.

Erythropoietin receptor: application in drug development.

Erythropoietin (EPO) is the primary hormone responsible for the growth and maturation of red blood cells in mammals. In contrast to many other growth factors, the specificity of EPO for mature erythroid cells has lead to its development as a safe and efficacious therapeutic, EPREX. The medical benefits of EPREX have been well established in the treatment of anaemic chronic renal failure patients, anaemic HIV patients treated with AZT, cancer chemotherapy patients, and patients wishing to donate their own blood prior to elective surgery (autologous predonation). Due to the chronic nature of EPO therapy, it would be desirable to have an orally administered 'second generation' molecule. An understanding of the structural basis of the interaction of EPO with its receptor will aid in the design of an oral anaemia drug. In this study, a series of mutations have been generated in a truncated form of the receptor comprising the extracellular region, termed EPO binding protein (EBP). One mutant, in which alanine replaces phenylalanine at position 93 (F93A) has a 500-fold reduction in binding compared to wild-type EBP. A neutralizing anti-EBP antibody binds poorly to the F93A mutant, while a non-neutralizing anti-EBP antibody binds wild-type and F93A equally well. Information from this mutational analysis can be applied to a receptor 3-D model and ultimately used in drug development.

Structural consequences of the replacement of Glu239 by Gln in the catalytic chain of Escherichia coli aspartate transcarbamylase.

Low-angle X-ray scattering in solution has been used to probe the quaternary structure of a mutant version of Escherichia coli aspartate transcarbamylase in which Glu239 of the catalytic chain was replaced by glutamine by site-directed mutagenesis. X-ray crystallographic studies of the wild-type enzyme have shown that one set of intersubunit interactions involving Glu239 are lost, and are replaced by another set of intrachain interactions when the enzyme undergoes the allosteric transition from the T to the R state. Functional analysis of the mutant enzyme with glutamine in place of Glu239 indicates that homotropic co-operativity is lost without altering the maximal specific activity. The radius of gyration of the unligated mutant enzyme is larger than the unligated wild-type, indicating an alteration in quaternary structure of the mutant. However, the radius of gyration of the mutant enzyme in the presence of N-(phosphonoacetyl)-L-aspartate (PALA) is identical with the value for the wild-type enzyme in the presence of PALA. X-ray scattering at larger angles indicates that the mutant enzyme is in a new structural state different from the wild-type T and R structures. The scattering pattern in the presence of saturating concentrations of PALA is identical with that of the wild-type R structure. Saturating concentrations of carbamyl phosphate alone are sufficient to convert most of the mutant enzyme to the R structure, in the absence of aspartate. CTP shifts the scattering pattern of the mutant enzyme in the presence of saturating carbamyl phosphate towards the scattering curve of the unligated enzyme, but CTP has no effect on the scattering curve in the absence of carbamyl phosphate or in the presence of subsaturating PALA. However, in the presence of subsaturating PALA, ATP causes a strong shift towards the R structure. Neither ATP nor CTP has any effect on the activity of the mutant enzyme. These data suggest that the replacement of Glu239 by glutamine results in a new quaternary structure. These data also explain, on a structural basis, why co-operativity is lost in this mutant enzyme.

Regulatory behavior of Escherichia coli aspartate transcarbamylase altered by site-specific mutation of Tyr240----Phe in the catalytic chain.

Isotopic exchange kinetics at chemical equilibrium have been used to identify changes in the regulatory properties of aspartate transcarbamylase (ATCase) caused by site-specific mutation of Tyr240----Phe (Y240F) in the catalytic chain. With both wild-type and the mutant enzymes, ATP activates both [14C]Asp in equilibrium N-carbamyl-L-aspartate (C-Asp) and the [32P]carbamyl phosphate (C-P) in equilibrium Pi exchanges. In contrast, with wild-type enzyme, CTP inhibits both exchanges, but with Y240F mutant enzyme CTP inhibits Asp in equilibrium C-Asp exchange and activates C-P in equilibrium Pi exchange. The bisubstrate analog N-(phosphonacetyl-L-aspartate), PALA, activates Asp in equilibrium C-Asp at a lower concentration with the Y240F enzyme, but the extent of activation is decreased, relative to wild-type enzyme. PALA activation of C-P in equilibrium Pi observed with wild-type enzyme disappears completely with the Y240F mutant enzyme. Analysis of perturbations of exchange rates by ATP and CTP were carried out by systematic methods plus computer-based simulations with the ISOBI program. These analyses indicate that (a) ATP increases the rates of association and dissociation for both C-P and Asp, but (b) CTP differentially increases the rate of C-P association to a greater degree than dissociation, but also decreases the rates for Asp association and dissociation in equal proportion. In addition, Arrhenius plots for Y240F ATCase suggest that ATP and CTP act by different mechanisms: ATP increases Vmax (decreases delta G not equal to) uniformly at all temperatures, whereas CTP does not alter either Vmax (delta G not equal to) or the Arrhenius slope (delta H not equal to).

Relationship between domain closure and binding, catalysis, and regulation in Escherichia coli aspartate transcarbamylase.

Previous evidence, from both crystallographic and biochemical studies, has indicated that profound tertiary and quaternary changes in the structure of Escherichia coli aspartate transcarbamylase occur upon the binding of the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate (PALA). In particular, within a single catalytic polypeptide chain, the aspartate binding domain relocates closer to the carbamyl phosphate binding domain, thereby resulting in a major reorganization of the interface between the two domains. Among the new interactions, salt bridges between Glu-50 and both Arg-167 and Arg-234 are formed. In the present study, site-directed mutagenesis is used to replace Glu-50 by glutamine in the catalytic chain. The Michaelis constant for aspartate of the mutant catalytic subunit is about 10-fold higher and the turnover number 10-fold lower than their respective counterparts in the wild-type catalytic subunit, whereas the dissociation constant for carbamyl phosphate is almost unchanged. For the holoenzyme, this substitution results in an 8-fold decrease in the specific activity, a 20-fold increase in the aspartate concentration that gives half of the maximal velocity, and a loss of cooperativity for both substrates. However, the mutant enzyme is not "frozen" in a low-affinity-low-activity conformation since PALA stimulates the activity severalfold and induces an increase in the sulfhydryl reactivity analogous to that of the wild-type enzyme. Together these results indicate that the stabilization of the aspartate binding domain near the carbamyl phosphate binding domain, through specific interdomain bridging interactions, is necessary for the high-affinity-high-activity configuration of the active site.(ABSTRACT TRUNCATED AT 250 WORDS)