Aluminum binding to phosphatidylcholine lipid bilayer membranes: aluminum exchange lifetimes from 31P NMR spectroscopy.

Two-dimensional (2D) (31)P magic angle spinning (MAS) nuclear magnetic resonance (NMR) exchange spectroscopy (EXSY) demonstrated that aluminum binds to the phosphate group of phosphatidylcholine (PC) in multilamellar vesicles at pH 3.2, forming preferentially 2/1, in addition to 1/1 (PC/Al) complexes in slow exchange with one another, and with free PC, on the NMR timescale. Exchange rate constants between these three co-existing species were measured as a function of temperature using one-dimensional (1D) selective inversion recovery (SIR) (31)P MAS NMR. Over the temperature range from 5 to 35 degrees C all three exchange rate constants increased by roughly an order of magnitude from k approximately 1-2 to 10-14s(-1), exhibiting Arrhenius behavior with activation energies on the order of 30-45 kJ mol(-1) and correspondingly positive enthalpies of activation. Entropies of activation were uniformly negative, consistent with an ordered transition state. From a biological perspective, the results demonstrate that aluminum binding to PC in biomembranes is transient on a biologically relevant time scale, so that the lipid bilayer portion of biomembranes is unlikely to act as a long term repository for aluminum, but rather should be viewed as a temporary reservoir of biologically available aluminum.

Expression and characterization of the FXYD ion transport regulators for NMR structural studies in lipid micelles and lipid bilayers.

The proteins PLM (phospholemman), CHIF (channel inducing factor), and Mat8 (mammary tumor protein 8 kDa) are members of the FXYD family of ion transport regulatory membrane proteins. Here we describe their cloning and expression in Escherichia coli, and their purification for NMR structural studies in lipid micelles and lipid bilayers. The molecular masses of the purified recombinant FXYD proteins, determined from SDS-PAGE and from MALDI TOF mass spectrometry, reflect monomeric species. The solution NMR and CD spectra in SDS micelles show that they adopt helical conformations. The solid-state NMR spectra in lipid bilayers give the first view of their transmembrane architecture.

Discovery of a novel class of reversible non-peptide caspase inhibitors via a structure-based approach.

In this paper, we report a simple structure-based iterative optimizations (SUBITO) strategy to identify and optimize new protein ligands and inhibitors. The approach is based on a combination of NMR-based screening and computational docking methods and enabled the identification of novel chemical leads among hundreds of thousands of commercially available compounds by screening only a few hundred compounds from a scaffold library followed by iterative screening steps where only few dozen compounds are tested. As an application, we report on the discovery of a novel class of non-peptide reversible caspase inhibitors, with IC(50) values in the low micromolar range.

Targeting apoptosis via chemical design: inhibition of bid-induced cell death by small organic molecules.

Bid is a key member of the Bcl-2 family proteins involved in the control of the apoptotic cascade in cells, leading to cell death. Uncontrolled cell death is associated with several human pathologies, such as neurodegenerative diseases and ischemic injuries. Therefore, Bid represents a potential yet unexplored and challenging target for strategies aimed at the development of therapeutic agents. Here we show that a multidisciplinary NMR-based approach that we named SAR by ILOEs (structure activity relationships by interligand nuclear Overhauser effect) allowed us to rationally design a series of 4-phenylsulfanyl-phenylamine derivatives that are capable of occupying a deep hydrophobic crevice on the surface of Bid. These compounds represent the first antiapoptotic small molecules targeting a Bcl-2 protein as shown by their ability to inhibit tBid-induced SMAC release, caspase-3 activation, and cell death.

Hydration-optimized oriented phospholipid bilayer samples for solid-state NMR structural studies of membrane proteins.

The preparation of oriented, hydration-optimized lipid bilayer samples, for NMR structure determination of membrane proteins, is described. The samples consist of planar phospholipid bilayers, containing membrane proteins, that are oriented on single pairs of glass slides, and are placed in the coil of the NMR probe with the bilayer plane perpendicular to the direction of the magnetic field. Lipid bilayers provide a medium that closely resembles the biological membrane, and sample orientation both preserves the intrinsic membrane-defined directional quality of membrane proteins, and provides the mechanism for resonance line narrowing. The hydration-optimized samples overcome some of the difficulties associated with multi-dimensional, high-resolution, solid-state NMR spectroscopy of membrane proteins. These samples have greater stability over the course of multi-dimensional NMR experiments, they have lower sample conductance for greater rf power efficiency, and enable greater rf coil filling factors to be obtained for improved experimental sensitivity. Sample preparation is illustrated for the membrane protein CHIF (channel inducing factor), a member of the FXYD family of ion transport regulators.

Aluminum binding to phosphatidylcholine lipid bilayer membranes: 27Al and 31P NMR spectroscopic studies.

27Al and 31P nuclear magnetic resonance (NMR) spectroscopies were used to investigate aluminum interactions at pH 3.4 with model membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). A solution state 27Al NMR difference assay was developed to quantify aluminum binding to POPC multilamellar vesicles (MLVs). Corresponding one-dimensional (1D) fast magic angle spinning (MAS) 31P NMR spectra showed that aluminum induced the appearance of two new isotropic resonances for POPC shifted to -6.4 ppm and -9.6 ppm upfield relative to, and in slow exchange with, the control resonance at -0.6 ppm. Correlation of the (27)Al and (31)P NMR binding data revealed a 1:2 aluminum:phospholipid stoichiometry in the aluminum-bound complex at -9.6 ppm and a 1:1 aluminum:phospholipid stoichiometry in that at -6.4 ppm. Slow MAS 31P NMR spectra demonstrated shifts in the anisotropic chemical shift tensor components of the aluminum-bound POPC consistent with a close coordination of aluminum with phosphorus. A model of the aluminum-bis-phospholipid complex is proposed on the basis of these findings.

The FRB domain of mTOR: NMR solution structure and inhibitor design.

The mammalian target of rapamycin (mTOR) is a protein that is intricately involved in signaling pathways controlling cell growth. Rapamycin is a natural product that binds and inhibits mTOR function by interacting with its FKBP-rapamycin-binding (FRB) domain. Here we report on the NMR solution structure of FRB and on further studies aimed at the identification and characterization of novel ligands that target the rapamycin binding pocket. The biological activity of the ligands, and that of rapamycin in the absence of FKBP12, was investigated by assaying the kinase activity of mTOR. While we found that rapamycin binds the FRB domain and inhibits the kinase activity of mTOR even in the absence of FKBP12 (in the low micromolar range), our most potent ligands bind to FRB with similar binding affinity but inhibit the kinase activity of mTOR at much higher concentrations. However, we have also identified one low-affinity compound that is also capable of inhibiting mTOR. Hence, we have identified compounds that can directly mimic rapamycin or can dissociate the FRB binding from the inhibition of the catalytic activity of mTOR. As such, these ligands could be useful in deciphering the complex regulation of mTOR in the cell and in validating the FRB domain as a possible target for the development of novel therapeutic compounds.

Structure-activity relationships by interligand NOE-based design and synthesis of antiapoptotic compounds targeting Bid.

Bcl-2 family proteins play a crucial role in tissue homeostasis and apoptosis (programmed cell death). Bid is a proapoptotic member of the Bcl-2 family, promoting cell death when activated by caspase-8. Following an NMR-based approach (structure-activity relationships by interligand NOE) we were able to identify two chemical fragments that bind on the surface of Bid. Covalent linkage of the two fragments led to high-affinity bidentate derivatives. In vitro and in-cell assays demonstrate that the compounds prevent tBid translocation to the mitochondrial membrane and the subsequent release of proapoptotic stimuli and inhibit neuronal apoptosis in the low micromolar range. Therefore, by using a rational chemical-biology approach, we derived antiapoptotic compounds that may have a therapeutic potential for disorders associated with Bid activation, e.g., neurodegenerative diseases, cerebral ischemia, or brain trauma.

NMR-based techniques in the hit identification and optimisation processes.

In this review, the use of general NMR spectroscopy techniques to detect ligand binding and to monitor enzyme kinetics and inhibition, which appear particularly useful in hit identification and validation, is reiterated. Furthermore, the use of NMR-based strategies for lead optimisations that are based on either iterative derivatisations of an initial core structure or on linking fragments that occupy adjacent pockets in the target's binding site will also be described. Several recent examples will be reported and the use of these techniques in cases when the three dimensional structure of the target protein is known will be discussed.