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.

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.

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.

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.

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.

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).

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.

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.

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.