Gregg Siegal

Perspectives on NMR in drug discovery: a technique comes of age

In the past decade, the potential of harnessing the ability of nuclear magnetic resonance (NMR) spectroscopy to monitor intermolecular interactions as a tool for drug discovery has been increasingly appreciated in academia and industry. In this Perspective, we highlight some of the major applications of NMR in drug discovery, focusing on hit and lead generation, and provide a critical analysis of its current and potential utility.

ATP-induced conformational changes of the nucleotide-binding domain of Na,K-ATPase.

The Na,K-ATPase hydrolyzes ATP to drive the coupled extrusion and uptake of Na+ and K+ ions across the plasma membrane. Here, we report two high-resolution NMR structures of the 213-residue nucleotide-binding domain of rat α1 Na,K-ATPase, determined in the absence and the presence of ATP. The nucleotide binds in the anti conformation and shows a relative paucity of interactions with the protein, reflecting the low-affinity ATP-binding state. Binding of ATP induces substantial conformational changes in the binding pocket and in residues located in the hinge region connecting the N- and P-domains. Structural comparison with the Ca-ATPase stabilized by the inhibitor thapsigargin, E2(TG), and the model of the H-ATPase in the E1 form suggests that the observed changes may trigger the series of events necessary for the release of the K+ ions and/or disengagement of the A-domain, leading to the eventual transfer of the γ-phosphate group to the invariant Asp369.

Fragment Screening of Stabilized G-Protein-Coupled Receptors Using Biophysical Methods

Biophysical studies with G-protein-coupled receptors (GPCRs) are typically very challenging due to the poor stability of these receptors when solubilized from the cell membrane into detergent solutions. However, the stability of a GPCR can be greatly improved by introducing a number of point mutations into the protein sequence to give a stabilized receptor or StaR®. Here, we present the utility of StaRs for biophysical studies and the screening of fragment libraries. Two case studies are used to illustrate the methods: first, the screening of a library of fragments by surface plasmon resonance against the adenosine A(2A) receptor StaR, demonstrating how very small and weakly active xanthine fragments can be detected binding to the protein on chips; second, the screening and detection of fragment hits of a larger fragment library in an NMR format called TINS (target-immobilized NMR screening) against the β(1) adrenergic StaR.

How to catch a membrane protein in action: a review of functional membrane protein immobilization strategies and their applications.

Analysis of the results from the human proteome project suggests >30% of proteins are membrane bound.1 Membrane proteins (MPs) are in the core group responsible for signal transduction, including the G-protein coupled receptors (GPCRs), which represent 30-40% of targets for marketed drugs.2 It is needless to stress, therefore, the great commercial, industrial, and research value of MPs. There is growing interest in immobilizing MPs to various surfaces to create new biosensors or platforms enabling the study of their biological functions and identification of new leads for drug discovery. Soluble proteins have been readily immobilized through cross-linking aldehydes or thiols with protein amines or carboxyl groups for applications such as microarraying by printing.3-6 Functional immobilization of MPs requires consideration of their physiological needs, often dictated by the quality and components of the natural hydrophobic environment surrounding this class of proteins. * To whom correspondence should be addressed. Leiden Institute of Chemistry, Leiden University, Postbus 9502, 2300RA Leiden, The Netherlands. Phone: +31 (0)71 527 4543. Fax: +31-(0)71-5274609. E-mail: G.Siegal@chem.leidenuniv.nl. † Leiden Amsterdam Center for Drug Research, Leiden University. ‡ Leiden Institute of Chemistry, Leiden University. Chem. Rev. 2011, 111, 640–656 640

Application of Fragment-Based Drug Discovery to Membrane Proteins: Identification of Ligands of the Integral Membrane Enzyme DsbB

Membrane proteins are important pharmaceutical targets, but they pose significant challenges for fragment-based drug discovery approaches. Here, we present the first successful use of biophysical methods to screen for fragment ligands to an integral membrane protein. The Escherichia coli inner membrane protein DsbB was solubilized in detergent micelles and lipid bilayer nanodiscs. The solubilized protein was immobilized with retention of functionality and used to screen 1071 drug fragments for binding using target immobilized NMR Screening. Biochemical and biophysical validation of the eight most potent hits revealed an IC(50) range of 7-200 microM. The ability to insert a broad array of membrane proteins into nanodiscs, combined with the efficiency of TINS, demonstrates the feasibility of finding fragments targeting membrane proteins.

Small-Molecule Binding Sites on Proteins Established by Paramagnetic NMR Spectroscopy

Determining the three-dimensional structure of a small molecule-protein complex with weak affinity can be a significant challenge. We present a paramagnetic NMR method to determine intermolecular structure restraints based on pseudocontact shifts (PCSs). Since the ligand must be in fast exchange between free and bound states and the fraction bound can be as low as a few percent, the method is ideal for ligands with high micromolar to millimolar dissociation constants. Paramagnetic tags are attached, one at a time, in a well-defined way via two arms at several sites on the protein surface. The ligand PCSs were measured from simple 1D (1)H spectra and used as docking restraints. An independent confirmation of the complex structure was carried out using intermolecular NOEs. The results show that structures derived from these two approaches are similar. The best results are obtained if the magnetic susceptibility tensors of the tags are known, but it is demonstrated that with two-armed probes, the magnetic susceptibility tensor can be predicted with sufficient accuracy to provide a low-resolution model of the ligand orientation and the location of the binding site in the absence of isotope-labeled protein. This approach can facilitate fragment-based drug discovery in obtaining structural information on the initial fragment hits.

Fragment Screening of GPCRs Using Biophysical Methods: Identification of Ligands of the Adenosine A2A Receptor with Novel Biological Activity

Fragment-based drug discovery (FBDD) has proven a powerful method to develop novel drugs with excellent oral bioavailability against challenging pharmaceutical targets such as protein-protein interaction targets. Very recently the underlying biophysical techniques have begun to be successfully applied to membrane proteins. Here we show that novel, ligand efficient small molecules with a variety of biological activities can be found by screening a small fragment library using thermostabilized (StaR) G protein-coupled receptors (GPCRs) and target immobilized NMR screening (TINS). Detergent-solubilized StaR adenosine A(2A) receptor was immobilized with retention of functionality, and a screen of 531 fragments was performed. Hits from the screen were thoroughly characterized for biochemical activity using the wild-type receptor. Both orthosteric and allosteric modulatory activity has been demonstrated in biochemical validation assays. Allosteric activity was confirmed in cell-based functional assays. The validated fragment hits make excellent starting points for a subsequent hit-to-lead elaboration program.

Conformational analysis of furanoid ε-sugar amino acid containing cyclic peptides by NMR spectroscopy, molecular dynamics simulation, and X-ray crystallography: Evidence for a novel turn structure

Sugar amino acids (SAAs) are useful building blocks for the design of peptidomimetics and peptide scaffolds. The three-dimensional structures of cyclic hybrid molecules containing the furanoid epsilon-SAA III and several amino acids were elucidated to study the preferred conformation of such an epsilon-SAA and its conformational influence on the backbone of cyclic peptides. NMR-based molecular dynamics simulations and empirical calculations of the cyclic tetramer 1, consisting of two copies of the SAA residue and two amino acids, revealed that it is conformationally restrained. The two SAA residues adopt different conformations. One of them forms an unusual turn, stabilized by an intraresidue nine-member hydrogen bond. The methylene functionalities of the other SAA residue are positioned in such a way that an intraresidue H bond is not possible. The X-ray crystal structure of 1 strongly resembles the solution conformation. Molecular dynamics calculations in combination with NMR analysis were also performed for compounds 2 and 3, which contain the RGD (Arg-Gly-Asp) consensus sequence and were previously shown to inhibit alpha(IIb)beta(3)-receptor-mediated platelet aggregation. The biologically most active compound 2 adopts a preferred conformation with the single SAA residue folded into the nine-member H bond-containing turn. Compound 3, containing an additional valine residue, as compared with compound 2, is conformational flexible. Our studies demonstrate that the furanoid epsilon-SAA III is able to introduce an unusual intraresidue hydrogen bond-stabilized beta-turn-like conformation in two of the three cyclic structures.

Complementarity between in Silico and Biophysical Screening Approaches in Fragment-Based Lead Discovery against the A2A Adenosine Receptor

Fragment-based lead discovery (FBLD) is becoming an increasingly important method in drug development. We have explored the potential to complement NMR-based biophysical screening of chemical libraries with molecular docking in FBLD against the A(2A) adenosine receptor (A(2A)AR), a drug target for inflammation and Parkinsons disease. Prior to an NMR-based screen of a fragment library against the A(2A)AR, molecular docking against a crystal structure was used to rank the same set of molecules by their predicted affinities. Molecular docking was able to predict four out of the five orthosteric ligands discovered by NMR among the top 5% of the ranked library, suggesting that structure-based methods could be used to prioritize among primary hits from biophysical screens. In addition, three fragments that were top-ranked by molecular docking, but had not been picked up by the NMR-based method, were demonstrated to be A(2A)AR ligands. While biophysical approaches for fragment screening are typically limited to a few thousand compounds, the docking screen was extended to include 328,000 commercially available fragments. Twenty-two top-ranked compounds were tested in radioligand binding assays, and 14 of these were A(2A)AR ligands with K(i) values ranging from 2 to 240 μM. Optimization of fragments was guided by molecular dynamics simulations and free energy calculations. The results illuminate strengths and weaknesses of molecular docking and demonstrate that this method can serve as a valuable complementary tool to biophysical screening in FBLD.

Target Immobilization as a Strategy for NMR-Based Fragment Screening Comparison of TINS, STD, and SPR for Fragment Hit Identification

Fragment-based drug discovery (FBDD) has become a widely accepted tool that is complementary to high-throughput screening (HTS) in developing small-molecule inhibitors of pharmaceutical targets. Because a fragment campaign can only be as successful as the hit matter found, it is critical that the first stage of the process be optimized. Here the authors compare the 3 most commonly used methods for hit discovery in FBDD: high concentration screening (HCS), solution ligand-observed nuclear magnetic resonance (NMR), and surface plasmon resonance (SPR). They selected the commonly used saturation transfer difference (STD) NMR spectroscopy and the proprietary target immobilized NMR screening (TINS) as representative of the array of possible NMR methods. Using a target typical of FBDD campaigns, the authors find that HCS and TINS are the most sensitive to weak interactions. They also find a good correlation between TINS and STD for tighter binding ligands, but the ability of STD to detect ligands with affinity weaker than 1 mM KD is limited. Similarly, they find that SPR detection is most suited to ligands that bind with KD better than 1 mM. However, the good correlation between SPR and potency in a bioassay makes this a good method for hit validation and characterization studies.