Eleanor W. W. Leung

Applications of 19F-NMR in Fragment-Based Drug Discovery

19F-NMR has proved to be a valuable tool in fragment-based drug discovery. Its applications include screening libraries of fluorinated fragments, assessing competition among elaborated fragments and identifying the binding poses of promising hits. By observing fluorine in both the ligand and the target protein, useful information can be obtained on not only the binding pose but also the dynamics of ligand-protein interactions. These applications of 19F-NMR will be illustrated in this review with studies from our fragment-based drug discovery campaigns against protein targets in parasitic and infectious diseases.

19F NMR as a Probe of Ligand Interactions with the iNOS Binding site of SPRY Domain‐Containing SOCS Box Protein 2

SPRY domain‐containing SOCS box protein 2 (SPSB2) regulates inducible nitric oxide synthase (iNOS) by targeting it for proteasomal degradation. Inhibiting this interaction prolongs the intracellular lifetime of iNOS, leading in turn to enhanced killing of infectious pathogens such as bacteria and parasites. SPSB2 recognizes a linear motif (DINNN) in the disordered N‐terminus of iNOS, and ligands that target the DINNN binding site on SPSB2 are potentially novel anti‐infective agents. We have explored 19F NMR as a means of probing ligand binding to SPSB2. All six Trp residues in SPSB2 were replaced with 5‐fluorotryptophan (5‐F‐Trp) by utilizing a Trp auxotroph strain of Escherichia coli. The labeled protein was well folded and bound a DINNN‐containing peptide with similar affinity to native SPSB2. Six well‐resolved 5‐F‐Trp resonances were observed in the 19F NMR spectrum and were assigned using site‐directed mutagenesis. The 19F resonance of W207 was significantly perturbed upon binding to DINNN‐containing peptides. Other resonances were perturbed to a lesser extent although in a way that was sensitive to the composition of the peptide. Analogues of compounds identified in a fragment screen also perturbed the W207 resonance, confirming their binding to the iNOS peptide‐binding site on SPSB2. 19F NMR promises to be a valuable approach in developing inhibitors that bind to the DINNN binding site.

Expression and isotopic labelling of the potassium channel blocker ShK toxin as a thioredoxin fusion protein in bacteria.

The polypeptide toxin ShK is a potent blocker of Kv1.3 potassium channels, which play a crucial role in the activation of human effector memory T-cells (T(EM)). Selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by T(EM) cells, such as multiple sclerosis, rheumatoid arthritis, and type-1 diabetes. We have established a recombinant peptide expression system in order to generate isotopically-labelled ShK and various ShK analogues for in-depth biophysical and pharmacological studies. ShK was expressed as a thioredoxin fusion protein in Escherichia coli BL21 (DE3) cells and purified initially by Ni²⁺ iminodiacetic acid affinity chromatography. The fusion protein was cleaved with enterokinase and purified to homogeneity by reverse-phase HPLC. NMR spectra of ¹⁵N-labelled ShK were similar to those reported previously for the unlabelled synthetic peptide, confirming that recombinant ShK was correctly folded. Recombinant ShK blocked Kv1.3 channels with a K(d) of 25 pM and inhibited the proliferation of human and rat T lymphocytes with a preference for T(EM) cells, with similar potency to synthetic ShK in all assays. This expression system also enables the efficient production of ¹⁵N-labelled ShK for NMR studies of peptide dynamics and of the interaction of ShK with Kv1.3 channels.

A potent cyclic peptide targeting SPSB2 protein as a potential anti-infective agent.

The protein SPSB2 mediates proteosomal degradation of inducible nitric oxide synthase (iNOS). Inhibitors of SPSB2-iNOS interaction may prolong the lifetime of iNOS and thereby enhance the killing of persistent pathogens. We have designed a cyclic peptide, Ac-c[CVDINNNC]-NH2, containing the key sequence motif mediating the SPSB2-iNOS interaction, which binds to the iNOS binding site on SPSB2 with a Kd of 4.4 nM, as shown by SPR, [(1)H,(15)N]-HSQC, and (19)F NMR. An in vitro assay on macrophage cell lysates showed complete inhibition of SPSB2-iNOS interactions by the cyclic peptide. Furthermore, its solution structure closely matched (backbone rmsd 1.21 Å) that of the SPSB2-bound linear DINNN peptide. The designed peptide was resistant to degradation by the proteases pepsin, trypsin, and chymotrypsin and stable in human plasma. This cyclic peptide exemplifies potentially a new class of anti-infective agents that acts on the host innate response, thereby avoiding the development of pathogen resistance.

Defining the interaction of perforin with calcium and the phospholipid membrane

Following its secretion from cytotoxic lymphocytes into the immune synapse, perforin binds to target cell membranes through its Ca(2+)-dependent C2 domain. Membrane-bound perforin then forms pores that allow passage of pro-apoptopic granzymes into the target cell. In the present study, structural and biochemical studies reveal that Ca(2+) binding triggers a conformational change in the C2 domain that permits four key hydrophobic residues to interact with the plasma membrane. However, in contrast with previous suggestions, these movements and membrane binding do not trigger irreversible conformational changes in the pore-forming MACPF (membrane attack complex/perforin-like) domain, indicating that subsequent monomer-monomer interactions at the membrane surface are required for perforin pore formation.

Redox‐stable cyclic peptide inhibitors of the SPSB2–iNOS interaction

SPSB2 mediates the proteasomal degradation of iNOS. Inhibitors of SPSB2–iNOS interaction are expected to prolong iNOS lifetime and thereby enhance killing of persistent pathogens. Here, we describe the synthesis and characterization of two redox‐stable cyclized peptides containing the DINNN motif required for SPSB2 binding. Both analogues bind with low nanomolar affinity to the iNOS binding site on SPSB, as determined by SPR and 19F NMR, and efficiently displace full‐length iNOS from binding to SPSB2 in macrophage cell lysates. These peptides provide a foundation for future development of redox‐stable, potent ligands for SPSB proteins as a potential novel class of anti‐infectives.

Structural Basis for Ca2+-mediated Interaction of the Perforin C2 Domain with Lipid Membranes

Background: Perforin is a critical component of immune homeostasis, responsible for clearing virally infected cells. Results: The molecular details of calcium binding by the perforin C2 domain are revealed. Conclusion: Calcium-mediated structural rearrangement activates perforin for membrane binding. Significance: The C2 domain regulates membrane binding by calcium-dependent events, which have now been defined for a mammalian perforin C2. Natural killer cells and cytotoxic T-lymphocytes deploy perforin and granzymes to kill infected host cells. Perforin, secreted by immune cells, binds target membranes to form pores that deliver pro-apoptotic granzymes into the target cell. A crucial first step in this process is interaction of its C2 domain with target cell membranes, which is a calcium-dependent event. Some aspects of this process are understood, but many molecular details remain unclear. To address this, we investigated the mechanism of Ca2+ and lipid binding to the C2 domain by NMR spectroscopy and x-ray crystallography. Calcium titrations, together with dodecylphosphocholine micelle experiments, confirmed that multiple Ca2+ ions bind within the calcium-binding regions, activating perforin with respect to membrane binding. We have also determined the affinities of several of these binding sites and have shown that this interaction causes a significant structural rearrangement in CBR1. Thus, it is proposed that Ca2+ binding at the weakest affinity site triggers changes in the C2 domain that facilitate its interaction with lipid membranes.

Design, Synthesis, and Characterization of Cyclic Peptidomimetics of the Inducible Nitric Oxide Synthase Binding Epitope That Disrupt the Protein-Protein Interaction Involving SPRY Domain-Containing Suppressor of Cytokine Signaling Box Protein (SPSB) 2 and Inducible Nitric Oxide Synthase

SPRY domain-containing suppressor of cytokine signaling box protein (SPSB) 2-deficient macrophages have been found to exhibit prolonged expression of inducible nitric oxide synthase (iNOS) and enhanced killing of persistent pathogens, suggesting that inhibitors of the SPSB2-iNOS interaction have potential as novel anti-infectives. In this study, we describe the design, synthesis, and characterization of cyclic peptidomimetic inhibitors of the SPSB2-iNOS interaction constrained by organic linkers to improve stability and druggability. SPR, ITC, and (19)F NMR analyses revealed that the most potent cyclic peptidomimetic bound to the iNOS binding site of SPSB2 with low nanomolar affinity (KD 29 nM), a 10-fold improvement over that of the linear peptide DINNN (KD 318 nM), and showed strong inhibition of SPSB2-iNOS interaction in macrophage cell lysates. This study exemplifies a novel approach to cyclize a Type II β-turn linear peptide and provides a foundation for future development of this group of inhibitors as new anti-infectives.

Structure and activity of contryphan-Vc2: Importance of the d-amino acid residue

Abstract In natural proteins and peptides, amino acids exist almost invariably as l‐isomers. There are, however, several examples of naturally‐occurring peptides containing d‐amino acids. In this study we investigated the role of a naturally‐occurring d‐amino acid in a small peptide identified in the transcriptome of a marine cone snail. This peptide belongs to a family of peptides known as contryphans, all of which contain a single d‐amino acid residue. The solution structure of this peptide was solved by NMR, but further investigations with molecular dynamics simulations suggest that its solution behaviour may be more dynamic than suggested by the NMR ensemble. Functional tests in mice uncovered a novel bioactivity, a depressive phenotype that contrasts with the hyperactive phenotypes typically induced by contryphans. Trp3 is important for bioactivity, but this role is independent of the chirality at this position. The d‐chirality of Trp3 in this peptide was found to be protective against enzymatic degradation. Analysis by NMR and molecular dynamics simulations indicated an interaction of Trp3 with lipid membranes, suggesting the possibility of a membrane‐mediated mechanism of action for this peptide. Graphical abstract Figure. No caption available. HighlightsContryphan‐Vc2 contains a natural d‐amino acid residue.Both d‐ and l‐analogues exhibit the same bioactivity.Both analogues interact with lipid membranes.Possible membrane‐mediated mechanism of action.

Structure and Functional Characterization of the Conserved JAK Interaction Region in the Intrinsically Disordered N-Terminus of SOCS5.

SOCS5 can negatively regulate both JAK/STAT and EGF-receptor pathways and has therefore been implicated in regulating both the immune response and tumorigenesis. Understanding the molecular basis for SOCS5 activity may reveal novel ways to target key components of these signaling pathways. The N-terminal region of SOCS5 coordinates critical protein interactions involved in inhibition of JAK/STAT signaling, and a conserved region within the N-terminus of SOCS5 mediates direct binding to the JAK kinase domain. Here we have characterized the solution conformation of this conserved JAK interaction region (JIR) within the largely disordered N-terminus of SOCS5. Using nuclear magnetic resonance (NMR) chemical shift analysis, relaxation measurements, and NOE analysis, we demonstrate the presence of preformed structural elements in the JIR of mouse SOCS5 (mSOCS5175-244), consisting of an α-helix encompassing residues 224-233, preceded by a turn and an extended structure. We have identified a phosphorylation site (Ser211) within the JIR of mSOCS5 and have investigated the role of phosphorylation in modulating JAK binding using site-directed mutagenesis.