Leszek Poppe

Hepcidin revisited, disulfide connectivity, dynamics, and structure.

Hepcidin is a tightly folded 25-residue peptide hormone containing four disulfide bonds, which has been shown to act as the principal regulator of iron homeostasis in vertebrates. We used multiple techniques to demonstrate a disulfide bonding pattern for hepcidin different from that previously published. All techniques confirmed the following disulfide bond connectivity: Cys1–Cys8, Cys3–Cys6, Cys2–Cys4, and Cys5–Cys7. NMR studies reveal a new model for hepcidin that, at ambient temperatures, interconverts between two different conformations, which could be individually resolved by temperature variation. Using these methods, the solution structure of hepcidin was determined at 325 and 253 K in supercooled water. X-ray analysis of a co-crystal with Fab appeared to stabilize a hepcidin conformation similar to the high temperature NMR structure.

Design and Synthesis of Conformationally Constrained Glucagon-Like Peptide-1 Derivatives with Increased Plasma Stability and Prolonged in Vivo Activity

A series of conformationally constrained derivatives of glucagon-like peptide-1 (GLP-1) were designed and evaluated. By use of [Gly (8)]GLP-1(7-37)-NH2 (2) peptide as a starting point, 17 cyclic derivatives possessing i to i + 4, i to i + 5, or i to i + 7 side chain to side chain lactam bridges from positions 18 to 30 were prepared. The effect of a helix-promoting alpha-amino-isobutyric acid (Aib) substitution at position 22 was also evaluated. The introduction of i to i + 4 glutamic acid-lysine lactam constraints in c[Glu (18)-Lys (22)][Gly (8)]GLP-1(7-37)-NH2 (6), c[Glu (22)-Lys (26)][Gly (8)]GLP-1(7-37)-NH2 (10), and c[Glu (23)-Lys (27)][Gly (8)]GLP-1(7-37)-NH2 (11) resulted in potent functional activity and receptor affinities comparable to native GLP-1. Selected GLP-1 peptides were chemoselectively PEGylated in order to prolong their in vivo activity. PEGylated peptides [Gly (8),Aib (22)]GLP-1(7-37)-Cys ((PEG))-Ala-NH2 (23) and c[Glu (22)-Lys (26)][Gly (8)]GLP-1(7-37)-Cys ((PEG))-Ser-Gly-NH2 (24) retained picomolar functional potency and avid receptor binding properties. Importantly, PEGylated GLP-1 peptide 23 exhibited sustained in vivo efficacy with respect to blood glucose reduction and decreased body weight for several days in nonhuman primates.

Ordering of the N-terminus of human MDM2 by small molecule inhibitors.

Restoration of p53 function through the disruption of the MDM2-p53 protein complex is a promising strategy for the treatment of various types of cancer. Here, we present kinetic, thermodynamic, and structural rationale for the remarkable potency of a new class of MDM2 inhibitors, the piperidinones. While these compounds bind to the same site as previously reported for small molecule inhibitors, such as the Nutlins, data presented here demonstrate that the piperidinones also engage the N-terminal region (residues 10-16) of human MDM2, in particular, Val14 and Thr16. This portion of MDM2 is unstructured in both the apo form of the protein and in MDM2 complexes with p53 or Nutlin, but adopts a novel β-strand structure when complexed with the piperidinones. The ordering of the N-terminus upon binding of the piperidinones extends the current model of MDM2-p53 interaction and provides a new route to rational design of superior inhibitors.

Engineering Potent and Selective Analogues of GpTx-1, a Tarantula Venom Peptide Antagonist of the NaV1.7 Sodium Channel

NaV1.7 is a voltage-gated sodium ion channel implicated by human genetic evidence as a therapeutic target for the treatment of pain. Screening fractionated venom from the tarantula Grammostola porteri led to the identification of a 34-residue peptide, termed GpTx-1, with potent activity on NaV1.7 (IC50 = 10 nM) and promising selectivity against key NaV subtypes (20× and 1000× over NaV1.4 and NaV1.5, respectively). NMR structural analysis of the chemically synthesized three disulfide peptide was consistent with an inhibitory cystine knot motif. Alanine scanning of GpTx-1 revealed that residues Trp(29), Lys(31), and Phe(34) near the C-terminus are critical for potent NaV1.7 antagonist activity. Substitution of Ala for Phe at position 5 conferred 300-fold selectivity against NaV1.4. A structure-guided campaign afforded additive improvements in potency and NaV subtype selectivity, culminating in the design of [Ala5,Phe6,Leu26,Arg28]GpTx-1 with a NaV1.7 IC50 value of 1.6 nM and >1000× selectivity against NaV1.4 and NaV1.5.

Profiling Formulated Monoclonal Antibodies by 1H NMR Spectroscopy

Nuclear magnetic resonance (NMR) is arguably the most direct methodology for characterizing the higher-order structure of proteins in solution. Structural characterization of proteins by NMR typically utilizes heteronuclear experiments. However, for formulated monoclonal antibody (mAb) therapeutics, the use of these approaches is not currently tenable due to the requirements of isotope labeling, the large size of the proteins, and the restraints imposed by various formulations. Here, we present a new strategy to characterize formulated mAbs using (1)H NMR. This method, based on the pulsed field gradient stimulated echo (PGSTE) experiment, facilitates the use of (1)H NMR to generate highly resolved spectra of intact mAbs in their formulation buffers. This method of data acquisition, along with postacquisition signal processing, allows the generation of structural and hydrodynamic profiles of antibodies. We demonstrate how variation of the PGSTE pulse sequence parameters allows proton relaxation rates and relative diffusion coefficients to be obtained in a simple fashion. This new methodology can be used as a robust way to compare and characterize mAb therapeutics.

PADLOC: A Powerful Tool to Assign Disulfide Bond Connectivities in Peptides and Proteins by NMR Spectroscopy

The determination of the disulfide bond connectivity in a peptide or protein represents a significant challenge. It is notoriously difficult to use NMR spectroscopy to assign disulfide connectivities because NMR spectra lack direct evidence for disulfide bonds. These bonds are typically inferred from three-dimensional structure calculations, which can result in ambiguous disulfide assignment. Here, we present a new NMR based methodology, in which the disulfide connectivity is obtained by applying Bayesian rules of inference to the local topology of cysteine residues. We illustrate how this approach successfully predicts the disulfide connectivity in proteins for which crystal structures are available in the protein data bank (PDB). We also demonstrate how this methodology is used with experimental NMR data for peptides with complex disulfide topologies, including hepcidin, Kalata-B1, and μ-Conotoxin KIIIA. In the case of μ-Conotoxin KIIIA, the PADLOC connectivity (1-15,2-9,4-16) differs from previously published results; additional evidence is presented demonstrating unequivocally that this newly proposed connectivity is correct.

Single Residue Substitutions That Confer Voltage-Gated Sodium Ion Channel Subtype Selectivity in the NaV1.7 Inhibitory Peptide GpTx-1

There is interest in the identification and optimization of new molecular entities selectively targeting ion channels of therapeutic relevance. Peptide toxins represent a rich source of pharmacology for ion channels, and we recently reported GpTx-1 analogs that inhibit NaV1.7, a voltage-gated sodium ion channel that is a compelling target for improved treatment of pain. Here we utilize multi-attribute positional scan (MAPS) analoging, combining high-throughput synthesis and electrophysiology, to interrogate the interaction of GpTx-1 with NaV1.7 and related NaV subtypes. After one round of MAPS analoging, we found novel substitutions at multiple residue positions not previously identified, specifically glutamic acid at positions 10 or 11 or lysine at position 18, that produce peptides with single digit nanomolar potency on NaV1.7 and 500-fold selectivity against off-target sodium channels. Docking studies with a NaV1.7 homology model and peptide NMR structure generated a model consistent with the key potency and selectivity modifications mapped in this work.

Different Modes of Inhibitor Binding to Prolyl Hydroxylase by Combined Use of X-ray Crystallography and NMR Spectroscopy of Paramagnetic Complexes

In aqueous solution, azaquinolone inhibitors bind to prolyl 4-hydroxylase in two different orientations, as first detected by (19)F spectroscopy. This contrasts with the crystallographic structure where only one orientation has been determined. Dissection of the metal binding properties of the enzyme allowed structures of both complexes to be obtained in solution from (19)F and (13)C dipolar shifts in a labeled ligand.

Discovery of Ligands for Nurr1 by Combined Use of NMR Screening with Different Isotopic and Spin-Labeling Strategies

A comprehensive approach to target screening, hit validation, and binding site determination by nuclear magnetic resonance (NMR) spectroscopy is presented. NMR 19F signal perturbation was used to screen a small compound library and identify candidate ligands to the target of interest. Ligand dissociation constants were measured using a pegylated form of the protein, which resulted in a 2-fold increase in the strength of the saturation transfer difference signal. The initial small-molecule hits were further optimized by combining a residue-specific labeling strategy, to identify the specific sites of interaction with the protein, with a second site screening approach based on relaxation enhancement using a paramagnetic probe. The advantages of this combination strategy in the identification and optimization of weak binding chemical entities early in a program are illustrated with the discovery of a low micromolar ligand (Kd = 20 µM) for Nurr1 and identification of the binding site location through residue-specific 15N isotope labeling and derivatization of Cys residues with 2-mercaptoethanol-1-13C. (Journal of Biomolecular Screening 2007:301-311)

Solubility prediction by recursive partitioning

AbstractPurpose. To build and test a computational model for predicting small molecule solubility, to improve the cost-effectiveness of the selection of vendor compounds suitable for nuclear magnetic resonance (NMR) screening. Methods. A simple recursive partitioning decision tree-based classification model was generated utilizing “off-the-shelf” commercial software from Accelrys Inc., with a training set of 1992 compounds based on a series of calculated topologic and physical properties. The predictive ability of the decision tree was then assessed by employing it to classify a test set of 2851 vendor compounds, and the classification was subsequently used to guide the purchase of 686 compounds for the purpose of NMR screening. Results. When the decision tree was used to guide purchasing, the percentage of “acceptable” compounds suitable for NMR screening doubled compared with the use of a simple cLogP cutoff, improving the successful selection rate from 25% to 50%. Conclusions. A simple recursive partitioning decision tree may successfully be used to improve cost-effectiveness by reducing the wastage associated with the unnecessary purchase of vendor compounds unsuitable for NMR screening because of insolubility.