Vincent S. Madison

Crystal Structures of MEK1 Binary and Ternary Complexes with Nucleotides and Inhibitors.

MEK1 is a member of the MAPK signal transduction pathway that responds to growth factors and cytokines. We have determined that the kinase domain spans residues 35-382 by proteolytic cleavage. The complete kinase domain has been crystallized and its X-ray crystal structure as a complex with magnesium and ATP-gammaS determined at 2.1 A. Unlike crystals of a truncated kinase domain previously published, the crystals of the intact domain can be grown either as a binary complex with a nucleotide or as a ternary complex with a nucleotide and one of a multitude of allosteric inhibitors. Further, the crystals allow for the determination of costructures with ATP competitive inhibitors. We describe the structures of nonphosphorylated MEK1 (npMEK1) binary complexes with ADP and K252a, an ATP-competitive inhibitor (see Table 1), at 1.9 and 2.7 A resolution, respectively. Ternary complexes have also been solved between npMEK1, a nucleotide, and an allosteric non-ATP competitive inhibitor: ATP-gammaS with compound 1 and ADP with either U0126 or the MEK1 clinical candidate PD325089 at 1.8, 2.0, and 2.5 A, respectively. Compound 1 is structurally similar to PD325901. These structures illustrate fundamental differences among various mechanisms of inhibition at the molecular level. Residues 44-51 have previously been shown to play a negative regulatory role in MEK1 activity. The crystal structure of the integral kinase domain provides a structural rationale for the role of these residues. They form helix A and repress enzymatic activity by stabilizing an inactive conformation in which helix C is displaced from its active state position. Finally, the structure provides for the first time a molecular rationale that explains how mutations in MEK may lead to the cardio-facio-cutaneous syndrome.

Application of Fragment-Based NMR Screening, X-ray Crystallography, Structure-Based Design, and Focused Chemical Library Design to Identify Novel μM Leads for the Development of nM BACE-1 (β-Site APP Cleaving Enzyme 1) Inhibitors

Fragment-based NMR screening, X-ray crystallography, structure-based design, and focused chemical library design were used to identify novel inhibitors for BACE-1. A rapid optimization of an initial NMR hit was achieved by a combination of NMR and a functional assay, resulting in the identification of an isothiourea hit with a K(d) of 15 microM for BACE-1. NMR data and the crystal structure revealed that this hit makes H-bond interactions with the two catalytic aspartates, occupies the nonprime side region of the active site of BACE-1, and extends toward the S3 subpocket (S3sp). A focused NMR-based search for heterocyclic isothiourea isosteres resulted in several distinct classes of BACE-1 active site directed compounds with improved chemical stability and physicochemical properties. The strategy for optimization of the 2-aminopyridine lead series to potent inhibitors of BACE-1 was demonstrated. The structure-based design of a cyclic acylguanidine lead series and its optimization into nanomolar BACE-1 inhibitors are the subject of the companion paper

Peptidomimetic compounds that inhibit antigen presentation by autoimmune disease-associated class II major histocompatibility molecules

We have identified a heptapeptide with high affinity to rheumatoid arthritis–associated class II major histocompatibility (MHC) molecules. Using a model of its interaction with the class II binding site, a variety of mimetic substitutions were introduced into the peptide. Several unnatural amino acids and dipeptide mimetics were found to be appropriate substituents and could be combined into compounds with binding affinities comparable to that of the original peptide. Compounds were designed that were several hundred-fold to more than a thousand-fold more potent than the original peptide in inhibiting T-cell responses to processed protein antigens presented by the target MHC molecules. Peptidomimetic compounds of this type could find therapeutic use as MHC-selective antagonists of antigen presentation in the treatment of autoimmune diseases.

NMR characterization of interleukin-2 in complexes with the IL-2Rα receptor component, and with low molecular weight compounds that inhibit the IL-2/IL-Rα interaction

Nuclear magnetic resonance (NMR) methods were employed to study the interaction of the cytokine Interleukin‐2 (IL‐2) with the α‐subunit of its receptor (IL‐2Rα), and to help understand the behavior of small molecule inhibitors of this interaction. Heteronuclear 1H‐15N HSQC experiments were used to identify the interaction surface of 15N‐enriched Interleukin‐2 (15N‐IL‐2) in complex with human IL‐2Rα. In these experiments, chemical shift and line width changes in the heteronuclear single‐quantum coherence (HSQC) spectra upon binding of 15N‐IL‐2 enabled classification of NH atoms as either near to, or far from, the IL‐2Rα interaction surface. These data were complemented by hydrogen/deuterium (H/D) exchange measurements, which illustrated enhanced protection of slowly‐exchanging IL‐2 NH protons near the site of interaction with IL‐2Rα. The interaction surface defined by NMR compared well with the IL‐2Rα binding site identified previously using mutagenesis of human and murine IL‐2. Two low molecular weight inhibitors of the IL‐2/IL‐2Rα interaction were studied: one (a cyclic peptide derivative) was found to mimic a part of the cytokine and bind to IL‐2Rα; the other (an acylphenylalanine derivative) was found to bind to IL‐2. For the interaction between IL‐2 and the acylphenylalanine, chemical shift perturbations of 15N and 15NH backbone resonances were tracked as a function of ligand concentration. The perturbation pattern observed for this complex revealed that the acylphenylalanine is a competitive inhibitor—it binds to the same site on IL‐2 that interacts with IL‐2Rα.

Identification of novel cannabinoid CB1 receptor antagonists by using virtual screening with a pharmacophore model.

CB1 receptor antagonists have proven to be clinically effective in treating obesity and related disorders. We report here the identification of a novel class of azetidinone CB1 antagonists by using virtual screening methods. For this purpose, we developed a pharmacophore model based on known representative CB1 antagonists and employed it to screen a database of about a half million Schering-Plough compounds. We applied a stepwise filtering protocol based on molecular weight, compound availability, and a modified rule-of-five to reduce the number of hits. We then combined Bayesian modeling and clustering techniques to select a final set of 420 compounds for in vitro testing. Five compounds were found to have >50% inhibition at 100 nM in a CB1 competitive binding assay and were further characterized by using both CB1 and CB2 assays. The most potent compound has a CB1 K i of 53 nM and >5-fold selectivity against the CB2 receptor.

Discovery of Narlaprevir (SCH 900518): A Potent, Second Generation HCV NS3 Serine Protease Inhibitor

Boceprevir (SCH 503034), 1, a novel HCV NS3 serine protease inhibitor discovered in our laboratories, is currently undergoing phase III clinical trials. Detailed investigations toward a second generation protease inhibitor culminated in the discovery of narlaprevir (SCH 900518), 37, with improved potency (∼10-fold over 1), pharmacokinetic profile and physicochemical characteristics, currently in phase II human trials. Exploration of synthetic sequence for preparation of 37 resulted in a route that required no silica gel purification for the entire synthesis.

Construction and characterization of a fully active PXR/SRC-1 tethered protein with increased stability

The nuclear xenobiotic receptor PXR is a ligand-inducible transcription factor regulating drug-metabolizing enzymes and transporters and a master switch mediating potentially adverse drug-drug interactions. In addition to binding a coactivator protein such as SRC-1, the C-terminal ligand-binding domain (LBD) is solely responsible for ligand recognition and thus the ligand-dependent downstream effects. In an effort to facilitate structural studies of PXR to understand and abolish the interactions between PXR and its ligands, several recombinant PXR/SRC-1 constructs were designed and evaluated for expression, stability and activity. Expression strategies employing either dual expression or translationally coupled bicistronic expression were found to be unsuitable for producing stable PXR in a stochiometric complex with a peptide derived from SRC-1 (SRC-1p). A single polypeptide chain encompassing PXR and SRC-1p tethered with a peptidyl linker was designed to promote intramolecular complex formation. This tethered protein was overexpressed as a soluble protein and required no additional SRC-1p for further stabilization. X-ray crystal structures in the presence and absence of the known PXR agonist SR-12813 were determined to high resolution. In addition, a circular dichroism-based binding assay was developed to allow rapid evaluation of PXR ligand affinity, making this tethered protein a convenient and effective reagent for the rational attenuation of drug-induced PXR-mediated metabolism.

Insertion of a Structural Domain of Interleukin (IL)-1β Confers Agonist Activity to the IL-1 Receptor Antagonist IMPLICATIONS FOR IL-1 BIOACTIVITY

We showed previously that replacement of Lys-145 in the IL-1 receptor antagonist (IL-1ra) with Asp resulted in an analog (IL-1ra K145D) with partial agonist activity. To identify additional amino acids that affect IL-1 bioactivity, we created second site mutations in IL-1ra K145D. Substitutions of single amino acids surrounding position 145 were made; none of these substitutions increased the bioactivity of IL-1ra K145D. However, the insertion of the β-bulge (QGEESN) of IL-1β at the corresponding region of IL-1ra K145D resulted in a 3-4-fold augmentation of bioactivity. An additional increase in agonist activity was observed when the β-bulge was coexpressed with a second substitution (His-54 Pro) in IL-1ra K145D. We also show that the bioactivity of both IL-1ra K145D and the triple mutant IL-1ra K145D/H54P/QGEESN is dependent on interaction with the newly cloned IL-1 receptor accessory protein.

Biochemical analysis of the transducin-phosphodiesterase interaction

In vertebrate rod cells, the activated α-subunit of rod transducin interacts with the γ (regulatory) subunits of phosphodiesterase to disinhibit the catalytic subunits. A 22-amino acid long region of rod transducin involved in phosphodiesterase activation has recently been identified. We have used peptides from this region of rod transducin and from several other G protein α-subunits to study the nature and specificity of the G protein α-effector interaction. Although peptides derived from rod transducin, cone transducin and gustducin are similar, only the rod peptide is capable of activating rod phosphodiesterase. Using substituted peptides we have identified five residues on one exposed face of rod transducin as important to phosphodiesterase activation. These results disagree with previous models which propose that loop regions of rod transducin interact with phosphodiesterase γ

Design, synthesis, and three-dimensional structural characterization of a constrained Ω-loop excised from interleukin-1α

Abstract The cyclic peptide 1 , containing a 2,7-disubstituted naphthalene spacer, was designed to mimic an exposed Ω-loop present in interleukin-1α, an important mediator of immune and inflammatory responses. The synthesis of this cyclic peptide was accomplished via solution phase fragment condensation methodology. The three dimensional characterization using 2D-NMR techniques revealed it to be an excellent mimic for the Ω-loop sequence 41–48 in interleukin -1α.