Design, synthesis, functional and structural characterization of an inhibitor of N-acetylneuraminate-9-phosphate phosphatase: observation of extensive dynamics in an enzyme/inhibitor complex.

The design, synthesis and characterization of a phosphonate inhibitor of N-acetylneuraminate-9-phosphate phosphatase (HDHD4) is described. Compound 3, where the substrate C-9 oxygen was replaced with a nonlabile CH2 group, inhibits HDHD4 with a binding affinity (IC50 11µM) in the range of the native substrate Neu5Ac-9-P (compound 1, Km 47µM). Combined SAR, modeling and NMR studies are consistent with the phosphonate group in inhibitor 3 forming a stable complex with native Mg(2+). In addition to this key interaction, the C-1 carboxylate of the sugar interacts with a cluster of basic residues, K141, R104 and R72. Comparative NMR studies of compounds 3 and 1 with Ca(2+) and Mg(2+) are indicative of a highly dynamic process in the active site for the HDHD4/Mg(2+)/3 complex. Possible explanations for this observation are discussed.

NMR structure of a potent small molecule inhibitor bound to human keratinocyte fatty acid-binding protein.

The NMR structure is presented for compound 1 (BMS-480404) (Ki = 33 (+/-2) nM) bound to keratinocyte fatty acid-binding protein. This article describes interactions between a high affinity drug-like compound and a member of the fatty acid-binding protein family. A benzyl group ortho to the mandelic acid in 1 occupies an area of the protein that fatty acids do not normally contact. Similar to that in the kFABP-palmitic acid structure, the acid moiety in 1 is proximal to R129 and Y131. Computational modeling indicates that the acid moiety in 1 interacts indirectly via a modeled water molecule to R109.

Structural and functional characterization of CFE88: evidence that a conserved and essential bacterial protein is a methyltransferase.

CFE88 is a conserved essential gene product from Streptococcus pneumoniae. This 227-residue protein has minimal sequence similarity to proteins of known 3D structure. Sequence alignment models and computational protein threading studies suggest that CFE88 is a methyltransferase. Characterization of the conformation and function of CFE88 has been performed by using several techniques. Backbone atom and limited side-chain atom NMR resonance assignments have been obtained. The data indicate that CFE88 has two domains: an N-terminal domain with 163 residues and a C-terminal domain with 64 residues. The C-terminal domain is primarily helical, while the N-terminal domain has a mixed helical/extended (Rossmann) fold. By aligning the experimentally observed elements of secondary structure, an initial unrefined model of CFE88 has been constructed based on the X-ray structure of ErmC' methyltransferase (Protein Data Bank entry 1QAN). NMR and biophysical studies demonstrate binding of S-adenosyl-L-homocysteine (SAH) to CFE88; these interactions have been localized by NMR to the predicted active site in the N-terminal domain. Mutants that target this predicted active site (H26W, E46R, and E46W) have been constructed and characterized. Overall, our results both indicate that CFE88 is a methyltransferase and further suggest that the methyltransferase activity is essential for bacterial survival.

Eleven amino acid glucagon-like peptide-1 receptor agonists with antidiabetic activity.

Glucagon-like peptide 1 (GLP-1) is a 30 or 31 amino acid peptide hormone that contributes to the physiological regulation of glucose homeostasis and food intake. Herein, we report the discovery of a novel class of 11 amino acid GLP-1 receptor agonists. These peptides consist of a structurally optimized 9-mer, which is closely related to the N-terminal 9 amino acids of GLP-1, linked to a substituted C-terminal biphenylalanine (BIP) dipeptide. SAR studies resulted in 11-mer GLP-1R agonists with similar in vitro potency to the native 30-mer. Peptides 21 and 22 acutely reduced plasma glucose excursions and increased plasma insulin concentrations in a mouse model of diabetes. These peptides also showed sustained exposures over several hours in mouse and dog models. The described 11-mer GLP-1 receptor agonists represent a new tool in further understanding GLP-1 receptor pharmacology that may lead to novel antidiabetic agents.

Multiple and single binding modes of fragment-like kinase inhibitors revealed by molecular modeling, residue type-selective protonation, and nuclear overhauser effects.

Fragment-like inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK2) include 5-hydroxyisoquinoline (IC50 approximately 85 microM). Modeling studies identified four possible binding modes for this compound. Two-dimensional (1)H-(1)H NOESY data obtained with selectively protonated samples of MK2 in complex with 5-hydroxyisoquinoline demonstrated that two of the four predicted binding modes are well populated. A second small isoquinoline was subsequently shown to bind in a single mode. NMR and modeling studies using this general approach are expected to facilitate "scaffold hopping" and structure-guided elaborations of fragment-like kinase inhibitor cores.

Observation of O-H...N scalar coupling across a hydrogen bond in nocathiacin I.

We report here the observation of O-H...N hydrogen-bond (1h)J(N,OH) scalar coupling in a biologically active natural product. The intramolecular hydrogen bond between the threonine hydroxyl (Thr-OH) group and the thiazolyl nitrogen at the second thiazole ring (Thz-2) in nocathiacin I was directly detected by a 1H-15N HMBC NMR experiment. The magnitude of the scalar coupling constant (1h)J(N,OH) was accurately measured to be 1.8 +/- 0.1 Hz by a J-resolved 1H-15N HMBC experiment. By adding the O-H...N distance restraint, the 3D solution structure of nocathiacin I was refined. The structure refinement indicated that the distance between the Thr-3 hydroxyl hydrogen and the Thz-2 nitrogen is or= 0.23 A. The presence of an intramolecular hydrogen bond in nocathiacin I is further supported by a number of NMR parameters and additional NMR experiments. This observation provides valuable information for characterizing molecular conformations, and for studying structure-activity relationships.

Protein-ligand NOE matching: a high-throughput method for binding pose evaluation that does not require protein NMR resonance assignments.

Given the three-dimensional (3D) structure of a protein, the binding pose of a ligand can be determined using distance restraints derived from assigned intra-ligand and protein-ligand nuclear Overhauser effects (NOEs). A primary limitation of this approach is the need for resonance assignments of the ligand-bound protein. We have developed an approach that utilizes data from 3D 13C-edited, 13C/15N-filtered HSQC-NOESY spectra for evaluating ligand binding poses without requiring protein NMR resonance assignments. Only the 1H NMR assignments of the bound ligand are essential. Trial ligand binding poses are generated by any suitable method (e.g., computational docking). For each trial binding pose, the 3D 13C-edited, 13C/15N-filtered HSQC-NOESY spectrum is predicted, and the predicted and observed patterns of protein-ligand NOEs are matched and scored using a fast, deterministic bipartite graph matching algorithm. The best scoring (lowest "cost") poses are identified. Our method can incorporate any explicit restraints or protein assignment data that are available, and many extensions of the basic procedure are feasible. Only a single sample is required, and the method can be applied to both slowly and rapidly exchanging ligands. The method was applied to three test cases: one complex involving muscle fatty acid-binding protein (mFABP) and two complexes involving the leukocyte function-associated antigen 1 (LFA-1) I-domain. Without using experimental protein NMR assignments, the method identified the known binding poses with good accuracy. The addition of experimental protein NMR assignments improves the results. Our "NOE matching" approach is expected to be widely applicable; i.e., it does not appear to depend on a fortuitous distribution of binding pocket residues.

Conformation and absolute configuration of nocathiacin I determined by NMR spectroscopy and chiral capillary electrophoresis.

Nocathiacin I (BMS-249524) is a highly cross-linked thiazolyl peptide that displays potent activity against Gram-positive bacteria, including a number of antibiotic-resistant strains. This natural product contains 10 chiral centers. NMR studies have been performed to characterize the solution structure of nocathiacin I. A uniformly 13C,15N-labeled sample was used to obtain NMR assignments. Restrained simulated annealing calculations were performed by using accurately determined NOE distance restraints. All of the chiral centers were allowed to float during the simulated annealing protocol. Two clusters of structures were obtained that satisfy the NOE restraints very well and that are reasonably consistent with vicinal J-coupling constants. Within each cluster, all 10 chiral centers are uniquely defined. The two clusters are effectively mirror images of each other: all chiral centers that have the R(S) configuration in one cluster have the S(R) configuration in the other. The single threonine residue in nocathiacin I was subsequently determined to be l-threonine by chiral capillary electrophoresis, allowing the absolute configurations of all 10 chiral centers to be defined.