Circularly polarized luminescence of Sm (III) and Eu (III) complexes with chiral ligand (R/S)-BINAPO.

Luminescent lanthanide (III) ions have been exploited for circularly polarized luminescence (CPL) for decades. However, very few of these studies have involved chiral samarium (III) complexes. Complexes are prepared by mixing axial chiral ligands (R/S))-2,2'-bis(diphenylphosphoryl)-1,1'-binaphthyl (BINAPO) with europium and samarium Tris (trifluoromethane sulfonate) (Eu (OTf)3 and Sm (OTf)3 ). Luminescence-based titration shows that the complex formed is Ln((R/S)-BINAPO)2 (OTf)3 , where Ln = Eu or Sm. The CPL spectra are reported for Eu((R/S)-BINAPO)2 (OTf)3 and Sm((R/S)-BINAPO)2 (OTf)3 . The sign of the dissymmetry factors, gem , was dependent upon the chirality of the BINAPO ligand, and the magnitudes were relatively large. Of all of the complexes in this study, Sm((S)-BINAPO)2 (OTf)3 has the largest gem  = 0.272, which is one of the largest recorded for a chiral Sm3+ complex. A theoretical three-dimensional structural model of the complex that is consistent with the experimental observations is developed and refined. This report also shows that (R/S)-BINAPO are the only reported ligands where gem (Sm3+ ) > gem (Eu3+ ).

Dual Exosite-binding Inhibitors of Insulin-degrading Enzyme Challenge Its Role as the Primary Mediator of Insulin Clearance in Vivo.

Insulin-degrading enzyme (IDE, insulysin) is the best characterized catabolic enzyme implicated in proteolysis of insulin. Recently, a peptide inhibitor of IDE has been shown to affect levels of insulin, amylin, and glucagon in vivo. However, IDE(-/-) mice display variable phenotypes relating to fasting plasma insulin levels, glucose tolerance, and insulin sensitivity depending on the cohort and age of animals. Here, we interrogated the importance of IDE-mediated catabolism on insulin clearance in vivo. Using a structure-based design, we linked two newly identified ligands binding at unique IDE exosites together to construct a potent series of novel inhibitors. These compounds do not interact with the catalytic zinc of the protease. Because one of these inhibitors (NTE-1) was determined to have pharmacokinetic properties sufficient to sustain plasma levels >50 times its IDE IC50 value, studies in rodents were conducted. In oral glucose tolerance tests with diet-induced obese mice, NTE-1 treatment improved the glucose excursion. Yet in insulin tolerance tests and euglycemic clamp experiments, NTE-1 did not enhance insulin action or increase plasma insulin levels. Importantly, IDE inhibition with NTE-1 did result in elevated plasma amylin levels, suggesting the in vivo role of IDE action on amylin may be more significant than an effect on insulin. Furthermore, using the inhibitors described in this report, we demonstrate that in HEK cells IDE has little impact on insulin clearance. In total, evidence from our studies supports a minimal role for IDE in insulin metabolism in vivo and suggests IDE may be more important in helping regulate amylin clearance.

Preparation and biological evaluation of conformationally constrained BACE1 inhibitors.

The BACE1 enzyme is a key target for Alzheimer's disease. During our BACE1 research efforts, fragment screening revealed that bicyclic thiazine 3 had low millimolar activity against BACE1. Analysis of the co-crystal structure of 3 suggested that potency could be increased through extension toward the S3 pocket and through conformational constraint of the thiazine core. Pursuit of S3-binding groups produced low micromolar inhibitor 6, which informed the S3-design for constrained analogs 7 and 8, themselves prepared via independent, multi-step synthetic routes. Biological characterization of BACE inhibitors 6-8 is described.

Identification of potent and selective hydantoin inhibitors of aggrecanase-1 and aggrecanase-2 that are efficacious in both chemical and surgical models of osteoarthritis.

A disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) and ADAMTS-5 are zinc metalloproteases commonly referred to as aggrecanase-1 and aggrecanase-2, respectively. These enzymes are involved in the degradation of aggrecan, a key component of cartilage. Inhibitors of these enzymes could be potential osteoarthritis (OA) therapies. A series of hydantoin inhibitors of ADAMTS-4 and ADAMTS-5 were identified from a screening campaign and optimized through structure-based drug design to give hydantoin 13. Hydantoin 13 had excellent selectivity over other zinc metalloproteases such as TACE, MMP2, MMP3, MMP13, and MMP14. The compound also produced efficacy in both a chemically induced and surgical model of OA in rats.

Metabolic-intermediate complex formation with cytochrome P450: theoretical studies in elucidating the reaction pathway for the generation of reactive nitroso intermediate.

Mechanism-based inhibition (MBI) of cytochrome P450 (CYP) can lead to drug-drug interactions and often to toxicity. Some aliphatic and aromatic amines can undergo biotransformation reactions to form reactive metabolites such as nitrosoalkanes, leading to MBI of CYPs. It has been proposed that the nitrosoalkanes coordinate with the heme iron, forming metabolic-intermediate complex (MIC), resulting in the quasi-irreversible inhibition of CYPs. Limited mechanistic details regarding the formation of reactive nitroso intermediate and its coordination with heme-iron have been reported. A quantum chemical analysis was performed to elucidate potential reaction pathways for the generation of nitroso intermediate and the formation of MIC. Elucidation of the energy profile along the reaction path, identification of three-dimensional structures of reactive intermediates and transition states, as well as charge and spin density analyses, were performed using the density functional B3LYP method. The study was performed using Cpd I [iron (IV-oxo] heme porphine with SH(-) as the axial ligand) to represent the catalytic domain of CYP, simulating the biotransformation process. Three pathways: (i) N-oxidation followed by proton shuttle, (ii) N-oxidation followed by 1,2-H shift, and (iii) H-abstraction followed by rebound mechanism, were studied. It was observed that the proton shuttle pathway was more favorable over the whole reaction leading to reactive nitroso intermediate. This study revealed that the MIC formation from a primary amine is a favorable exothermic process, involving eight different steps and preferably takes place on the doublet spin surface of Cpd I. The rate-determining step was identified to be the first N-oxidation of primary amine.

Metabolism of LY654322, a growth hormone secretagogue, to an unusual diimidazopyridine metabolite.

2-Methylalanyl-N-{1-[(1R)-1-(4-fluorophenyl)-1-methyl-2-oxo-2-pyrrolidin-1-ylethyl]-1H-imidazol-4-yl}-5-phenyl-D-norvalinamide (LY654322) was rapidly cleared in rats and dogs by renal excretion of parent and metabolism (oxidative and hydrolytic). Among the metabolites identified in the urine of rats and dogs was M25, which was structurally unusual. Indeed, the characterization of M25 and investigation into its disposition relied on the convergence of diverse analytical methodologies. M25 eluted after the parent on reverse-phase chromatography with an MH(+) at m/z 598 (parent + 35 Da). Given its increased lipophilicity and its mass difference compared with the parent, it was evident that M25 was not a phase 2 conjugate. Subsequent liquid chromatography with multiple-stage tandem mass spectrometry and accurate mass experiments identified the structure of M25 as having two replicates of the 1-(4-fluorophenyl)-1-methyl-2-oxo-2-pyrrolidinyl substructure flanking a central aromatic core of composition C(7)H(3)N(5) that was refractory to fragmentation. Compared with the UV spectrum of the parent (λ(max) = 213 nm), M25 displayed a bathochromic shift (λ(max) = 311 nm), which substantiated extensive conjugation within the central core. Subsequent NMR analysis of M25 isolated from dog urine coupled with molecular modeling revealed the structure to be consistent with a diimidazopyridine core with two symmetrically substituted 1-(4-fluorophenyl)-1-methyl-2-oxo-2-pyrrolidinyl moieties. Using a structural analog with a chromophore similar to M25, LC-UV was used to quantitate M25 and determine its urinary disposition. The formation of M25 appears consistent with hydrolysis of LY654322 to an aminoimidazole, dimerization of the latter with the loss of NH(3), C-formylation, and subsequent ring closure and aromatization with loss of H(2)O.

Investigation of the terminal P4 domain in a series of D-phenylglycinamide-based factor Xa inhibitors.

Several P4 domain derivatives of the general d-phenylglycinamide-based scaffold (2) were synthesized and evaluated for their ability to bind to the serine protease factor Xa. Some of the more potent compounds were evaluated for their anticoagulant effects in vitro. A select subset containing various P1 indole constructs was further evaluated for their pharmacokinetic properties after oral administration to rats.

D-phenylglycinol-derived non-covalent factor Xa inhibitors: effect of non-peptidic S4 linkage elements on affinity and anticoagulant activity.

Analogs to a series of D-phenylglycinamide-derived factor Xa inhibitors were discovered. It was found that the S4 amide linkage can be replaced with an ether linkage to reduce the peptide character of the molecules and that this substitution leads to an increase in binding affinity that is not predicted based on modeling. Inhibitors which incorporate ether, amino, or alkyl S4 linkage motifs exhibit similar levels of binding affinity and also demonstrate potent in vitro functional activity, however, binding affinity in this series is strongly dependent on the nature of the S1 binding element.

Anthranilamide inhibitors of factor Xa.

SAR about the B-ring of a series of N(2)-aroyl anthranilamide factor Xa (fXa) inhibitors is described. B-ring o-aminoalkylether and B-ring p-amine probes of the S1' and S4 sites, respectively, afforded picomolar fXa inhibitors that performed well in in vitro anticoagulation assays.

Discovery of non-covalent dipeptidyl peptidase IV inhibitors which induce a conformational change in the active site.

A series of non-covalent inhibitors of the serine protease dipeptidyl peptidase IV (DPP-IV) were found to adopt a U-shaped binding conformation in X-ray co-crystallization studies. Remarkably, Tyr547 undergoes a 70 degrees side-chain rotation to accommodate the inhibitor and allows access to a previously unexposed area of the protein backbone for hydrogen bonding.

Investigation of factor Xa inhibitors containing non-amidine S1 elements.

Several non-amidino S1 derivatives of the 1,2-diaminobenzene-based scaffold (4) were synthesized and evaluated for their ability to bind to the active site and inhibit the human protease factor Xa. A subset of these compounds were also evaluated for their anticoagulant effects in human plasma as measured by prothrombin time (PT).

Inhibition of thrombin activatable fibrinolysis inhibitor by cysteine derivatives.

Thrombin Activatable Fibrinolysis Inhibitor (TAFI) is a basic carboxypeptidase that functions as a fibrinolysis inhibitor through the cleavage of C-terminal lysine on partially degraded fibrin. Modulation of TAFI activity provides a potential therapy for thrombosis complications by potentiating fibrinolysis. In our study, we identified three novel TAFI inhibitors containing a cysteine backbone. Three cysteine derivatives, guanidinyl-L-cysteine, glycyl-L-cysteine, and glycyl-glycyl-L-cysteine were tested in TAFI substrate assays and showed K(app)(i)=0.08, 0.14, and 0.99 microM, respectively. Subsequent fibrinolysis assays confirmed their TAFI inhibitory activities. Guanidinyl-L-cysteine showed inhibitory activity in a human plasma clot lysis assay (IC(50)=9.4 microM). Identification of these cysteine derivatives represents an opportunity to develop potent and specific TAFI inhibitors.

Novel lipoglycopeptides as inhibitors of bacterial signal peptidase I.

Signal peptidase (SPase) I is responsible for the cleavage of signal peptides of many secreted proteins in bacteria. Because of its unique physiological and biochemical properties, it serves as a potential target for development of novel antibacterial agents. In this study, we report the production, isolation, and structure determination of a family of structurally related novel lipoglycopeptides from a Streptomyces sp. as inhibitors of SPase I. Detailed spectroscopic analyses, including MS and NMR, revealed that these lipoglycopeptides share a common 14-membered cyclic peptide core, an acyclic tripeptide chain, and a deoxy-alpha-mannose sugar, but differ in the degree of oxidation of the N-methylphenylglycine residue and the length and branching of the fatty acyl chain. Biochemical analysis demonstrated that these peptides are potent and competitive inhibitors of SPase I with K(i) 50 to 158 nm. In addition, they showed modest antibacterial activity against a panel of pathogenic Gram-positive and Gram-negative bacteria with minimal inhibitory concentration of 8-64 microm against Streptococcus pneumonniae and 4-8 microm against Escherichia coli. Notably, they mechanistically blocked the protein secretion in whole cells as demonstrated by inhibiting beta-lactamase release from Staphylococcus aureus. Taken together, the present discovery of a family of novel lipoglycopeptides as potent inhibitors of bacterial SPase I may lead to the development of a novel class of broad-spectrum antibiotics.