Discovery and characterization of [(cyclopentyl)ethyl]benzoic acid inhibitors of microsomal prostaglandin E synthase-1.

We describe a novel class of acidic mPGES-1 inhibitors with nanomolar enzymatic and human whole blood (HWB) potency. Rational design in conjunction with structure-based design led initially to the identification of anthranilic acid 5, an mPGES-1 inhibitor with micromolar HWB potency. Structural modifications of 5 improved HWB potency by over 1000×, reduced CYP2C9 single point inhibition, and improved rat clearance, which led to the selection of [(cyclopentyl)ethyl]benzoic acid compound 16 for clinical studies. Compound 16 showed an IC80 of 24nM for inhibition of PGE2 formation in vitro in LPS-stimulated HWB. A single oral dose resulted in plasma concentrations of 16 that exceeded its HWB IC80 in both rat (5mg/kg) and dog (3mg/kg) for over twelve hours.

Characterization of 3,3-dimethyl substituted N-aryl piperidines as potent microsomal prostaglandin E synthase-1 inhibitors.

Here we report on novel, potent 3,3-dimethyl substituted N-aryl piperidine inhibitors of microsomal prostaglandin E synthases-1(mPGES-1). Example 14 potently inhibited PGE2 synthesis in an ex vivo human whole blood (HWB) assay with an IC50 of 7nM. In addition, 14 had no activity in human COX-1 or COX-2 assays at 30µM, and failed to inhibit human mPGES-2 at 62.5µM in a microsomal prep assay. These data are consistent with selective mPGES-1-mediated reduction of PGE2. In dog, 14 had oral bioavailability (74%), clearance (3.62mL/(min*kg)) and volume of distribution (Vd,ss=1.6L/kg) values within our target ranges. For these reasons, 14 was selected for further study.

Structure-based, multi-targeted drug discovery approach to eicosanoid inhibition: Dual inhibitors of mPGES-1 and 5-lipoxygenase activating protein (FLAP).

BACKGROUND: Due to the importance of both prostaglandins (PGs) and leukotrienes (LTs) as pro-inflammatory mediators, and the potential for eicosanoid shunting in the presence of pathway target inhibitors, we have investigated an approach to inhibiting the formation of both PGs and LTs as part of a multi-targeted drug discovery effort. METHODS: We generated ligand-protein X-ray crystal structures of known inhibitors of microsomal prostaglandin E2 synthase-1 (mPGES-1) and the 5-Lipoxygenase Activating Protein (FLAP), with their respective proteins, to understand the overlapping pharmacophores. We subsequently used molecular modeling and structure-based drug design (SBDD) to identify hybrid structures intended to inhibit both targets. RESULTS: This work enabled the preparation of compounds 4 and 5, which showed potent in vitro inhibition of both targets. SIGNIFICANCE: Our findings enhance the structural understanding of mPGES-1 and FLAP's unique ligand binding pockets and should accelerate the discovery of additional dual inhibitors for these two important integral membrane protein drug targets.

Statistical design of experiments as a tool in mass spectrometry.

This Tutorial is an introduction to statistical design of experiments (DOE) with focus on demonstration of how DOE can be useful to the mass spectrometrist. In contrast with the commonly used one factor at a time approach, DOE methods address the issue of interaction of variables and are generally more efficient. The complex problem of optimizing data-dependent acquisition parameters in a bottom-up proteomics LC-MS/MS analysis is used as an example of the power of the technique. Using DOE, a new data-dependent method was developed that improved the quantity of confidently identified peptides from rat serum.

High throughput screening of library compounds against an oligonucleotide substructure of an RNA target.

Approximately 300,000 compounds from selected libraries were screened against a subdomain of a hepatitis C viral (HCV) RNA using a high throughput flow injection mass spectrometry (FIA-MS) method with automated data storage and analysis. Samples contained 2 microM RNA target and 10 microM of each of up to ten ligands. Preliminary studies to optimize operational parameters used the binding of aminoglycosides to the A44 subdomain of bacterial RNA. Binding (confirmed by titration) and sensitivity were maximized within the constraints of the library and throughput. The mobile phase of 5 mM ammonium acetate in 50% isopropanol maintained the noncovalent complexes and provided good detection by electrospray mass spectrometry. Additionally, this composition maximized general solubility of the various classes of compounds including the oligonucleotide and organic library molecules. Cation adduction was insignificant in this screen although some solute and target dependent acetate adduction was observed. The ion trap mass spectrometer provided sufficient mass resolution to identify complexes of RNA with known components of the library. Converted mass spectral data (netCDF) were subjected to two types of statistical evaluation based on binding. The first algorithm identified noncovalent complexes that correlated with the molecular weights of the injected compounds. The second yielded the largest peak in the noncovalent complex region of the spectrum; this spectrum may or may not correlate with expected well components. Sixty-three compounds were confirmed to bind by more stringent secondary testing. Titrations, which were carried out with selected binding compounds, yielded a range of dissociation constants. Biological activity was observed for eleven confirmed binders.

Comparison of the Paul ion trap to the linear ion trap for use in global proteomics.

A critical evaluation of the performance of a 2-D linear ion trap (IT) instrument to two 3-D quadrupole IT instruments with emphasis on identification of rat serum proteins by bottom-up LC-MS/MS is presented. The speed and sensitivity of each of the instruments were investigated, and the effects that each of these have on the bottom-up proteomics identification approach are discussed.

Use of lysozyme as a standard for evaluating the effectiveness of a proteomics process.

Automated sequencing of unknowns in bottom-up proteomics makes the data produced susceptible to process control errors, which can be propagated into mistakes in analyte identification. Inclusion of an unintrusive internal standard, such as lysozyme, allows monitoring all phases of the proteomics process including sample preparation, enzymatic digestion, HPLC, mass spectrometry, and database searching. By using this internal standard, digestion issues including rearrangements, semi-tryptic fragments, and modifications were monitored. In addition, control of the HPLC process including column performance was achieved. The use of the lysozyme standard allowed easy optimization of mass spectral conditions including data dependent and collision induced dissociation settings. The use of this internal standard in a study of differential protein expression in rat serum samples is presented.