Elizabeth Cauchon

Molecular pharmacology of the human prostaglandin D2 receptor, CRTH2

The recombinant human prostaglandin D2 (PGD2) receptor, hCRTH2, has been expressed in HEK293(EBNA) and characterized with respect to radioligand binding and signal transduction properties. High and low affinity binding sites for PGD2 were identified in the CRTH2 receptor population by saturation analysis with respective equilibrium dissociation constants (KD) of 2.5 and 109 nM. This revealed that the affinity of PGD2 for CRTH2 is eight times less than its affinity for the DP receptor. Equilibrium competition binding assays revealed that of the compounds tested, only PGD2 and several related metabolites bound with high affinity to CRTH2 (Ki values ranging from 2.4 to 34.0 nM) with the following rank order of potency: PGD2>13,14‐dihydro‐15‐keto PGD2>15‐deoxy‐Δ12,14‐PGJ2>PGJ2>Δ12‐PGJ2>15(S)‐15 methyl‐PGD2. This is in sharp contrast with the rank order of potency obtained at DP : PGD2>PGJ2>Δ12‐PGJ2>15‐deoxy‐Δ12,14‐PGJ2 >>>13,14‐dihydro‐15‐keto‐PGD2. Functional studies demonstrated that PGD2 activation of recombinant CRTH2 results in decrease of intracellular cAMP in a pertussis toxin‐sensitive manner. Therefore, we showed that CRTH2 can functionally couple to the G‐protein Gαi/o. PGD2 and related metabolites were tested and their rank order of potency followed the results of the membrane binding assay. By Northern blot analysis, we showed that, besides haemopoietic cells, CRTH2 is expressed in many other tissues such as brain, heart, thymus, spleen and various tissues of the digestive system. In addition, in situ hybridization studies revealed that CRTH2 mRNA is expressed in human eosinophils. Finally, radioligand binding studies demonstrated that two eosinophilic cell lines, butyric acid‐differentiated HL‐60 and AML 14.3D10, also endogenously express CRTH2.

Development of a homogeneous immunoassay for the detection of angiotensin I in plasma using AlphaLISA acceptor beads technology

Plasma renin activity (PRA) is a well-established biomarker for assessing the efficacy of various antihypertensive agents such as direct renin inhibitors, angiotensin receptor blockers, and angiotensin-converting enzyme inhibitors (ACEIs). PRA measurements are obtained through the detection and quantification of angiotensin I (Ang I) produced by the action of renin on its natural substrate angiotensinogen. The most accepted and reproducible method for PRA measurement uses an antibody capture Ang I methodology that employs specific antibodies that recognize and protect Ang I against angiotensinase activities contained in plasma. The amount of Ang I is then quantified by either radioimmunoassay (RIA) or enzyme immunoassay (EIA). In the current report, we describe the optimization of a novel homogeneous immunoassay based on the AlphaScreen technology for the detection and quantification of antibody-captured Ang I using AlphaLISA acceptor beads in buffer and in the plasma of various species (human, rat, and mouse). Ex vivo measurements of renin activity were performed using 10 microl or less of a reaction mixture, and concentrations as low as 1 nM Ang I were quantified. Titration curves obtained for the quantification of Ang I in buffer and plasma gave similar EC(50) values of 5.6 and 14.4 nM, respectively. Both matrices generated an equivalent dynamic range that varies from approximately 1 to 50 nM. Renin inhibitors have been successfully titrated and IC(50) values obtained correlated well with those obtained using EIA methodology (r(2)=0.80). This assay is sensitive, robust, fast, and less tedious than measurements performed using nonhomogeneous EIA. The AlphaLISA methodology is homogeneous, does not require wash steps prior to the addition of reagents, and does not generate radioactive waste.

In Vitro Characterization of MK-1439, a Novel HIV-1 Nonnucleoside Reverse Transcriptase Inhibitor

ABSTRACT Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are a mainstay of therapy for treating human immunodeficiency type 1 virus (HIV-1)-infected patients. MK-1439 is a novel NNRTI with a 50% inhibitory concentration (IC50) of 12, 9.7, and 9.7 nM against the wild type (WT) and K103N and Y181C reverse transcriptase (RT) mutants, respectively, in a biochemical assay. Selectivity and cytotoxicity studies confirmed that MK-1439 is a highly specific NNRTI with minimum off-target activities. In the presence of 50% normal human serum (NHS), MK-1439 showed excellent potency in suppressing the replication of WT virus, with a 95% effective concentration (EC95) of 20 nM, as well as K103N, Y181C, and K103N/Y181C mutant viruses with EC95 of 43, 27, and 55 nM, respectively. MK-1439 exhibited similar antiviral activities against 10 different HIV-1 subtype viruses (a total of 93 viruses). In addition, the susceptibility of a broader array of clinical NNRTI-associated mutant viruses (a total of 96 viruses) to MK-1439 and other benchmark NNRTIs was investigated. The results showed that the mutant profile of MK-1439 was superior overall to that of efavirenz (EFV) and comparable to that of etravirine (ETR) and rilpivirine (RPV). Furthermore, E138K, Y181C, and K101E mutant viruses that are associated with ETR and RPV were susceptible to MK-1439 with a fold change (FC) of <3. A two-drug in vitro combination study indicated that MK-1439 acts nonantagonistically in the antiviral activity with each of 18 FDA-licensed drugs for HIV infection. Taken together, these in vitro data suggest that MK-1439 possesses the desired properties for further development as a new antiviral agent.

Discovery of MK-1439, an orally bioavailable non-nucleoside reverse transcriptase inhibitor potent against a wide range of resistant mutant HIV viruses.

The optimization of a novel series of non-nucleoside reverse transcriptase inhibitors (NNRTI) led to the identification of pyridone 36. In cell cultures, this new NNRTI shows a superior potency profile against a range of wild type and clinically relevant, resistant mutant HIV viruses. The overall favorable preclinical pharmacokinetic profile of 36 led to the prediction of a once daily low dose regimen in human. NNRTI 36, now known as MK-1439, is currently in clinical development for the treatment of HIV infection.

An Automated Multistep High-Throughput Screening Assay for the Identification of Lead Inhibitors of the Inducible Enzyme mPGES-1

Prostaglandin E2 synthase (mPGES-1), the enzyme which catalyzes the synthesis of PGE2, is induced during the inflammatory response. For this reason, mPGES-1 could be a potential therapeutic target. A high-throughput screening assay was developed to identify potential inhibitors of mPGES-1. The assay consisted of a 30-s mPGES-1 enzymatic reaction followed by the detection of PGE2 by enzyme immunoassay (EIA). The enzymatic reaction was performed in a batch mode because the instability of the substrate (10 min) limited the number of plates assayed within a working day. The detection of the product by EIA was performed on 3 instruments requiring 14 different steps for complete automation. The authors describe here the optimization and implementation of a 2-part assay on a Thermo CRS robotic system. More than 315,000 compounds were tested, and a hit rate of 0.84% was obtained for this assay. Although the entire assay required multiple steps, the assay was successfully miniaturized and automated for a high-throughput screening campaign.

Renin inhibitors for the treatment of hypertension: Design and optimization of a novel series of pyridone-substituted piperidines

An SAR campaign aimed at decreasing the overall lipophilicity of renin inhibitors such as 1 is described herein. It was found that replacement of the northern appendage in 1 with an N-methyl pyridone and subsequent re-optimization of the benzyl amide handle afforded compounds with in vitro and in vivo profiles suitable for further profiling. An unexpected CV toxicity in dogs observed with compound 20 led to the employment of a time and resource sparing rodent model for in vivo screening of key compounds. This culminated in the identification of compound 31 as an optimized renin inhibitor.

Renin inhibitors for the treatment of hypertension: Design and optimization of a novel series of tertiary alcohol-bearing piperidines

The design and optimization of a novel series of renin inhibitor is described herein. Strategically, by committing the necessary resources to the development of synthetic sequences and scaffolds that were most amenable for late stage structural diversification, even as the focus of the SAR campaign moved from one end of the molecule to another, highly potent renin inhibitors could be rapidly identified and profiled.

Renin inhibitors for the treatment of hypertension: design and optimization of a novel series of spirocyclic piperidines.

The discovery and SAR of a novel series of spirocyclic renin inhibitors are described herein. It was found that by restricting the northern aromatic plate to the bioactive conformation through spirocyclization, increase in renin potency and decrease in hERG affinity could both be realized. When early members of this series were found to be potent time-dependent CYP3A4 inhibitors, two distinct strategies to address this liability were explored and this effort culminated in the identification of compound 31 as an optimized renin inhibitor.

The discovery and synthesis of potent zwitterionic inhibitors of renin

The incorporation of a carboxylic acid within in a series of 3-amido-4-aryl substituted piperidines (represented by general structure 32) led to the discovery of potent, zwitterionic, renin inhibitors with improved off-target profiles (CYP3A4 time-dependent inhibition and hERG affinity) relative to analogous non-zwitterionic inhibitors of the past (i.e., 3). Strategies to address the oral absorption of these zwitterions are also discussed within.

3,4-Diarylpiperidines as potent renin inhibitors.

The discovery and SAR of a series of potent renin inhibitors possessing a novel 3,4-diarylpiperidine scaffold are described herein. The resulting compound 38 exhibit low nanomolar plasma renin IC(50), had a clean CYP 3A4 profile and displayed micromolar affinity for the hERG channel. Furthermore, it was found to be efficacious in the double transgenic rat hypertension model and show good to moderate oral bioavailability in two animal species.