Optimization of physicochemical properties of pyrrolidine GPR40 AgoPAMs results in a differentiated profile with improved pharmacokinetics and reduced off-target activities.

GPR40 AgoPAMs are highly effective antidiabetic agents that have a dual mechanism of action, stimulating both glucose-dependent insulin and GLP-1 secretion. The early lipophilic, aromatic pyrrolidine and dihydropyrazole GPR40 AgoPAMs from our laboratory were highly efficacious in lowering plasma glucose levels in rodents but possessed off-target activities and triggered rebound hyperglycemia in rats at high doses. A focus on increasing molecular complexity through saturation and chirality in combination with reducing polarity for the pyrrolidine AgoPAM chemotype resulted in the discovery of compound 46, which shows significantly reduced off-target activities as well as improved aqueous solubility, rapid absorption, and linear PK. In vivo, compound 46 significantly lowers plasma glucose levels in rats during an oral glucose challenge yet does not demonstrate the reactive hyperglycemia effect at high doses that was observed with earlier GPR40 AgoPAMs.

Development and Validation of a Sensitive and Robust Multiplex Antigen Capture Assay to Quantify Streptococcus pneumoniae Serotype-Specific Capsular Polysaccharides in Urine.

Streptococcus pneumoniae is a major cause of community-acquired pneumonia (CAP) in young children, older adults, and those with immunocompromised status. Since the introduction of pneumococcal vaccines, the burden of invasive pneumococcal disease caused by vaccine serotypes (STs) has decreased; however, the effect on the burden of CAP is unclear, potentially due to the lack of testing for pneumococcal STs. We describe the development, qualification, and clinical validation of a high-throughput and multiplex ST-specific urine antigen detection (SSUAD) assay to address the unmet need in CAP pneumococcal epidemiology. The SSUAD assay is sensitive and specific to the 15 STs in the licensed pneumococcal conjugate vaccine V114 (STs 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F) and uses ST-specific monoclonal antibodies for rapid and simultaneous quantification of the 15 STs using a Luminex microfluidics system. The SSUAD assay was optimized and qualified using healthy adult urine spiked with pneumococcal polysaccharides and validated using culture-positive clinical urine samples (n = 34). Key parameters measured were accuracy, precision, sensitivity, specificity, selectivity, and parallelism. The SSUAD assay met all prespecified validation acceptance criteria and is suitable for assessments of disease burden associated with the 15 pneumococcal STs included in V114. IMPORTANCE Streptococcus pneumoniae has more than 90 serotypes capable of causing a range of disease manifestations, including otitis media, pneumonia, and invasive diseases, such as bacteremia or meningitis. Only a minority (<10%) of pneumococcal diseases are bacteremic with known serotype distribution. Culture and serotyping of respiratory specimens are neither routine nor reliable. Hence, the serotype-specific disease burden of the remaining (>90%) noninvasive conditions is largely unknown without reliable laboratory techniques. To address this need, a 15-plex urine antigen detection assay was developed and validated to quantify pneumococcal serotype-specific capsular polysaccharides in urine. This assay will support surveillance to estimate the pneumococcal disease burden and serotype distribution in nonbacteremic conditions. Data obtained from this assay will be critical for understanding the impact of pneumococcal vaccines on noninvasive pneumococcal diseases and to inform the choice of pneumococcal serotypes for next-generation vaccines.

Discovery of Potent and Orally Bioavailable Dihydropyrazole GPR40 Agonists.

G protein-coupled receptor 40 (GPR40) has become an attractive target for the treatment of diabetes since it was shown clinically to promote glucose-stimulated insulin secretion. Herein, we report our efforts to develop highly selective and potent GPR40 agonists with a dual mechanism of action, promoting both glucose-dependent insulin and incretin secretion. Employing strategies to increase polarity and the ratio of sp3/sp2 character of the chemotype, we identified BMS-986118 (compound 4), which showed potent and selective GPR40 agonist activity in vitro. In vivo, compound 4 demonstrated insulinotropic efficacy and GLP-1 secretory effects resulting in improved glucose control in acute animal models.

Discovery of BI 135585, an in vivo efficacious oxazinanone-based 11ß hydroxysteroid dehydrogenase type 1 inhibitor.

A potent, in vivo efficacious 11ß hydroxysteroid dehydrogenase type 1 (11ß HSD1) inhibitor (11j) has been identified. Compound 11j inhibited 11ß HSD1 activity in human adipocytes with an IC50 of 4.3nM and in primary human adipose tissue with an IC80 of 53nM. Oral administration of 11j to cynomolgus monkey inhibited 11ß HSD1 activity in adipose tissue. Compound 11j exhibited >1000× selectivity over other hydroxysteroid dehydrogenases, displays desirable pharmacodynamic properties and entered human clinical trials in 2011.

Discovery of Pyrrolidine-Containing GPR40 Agonists: Stereochemistry Effects a Change in Binding Mode.

A novel series of pyrrolidine-containing GPR40 agonists is described as a potential treatment for type 2 diabetes. The initial pyrrolidine hit was modified by moving the position of the carboxylic acid, a key pharmacophore for GPR40. Addition of a 4-cis-CF3 to the pyrrolidine improves the human GPR40 binding Ki and agonist efficacy. After further optimization, the discovery of a minor enantiomeric impurity with agonist activity led to the finding that enantiomers (R,R)-68 and (S,S)-68 have differential effects on the radioligand used for the binding assay, with (R,R)-68 potentiating the radioligand and (S,S)-68 displacing the radioligand. Compound (R,R)-68 activates both Gq-coupled intracellular Ca2+ flux and Gs-coupled cAMP accumulation. This signaling bias results in a dual mechanism of action for compound (R,R)-68, demonstrating glucose-dependent insulin and GLP-1 secretion in vitro. In vivo, compound (R,R)-68 significantly lowers plasma glucose levels in mice during an oral glucose challenge, encouraging further development of the series.

384-Well Multiplexed Luminex Cytokine Assays for Lead Optimization.

Cytokines serve as a major mechanism of communication between immune cells and are the functional molecules at the end of immune pathways. Abnormalities in cytokines are involved in a wide variety of diseases, including chronic inflammation, autoimmune diseases, and cancer. Cytokines are not only direct targets of therapeutics but also important biomarkers for assessing drug efficacy and safety. Traditionally, enzyme-linked immunosorbent assays (ELISA) were most popular for identifying and quantifying cytokines. However, ELISA is expensive, labor intensive, and low throughput. Here, we report the development of a miniaturized Luminex (Austin, TX) assay platform to establish a panel of high-throughput, multiplexed assays for measuring cytokines in human whole blood. The miniaturized 384-well Luminex assay uses <25% of the assay reagents compared with the 96-well assay. The development and validation of the 384-well Luminex cytokine assays enabled high-throughput screening of compounds in primary cells using cytokines as physiologically relevant readouts. Furthermore, this miniaturized multiplexed technology platform allows for high-throughput biomarker profiling of biofluids from animal studies and patient samples for translational research.

Biphenyl/diphenyl ether renin inhibitors: filling the S1 pocket of renin via the S3 pocket.

Structure-based design led to the discovery of a novel class of renin inhibitors in which an unprecedented phenyl ring filling the S1 site is attached to the phenyl ring filling the S3 pocket. Optimization for several parameters including potency in the presence of human plasma, selectivity against CYP3A4 inhibition and improved rat oral bioavailability led to the identification of 8d which demonstrated antihypertensive efficacy in a transgenic rat model of human hypertension.

Structure-based design and synthesis of 1,3-oxazinan-2-one inhibitors of 11ß-hydroxysteroid dehydrogenase type 1.

Structure based design led directly to 1,3-oxazinan-2-one 9a with an IC(50) of 42 nM against 11ß-HSD1 in vitro. Optimization of 9a for improved in vitro enzymatic and cellular potency afforded 25f with IC(50) values of 0.8 nM for the enzyme and 2.5 nM in adipocytes. In addition, 25f has 94% oral bioavailability in rat and >1000× selectivity over 11ß-HSD2. In mice, 25f was distributed to the target tissues, liver, and adipose, and in cynomolgus monkeys a 10 mg/kg oral dose reduced cortisol production by 85% following a cortisone challenge.

Discovery and optimization of adamantyl carbamate inhibitors of 11ß-HSD1.

Synthesis of 2-adamantyl carbamate derivatives of piperidines and pyrrolidines led to the discovery of 9a with an IC(50) of 15.2 nM against human 11ß-HSD1 in adipocytes. Optimization for increased adipocyte potency, metabolic stability and selectivity afforded 11k and 11l, both of which were >25% orally bioavailable in rat.

Spirocyclic ureas: orally bioavailable 11 beta-HSD1 inhibitors identified by computer-aided drug design.

Structure-guided drug design led to the identification of a class of spirocyclic ureas which potently inhibit human 11beta-HSD1 in vitro. Lead compound 10j was shown to be orally bioavailable in three species, distributed into adipose tissue in the mouse, and its (R) isomer 10j2 was efficacious in a primate pharmacodynamic model.

Optimization of orally bioavailable alkyl amine renin inhibitors.

Structure-guided drug design led to new alkylamine renin inhibitors with improved in vitro and in vivo potency. Lead compound 21a, has an IC(50) of 0.83nM for the inhibition of human renin in plasma (PRA). Oral administration of 21a at 10mg/kg resulted in >20h reduction of blood pressure in a double transgenic rat model of hypertension.

Design and optimization of renin inhibitors: Orally bioavailable alkyl amines.

Structure-based drug design led to the identification of a novel class of potent, low MW alkylamine renin inhibitors. Oral administration of lead compound 21l, with MW of 508 and IC(50) of 0.47nM, caused a sustained reduction in mean arterial blood pressure in a double transgenic rat model of hypertension.

A simple homogeneous scintillation proximity assay for acyl-coenzyme A:diacylglycerol acyltransferase.

Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) is a key enzyme in triacylglycerol synthesis, and inhibiting this enzyme is a promising approach for treating obesity, type II diabetes, and dyslipidemia. There are two distinct DGAT enzymes: DGAT1 and DGAT2. The conventional assay for measuring DGAT activity is a thin layer chromatography (TLC) method, which is not amenable to screening a large number of compounds. To increase the throughput, we have developed a novel, homogeneous scintillation proximity assay (SPA) for DGAT. In this assay, when (3)H-labeled acyl-CoA is used as the acyl donor and diacylglycerol is used as the acyl acceptor, the (3)H-labeled triacylglycerol product formed in the reaction binds to polylysine SPA beads, producing a signal that is measured in a TopCount or LEADseeker. The apparent Michaelis-Menten kinetic parameters determined by this DGAT SPA method agreed well with the values determined with the conventional TLC assay. The statistical values also indicate that the DGAT SPA is a robust assay, with a Z' of more than 0.60 and a signal/background ratio of approximately 9. These results suggest that the current assay provides high-throughput capacity for the identification of DGAT inhibitors.

Cloning, characterization and site-directed mutagenesis of canine renin.

Inhibition of renin has been shown to be successful in managing hypertension and maintaining cardiac health. Canine models have played a key role in preclinical assessment of renin inhibitors. Here we report the cloning of canine prorenin gene. The amino acid sequence of mature canine renin was approximately 70% identical to that of human renin. The full-length prorenin was expressed in HEK 293 cells, purified and converted to its active form by trypsin-mediated cleavage of the 43 residue propeptide. The mature enzyme was characterized by steady-state kinetics using a peptide corresponding to the canine angiotensinogen sequence, Ac-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser-OH (cleavage between Leu(10)-Leu(11)). The reaction followed Michaelis-Menten kinetics with a K(M) of 120 microM and a second-order rate constant (k(cat)/K(M)) of 1.7 x 10(5) M(-)(1)s(-)(1). The enzyme was inhibited by various human renin inhibitors, but at reduced potency compared to the human renin. The basis of the species specificity was investigated by mutagenesis. Based on primary sequence and structural alignments, three mutants were prepared (G149S-S150T, V286L, G149S-S150T-V286L). Each mutant yielded catalytically active enzymes with lower specific activities than native canine renin. V286L had the greatest effect on substrate specificity, while G149S, S150T mutations produced enzymes with inhibitor profiles similar to human renin.

Persistent anti-gag, -Nef, and -Rev IgM levels as markers of the impaired functions of CD4+ T-helper lymphocytes during SIVmac251 infection of cynomolgus macaques.

This study analyzed the antigen-specific (Gag, Nef, Rev, and Tat) IgM, IgG, and IgA humoral responses during the first 200 days of SIVmac251 infection in cynomolgus macaques. These responses were tested for correlation with the CD4(+) T-cell-related hematologic parameters and viral load throughout the course of the study (acute and chronic infection, during and after antiretroviral therapy). Strong inverse correlations were observed between the percentage of CD4(+) T cells at almost every timepoint of the study and the levels of IgM (but not IgG and IgA) against Gag, Nef, and Rev (but not Tat) measured after, but not during, the primary peak of IgM response. Significant levels of persistent antigen-specific IgMs may reflect the prevalence of mature plasma cells that have not undergone immunoglobulin class switching, possibly due to defects in helper T-cell function. Strong correlations were observed between the preinfection CD4(+) T-cell count or CD4/CD8 ratio and the same parameters measured throughout the study, suggesting the importance of preinfection immune status as a determinant of disease progression. The negative correlations between the post-acute-phase IgM levels and the percentage of CD4(+) T cells at later times during the study suggest the potential prognostic value of this measurement.