Identification of a Potent, Selective, and Efficacious Phosphatidylinositol 3-Kinase δ (PI3Kδ) Inhibitor for the Treatment of Immunological Disorders.

PI3Kδ plays an important role controlling immune cell function and has therefore been identified as a potential target for the treatment of immunological disorders. This article highlights our work toward the identification of a potent, selective, and efficacious PI3Kδ inhibitor. Through careful SAR, the successful replacement of a polar pyrazole group by a simple chloro or trifluoromethyl group led to improved Caco-2 permeability, reduced Caco-2 efflux, reduced hERG PC activity, and increased selectivity profile while maintaining potency in the CD69 hWB assay. The optimization of the aryl substitution then identified a 4'-CN group that improved the human/rodent correlation in microsomal metabolic stability. Our lead molecule is very potent in PK/PD assays and highly efficacious in a mouse collagen-induced arthritis model.

Development and evaluation of a multiple-plate fraction collector for sample processing: application to radioprofiling in drug metabolism studies.

Microplate scintillation counters are utilized routinely in drug metabolism laboratories for the off-line radioanalysis of fractions collected during HPLC radioprofiling. In this process, the current fraction collection technology is limited by the number of plates that can be used per injection as well as the potential for sample loss due to dripping or spraying as the fraction collector head moves from well to well or between plates. More importantly, sample throughput is limited in the conventional process, since the collection plates must be manually exchanged after each injection. The Collect PAL, an innovative multiple-plate fraction collector, was developed to address these deficiencies and improve overall sample throughput. It employs a zero-loss design and has sub-ambient temperature control. Operation of the system is completely controlled with software and up to 24 (96- or 384-well) fraction collection plates can be loaded in a completely automated run. The system may also be configured for collection into various-sized tubes or vials. At flow rates of 0.5 or 1.0 mL/min and at collection times of 10 or 15s, the system precisely delivered 83-µL fractions (within 4.1% CV) and 250-µL fractions (within 1.4% CV), respectively, of three different mobile phases into 12 mm × 32 mm vials. Similarly, at a flow rate of 1 mL/min and 10s collection times, the system precisely dispensed mobile phase containing a [(14)C]-radiolabeled compound across an entire 96-well plate (% CV was within 5.3%). Triplicate analyses of metabolism test samples containing [(14)C]buspirone and its metabolites, derived from three different matrices (plasma, urine and bile), indicated that the Collect PAL produced radioprofiles that were reproducible and comparable to the current technology; the % CV for 9 selected peaks in the radioprofiles generated with the Collect PAL were within 9.3%. Radioprofiles generated by collecting into 96- and 384-well plates were qualitatively comparable; however, the peak resolution was greater in the profiles that were collected in 384-well plates due to the collection of a larger number of fractions per minute. In conclusion, this new and innovative fraction collector generated radioprofile results that were comparable to current technology and should provide a major improvement in capacity and throughput for radioprofiling studies.

Rapid screening of glutathione-trapped reactive metabolites by linear ion trap mass spectrometry with isotope pattern-dependent scanning and postacquisition data mining.

The present study describes a novel integrated approach for rapid analysis of reactive metabolites with a linear ion trap mass spectrometer (LTQ). In this approach, an isotope pattern-dependent scanning method was applied to the data acquisition of glutathione (GSH)-trapped reactive metabolites. Recorded full-scan MS and MS/MS data sets were further processed with neutral loss filtering, product ion filtering, and extracted ion chromatographic analysis to search for protonated molecules and MS/MS spectra of GSH adducts. To evaluate the effectiveness and reliability of the approach, GSH adducts of carbamazepine, diclofenac, 4-ethylphenol, acetaminophen, p-cresol, and omeprazole were analyzed, which were formed in human liver microsome incubations fortified with a mixture of nonlabeled GSH and stable isotope-labeled GSH at a 1:0.8 ratio. Results demonstrate that the combination of the isotope pattern-dependent scanning with the postacquisition data mining was very effective in detecting low levels of GSH adducts, regardless of their fragmentation patterns. As compared to a neutral loss scanning method performed with a triple quadrupole mass spectrometer, the LTQ-based approach had several major advantages, including the superior selectivity and sensitivity in detecting different classes of GSH adducts and the higher throughput capability of the detection and MS/MS spectral acquisition of GSH adducts in a single LC/MS run. Overall, this analytical approach provides a simple and efficient means for screening for reactive metabolites using a linear ion trap LC/MS platform.

Synthesis and characterization of site-specific and stereoisomeric fjord dibenzo[a,l]pyrene diol epoxide-N(6)-adenine adducts: unusual thermal stabilization of modified DNA duplexes.

The fjord polycyclic aromatic hydrocarbon compound dibenzo[a,l]pyrene (DB[a,l]P) is significantly more tumorigenic than the bay region benzo[a]pyrene in animal model systems. The molecular origins of the unusually strong genotoxic properties of DB[a,l]P and its fjord region diol epoxide metabolites are of great interest and are believed to be related to the structural characteristics of the DNA adducts formed. Site-specifically modified oligonucleotides were prepared by reacting the single adenine residue in 5'-d(CTCTCACTTCC) (I) with the racemic fjord diol epoxide r11,t12-dihydrodiol-t13,14-epoxide-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (anti-DB[a,l]PDE) in aqueous solutions. Four different oligonucleotides I with the single adenosine residues involving a covalent bond between the C14 position of DB[a,l]PDE and N(6)-dA are identified and purified. The CD spectra of the mononucleotide adducts are similar to those of Li et al. [Li et al. (1999) Chem. Res. Toxicol. 12, 758] who characterized DB[a,l]PDE-N(6)-dA adducts by a combination of CD and NMR methods. The stereochemical properties of each of the four DB[a,l]PDE-modified oligonucleotides were assigned on the basis of a combination of empirical CD rules and other approaches and differ from those of Li et al. The thermal melting points, T(m), of the unmodified duplex of I with its complementary strand (IC), T(m) = 43.8 +/- 0.5 degrees C, were compared with the same duplexes containing stereoisomeric anti-DB[a,l]PDE-N(6)-dA lesions. The T(m) of duplexes I.IC containing lesions with R absolute configurations at C14 of the DB[a,l]PDE residues are greater by 6-8 degrees C, while those with S configuration are lower by 6-10 degrees C. Similar effects are observed with adducts in the same sequence context derived from the fjord PAH anti-diol epoxides of benzo[g]chrysene, while duplexes containing lesions derived from benzo[c]phenanthrene diol epoxides with 1R and 1S configurations exhibit unchanged T(m) values. In contrast, the T(m) values of duplexes with lesions derived from the bay region benzo[a]pyrene diol epoxides (B[a]PDE) in the same sequence are lower by 12 degrees (10R adducts) and by 19 degrees (10S adducts). The greater thermal stabilities of duplexes with fjord PAH-N(6)-dA lesions relative to those with bay region B[a]PDE-N(6)-dA adducts, are correlated with lower susceptibilities of excision by human nucleotide excision repair enzymes [Buterin et al. (2000) Cancer Res. 60, 1849]. The implications of these relationships are discussed in terms of present knowledge of the conformations of fjord and bay region PAH diol epoxide-N(6)-dA lesions in double stranded DNA.