The determination of human peripheral blood mononuclear cell counts using a genomic DNA standard and application in tenofovir diphosphate quantitation.

A fluorescent quantitation method to determine PBMC-derived DNA amounts using purified human genomic DNA (gDNA) as the reference standard was developed and validated. gDNA was measured in a fluorescence-based assay using a DNA intercalant, SYBR green. The fluorescence signal was proportional to the amount (mass) of DNA in the sample. The results confirmed a linear fit from 0.0665 to 1.17 µg/µL for gDNA, corresponding to 2.0 × 106 to 35.0 × 106 cells/PBMC sample. Intra-batch and inter-batch accuracy (%RE) was within ±15%, and precision (%CV) was <15%. Benchtop stability, freeze/thaw stability and long term storage stability of gDNA in QC sample matrix, PBMC pellets samples, and pellet debris samples, respectively, as well as dilution linearity had been established. Consistency between hemocytometry cell counting method and gDNA-based counting method was established. 6 out of 6 evaluated PBMC lots had hemocytometry cell counts that were within ±20% of the cell counts determined by the gDNA method. This method was used in conjunction with a validated LC-MS/MS method to determine the level of tenofovir diphosphate (TFV-DP), the active intracellular metabolite of the prodrugs tenofovir alafenamide (TAF) and tenofovir disoproxil fumarate (TDF), measured in PBMCs in clinical trials of TAF or TDF-containing fixed dose combinations.

Design of an encodable tyrosine kinase-inducible domain: detection of tyrosine kinase activity by terbium luminescence.

Tyrosine kinases are critical mediators of intracellular signaling and of intracellular responses to extracellular signaling. Changes in tyrosine kinase activity are implicated in numerous human diseases, including cancers, diabetes, and pathogen infectivity. To address questions in tyrosine phosphorylation, we have designed a protein tyrosine kinase-inducible domain, a small, genetically encodable protein motif whose structure is dependent on its tyrosine phosphorylation state. Tyrosine kinase-inducible domain peptides are based on EF-hand loops in which a structurally critical Glu12 residue is replaced by tyrosine at residue 11 or at residue 15 of the protein. Tyrosine kinase-inducible domain peptides bind terbium(III) in a phosphorylation-dependent manner, showing strong terbium luminescence when phosphorylated but weak terbium luminescence when not phosphorylated. Lanthanide binding was confirmed by NMR. A tyrosine kinase-inducible domain peptide, pKID-Abl, was designed to incorporate a recognition sequence of the Abl kinase. Incubation of pKID-Abl with Abl kinase resulted in a large increase in terbium luminescence. This increase in luminescence was abolished when pKID-Abl and Abl kinase were incubated with the Abl kinase inhibitor Gleevec. In addition, incubation of phosphorylated pKID-Abl with the tyrosine phosphatase YOP resulted in a large reduction in terbium luminescence. pKID-Abl was employed as a fluorescent sensor of Abl tyrosine kinase activity in HeLa cell extracts, exhibiting low luminescence with extracts from serum-starved cells and increased luminescence using extracts from EGF-treated cells. These results indicate that tyrosine kinase-inducible domains may be used as sensors of tyrosine kinase and tyrosine phosphatase activity and in the detection of tyrosine kinase inhibitors.

Workshop on Recent Issues in Bioanalysis (WRIB) Poster Award winners 2018.

The 12th WRIB held in Philadelphia, USA in April 2018. It drew over 900 professionals representing large Pharmas, Biotechs, Contract Research Organizations and multiple regulatory agencies from around the world, from the global bioanalytical community. Bioanalysis and Bioanalysis Zone are very proud to be supporting the WRIB Poster Awards again this year, and we feature the profiles of the authors of the winning posters. Visit www.bioanalysis-zone.com to see the winning posters in full.

Differential effect of the GPIIb/IIIa antagonist orbofiban on human platelet aggregate formation in vitro.

Platelet microaggregates have been demonstrated in the systemic circulation of patients with atherosclerotic cardiovascular diseases. Glycoprotein IIb/IIIa antagonists have been reported to play roles in platelet activation, which may be associated with pro-thrombotic events. We report the effect of the orally active GPIIb/IIIa antagonist, orbofiban, on human platelet microaggregate formation in vitro. Laser light scatter (LSS) technology was used to monitor small, medium and large platelet aggregates formed in platelet-rich plasma in response to ADP or thrombin receptor activating peptide (TRAP)-6. ADP at 0.5 microM induced only small platelet aggregates that were prevented by orbofiban in a concentration-dependent manner. Likewise, orbofiban dissolved small aggregates after their formation. In marked contrast, in the presence of strong agonist stimulation (20 microM ADP or 3 microM TRAP-6) which generated primarily large platelet aggregates, orbofiban blocked the large aggregates while significantly augmenting small aggregates by three- to sixfold. The data suggest that GPIIb/IIIa antagonists do not induce platelet microaggregation directly but may augment small platelet microaggregate formation indirectly at conditions of strong stimuli. The percentage of the microaggregate population expressing P-selectin remained the same in the presence of orbofiban, indicating that these small microaggregates remain activated, although the mean intensity of expression was diminished, possibly reflecting the reduced size of the particles or density of P-selectin molecules. In summary, an increase in small platelet microaggregates might have contributed to pro-thrombotic events in orbofiban-treated patients.

Determinants of specificity of MDM2 for the activation domains of p53 and p65: proline27 disrupts the MDM2-binding motif of p53.

Transcriptional activation and repression via the transcription factors p53 and p65 are mediated by hydrophobic short linear motifs (FXX phi phi) in their activation domains (ADs). To understand the molecular basis for specificity in binding to disparate biological targets, a series of chimeric peptides was synthesized, with sequences derived from the ADs of p53, which binds both the general transcriptional machinery and the repressor protein MDM2, and p65, which is reported to bind the general transcriptional machinery but not MDM2. The FXX phi phi motifs of p53 and p65 differ by only two residues, whereas the flanking sequences have no sequence identity. The affinities of the chimeric peptides to MDM2(25-117) and hTAF(II)31(1-140) were determined. Specificity for binding MDM2 via FXX phi phi motifs derives almost entirely from Trp23 of p53, with a 3.0 kcal mol(-1) loss of binding energy when Trp23 is changed to p65-derived Leu. The identity of the N-terminal flanking sequence did not significantly affect binding to MDM2. In contrast, replacement of the C-terminal sequence of p53 with that of p65 increased the affinity of the chimera for MDM2 by 1.1 kcal mol(-1), contrary to expectations. Replacement of the highly conserved residue Pro27 of p53 with Ser from p65 resulted in a 2.3 kcal mol(-1) improvement in binding to MDM2, generating a ligand (p53-P27S) (Kd = 4.7 nM) that exhibits the highest MDM2 affinity observed for a genetically encodable ligand. The basis for the increased affinity of p53-P27S over p53 was examined by circular dichroism and nuclear magnetic resonance. Pro27 disrupts the recognition alpha-helix of p53, with p53-P27S significantly more alpha-helical than p53.

Raising HDL cholesterol without inducing hepatic steatosis and hypertriglyceridemia by a selective LXR modulator.

Liver X receptors (LXRs) are ligand-activated transcription factors that belong to the nuclear receptor superfamily. LXRs activate transcription of a spectrum of genes that regulate reverse cholesterol transport, including the ATP binding cassette transporter A1 (ABCA1), and raise HDL cholesterol (HDL-C) levels. However, LXR agonists also induce genes that stimulate lipogenesis, including the sterol response element binding protein (SREBP1-c) and fatty acid synthetase (FAS). The induction of these genes in the liver cause increased hepatic triglyceride synthesis, hypertriglyceridemia, and hepatic steatosis. As LXR response elements have been identified in these promoters, it is not clear if these two processes can be separated. Herein, we demonstrate that plasma HDL-C elevation and intestinal ABCA1 induction can occur with relatively little induction of FAS and SREBP1-c in mouse liver via a selective LXR modulator GW3965. This is in contrast to the strong induction of hepatic lipogenic genes by the well-characterized LXR agonist T0901317 (T317). Consistent with the in vivo results, GW3965 is a very weak LXR activator compared with T317 in human hepatoma cells. GW3965-liganded LXR recruits selected coactivators less effectively than T317 and may explain in part the tissue selective gene induction. This demonstration that tissue and gene selective modulation is possible with selective LXR modulators has positive implications for the development of this class of antiatherosclerotic agents.