Characterization of the Analgesic and Anti-Inflammatory Activities of Ketorolac and Its Enantiomers in the Rat

ABSTRACT The marked analgesic efficacy of ketorolac in humans, relative to other nonsteroidal anti-inflammatory drugs (NSAIDs), has lead to speculation as to whether additional non-NSAID mechanism(s) contribute to its analgesic actions. To evaluate this possibility, we characterized (R,S)-ketorolac's pharmacological properties in vivo and in vitro using the nonselective cyclooxygenase (COX) inhibitors [indomethacin (INDO) and diclofenac sodium (DS)] as well as the selective COX-2 inhibitor, celecoxib, as references. The potency of racemic (R,S)-ketorolac was similar in tests of acetic acid-induced writhing, carrageenaninduced paw hyperalgesia, and carrageenan-induced edema formation in rats; ID 50 values ϭ 0.24, 0.29, and 0.08 mg/kg, respectively. (R,S)-ketorolac's actions were stereospecific, with (S)-ketorolac possessing the biological activity of the racemate in the above tests. The analgesic potencies for (R,S)-, (S)-, and (R)-ketorolac, INDO, and DS were highly correlated with their anti-inflammatory potencies, suggesting a common mechanism. (R,S)-ketorolac was significantly more potent than INDO or DS in vivo. Neither difference in relative potency of COX inhibition for (R,S)-ketorolac over INDO and DS nor activity of (S)-ketorolac at a number of other enzymes, channels, or receptors could account for the differences in observed potency. The distribution coefficient for (R,S)-ketorolac was approximately 30-fold less than for DS or INDO, indicating that (R,S)-ketorolac is much less lipophilic than these NSAIDs. Therefore, the physicochemical and pharmacokinetics properties of (R,S)-ketorolac may optimize the concentrations of (S)-ketorolac at its biological target(s), resulting in greater efficacy and potency in vivo

A methodology paper for histone variant peptide quantitation

Co-existing Post-Translational Modifications (PTMs) on histone H3 were shown to have positive or negative interplay between each other, indicating their dependency and crosstalk in the epigenetic regulation pathways. Recent reports have shown that an important activating mark, H3K36 methylation, is antagonizing with PRC2 mediated K27 methylation with interesting crosstalk mechanism during transcription regulation.(1) Our previous study demonstrated that a multiple reaction monitoring (MRM) based LC-MS/MS method can be used to quantitatively profile global histone H3 post-translational modifications (PTMs) with stable isotopic labeled peptides as internal standards.(30) To further develop this method for in-depth understanding of different histone variants with distinct epigenetic roles, we herein report an integrated MRM method with RP/HILIC combination to analyze histone peptides, especially the combinatorial K27/K36 peptides of all three major H3 variants, from various mouse tissues/organs. Despite subtle biochemical differences in H3 variants, we successfully obtained decent separation and high sensitivity for histone H3.3 specific peptides and histone H3.1/3.2 specific peptides by optimizing the column selection and MS response in the combined HILIC/RP-MRM methods. Meanwhile, the sensitivity for low-abundant K4 methylation peptides was greatly improved. From the quantitative results of 4 mouse tissues/organs, we observed unique combinatorial K27/K36 modification patterns of histone variants indicating distinct histone codes in epigenetic regulation during cell differentiation and developmental process. The expression abundance of H3.3 was simultaneously measured in these mouse samples suggesting its different molecular functions in different functional organs

Quantitative Profiling of Combinational K27/K36 Modifications on Histone H3 Variants in Mouse Organs

The coexisting post-translational modifications (PTMs) on histone H3 N-terminal tails were known to crosstalk between each other, indicating their interdependency in the epigenetic regulation pathways. H3K36 methylation, an important activating mark, was recently reported to antagonize with PRC2-mediated H3K27 methylation with possible crosstalk mechanism during transcription regulation process. On the basis of our previous studies, we further integrated RP/HILIC liquid chromatography with MRM mass spectrometry to quantify histone PTMs from various mouse organs, especially the combinatorial K27/K36 marks for all three major histone H3 variants. Despite their subtle difference in physicochemical properties, we successfully obtained decent separation and high detection sensitivity for both histone H3.3 specific peptides and histone H3.1/3.2 specific peptides. In addition, the overall abundance of H3.3 can be quantified simultaneously. We applied this method to investigate the pattern of the combinatorial K27/K36 marks for all three major histone H3 variants across five mouse organs. Intriguing distribution differences were observed not only between different H3 variants but also between different organs. Our data shed the new insights into histone codes functions in epigenetic regulation during cell differentiation and developmental process

RO1138452 and RO3244794: characterization of structurally distinct, potent and selective IP (prostacyclin) receptor antagonists

1. Prostacyclin (PGI(2)) possesses various physiological functions, including modulation of nociception, inflammation and cardiovascular activity. Elucidation of these functions has been hampered by the absence of selective IP receptor antagonists. 2. Two structurally distinct series of IP receptor antagonists have been developed: 4,5-dihydro-1H-imidazol-2-yl)-[4-(4-isopropoxy-benzyl)-phenyl]-amine (RO1138452) and R-3-(4-fluoro-phenyl)-2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-propionic acid (RO3244794). 3. RO1138452 and RO3244794 display high affinity for IP receptors. In human platelets, the receptor affinities (pK(i)) were 9.3±0.1 and 7.7±0.03, respectively; in a recombinant IP receptor system, pK(i) values were 8.7±0.06 and 6.9±0.1, respectively. 4. Functional antagonism of RO1138452 and RO3244794 was studied by measuring inhibition of carbaprostacyclin-induced cAMP accumulation in CHO-K1 cells stably expressing the human IP receptor. The antagonist affinities (pK(i)) of RO1138452 and RO3244794 were 9.0±0.06 and 8.5±0.11, respectively. 5. Selectivity profiles for RO1138452 and RO3244794 were determined via a panel of receptor binding and enzyme assays. RO1138452 displayed affinity at I(2) (8.3) and PAF (7.9) receptors, while RO3244794 was highly selective for the IP receptor: pK(i) values for EP(1) (<5), EP(3) (5.38), EP(4) (5.74) and TP (5.09). 6. RO1138452 (1–10 mg kg(−1), i.v.) and RO3244794 (1–30 mg kg(−1), i.v.) significantly reduced acetic acid-induced abdominal constrictions. RO1138452 (3–100 mg kg(−1), p.o.) and RO3244794 (0.3–30 mg kg(−1), p.o.) significantly reduced carrageenan-induced mechanical hyperalgesia and edema formation. RO3244794 (1 and 10 mg kg(−1), p.o.) also significantly reduced chronic joint discomfort induced by monoiodoacetate. 7. These data suggest that RO1138452 and RO3244794 are potent and selective antagonists for both human and rat IP receptors and that they possess analgesic and anti-inflammatory potential

Absolute Quantification of Histone PTM Marks by MRM-Based LC-MS/MS

The N-terminal tails of core histones harbor the sites of numerous post-translational modifications (PTMs) with important roles in the regulation of chromatin structure and function. Profiling histone PTM marks provides data that help understand the epigenetics events in cells and their connections with cancer and other diseases. Our previous study demonstrated that specific derivatization of histone peptides by NHS propionate significantly improved their chromatographic performance on reversed phase columns for LC/MS analysis. As a step forward, we recently developed a multiple reaction monitoring (MRM) based LC-MS/MS method to analyze 42 targeted histone peptides. By using stable isotopic labeled peptides as internal standards that are spiked into the reconstituted solutions, this method allows to measure absolute concentration of the tryptic peptides of H3 histone proteins extracted from cancer cell lines. The method was thoroughly validated for the accuracy and reproducibility through analyzing recombinant histone proteins and cellular samples. The linear dynamic range of the MRM assays was achieved in 3 orders of magnitude from 1 nM to 1 μM for all targeted peptides. Excellent intrabatch and interbatch reproducibility (<15% CV) was obtained. This method has been used to study translocated NSD2 (a histone lysine methyltransferase that catalyzes the histone lysine 36 methylation) function with its overexpression in KMS11 multiple myeloma cells. From the results we have successfully quantitated both individual and combinatorial histone marks in parental and NSD2 selective knockout KMS11 cells

Discovery of the Clinical Candidate MAK683: An EED-Directed, Allosteric, and Selective PRC2 Inhibitor for the Treatment of Advanced Malignancies.

Polycomb Repressive Complex 2 (PRC2) plays an important role in transcriptional regulation during animal development and in cell differentiation, and alteration of PRC2 activity has been associated with cancer. On a molecular level, PRC2 catalyzes methylation of histone H3 lysine 27 (H3K27), resulting in mono-, di-, or trimethylated forms of H3K27, of which the trimethylated form H3K27me3 leads to transcriptional repression of polycomb target genes. Previously, we have shown that binding of the low-molecular-weight compound EED226 to the H3K27me3 binding pocket of the regulatory subunit EED can effectively inhibit PRC2 activity in cells and reduce tumor growth in mouse xenograft models. Here, we report the stepwise optimization of the tool compound EED226 toward the potent and selective EED inhibitor MAK683 (compound 22) and its subsequent preclinical characterization. Based on a balanced PK/PD profile, efficacy, and mitigated risk of forming reactive metabolites, MAK683 has been selected for clinical development

DS_DISC766278 – Supplemental material for Discovery of Small-Molecule Antagonists of the H3K9me3 Binding to UHRF1 Tandem Tudor Domain

<p>Supplemental material, DS_DISC766278 for Discovery of Small-Molecule Antagonists of the H3K9me3 Binding to UHRF1 Tandem Tudor Domain by Guillermo Senisterra, Hugh Y. Zhu, Xiao Luo, Hailong Zhang, Guoliang Xun, Chunliang Lu, Wen Xiao, Taraneh Hajian, Peter Loppnau, Irene Chau, Fengling Li, Abdellah Allali-Hassani, Peter Atadja, Counde Oyang, En Li, Peter J. Brown, Cheryl H. Arrowsmith, Kehao Zhao, Zhengtian Yu, and Masoud Vedadi in SLAS Discovery</p

Discovery of First-in-Class, Potent, and Orally Bioavailable Embryonic Ectoderm Development (EED) Inhibitor with Robust Anticancer Efficacy

Overexpression and somatic heterozygous mutations of EZH2, the catalytic subunit of polycomb repressive complex 2 (PRC2), are associated with several tumor types. EZH2 inhibitor, EPZ-6438 (tazemetostat), demonstrated clinical efficacy in patients with acceptable safety profile as monotherapy. EED, another subunit of PRC2 complex, is essential for its histone methyltransferase activity through direct binding to trimethylated lysine 27 on histone 3 (H3K27Me3). Herein we disclose the discovery of a first-in-class potent, selective, and orally bioavailable EED inhibitor compound <b>43</b> (EED226). Guided by X-ray crystallography, compound <b>43</b> was discovered by fragmentation and regrowth of compound <b>7</b>, a PRC2 HTS hit that directly binds EED. The ensuing scaffold hopping followed by multiparameter optimization led to the discovery of <b>43</b>. Compound <b>43</b> induces robust and sustained tumor regression in EZH2^MUT preclinical DLBCL model. For the first time we demonstrate that specific and direct inhibition of EED can be effective as an anticancer strategy

Pyrrolopyrazines as Selective Spleen Tyrosine Kinase Inhibitors

We describe the discovery of several pyrrolopyrazines as potent and selective Syk inhibitors and the efforts that eventually led to the desired improvements in physicochemical properties and human whole blood potencies. Ultimately, our mouse model revealed unexpected toxicity that precluded us from further advancing this series