Biochemical Characterization of Selective Inhibitors of Human Group IIA Secreted Phospholipase A₂ and Hyaluronic Acid-Linked Inhibitor Conjugates

We explored the inhibition mode of group IIA secreted phospholipase A₂ (GIIA sPLA₂) selective inhibitors and tested their ability to inhibit GIIA sPLA₂ activity as chemical conjugates with hyaluronic acid (HA). Analogues of a benzo-fused indole sPLA₂ inhibitor were developed in which the carboxylate group on the inhibitor scaffold, which has been shown to coordinate to a Ca²⁺ ligand in the enzyme active site, was replaced with other functionality. Replacing the carboxylate group with amine, amide, or hydroxyl groups had no effect on human GIIA (hGIIA) sPLA₂ inhibition potency but dramatically lowered inhibition potency against hGV and hGX sPLA₂s. An alkylation protection assay was used to probe active site binding of carboxylate and noncarboxylate inhibitors in the presence and absence of Ca²⁺ and/or lipid vesicles. We observed that carboxylate-containing inhibitors bind the hGIIA sPLA₂ active site with low nanomolar affinity, but only when Ca²⁺ is present. Noncarboxylate, GIIA sPLA₂ selective inhibitors also bind the hGIIA sPLA₂ active site in the nanomolar range. However, binding for GIIA sPLA₂ selective inhibitors was dependent on the presence of a lipid membrane and not Ca²⁺. These results indicate that GIIA sPLA₂ selective inhibitors exert their inhibitory effects by binding to the hGIIA sPLA₂ active site. An HA-linked GIIA inhibitor conjugate was developed using peptide coupling conditions and found to be less potent and selective against hGIIA sPLA₂ than the unconjugated inhibitor. Compounds reported in this study are some of the most potent and selective GIIA sPLA₂ active site binding inhibitors reported to date

The unspecific effect of PP on thymocyte maturation is not reversed by exogenous AA and PGE₂.

<p><b>A.</b> Representative thymocyte subpopulation distribution in WT cPLA₂α FTOC after 5 days of culture in absence or presence of 1μM of PP and exogenous (1μM) AA and PGE₂. Thymocytes were identified cytofluorometrically using fluorochrome-conjugated antibodies directed against CD3, CD4 and CD8. <b>B.</b> WT cPLA₂α fetal thymuses were cultured during 5 days as FTOCs in absence or presence of 1μM of PP, and exogenous (1μM) AA and PGE₂. After mechanical dissociation of fetal thymuses, the thymocytes were identified with fluorochrome-conjugated antibodies directed against CD3, CD4, and CD8 and analyzed by flow cytometry. Data are mean ± SEM of 3 independent experiments and the number of fetal thymuses for each condition is: Diluent (n = 5); 1μM PP (n = 6); 1μM PP and 1μM AA (n = 5); 1μM PP and 1μM PGE₂ (n = 5). * P< .05; ** P< .01; *** P< .001.</p

The disruption of the cPLA₂α gene does not impact thymocyte maturation in FTOC.

<p><b>A.</b> Representative thymocyte subpopulation distribution in WT and KO cPLA₂α FTOC. After 5 days of culture, the identification of thymocytes with fluorochrome-conjugated antibodies directed against CD3, CD4 and CD8 was determined by flow cytometry. <b>B.</b> WT and KO cPLA₂α fetal thymuses were cultured during 5 days as FTOCs. After mechanical dissociation of fetal thymuses, the thymocytes were labeled with fluorochrome-conjugated antibodies directed against CD3, CD4, and CD8, and analyzed by flow cytometry. Data are mean ± SEM of 6 independent experiments and the number of fetal thymuses for each genotype is: cPLA₂^+/+ (n = 17); cPLA₂^-/- (n = 9). NS (non significant).</p

Eicosanoid profiles of cPLA₂α WT and KO FTOC supernatants and adult mouse thymuses.

<p><b>A.</b> Expression distribution of the eicosanoids present in cPLA₂α WT FTOCs. The supernatants of FTOCs were collected at the indicated time of culture and the eicosanoid profiles were determined by combined liquid chromatography/tandem mass spectrometry. Data are mean of 3 different supernatants. <b>B.</b> Eicosanoid profiles of cPLA₂α WT and KO FTOC supernatants. The supernatants of FTOCs were collected at the indicated time of culture and eicosanoid profiles were determined by combined liquid chromatography/tandem mass spectrometry. Data are mean ± SEM of 3 different supernatants. <b>C.</b> Eicosanoid profiles of cPLA₂α WT and KO thymuses from adult mice. Adult thymuses were mechanically disrupted and eicosanoid profiles were determined by combined liquid chromatography/tandem mass spectrometry. ** P< .01, data are means ± SEM of 3 cPLA₂α WT thymuses and 2 cPLA₂α KO thymuses.</p

Tandem Mass Spectrometry Assays of Palmitoyl Protein Thioesterase 1 and Tripeptidyl Peptidase Activity in Dried Blood Spots for the Detection of Neuronal Ceroid Lipofuscinoses in Newborns

We report new substrates for quantitative enzyme activity measurements of human palmitoyl protein thioesterase (PPT1) and tripeptidyl peptidase (TPP1) in dried blood spots from newborns using tandem mass spectrometry. Deficiencies in these enzyme activities due to inborn errors of metabolism cause neuronal ceroid lipofuscinoses. The assays use synthetic compounds that were designed to mimic the natural substrates. Incubation produces nanomole quantities of enzymatic products per a blood spot that are quantified by tandem mass spectrometry using synthetic internal standards and selected reaction monitoring. The assays utilize a minimum steps for sample workup and can be run in a duplex format for the detection of neuronal ceroid lipofuscinoses or potentially multiplexed with other mass spectrometry-based assays for newborn screening of lysosomal storage disorders

Pharmacological blockade of cPLA₂α does not affect thymocyte maturation in FTOC.

<p><b>A.</b> Representative thymocyte subpopulation distribution in WT cPLA₂α FTOC after 5 days of culture in absence or presence of indicated concentrations of PP. Thymocytes were identified with fluorochrome-conjugated antibodies directed against CD3, CD4 and CD8 by flow cytometry. <b>B.</b> WT cPLA₂α fetal thymuses were cultured during 5 days as FTOCs in absence or presence of indicated concentrations of PP. After mechanical dissociation of fetal thymuses, the thymocytes were labeled with fluorochrome-conjugated antibodies directed against CD3, CD4, and CD8 and analyzed by flow cytometry. Data are mean ± SEM of 5 independent experiments and the number of fetal thymuses for each condition is: Diluent (n = 10); 10nM PP (n = 8); 100nM PP (n = 12). NS (non significant).</p

cPLA₂α inhibition by high concentration of PP impacts thymocyte maturation in FTOC.

<p><b>A.</b> Representative thymocyte subpopulation distribution in WT cPLA₂α FTOC after 5 days of culture in absence or presence of 1μM of PP. Thymocytes were identified cytofluorometrically using fluorochrome-conjugated antibodies directed against CD3, CD4 and CD8. <b>B.</b> WT cPLA₂α fetal thymuses were cultured during 5 days as FTOCs in absence or presence of 1μM of PP. After mechanical dissociation of fetal thymuses, the thymocytes were labeled with fluorochrome-conjugated antibodies directed against CD3, CD4, and CD8 and analyzed by flow cytometry. Data are mean ± SEM of 9 independent experiments and the number of fetal thymuses for each condition is: Diluent (n = 22); 1μM PP (n = 13). *** P< .001.</p

cPLA₂α inhibition does not impact human thymocyte maturation.

<p><b>A.</b> Representative thymocyte subpopulation distribution in human FTOC after 5 days of culture in absence or presence of indicated concentrations of PP. Thymocytes were identified cytofluorometrically using fluorochrome-conjugated antibodies directed against CD3, CD4 and CD8. <b>B.</b> Human FTOCs were cultured during 5 days in absence or presence of indicated concentrations of PP. After mechanical dissociation of human FTOCs, the thymocytes were labeled with fluorochrome-conjugated antibodies directed against CD3, CD4, and CD8 and analyzed by flow cytometry. Data are mean ± SEM of 3 independent experiments and the number of thymuses for each condition is: Diluent (n = 6); 10nM PP (n = 6); 100nM PP (n = 6); 1000nM PP (n = 6). NS (non significant).</p

1‑Benzyl-3-aryl-2-thiohydantoin Derivatives as New Anti-<i>Trypanosoma brucei</i> Agents: SAR and in Vivo Efficacy

A high throughput screening and subsequent hit validation identified compound <b>1</b> as an inhibitor of <i>Trypanosoma brucei</i> parasite growth. Extensive structure–activity relationship optimization based on antiparasitic activity led to the highly potent compounds, 1-(4-fluorobenzyl)-3-(4-dimethylamino-3-chlorophenyl)-2-thiohydantoin (<b>68</b>) and 1-(2-chloro-4-fluorobenzyl)-3-(4-dimethylamino-3-methoxyphenyl)-2-thiohydantoin (<b>76</b>), with a <i>T. brucei</i> EC₅₀ of 3 and 2 nM, respectively. This represents >100-fold improvement in potency compared to compound <b>1</b>. In vivo efficacy experiments of <b>68</b> and <b>76</b> in an acute mouse model of Human African Trypanosomiasis showed a 100% cure rate after 4 days of oral treatment at 50 mg/kg twice per day