Measuring Rubisco activity: challenges and opportunities of NADH-linked microtiter plate-based and 14C-based assays

Rubisco is central to carbon assimilation, and efforts to improve the efficiency and sustainability of crop production have spurred interest in phenotyping Rubisco activity. We tested the hypothesis that microtiter plate-based methods provide comparable results to those obtained with the radiometric assay that measures the incorporation of 14CO2 into 3-phosphoglycerate (3-PGA). Three NADH-linked assays were tested that use alternative coupling enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glycerolphosphate dehydrogenase (GlyPDH); phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH); and pyruvate kinase (PK) and lactate dehydrogenase (LDH). To date there has been no thorough evaluation of their reliability by comparison with the 14C-based method. The three NADH-linked assays were used in parallel to estimate (i) the 3-PGA concentration–response curve of NADH oxidation, (ii) the Michaelis–Menten constant for ribulose-1,5-bisphosphate, (iii) fully active and inhibited Rubisco activities, and (iv) Rubisco initial and total activities in fully illuminated and shaded leaves. All three methods correlated strongly with the 14C-based method, and the PK–LDH method showed a strong correlation and was the cheapest method. PEPC–MDH would be a suitable option for situations in which ADP/ATP might interfere with the assay. GAPDH–GlyPDH proved more laborious than the other methods. Thus, we recommend the PK–LDH method as a reliable, cheaper, and higher throughput method to phenotype Rubisco activity for crop improvement efforts.

Synthesis & Evaluation of Antitubercular Activity of Novel Mannich Bases of imidazo[2,1-b][1,3,4]thiadiazoles

A series of 2-(4-methoxybenzyl)-6-aryl-5-pyrrolidin/piperidin/morpholin-1-ylmethyl-imidazo [2,1-[1,3, thiadiazoles (3a-e, 4a-e & 5a-e) were synthesized by Mannich reaction by condensing 2-(4-methoxybenzyl)-6-arylimidazo [2,1-[1,3,thiadiazoles with pyrrolidine, piperidine and morpholine. The title compounds were screened for antitubercular activity againstMycobacterium tuberculosisH37Rv using the BACTEC 460 radiometric assay. Mannich bases with pyrrolidine substitution were found to be most active antitubercular agents. It proves that as the ring size decreases it becomes much potent in its activity. Pyrrolidine being 5 membered ring structure & further combined with imidazothiadiazole nucleus enhances the pharmacological activity.

Selective Inhibitors of Arginine Methyl Transferase 5 (PRMT5) As a Novel Treatment for β-Thalassemia and Sickle Cell Disease.

Abstract 2129 The developmental switch in human β-like globin gene subtype from fetal (γ) to adult (β) that begins at birth foreshadows the onset of the hemoglobinopathies, β-thalassemia and sickle cell disease (SCD). In the clinical setting it is established that β-thalassemia and SCD patients with hereditary persistence of fetal hemoglobin mutations enjoy a significant amelioration of disease severity due to the continued expression of γ-globin. This has prompted the search for therapeutic strategies to reverse γ-globin gene silencing. Central to the mechanism of γ-gene silencing is DNA methylation, which marks critical CpG dinucleotides flanking the γ-gene transcriptional initiation site in adult bone marrow erythroid cells. These marks are established by recruitment of DNMT3A, a DNA methyltransferase, to the γ-globin promoter by protein arginine methyltransferase 5 (PRMT5)[Zhao Q et al. Nat Struct Mol Biol. 2009;16(3):304–311]. PRMT5 catalyses the symmetric dimethylation of arginine 3 of Histone 4 (H4R3me2), which serves as a template for direct binding of DNMT3A and the subsequent DNA methylation of the γ-gene promoter. Loss of PRMT5 or its enzymatic activity is sufficient to induce demethylation of the CpG dinucleotides and reactivation of γ-globin gene expression [Rank, G., et al. Blood, 116(9), 1585–92]. Based on these observations we hypothesize that small molecule inhibitors of PRMT5 activity could provide a beneficial treatment for β-thalassemia and SCD. To identify small molecule inhibitors of PRMT5 a high throughput screen (HTS) was performed. Both radiometric and non-radiometric assay formats were developed to support the screening campaign. The radiometric assay format measures the ability of PRMT5 purified from K562 cells to catalyse the labelling of a short peptide based on the N-terminal sequence of Histone H4 by 3H-Methyl-S-Adenosyl-L-methionine (SAM). In contrast, the non-radiometric assay format employs recombinant PRMT5/MEP50 and measures the production of S-adenosyl-L-homocysteine (SAH), which is generated by PRMT5-catalysed methylation of H4 peptide. SAH is measured with Transcreener EPIGEN” and the assay is formatted in 1536-well microtitre plates in a total assay volume of 4 μL. Using these assays, a chemical library of 350,000 lead-like molecules and known pharmacologically active agents was screened to identify inhibitors of PRMT5 methyltransferase activity. A number of compounds with low micromolar or submicromolar inhibitory activity were identified by the HTS campaign, and six were selected for re-synthesis. The inhibitory activity of five of the six compounds was confirmed. To provide an initial appraisal of inhibitor selectivity the five active compounds were subsequently tested against a panel of enzymes consisting of 23 protein and DNA methyltransferases and 12 kinases. These compounds were found to be remarkably selective PRMT5 inhibitors, inhibition of MLL4 being the only significant off-target activity noted for one of the scaffolds. We have established a critical path for selection and progression of new chemical analogues which entails testing the compounds for: i) inhibition of PRMT5, other protein methyl transferases and kinases; ii) the ability to induce expression of γ-globin mRNA in the K562 erythroleukemic cell line; iii) the ability to induce expression of γ-globin mRNA in adult bone marrow erythroid cells; and iv) the induction of γ-globin in vivo in β-YAC mice, a transgenic model which carries the 250-kb human globin locus. In parallel, the physicochemical, metabolism, and pharmacokinetic properties of the most promising compounds are also determined. Medicinal chemistry efforts have now produced molecules with > 100-fold increased inhibitory potency against PRMT5 compared to the original hits, and preliminary results indicate that the more potent compounds have the ability to induce γ-globin mRNA in our cell based models. These early results illustrate the potential of PRMT5 inhibitors as a novel approach for the treatment of β-thalassemia and sickle cell disease. Disclosures: No relevant conflicts of interest to declare.

Abstract 594: Selective Inhibition Of Lipoprotein-associated Phospholipase A 2 Attenuates Markers Of Plaque Vulnerability In Humans

Background: Lp-PLA 2 , an emerging marker of CV risk, generates products of oxidized LDL that may contribute to inflammation and apoptosis. Although Lp-PLA 2 is highly expressed in thin-cap fibroatheroma and ruptured plaques, its role in mediating lesion vulnerability remains unknown. Material and Methods: Patients were randomized to receive placebo or the selective Lp-PLA 2 inhibitor, darapladib, (40 or 80 mg po) for 14 days prior to carotid endartherectomy. In the post hoc analysis, caspase activities were measured in carotid plaques (colorimetric assays: AU/mg of protein) and correlated with inhibition of Lp-PLA 2 activity in plasma (radiometric assay) using Spearman’s rank method. Results: Caspase-8 (initiator) and -3 (effector) activities were measured as the index of apoptosis in carotid atheroma specimens. In the validation study, carotid atheroma (n=10) exhibited augmented levels of caspase activities compared to non-diseased vessels (n=8) (caspase-8: 4.74±0.3 vs 2.59±0.2; caspase-3: 10.2±0.9 vs 4.15±0.4 p<0.001). Next, caspase-8 (n=39) and -3 (n=59) were measured in plaque samples derived from the dose-ranging study with darapladib. In patients treated with darapladib, caspase-8 activity within the plaques was significantly reduced [placebo: 4.66±0.48; 40 mg: 3.4±0.4, NS; 80 mg: 3.0±0.52, p=0.042 versus placebo (ANOVA with Bonferroni adjustment)]. Similarly, downstream caspase-3 was also decreased in the high dose group (placebo: 11.8±0.84; 40 mg: 10.4±0.88, NS; 80 mg: 6.5±1.0 p=0.0004 versus placebo). To further explore the relationship between Lp-PLA 2 activity and markers of apoptosis, plaque caspase activity was correlated with Lp-PLA 2 inhibition in plasma. Plaque caspase-8 and -3 correlated with inhibition of Lp-PLA 2 activity in plasma (caspase-8: r=-0.34, p=0.039; caspase-3: r=−0.51, p<0.0001). Conclusions: Inhibition of Lp-PLA 2 reduced activity of proapoptotic caspases in human atheroma. The results suggest that Lp-PLA 2 inhibition may attenuate noxious effects of oxidized LDL and promote lesion stabilization in high risk patients.