Radiometric Assay Research Articles

Comparison of the Profile of the AML Drug Midostaurin (Rydapt®) As a Kinase Inhibitor with Those of Its Predominant Primary Human Metabolites

Background: The multi-targeted kinase inhibitor midostaurin (M) has single agent activity in internal tandem duplication and tyrosine kinase domain mutant FLT3 acute myeloid leukemia (AML). When added to standard chemotherapy M (50 mg, BID) significantly improved both event-free and overall survival in AML patients harboring activating FLT3 mutations (Stone RM, NEJM 2017). In humans, M is converted into 3 primary metabolites: the 10-O-demethylated compound CGP62221 and two epimeric C3-hydroxylated compounds, e1 and e2 (Fig 1). Following the administration of a single 50 mg dose of [14C]-M, the plasma AUC0-96hr of M, CGP62221, e1 and e2 were 18.0, 22.5, 4.12 and 25.8 µg Eq*h/mL respectively (He H, Drug Metab Dispos 2017) and following 50 mg BID the steady-state plasma trough levels of M, CGP62221 and e2 were 0.82, 1.48 and 6.73 µM (Wang Y, J Clin Pharmacol 2008), indicating substantial accumulation of the latter metabolite. Here we compare the kinase profile of M with those of its predominant metabolites.Methods: Reaction of M with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone afforded a mixture of oxidation products. After purification by chromatography (silica gel) and recrysallisation, X-ray crystallography of the more lipophilic epimer (VE12.094; m.p. 298-301°C) confirmed it to have (S)-stereochemistry (Fig 1). Comparison of this epimer with the products from cryopreserved human hepatocyte (BioreclamationIVT, Baltimore, MD, USA)-mediated metabolism of M showed it to be identical (ultra high performance liquid chromatography - mass spectrometry) to e1; the corresponding 3-(R)-epimer (e2) could not be purified. CGP58546 (Hoehn P, J Antibiot 1995) was treated with benzoic anhydride to give CGP62221 (purified by chromatography; m.p. 246°C). The profiles of these compounds as kinase inhibitors were determined using recombinant enzymes (13 lipid and 320 protein kinases) in radiometric transphosphorylation assays (ProQinase GmbH, Freiburg, Germany) and where >50% inhibition was observed at 10 µM, concentrations required to inhibit activity by 50% (IC50 value) were calculated from the dose-response curves.Results: All four compounds showed inhibition of both serine-threonine and tyrosine protein kinases, consistent with their binding in a type-1 ATP-competitive binding mode. M and CGP62221 showed both a similar pattern of selectivity against 320 protein kinases, as well as similar degrees of potency, although CGP62221 (the most potent inhibitor of PRKG2) was less active against LCK (Tables 1 and 2). The presence of a hydroxyl-group in the pyrrole-ring (e1 and e2) generally reduced potency resulting in increased selectivity towards VEGFR2 (none of the compounds evaluated inhibited VEGFR1 by >50% at 10 µM). PDK1 was the only kinase substantially more sensitive to e2 compared with e1 (>3-fold). None of the compounds substantially (>50%) inhibited the activity of any lipid kinase, nor of AKT, nor of any cyclin-dependent protein kinase at 10 µM.Conclusion: The disparate effects of FLT3-targeting drugs in AML clinical trials has been commented upon (Stone RM, Blood 2017). However, the efficacy and side-effects of drugs do not just reflect the biochemical and pharmacodynamic properties of the parent, but often comprise of complex cooperative effects between the properties of the parent and active metabolites. AML is a heterogeneous malignancy and activating mutations in the FLT3 and KIT receptor kinases are particularly implicated among the mutations driving the malignant phenotype (Patel JP, NEJM 2012). In the case of M, the major circulating metabolites are multi-targeted protein kinase inhibitors with activity against mutant forms of FLT3 and KIT, as well as several kinases involved in their signaling pathways or implicated in AML via stromal support, such as IGF1R (Chapuis N, Haematologica 2010), JAK (Furqan M, Biomarker Res 2013), LYN (Robinson LJ, Exp Hematol 2005), PDK1 (Zabkiewicz J, Haematologica 2014), RET (Rudat S, ASH 2016), SRC (Leischner H, Blood 2012) and SYK (Puissant A, Cancer Cell 2014). Notably, CGP62221, e1 and e2, which are major circulating components following chronic dosing of M, exhibited substantial kinase activity at physiologically relevant doses. A complex interplay between the kinase activities of M and its metabolites may contribute to the efficacy of M in AML and help engender the distinctive effects of M compared to other FLT3 inhibitors in this malignancy. [Display omitted] DisclosuresManley:Novartis International: Employment. Caravatti:Novartis International: Employment. Furet:Novartis International: Employment. Roesel:Novartis International AG: Employment. Tran:Novartis International: Employment. Wagner:Novartis Pharmaceuticals: Employment.

Enzymes of acetylcholine metabolism in the rat inferior colliculus

Although there is strong evidence for cholinergic projections to the rat inferior colliculus, especially from the pedunculopontine tegmental nucleus (Noftz et al., 2020), there is a lack of information about the quantitative prevalence of the enzymes of acetylcholine metabolism in its various portions. We have used microdissection of freeze-dried sections combined with radiometric assays to map the distributions in the rat inferior colliculus of the activities of choline acetyltransferase (ChAT), which catalyzes synthesis of acetylcholine, and acetylcholinesterase (AChE), which catalyzes its breakdown by hydrolysis. Both enzyme activities were present throughout the inferior colliculus. Average ChAT activity was consistently somewhat higher in the external cortex, excluding its most superficial layer, than in the dorsal cortex or central nucleus. Within the external cortex, ChAT activity was about half as high laterally in its most superficial layer as elsewhere. The distribution of AChE activity was more uniform than that of ChAT. Overall, ChAT activity in the rat inferior colliculus was relatively low, about a fifth of that in whole brain of rat and lower than in other central auditory regions, whereas AChE activity was about two-thirds that of rat whole brain and about average for central auditory regions. The results are compared to previous measurements for cat and hamster inferior colliculus. They are consistent with a modest role for cholinergic neurotransmission in the inferior colliculus, to modulate the activity of its major neuronal types.

The mechanism of leptin and IGF-1 in the diabetic rheumatoid arthritis Iraqi patients

We aimed here to study the impact of leptin on insulin like growth factor-1 (IGF-1) level to imply its antidiabetic effect on Iraqi rheumatoid arthritis’s, who was with and without diabetes mellitus.Rheumatoid arthritis is a chronic inflammatory disease, primarily targets the synovium and articular cartilage, which causes joint damage. Although the role of adipocytokines in mediating damages of joint has recently suggested, it is still a matter of considerable debate.METHODS: Patients diagnosed as diabetic rheumatoid arthritis aged from 33-60 years and others diagnosed as non-diabetic rheumatoid arthritis were compared with healthy control (aged 33-46 years). Some biochemical parameters have determined, such as fasting serum glucose, glycated hemoglobin, lipid profile, serum fasting insulin, IGF-1, and leptin, using ELISA and immune radiometric assay. Results indicated an elevation in some biochemical parameters in diabetic rheumatoid arthritis’s when compared with patients without diabetes. There was a significant increase in fasting serum glucose and some of the lipid components in diabetic rheumatoid arthritis patients compared to that non-diabetic. A significant elevation of leptin in diabetic rheumatoid arthritis patients compared to control (p<0.003). However, a non-significant difference of leptin was detected between diabetic rheumatoid arthritis and non-diabetic patients. In addition, some other parameters were significantly reduced in diabetic rheumatoid arthritis patients when compared to non-diabetic patients, such as high-density lipoprotein and IGF-1. This study highlights that leptin could act as pro-inflammatory mediator in rheumatoid arthritis. The negative correlation between leptin level and IGF-1 plays an important role in understanding the metabolic pathogenesis of rheumatoid arthritis.

Imaging of HER2-Positive Tumors in NOD/SCID Mice with Pertuzumab Fab-Hexahistidine Peptide Immunoconjugates Labeled with [99mTc]-(I)-Tricarbonyl Complex.

Molecular imaging of tumor HER2 expression may allow patient selection for HER2-targeted therapies. Our aim was to introduce hexahistidine (His6) peptides into pertuzumab Fab to enable labeling with the [99mTc(CO)3(H2O)3]+ complex and study these radioimmunoconjugates for microSPECT/CT imaging of HER2-positive tumor xenografts in mice. Fab were produced by papain digestion of pertuzumab and reacted with sulfo-SMCC for conjugation to His6-containing peptides (CGYGGHHHHHH). His6-peptide conjugation was measured by a radiometric assay. His6-pertuzumab Fab were labeled at 0.4-1.0MBq/μg with [99mTc(CO)3(H2O)3]+ for 1h at 37°C. HER2 immunoreactivity was assessed in a direct (saturation) binding assay using HER2-overexpressing SK-BR-3 human breast cancer (BC) cells. MicroSPECT/CT and biodistribution studies were performed in NOD/SCID mice with HER2-positive s.c. SK-OV-3 human ovarian cancer, or MDA-MB-361 or MDA-MB-231 human BC xenografts at 4 or 24h post i.v. injection of [99mTc]His6-pertuzumab Fab (29-49MBq, 70μg). The specificity of tumor uptake was assessed by comparison to irrelevant [99mTc]Fab 3913 in SK-OV-3 tumor-bearing mice. SDS-PAGE analysis demonstrated cleavage of pertuzumab to produce Fab, which eluted as a single peak with a retention time of 13.8min on SE-HPLC. Fab were conjugated to 2.1 ± 0.5 His6 peptides and labeled with [99mTc(CO)3(H2O)3]+ to a radiochemical purity of 92-97 % at 0.4-0.8MBq/μg. [99mTc]His6-pertuzumab Fab exhibited saturable and specific binding to SK-BR-3 cells with a KD = 51.3 ± 5.2 × 10-9M and Bmax = 3.5 ± 0.1 × 106 receptors/cell. SK-OV-3 tumors were imaged at 4 and 24h p.i [99mTc]His6-pertuzumab Fab. Tumor uptake at 24h p.i. was 4.1 ± 0.6 %ID/g, which was 13-fold significantly greater than [99mTc]Fab 3913 (0.3 ± 0.0 %ID/g; P < 0.01). MicroSPECT/CT imaged HER2-overexpressing MDA-MB-361 tumors but not MDA-MB-231 tumors with low HER2 expression. Tumor uptake was 5.2-fold significantly greater at 24h p.i. in MDA-MB-361 than MDA-MB-231 tumors (3.2 ± 0.1 %ID/g vs. 0.8 ± 0.1 %ID/g; P < 0.05). MicroSPECT/CT with [99mTc]His6-pertuzumab Fab imaged tumors in NOD/SCID mice that exhibited intermediate or high HER2 expression, but not tumors with low HER2. [99mTc]His6-pertuzumab Fab is promising for SPECT imaging of tumor HER2 expression.

Structural and Biochemical Studies Confirming the Mechanism of Action of Asciminib, an Agent Specifically Targeting the ABL Myristoyl Pocket (STAMP)

Tyrosine kinase inhibitors (TKIs) that inhibit the transphosphorylation activity of the BCR-ABL1 oncoprotein by binding the ATP-binding site of the catalytic domain of protein kinases are well established as being effective drugs for the treatments of chronic myeloid leukemia (CML). However, the off-target kinase activities of these non-specific TKIs are associated with adverse events that can limit their suitability for the treatment of patients and can negatively impact quality of life. Therefore, a new drug combining high efficacy with minimal side-effects could provide substantial therapeutic advantages. Asciminib is a new investigational agent that at concentrations &amp;lt;10 micromolar has very little effect (residual activities ≥66%) on the transphosphorylation activity of a large panel comprising of 335wild-typeprotein kinases (including ABL1 and ABL2). In this study (33PanQinase® Activity Assay, ProQinase GmbH, Freiburg, Germany) the radiometric kinase assay employed recombinant kinase constructs of the catalytic domains including the ATP-binding site. However, in isothermal calorimetry studies asciminib showed strong binding affinity (KD 0.5 nM) to a much larger construct ofwild-typeABL1 that contains the catalytic, SH2 and SH3 domains (residues 46-534, ABL46-534), with thermodynamic parameters (ΔH = -72.8 kJ/mol, ΔS = -65.3 J/mol/K, resulting in ΔG = -53.2 kJ/mol) indicating strong enthalpy-driven, entropically unfavorable binding. This binding translates to inhibition of tyrosine phosphorylation catalysed by the ABL64-515 construct with a mean IC50 value of 2.6 ± 0.8 nM (radiometric filter binding assay) and 0.5 ± 0.1 nM (fluorescence resonance energy transfer assay). This data shows that asciminib inhibits the kinase activity of ABL1 by an allosteric mechanism which does not involve direct interaction with the ATP-binding pocket. X-ray crystallographic studies of a ternary complex between asciminib, nilotinib and the ABL146-534 protein possessing Thr315Ile and Asp382Asn substitutions show that asciminib binds to ABL1 in a deep-pocket on the C-lobe of the kinase domain, referred to as the myristate (or myristoyl) pocket. Nuclear magnetic resonance studies confirm that asciminib can also form asciminib-ABL1-TKI ternary complexes with thewild-typeABL83-534 protein in solution. Native ABL1 kinase is post-translationally myristoylated at the N-terminal glycine residue and this myristate group plays an important role in autoregulating the kinase by binding to a pocket (myristate pocket) on the catalytic domain of the protein to induce the formation of an inactive conformation of the enzyme. This regulatory mechanism is lost in BCR-ABL1 since the N-terminal region is replaced in the fusion protein with a fragment of the BCR protein, thus rendering the ABL1 kinase constitutively active. The binding of asciminib in this pocket therefore mimics that of myristate, thus stabilizing the inactive state of the ABL1 kinase. Consistent with this binding mode to the ABL1 kinase, asciminib potently inhibits BCR-ABL1 driven proliferation of leukemia-derived cell lines, while having little effect on cells that do not express BCR-ABL1 (Figure 1). Thein vitrodata translates into anti-tumor activity in mouse models of CML where asciminib dose-dependently inhibited the growth of subcutaneous KCL22 cell xenografts, with 30 mg/kg administered twice-daily by oral gavage affording 92% tumor regression (Figure 2). Asciminib, the first-in-class STAMP (Specifically Targeting the ABL Myristoyl Pocket) inhibitor, has several important features as a potential treatment of CML. At physiologically achievable concentrations asciminib can overcome mutations on the ATP-binding site of BCR-ABL1 that impede the binding of TKIs which lead to drug resistance in patients with CML. Secondly, by not inhibiting kinases such as EGFR, KIT, CSF1R, PDGFR or the sSRC-family kinases that are associated with off-target activities of TKIs such as bosutinib, dasatinib, imatinib, nilotinib and ponatinib, asciminib is not expected to be associated with cross-intolerance. Thirdly, asciminib can bind to the ABL1 kinase domain together with ATP-competitive TKIs to form ternary complexes, such that appropriate drug combinations should greatly impede the emergence of drug resistant kinase mutations. Disclosures Schuld: Novartis Pharma AG:Current Employment, Current equity holder in publicly-traded company.Grzesiek:Novartis Pharma:Research Funding.Jahnke:Novartis:Current Employment, Current equity holder in publicly-traded company, Patents &amp; Royalties, Research Funding.Barys:Novartis Pharma AG:Current Employment.Cowan-Jacob:Novartis Pharma AG:Current Employment.Loo:Novartis Pharma AG:Current Employment.Wiget:Novartis Pharma AG:Current Employment.Manley:Novartis Pharma AG:Current Employment.

Selective Phosphodiesterase 1 Inhibitor BTTQ Reduces Blood Pressure in Spontaneously Hypertensive and Dahl Salt Sensitive Rats: Role of Peripheral Vasodilation.

Regulation of the peripheral vascular resistance via modulating the vessel diameter has been considered as a main determinant of the arterial blood pressure. Phosphodiesterase enzymes (PDE1-11) hydrolyse cyclic nucleotides, which are key players controlling the vessel diameter and, thus, peripheral resistance. Here, we have tested and reported the effects of a novel selective PDE1 inhibitor (BTTQ) on the cardiovascular system. Normal Sprague Dawley, spontaneously hypertensive (SHR), and Dahl salt-sensitive rats were used to test in vivo the efficacy of the compound. Phosphodiesterase radiometric enzyme assay revealed that BTTQ inhibited all three isoforms of PDE1 in nanomolar concentration, while micromolar concentrations were needed to induce effective inhibition for other PDEs. The myography study conducted on mesenteric arteries revealed a potent vasodilatory effect of the drug, which was confirmed in vivo by an increase in the blood flow in the rat ear arteriols reflected by the rise in the temperature. Furthermore, BTTQ proved a high efficacy in lowering the blood pressure about 9, 36, and 24 mmHg in normal Sprague Dawley, SHR and, Dahl salt-sensitive rats, respectively, compared to the vehicle-treated group. Moreover, additional blood pressure lowering of about 22 mmHg could be achieved when BTTQ was administered on top of ACE inhibitor lisinopril, a current standard of care in the treatment of hypertension. Therefore, PDE1 inhibition induced efficient vasodilation that was accompanied by a significant reduction of blood pressure in different hypertensive rat models. Administration of BTTQ was also associated with increased heart rate in both models of hypertension as well as in the normotensive rats. Thus, PDE1 appears to be an attractive therapeutic target for the treatment of resistant hypertension, while tachycardia needs to be addressed by further compound structural optimization.

Abstract 2915: Preclinical characterization of PRT543, a potent and selective inhibitor of protein arginine methyltransferase 5 (PRMT5), with broad antitumor activity in in vitro and in vivo models

Abstract Protein arginine methyltransferase 5 (PRMT5) is the major type II PRMT that catalyzes the formation of symmetrical dimethyl arginine (SDMA) on protein substrates and plays important roles in various cellular pathways including tumorigenesis. PRMT5 can methylate histones H3 and H4, thereby leading to repression of tumor suppressor genes at specific loci. PRMT5 also regulates gene expression on a global level by methylating and altering the function of transcription factors such as p53, E2F-1, NF-κB and others. Additionally, PRMT5 has been shown to interact with members of the spliceosome complex and regulate pre-mRNA processing of key target genes. In this study, we describe the in vitro and in vivo activity of PRT543, a novel, potent and selective inhibitor of PRMT5, in hematological and solid tumor models. In a scintillation proximity based radiometric assay, PRT543 inhibited the methyltransferase activity of the PRMT5/MEP50 complex with an IC50 of 10.8 nM. It was also highly selective against a panel of 36 other methyltransferases. PRT543 robustly inhibited cell proliferation in a panel of &amp;gt;50 cancer cell lines, representing both solid tumors and cancers of hematological origin, in a concentration-dependent manner with IC50 values ranging from 10 - 1000 nM. This anti-proliferative activity correlated with a reduction of symmetrically dimethylated SmD3 protein, a known PRMT5 substrate, demonstrating effective inhibition of PRMT5 enzymatic activity in cells. PRT543 demonstrated good oral bioavailability and favorable pharmacokinetic properties. Oral administration of this compound led to significant tumor growth inhibition in several subcutaneous mouse xenograft models including mantle cell lymphoma (Granta-519, Z-138), acute myeloid leukemia (MV4-11, SET2, HEL), small cell lung cancer (NCI-H1048) and bladder cancer (5637) at well-tolerated doses. This in vivo activity was accompanied by a corresponding decrease in symmetrically dimethylated SmD3 in tumor tissue collected from the treated animals. PRT543 was also tested in combination with other approved targeted therapies both in vitro and in vivo. Combining PRT543 with the BCL2 inhibitor, venetoclax, revealed a potent synergistic interaction in models of leukemia and lymphoma. Further ex vivo testing in genetically-defined primary patient tumor samples is ongoing. PRT543 is currently under evaluation in a Phase I clinical trial in patients with advanced solid tumors and hematological malignancies (NCT03886831). Citation Format: Neha Bhagwat, Yang Zhang, Hong Lin, Min Wang, Dave Rominger, Tom Emm, Divya Chugani-Mahtani, Dimitrios Angelis, Rupa Shetty, Raul Leal, William Gowen-MacDonald, Alexander Grego, Juan Luengo, Taghi Manshouri, Friederike Pastore, Ross L. Levine, Srdan Verstovsek, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. Preclinical characterization of PRT543, a potent and selective inhibitor of protein arginine methyltransferase 5 (PRMT5), with broad antitumor activity in in vitro and in vivo models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2915.

PKAN neurodegeneration and residual PANK2 activities in patient erythrocytes

ObjectivePantothenate kinase 2‐associated neurodegeneration (PKAN) is a rare neurodegenerative disease caused by mutations in the pantothenate kinase 2 (PANK2) gene. PKAN is associated with iron deposition in the basal ganglia and, occasionally, with the occurrence of misshaped erythrocytes (acanthocytes). The aim of this study was to assess residual activity of PANK2 in erythrocytes of PKAN patients and to correlate these data with the type of PANK2 mutations and the progression of neurodegeneration.MethodsResidual PANK2 activities in erythrocytes of 14 PKAN patients and 14 related carriers were assessed by a radiometric assay. Clinical data on neurodegeneration included the Barry–Albright Dystonia Scale (BAD‐Scale) besides further general patient features. A molecular visualization and analysis program was used to rationalize the influence of the PKAN causing mutations on a molecular level.ResultsErythrocytes of PKAN patients had markedly reduced or no PANK2 activity. However, patients with at least one allele of the c.1583C > T (T528M) or the c.833G > T (R278L) variant exhibited 12–56% of residual PANK2 activity. In line, molecular modeling indicated only minor effects on enzyme structure for these point mutations. On average, these patients with c.1583C > T or c.833G > T variant had lower BAD scores corresponding to lower symptom severity than patients with other PANK2 point mutations.InterpretationResidual erythrocyte PANK2 activity could be a predictor for the progression of neurodegeneration in PKAN patients. Erythrocytes are an interesting patient‐derived cell system with still underestimated diagnostic potential.

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.

The 2-/16α-Hydroxylated Estrogen Ratio-Breast Cancer Risk Hypothesis: Insufficient Evidence for its Support

During the past 25 years or so a number of studies have been carried out to address the hypothesis that the ratio of 2-hydroxyestrone (2-hydroxy-E1) to 16α-hydroxyestrone (16α-hydroxy-E1) is associated with breast cancer risk. The rationale for this hypothesis is based on data from studies that suggest a tumorigenic and genotoxic effect of 16α-hydroxy-E1 and a protective effect of 2-hydroxy-E1 regarding breast cancer risk. The adverse effect of 16α-hydroxy-E1 has been attributed to its potential to form covalent adducts with macromolecules.Initial studies used radiometric assays and enzyme immunoassays to test the hypothesis. However, concerns about the accuracy of these assays led to the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that is capable of measuring 5 unconjugated and 15 conjugated endogenous estrogens, which include 2- and 16-hydroxylated estrogen metabolites, in serum or urine. The conjugated estrogens are quantified following a deconjugation (hydrolysis) step to remove the sulfate and glucuronide groups.Epidemiologic studies have been using the LC-MS/MS assay to determine whether there is an association between breast cancer risk and the ratio of the sum of the concentrations of metabolites in the 2-hydroxylated estrogen pathway and in the 16-hydroxylated estrogen pathway. However, the validity of the pathways as biomarkers was not evaluated. The 16-hydroxylated estrogen pathway includes estriol, 16-epiestriol, 17-epiestriol and 16-ketoestradiol, in addition to 16α-hydroxy-E1. However, with the exception of 16α-hydroxy-E1, there is no evidence that any of the other estrogens in the pathway have tumorigenic or genotoxic properties, and they do not form covalent adducts with macromolecules.Another deficiency in the epidemiological studies pertains to the accuracy of estrogen metabolite measurements obtained after the hydrolysis step in the LC-MS/MS assays. No validation was performed to demonstrate that a constant efficiency of hydrolysis is found for all the different structural forms of sulfated and glucuronidated conjugates.Other deficiencies in the assays include the need for greater sensitivity so that the very low concentrations of unconjugated 2-hydroxy-E1, 2-hydroxy-E2, and 16α-hydroxy-E1 can be measured in serum. There is also a need to develop assays to measure intact forms of conjugated estrogens in both serum and urine, particularly the sulfates and glucuronides of 2-hydroxylated, 2-methoxylated, and 16α-hydroxylated E1 and E2. This will avoid inaccuracies that stem from hydrolysis procedures.Improvements in LC-MS/MS assay methodology to obtain accurate measurements of unconjugated and conjugated 2-hydroxylated, 2-methoxylated, and 16α-hydroxylated estrogen metabolites are needed. This should provide valuable data for testing the 2-/16α-hydroxylated estrogen-breast cancer risk hypothesis.

Endocannabinoid hydrolases are differentially distributed in human blood fractions and differentially influenced by thrombin

Plasma levels of the endocannabinoids anandamide and 2‐arachidonoylglycerol have been reported in a number of studies using both humans and laboratory animals, with typically much higher levels of 2‐arachidonoylglycerol than anandamide. In this investigation, we sought to characterize activities of the two primary endocannabinoid hydrolases, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) responsible for anandamide and 2‐arachidonoylglycerol hydrolysis, respectively, in different fractions from human blood.Following approval from the School of Life Sciences Ethics Committee, University of Nottingham, overnight fasting blood samples were taken in the morning from otherwise healthy volunteers. Blood fractions were isolated, including platelet‐rich plasma, packed erythrocytes (RBC), washed platelets, plasma and serum. Thrombin was employed to trigger clot formation, particularly with RBC and platelet‐rich plasma. Following the generation of a clot, the supernatant layer from a low speed centrifugation, equivalent to serum, was harvested and assayed for endocannabinoid hydrolase activity. The activities of FAAH and MAGL were quantified using both radiometric and ABPP assays.Of the blood fractions examined, MAGL activity was highest in platelets (92 % of the MAGL activity in whole blood), while FAAH activity was highest in RBC. Intriguingly, expressing the FAAH activity of various fractions relative to whole blood summated to 854 %, with RBC constituting 824 % of the whole blood activity. There were no differences between male and female volunteers in the levels and pattern of enzyme activities in the different blood fractions. Assessing enzyme activities from individual donors in these fractions on three visits separated by two week intervals showed no significant differences. The level of FAAH and MAGL activities was relatively stable in washed platelet and RBC samples after being stored in liquid nitrogen, while more than 50 % of their activities were lost after 3 days of storage at −80 °C.Interestingly, FAAH activity was low but detectable, while MAGL was essentially undetectable in serum. Thrombin addition to a washed platelet or RBC fraction caused clot formation within 5 min. FAAH activity in the supernatant fraction following thrombin exposure was much higher from RBC relative to platelets, in accordance with the activities in those fractions. In contrast, the activity of MAGL was significantly reduced in RBC and platelet fractions induced by thrombin compared to nonactivated erythrocytes and platelets, with negligible activities present in the releasate.In summary, we have identified a differential distribution of endocannabinoid hydrolases across different fractions of human blood, and identified the potential for FAAH, but not MAGL, release from RBC and platelets during thrombus formation.Support or Funding InformationFunded by the Saudi Cultural Bureau

Exploration of aurora B and cyclin-dependent kinase 4 inhibitors isolated from Scorzonera tortuosissima boiss. and their docking studies

Background: Flavonoids are components of the daily human diet (fruits and vegetables), and it has been shown to inhibit several kinase enzymes. Due to its kinase inhibitory activity, they are expected to be of great importance in the discovery of new anticancer drugs. Objectives: The objective was to study the cytotoxicity, kinase inhibitory activity, and docking of the isolated aglycones. Materials and Methods: Ultraviolet, high-performance liquid chromatography (LC), nuclear magnetic resonance, and LC-mass spectrometry were used for the identification of the isolated metabolites. Antiproliferative (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and radiometric protein kinase (PK) assays were used to measure the cytotoxicity and PK inhibitory effect of the isolated flavonoids. The docking study on both Aurora B and cyclin-dependent kinase 4 (CDK4)/CycD1 was performed by molecular operating environment (MOE). Results: Luteolin (1), quercetin (2), myricetin (3), apigenin-7-O -β-D-glucopyranoside (4), and kaempferol-7-O -β-D-glucopyranoside (5) were isolated from Scorzonera tortuosissima . Quercetin and myricetin exhibited the highest cytotoxicity against the Michigan Cancer Foundation-7 (MCF-7) (IC50: 5.56 and 7.14 μM, respectively) and against human hepatocellular carcinoma (HepG2) (IC50: 8.61 and 10.31 μM, respectively), while compounds luteolin and apigenin-7-O-β-D-glucopyranoside showed the least cytotoxicity compared to doxorubicin against to MCF-7 and HepG2 (IC50: 2.24 ± 0.85 and 1.82 ± 0.34 μM, respectively). The radiometric PK assay was applied for measurement of kinase inhibitory activity against Aurora B, CDK4/D1, cancer Osaka thyroid, IGF1-R, and FAK kinases, where the aglycone myricetin showed the highest inhibitory activity against Aurora B (IC50: 2.82 μM) and against CDK4/cyclin D1 (IC50: 3.16 μM), while the isolated glycosides 4 and 5 revealed the lowest activity. Docking of the most active compounds 1, 2, and 3 against Aurora B and CDK4/cyclin D1 confirmed its cytotoxic profile. Conclusion: The isolated flavonoids were firstly isolated from S. tortuosissima . The hypothetical mechanism of cytotoxic activity of 1, 2, and 3 on Aurora B and CDK4/cyclin D1 kinases was estimated by in silico study with these enzymes using MOE program.

Selective Phosphodiesterase 1 Inhibitor LY1 Reduces Blood Pressure in Spontaneously Hypertensive and Dahl Salt Sensitive Rats

Regulation of the peripheral vascular resistance via modulating the vessel diameter has been considered as a main determinant of the arterial blood pressure. Phosphodiesterase enzymes (PDE1‐11) hydrolyse cyclic nucleotides, which are key players controlling the vessel diameter and thus peripheral resistance, aiming at achieving and maintaining a homeostatic balance between their production and destruction. On the contrary, pathological shift in the balance to words the destruction leads to depletion of the cyclic nucleotides and dysregulation of the blood pressure. Here, we have tested and reported the effects of a novel selective PDE1 inhibitor (LY1) on the cardiovascular system. In vitro experiments have been conducted to determine the IC50 of LY1 using phosphodiesterase radiometric enzyme assay. We used rat mesenteric artery to conduct myogrphy study to determine the effect of the drug on the vessel diameter. Also, short as well as long term telemetric studies were done on normal Sprague Dawley (SD), spontaneously hypertensive (SHR), and Dahl salt sensitive (SS) rats to document the in vivo efficacy of the compound. LY1 inhibited all three isoforms of PDE1 in nanomolar concentration (1.74 nM, 2.44 nM and 1.20 nM for PDE1A, PDE1B and PDE1C, respectively), while micromolar concentrations were needed to induce effective inhibition for other PDEs. The myography study revealed a potent vasodilatory effect of the drug, which was confirmed in vivo by an increase in the blood flow in the rat ear arteriole reflected by the rise in the temperature. LY1 proved a high efficacy in lowering the blood pressure in two animal models for hypertension i.e. SHR and SS rats. 3 mg/kg of LY1 twice daily through oral gavage could efficiently reduce the blood pressure in both SHR and SS rats approximately 40 mmHg compared to the untreated control group. Therefore, LY1 induced an efficient vasodilation that was accompanied by a significant reduction of blood pressure in different hypertensive rat models. Thus, PDE1 appears to be a tempting therapeutic target for treatment of resistant hypertension.

Measurement of ATPase Activity of Valosin-containing Protein/p97.

Valosin-containing protein (VCP; also known as p97) is a type II ATPase regulating several cellular processes. Using proteomic techniques, we identified a chemical compound that binds to the D1 ATPase domain of VCP. The protocol described here was to study the effect of the compound on ATPase activity in vitro of purified VCP protein. ATPases are enzymes that hydrolyze ATP in a reaction resulting the release of an inorganic phosphate. This reaction can be measured using several methods, such as colorimetric, fluorescence, and radiometric assays, in addition to the bioluminescence assay mentioned here. Since the remaining ATP level after the reaction was detected using a luciferase assay, the luminescent signal indicates the ATPase activity inversely. This protocol is sensitive, rapid, and can be used for high-throughput screening assays to study the effect of compounds on ATPase function.

Characterization of a Unique Interrupted Adenylation Domain That Can Catalyze Three Reactions.

Interrupted adenylation (A) domains contain auxiliary domains within their structure and are a subject of growing interest in the field of nonribosomal peptide biosynthesis. They have been shown to possess intriguing functions and structure as well as promising engineering potential. Here, we present the characterization of an unprecedented type of interrupted A domain from the columbamides biosynthetic pathway, ColG(AMsMbA). This interrupted A domain contains two back-to-back methylation (M) domains within the same interruption site in the A domain, whereas previously, naturally occurring reported and characterized interrupted A domains harbored only one M domain. By a series of radiometric and mass spectrometry assays, we show that the first and second M domains site specifically methylate the side-chain oxygen and backbone nitrogen of l-Ser after the substrate is transferred onto a carrier thiolation domain, ColG(T). This is the first reported characterization of a dimethylating back-to-back interrupted A domain. The insights gained by this work lay the foundation for future combinatorial biosynthesis of site specifically methylated nonribosomal peptides.

Targeting Phosphatidylethanolamine with Fluorine-18 Labeled Small Molecule Probe for Apoptosis Imaging.

Externalization of phosphatidylethanolamine (PE) in dying cells makes the phospholipid an attractive target for apoptosis imaging. However, no ideal PE-targeted positron emission tomography (PET) radiotracer was developed. The goal of the study was to develop a novel PE-targeted radiopharmaceutical to imaging apoptosis. In this study, we have radiolabeled PE-binding polypeptide duramycin with fluorine-18 for PET imaging of apoptosis. Al[18F]F-NOTA-PEG3-duramycin was synthesized via chelation reaction of NOTA-PEG3-duramycin with Al[18F]F. PE-binding capacity of Al[18F]F-NOTA-PEG3-duramycin was determined in a competitive radiometric PE-binding assay. The pharmacokinetic profile was evaluated in Kunming mice. The apoptosis imaging capacity of Al[18F]F-NOTA-PEG3-duramycin was evaluated using in vitro cell uptake assay with camptothecin-treated Jurkat cells, along with in vivo PET imaging using erlotinib-treated nude mice. The total synthesis procedure lasted for 30min, with a decay-uncorrected radiochemical yield of 21.3 ± 2.6% (n = 10). Compared with the control cells, the binding of Al[18F]F-NOTA-PEG3-duramycin with camptothecin-induced apoptotic cells resulted in a tripling increase. A competitive radiometric PE-binding assay strongly confirmed the binding of Al[18F]F-NOTA-PEG3-duramycin to PE. The biodistribution study showed rapid blood clearance, prominent kidney retention, and low liver uptake. In the in vivo PET/CT imaging, Al[18F]F-NOTA-PEG3-duramycin demonstrated 2-fold increase in erlotinib-treated HCC827 tumors in nude mice. Considering the facile preparation and improved biological properties, Al[18F]F-NOTA-PEG3-duramycin seems to be a promising PET tracer candidate for imaging apoptosis in the monitoring of cancer treatment.

Chemical probes for protein arginine methyltransferases.

Protein arginine methyltransferases (PRMTs) catalyze the transfer of methyl groups to specific arginine residues of their substrates using S-adenosylmethionine as a methyl donor, contributing to regulation of many biological processes including transcription, and DNA damage repair. Dysregulation of PRMT expression is often associated with various diseases including cancers. Different methods have been used to characterize the activities of PRMTs and determine their kinetic parameters including mass spectrometry, radiometric, and antibody-based assays. Here, we present kinetic characterization of PRMTs using a radioactivity-based assay for better comparison along with previously reported values. We also report on full characterization of PRMT9 activity with SAP145 peptide as substrate. We further review the potent, selective and cell-active PRMT inhibitors discovered in recent years to provide a better understanding of available tools to investigate the roles these proteins play in health and disease.

Abstract LB-C12: Derazantinib (DZB): A dual FGFR/CSF1R-inhibitor active in PDX-models of urothelial cancer

Abstract Background: DZB is an oral small-molecule Fibroblast Growth Factor Receptor 1/2/3 inhibitor (FGFRi) with clinically relevant activity in FGFR2-fusion cholangiocarcinoma. Extensive kinase profiling identified Colony-Stimulating Factor 1 receptor (CSF1R) as an additional anti-cancer target for DZB. CSF1R plays a role in the maintenance of tumor-promoting M2-macrophages; inhibition facilitates repolarization to M1-type thus restoring tumor T cell activity. Screening of urothelial cancer (UC) models both in vitro and in vivo has provided information on potential response biomarkers additional to FGFR genetic aberrations. Methods: Kinase assays used a radiometric assay and anti-proliferative activity was assessed using crystal-violet (72h incubation). Bone-marrow derived mouse macrophages were CSF1 starved (12h), pre-incubated with DZB/BLZ945 (30/10m) and stimulated with 0.3 μM CSF1 (3m). CSF1R phosphorylation (pCSF1R) was analyzed by immunoblotting. Compound docking experiments used MOE software and public X-ray structures. DZB was tested at MTD (75 mg/kg, po, qd) in UC-CDX (8 mice/group) and -PDX (3 mice/group) models with FGFR-mutations and/or differing FGFR copy-number (CN)/RNA-seq expression levels. Efficacy and tolerability were quantified at the 3-week endpoint as a dT/C (treated/control). Results: Comparative kinase IC50s showed that DZB had 1:1 nM activity against FGFR1/2/3 and CSF1R, a potency not observed for other clinically relevant FGFRi’s. Structural analyses suggested a different size of the inhibitor binding-site of FGFR- and CSF1R-structures, with DZB efficiently occupying the smaller CSF1R kinase sub-pocket. Indeed, DZB reduced ligand-stimulated pCSF1R in mouse macrophages in a concentration-dependent manner, with a maximal effect similar to the selective CSF1R inhibitor BLZ945. Based on an in vitro anti-proliferative screen across 14 UC-lines, DZB had a mean GI50 of 1.7±0.2 μM (range 0.4-3.4 μM). The most sensitive lines were RT4 and RT112/84, both of which had FGFR3-TACC3 fusions, a known oncogenic-driver. In mice bearing s.c. RT4 tumors, DZB induced tumor-stasis (dT/C&amp;lt;0.1) and was well tolerated (dT/C&amp;gt;1.0) but no response was observed in the RT112/84 model suggesting that not only FGFR mutations contribute to DZB response. An unbiased UC-PDX screen indicated efficacy in 4/17 models (dT/C≤0.4; median=0.81) with DZB-response significantly positively-associated with high FGFR expression. The most sensitive tumor had high FGFR2 RNA-expression yet low CN. Data will be presented from confirmatory efficacy experiments and bioinformatic analyses. Conclusion: DZB is a potent FGFRi and CSF1R inhibitor. Screens in UC models indicate that DZB efficacy is driven by FGFR mutation and expression and, potentially, CSF1R modulation. A clinical trial is ongoing in UC patients (NCT04045613) to assess DZB monotherapy, and combination with the PD-L1 antibody atezolizumab. Citation Format: Paul McSheehy, Felix Bachmann, Nicole Forster-Gross, Marc Lecoultre, Mahmoud El Shemerly, Mila Roceri, Stefan Reinelt, Laurenz Kellenberger, Paul R Walker, Heidi Lane. Derazantinib (DZB): A dual FGFR/CSF1R-inhibitor active in PDX-models of urothelial cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-C12. doi:10.1158/1535-7163.TARG-19-LB-C12

Bioorthogonal Reporters for Detecting and Profiling Protein Acetylation and Acylation.

Protein acylation, exemplified by lysine acetylation, is a type of indispensable and widespread protein posttranslational modification in eukaryotes. Functional annotation of various lysine acetyltransferases (KATs) is critical to understanding their regulatory roles in abundant biological processes. Traditional radiometric and immunosorbent assays have found broad use in KAT study but have intrinsic limitations. Designing acyl-coenzyme A (CoA) reporter molecules bearing chemoselective chemical warhead groups as surrogates of the native cofactor acetyl-CoA for bioorthogonal labeling of KAT substrates has come into a technical innovation in recent years. This chemical biology platform equips molecular biologists with empowering tools in acyltransferase activity detection and substrate profiling. In the bioorthogonal labeling, protein substrates are first enzymatically modified with a functionalized acyl group. Subsequently, the chemical warhead on the acyl chain conjugates with either an imaging chromophore or an affinity handle or any other appropriate probes through an orthogonal chemical ligation. This bioorganic strategy reformats the chemically inert acetylation and acylation marks into a chemically maneuverable functionality and generates measurable signals without recourse to radioisotopes or antibodies. It offers ample opportunities for facile sensitive detection of KAT activity with temporal and spatial resolutions as well as allows for chemoproteomic profiling of protein acetylation pertaining to specific KATs of interest on the global scale. We reviewed here the past and current advances in bioorthogonal protein acylations and highlighted their wide-spectrum applications. We also discussed the design of other related acyl-CoA and CoA-based chemical probes and their deployment in illuminating protein acetylation and acylation biology.

Hormonal Regulation of Glucocorticoid Inactivation and Reactivation in αT3-1 and LβT2 Gonadotroph Cells.

The regulation of reproductive function by glucocorticoids occurs at all levels of the hypothalamo-pituitary-gonadal axis. Within the pituitary, glucocorticoids have been shown to directly alter gene expression in gonadotrophs, indicating that these cell types are sensitive to regulation by the glucocorticoid receptor. Whilst the major glucocorticoid metabolising enzymes, 11β-hydroxysteroid dehydrogenase (11βHSD; HSD11B1 and HSD11B2), have been described in human pituitary adenomas, the activity of these enzymes within different pituitary cell types has not been reported. Radiometric conversion assays were performed in αT3-1, LβT2 (gonadotrophs), AtT-20 (corticotrophs) and GH3 (somatolactotrophs) anterior pituitary cell lines, using tritiated cortisol, corticosterone, cortisone or 11-dehydrocorticosterone as substrates. The net oxidation of cortisol/corticosterone and net reduction of cortisone/11-dehydrocorticosterone were significantly higher in the two gonadotroph cells lines compared with the AtT-20 and GH3 cells after 4 h. Whilst these enzyme activities remained the same in αT3-1 and LβT2 cells over a 24 h period, there was a significant increase in glucocorticoid metabolism in both AtT-20 and GH3 cells over this same period, suggesting cell-type specific activity of the 11βHSD enzyme(s). Stimulation of both gonadotroph cell lines with either 100 nM GnRH or PACAP (known physiological regulators of gonadotrophs) resulted in significantly increased 11β-dehydrogenase (11βDH) and 11-ketosteroid reductase (11KSR) activities, over both 4 and 24 h. These data reveal that gonadotroph 11βHSD enzyme activity can act to regulate local glucocorticoid availability to mediate the influence of the HPA axis on gonadotroph function.