Bhavani Gopalakrishnan

Synthesis and Electrochemical and Biological Studies of Novel Coumarin–Chalcone Hybrid Compounds

A series of novel hydroxy-coumarin-chalcone hybrid compounds 2a-i has been synthesized by employing a simple and efficient methodology. An electrochemical characterization using cyclic voltammetry and ESR spectroscopy were carried out to characterize the oxidation mechanism for the target compounds. The antioxidant capacity and reactivity were determined by ORAC and ESR assays, respectively. Biological assays were assessed to evaluate the cytotoxicity and cytoprotection capacity against ROS/RNS on BAEC. The results revealed that all tested compounds present ORAC values that are much higher than other well-known antioxidant compounds such as quercetin and catechin. Compound 2e showed the highest ORAC value (14.1) and also presented a low oxidation potential, good scavenging capacity against hydroxyl radicals, low cytotoxicity, and high cytoprotective activity.

CD36-Dependent 7-Ketocholesterol Accumulation in Macrophages Mediates Progression of Atherosclerosis in Response to Chronic Air Pollution Exposure

Rationale: Air pollution exposure has been shown to potentiate plaque progression in humans and animals. Our previous studies have suggested a role for oxidized lipids in mediating adverse vascular effect of air pollution. However, the types of oxidized lipids formed in response to air pollutants and how this occurs and their relevance to atherosclerosis are not fully understood. Objective: To investigate the mechanisms by which particulate matter <2.5 &mgr;m (PM2.5) induces progression of atherosclerosis. Methods and Results: Atherosclerosis-prone ApoE−/− or LDLR−/− mice were exposed to filtered air or concentrated ambient PM2.5 using a versatile aerosol concentrator enrichment system for 6 months. PM2.5 increased 7-ketocholesterol (7-KCh), an oxidatively modified form of cholesterol, in plasma intermediate density lipoprotein/low-density lipoprotein fraction and in aortic plaque concomitant with progression of atherosclerosis and increased CD36 expression in plaque macrophages from PM2.5-exposed mice. Macrophages isolated from PM2.5-exposed mice displayed increased uptake of oxidized lipids without alterations in their efflux capacity. Consistent with these finding, CD36-positive macrophages displayed a heightened capacity for oxidized lipid uptake. Deficiency of CD36 on hematopoietic cells diminished the effect of air pollution on 7-KCh accumulation, foam cell formation, and atherosclerosis. Conclusions: Our results suggest a potential role for CD36-mediated abnormal accumulations of oxidized lipids, such as 7-KCh, in air pollution–induced atherosclerosis progression.

Inhibition of ROS-induced apoptosis in endothelial cells by nitrone spin traps via induction of phase II enzymes and suppression of mitochondria-dependent pro-apoptotic signaling

Oxidative stress is the main etiological factor behind the pathogenesis of various diseases including inflammation, cancer, cardiovascular and neurodegenerative disorders. Due to the spin trapping abilities and various pharmacological properties of nitrones, their application as therapeutic agent has been gaining attention. Though the antioxidant properties of the nitrones are well known, the mechanism by which they modulate the cellular defense machinery against oxidative stress is not well investigated and requires further elucidation. Here, we have investigated the mechanisms of cytoprotection of the nitrone spin traps against oxidative stress in bovine aortic endothelial cells (BAEC). Cytoprotective properties of both the cyclic nitrone 5,5-dimethyl-pyrroline N-oxide (DMPO) and linear nitrone α-phenyl N-tert-butyl nitrone (PBN) against H₂O₂-induced cytotoxicity were investigated. Preincubation of BAEC with PBN or DMPO resulted in the inhibition of H₂O₂-mediated cytotoxicity and apoptosis. Nitrone-treatment resulted in the induction and restoration of phase II antioxidant enzymes via nuclear translocation of NF-E2-related factor 2 (Nrf-2) in oxidatively-challenged cells. Furthermore, the nitrones were found to inhibit the mitochondrial depolarization and subsequent activation of caspase-3 induced by H₂O₂. Significant down-regulation of the pro-apoptotic proteins p53 and Bax, and up-regulation of the anti-apoptotic proteins Bcl-2 and p-Bad were observed when the cells were preincubated with the nitrones prior to H₂O₂-treatment. It was also observed that Nrf-2 silencing completely abolished the protective effects of nitrones. Hence, these findings suggest that nitrones confer protection to the endothelial cells against oxidative stress by modulating phase II antioxidant enzymes and subsequently inhibiting mitochondria-dependent apoptotic cascade.

Decitabine enhances anti-CD33 monoclonal antibody BI 836858–mediated natural killer ADCC against AML blasts

Acute myeloid leukemia (AML) is the most common type of acute leukemia, affecting older individuals at a median age of 67 years. Resistance to intensive induction chemotherapy is the major cause of death in elderly AML; hence, novel treatment strategies are warranted. CD33-directed antibody-drug conjugates (gemtuzumab ozogamicin) have been shown to improve overall survival, validating CD33 as a target for antibody-based therapy of AML. Here, we report the in vitro efficacy of BI 836858, a fully human, Fc-engineered, anti-CD33 antibody using AML cell lines and primary AML blasts as targets. BI 836858-opsonized AML cells significantly induced both autologous and allogeneic natural killer (NK)-cell degranulation and NK-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). In vitro treatment of AML blasts with decitabine (DAC) or 5-azacytidine, 2 hypomethylating agents that show efficacy in older patients, did not compromise BI 836858-induced NK-cell-mediated ADCC. Evaluation of BI 836858-mediated ADCC in serial marrow AML aspirates in patients who received a 10-day course of DAC (pre-DAC, days 4, 11, and 28 post-DAC) revealed significantly higher ADCC in samples at day 28 post-DAC when compared with pre-DAC treatment. Analysis of ligands to activating receptors (NKG2D) showed significantly increased NKG2D ligand [NKG2DL] expression in day 28 post-DAC samples compared with pre-DAC samples; when NKG2DL receptor was blocked using antibodies, BI 836858-mediated ADCC was significantly decreased, suggesting that DAC enhances AML blast susceptibility to BI 836858 by upregulating NKG2DL. These data provide a rationale for combination therapy of Fc-engineered antibodies such as BI 836858 with azanucleosides in elderly patients with AML.

Detection of nitric oxide and superoxide radical anion by electron paramagnetic resonance spectroscopy from cells using spin traps.

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation, and ultimately to oxidative stress leading to cell injury or death. Superoxide radical anion (O2•-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2•- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2•- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2•- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2•- in a process called uncoupling, which is believed to be caused by oxidation of heme or the co-factor, tetrahydrobiopterin (BH4). There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production. The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO, the phosphoryl-substituted DEPMPO, and the ester-substituted, EMPO and BMPO, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2•- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct.

Reversal of SIN-1-induced eNOS dysfunction by the spin trap, DMPO, in bovine aortic endothelial cells via eNOS phosphorylation.

Nitric oxide (NO) derived from eNOS is mostly responsible for the maintenance of vascular homeostasis and its decreased bioavailability is characteristic of reactive oxygen species (ROS)‐induced endothelial dysfunction (ED). Because 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO), a commonly used spin trap, can control intracellular nitroso‐redox balance by scavenging ROS and donating NO, it was employed as a cardioprotective agent against ED but the mechanism of its protection is still not clear. This study elucidated the mechanism of protection by DMPO against SIN‐1‐induced oxidative injury to bovine aortic endothelial cells (BAEC).

Polo-like kinase inhibitor volasertib marginally enhances the efficacy of the novel Fc-engineered anti-CD33 antibody BI 836858 in acute myeloid leukemia

Acute myeloid leukemia (AML) is the second most common type of leukemia in adults. Incidence of AML increases with age with a peak incidence at 67 years. Patients older than 60 years have an unfavorable prognosis due to resistance to conventional chemotherapy. Volasertib (BI 6727) is a cell-cycle regulator targeting polo-like kinase which has been evaluated in clinical trials in AML. We evaluated effects of volasertib in primary patient samples and NK cells. At equivalent doses, volasertib is cytotoxic to AML blasts but largely spares healthy NK cells. We then evaluated the effect of volasertib treatment in combination with BI 836858 on primary AML blast samples using antibody-dependent cellular cytotoxicity (ADCC) assays. Volasertib treatment of NK cells did not impair NK function as evidenced by comparable levels of BI 836858 mediated ADCC in both volasertib-treated and control-treated NK cells. In summary, volasertib is cytotoxic to AML blasts while sparing NK cell viability and function. Higher BI 836858 mediated ADCC was observed in patient samples pretreated with volasertib. These findings provide a strong rationale to test combination of BI 836858 and volasertib in AML.

The interleukin-3 receptor CD123 targeted SL-401 mediates potent cytotoxic activity against CD34 CD123 cells from acute myeloid leukemia / myelodysplastic syndrome patients and healthy donors.

Diseases with clonal hematopoiesis such as myelodysplastic syndrome and acute myeloid leukemia have high rates of relapse. Only a small subset of acute myeloid leukemia patients are cured with chemotherapy alone. Relapse in these diseases occurs at least in part due to the failure to eradicate leukemic stem cells or hematopoietic stem cells in myelodysplastic syndrome. CD123, the alpha chain of the interleukin-3 receptor heterodimer, is expressed on the majority of leukemic stem cells and myelodysplastic syndrome hematopoietic stem cells and in 80% of acute myeloid leukemia. Here, we report indiscriminate killing of CD123+ normal and acute myeloid leukemia / myelodysplastic syndrome cells by SL-401, a diphtheria toxin interleukin-3 fusion protein. SL-401 induced cytotoxicity of CD123+ primary cells/blasts from acute myeloid leukemia and myelodysplastic syndrome patients but not CD123− lymphoid cells. Importantly, SL-401 was highly active even in cells expressing low levels of CD123, with minimal effect on modulation of the CD123 target in acute myeloid leukemia. SL-401 significantly prolonged survival of leukemic mice in acute myeloid leukemia patient-derived xenograft mouse models. In addition to primary samples, studies on normal cord blood and healthy marrow show that SL-401 has activity against normal hematopoietic progenitors. These findings indicate potential use of SL-401 as a “bridge-to-transplant” before allogeneic hematopoietic cell transplantation in acute myeloid leukemia / myelodysplastic syndrome patients.

Abstract 1691: SL-401 activity against AML blasts correlates with CD123 levels and does not down-regulate CD123 expression

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Despite the progress, the current therapies for acute myeloid leukemia (AML) are not curative partly due to failure to eradicate the leukemic stem cells (LSCs) that repopulate the leukemia. CD123 is the alpha-chain of interleukin-3 receptor (IL-3R) that is expressed on AML and AML LSCs, while it is undetectable in normal hematopoietic stem cells. We report here time and dose dependent cytotoxic activity of SL-401(Stemline Therapeutics, Inc), a diphtheria toxin interleukin-3 fusion protein which is designed to selectively bind, internalize and induce apoptosis of CD123+ primary AML cells isolated from patients. AML blasts were obtained from leukapheresis of patients and cultured in RPMI1640 with 20% fetal bovine serum and antibiotics and supplemented with growth factors GM-CSF and SCF (10ng/ml) overnight before treatment with increasing concentrations of SL-401. CD123 expression of AML blasts was analyzed using multicolor flow cytometry before and 24 hours after treatment with SL-401. The viability of the cells before and after treatment with increasing concentrations of SL-401 was measured by annexinV-FITC and propidium iodide staining. SL-401 induced dose-dependent (SL-401: 10, 100, 1000ng/ml) cytotoxicity in primary AML in nanomolar range (trend p = 0.0002 at 48hr), which correlated with surface expression levels of CD123. AML CD34+CD33+ blasts showed high CD123 expression levels and were susceptible to cytotoxicity with SL-401 (P<0.0001,1000ng/ml at 48hr). CD34−CD33+ committed leukemic cells were also CD123+ and responded to SL-401. To determine if SL-401 modulated CD123 levels after exposure, AML blasts pre-treated with SL-401 for 24hr were analyzed by flow cytometry for receptor expression. Importantly, SL-401 treatment after 24 hours did not down-regulate CD123 expression on primary AML blasts indicating a lack of an escape mechanism for malignant cells. These results provide evidence that time- and dose-dependent activity of SL-401 against CD123+ AML blasts correlates with CD123 expression. Moreover, treatment does not down-regulate IL-3R expression. Clinical studies of SL-401 in patients with relapsed/refractory AML with evaluation of CD123 expression and blastic plasmacytoid dendritic cell neoplasm (BPDCN) are ongoing. Citation Format: Rajeswaran Mani, Bhavani Gopalakrishnan, Xiaokui Mo, Chris Brooks, William Blum, Gerard Lozanski, Sumithira Vasu, Natarajan Muthusamy. SL-401 activity against AML blasts correlates with CD123 levels and does not down-regulate CD123 expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1691. doi:10.1158/1538-7445.AM2015-1691