204 New Silicon Containing Ibuprofen Derivatives as a New Source of Safer Anti-Inflammatory and Secondary Anti-Oxidant Compounds David J. Pérez1, Uzma I. Zakai2, Irene Díaz-Reval1, Teresa Sumaya Martínez 3, Fernando Obledo-Benicio1, and Ángel Andrés Ramos-Organillo1

1Universidad de Colima, Mexico, 2University of Wisconsin-Madison, USA, 3Universidad Autónoma de Nayarit, Mexico The linking between inflammation and the increment in the releasing of free radicals is well known thus the search for a drug which is able to exert both: anti-inflammatory and anti-oxidant activity has become an important field of investigation. Ibuprofen (IBU) inhibits the enzymatic activity of the Cyclooxigenase (COX), which catalyses the conversion of arachidonic acid into prostaglandins, IBU is considered a biological anti-oxidant because of its capacity to inhibit the activity of a pro-oxidant enzyme (as COX has been classified), its properties to scavenge and prevent formation of certain free radical species have been reported, in addition it has shown capability of inhibiting the inflammation related nuclear factor (NF- ) that has been linked to the development of inflammation, chronic inflammatory diseases and cancer. Anti-oxidant activity is not only related to the scavenging of free radicals but also to the prevention of its formation, transition metals such as Fe(II) catalyze the formation of the free radicals, thus compounds that are able to act as Fe(II) chelators are considered secondary anti-oxidants. Silicon insertion into known drug scaffolds is a suitable strategy to increment or potentiate biological activity. We have designed

of anti-inflammatory and anti-oxidant compounds. Esters 4a and 4d displayed significant in vivo anti-inflammatory activity in the carrageenan rat paw edema model (inhibited up to 87% and 78% of edema formation) also resulted better Fe(II) chelating compounds (up to 60%) and more importantly they are endowed with a significant reduced gastro-toxic effect compared to that of IBU. The SCIDs amides 10a and 10b have incremented their potency to inhibit NF activation (IC50=45 and IC50=92) with respect to that of IBU (IC50=241) also are more effective free radical scavengers in the ABTS + test and better Fe(II) chelators; for instance 10a scavenged 45% of ABTS + radical and chelated 60% of Fe(II), similar values were achieved by 10b.

4a: n=1, R 1=R 2=Me, R 3=Ph 4d: n=3, R 1=R 2=R 3=Me

10a: n=1, R 1=R 2=Me, R 3=Ph 10b: n=1, R 1=R 2=Me, R 3=PhOMe

n n

Figure 1. Designed SCID´s

205 Using Protein Engineering to Develop a Bacterial-Human Heme Scavenging Protein for the Treatment of Sickle Cell Disease Ruslan Rafikov1, Diana Gutsaeva1, Alvin Head1, and Steve M Black1

1Georgia Regents University, USA Sickle Cell Disease (SCD) is a fatal hemolytic disorder resulting in multiple organ failure, poor quality of life and shortened life expectancy. Rupture of Red Blood Cells (RBC) in SCD releases intracellular components including hemoglobin (Hb) into the blood stream. Extracellular hemoglobin exhibits a highly toxic nature by

scavenging nitric oxide (NO) reducing its bioavailability. Hemoglobin and its degradation products free heme and iron perpetuate oxidative stress further decreasing NO availability. In addition, these degradation products promote microvascular occlusions and chronic inflammation through Toll-Like Receptor 4 activation on endothelium. Currently there is no viable therapy designed to stop the toxic effects of free Hb. Thus, the overall goal of this study was to apply a protein engineering approach, using a bacterial heme scavenging system, to eliminate the toxic effects of extracellular hemoglobin (Hb). Our hypothesis was that we could use the bacterial NEAr heme Transporter domain NEAT from S. aureus to extract the heme moiety from extracellular Hb molecules and, thus, eliminate extracellular Hb toxicity and preventing its degradation into free heme and iron. Our data indicate that a chimeric construct (NEAT-HP) where the bacterial NEAT domain was fused to the human haptoglobin (HP), which has a natural higher affinity to Hb, markedly reduced NO scavenging in the bloodstream, diminished free hemoglobin mediated peroxidase activity in plasma and attenuated white blood cells activation in a mouse model of SCD. We also found that NEAT-HP attenuated organ damage and inflammatory cell infiltration. Thus, we have developed an artificially engineered bacterial-human protein that targets the toxic effects of extracellular hemoglobin and prevents its further degradation into harmful free heme and iron. We propose that this protein could be effective not only in SCD, but also in other hemolytic disorders and hemorrhagic stroke.

206 Novel Small Molecule Inhibitors of Cytochrome B5 Reductase 3 Were Identified by Structure Guided Chemical Modifications of Propylthiouracil Md. Mizanur Rahaman1, Fabio Reinders1, Courtney Elaine Watkins1,Anh Nguyen1, Megan P. Miller1, David Koes1, Mark T. Gladwin1, Carlos J. Camacho1, and Adam C. Straub1

1University of Pittsburgh, USA NADH cytochrome B5 reductase 3 (CytB5R3) has recently gained

was demonstrated that the oxidation state of -or-

(NO) signaling through Specifically, it was shown that genetic loss of CytB5R3 protein enhanced NO signaling, suggesting that it could serve as a potential therapeutic. However, the lack of specific CytB5R3 small molecule inhibitors is a major drawback for studying its function in physiology and disease. Based on a previous report, it was found that propylthiouracil (PTU), a drug designed to treat hyperthyroidism, acts as a partial inhibitor of CytB5R3 by presumably blocking the NADH binding site. However, the IC50 is

of this high IC50, we used a computational, rational based drug design strategy to screen commercially available libraries for modified PTU derivatives. To measure efficacy, recombinant CytB5R3 was generated and subjected to a ferricyanide reduction assay in the presence of 19 different PTU derivatives and NADH. Our data demonstrate for the first time three new small molecule inhibitors: ZINC 05626394, ZINC39395747 and ZINC0562626. These irreversible inhibitors significantly improved efficacy of the inhibition of CytB5R3 activity by more than 20-fold. Importantly, these data provide the first steps toward uncovering potential therapies to increase NO bioavailability in order to treat or reverse cardiovascular diseases such as hypertension.

SFRBM 2014 S89

doi: 10.1016/j.freeradbiomed.2014.10.310

doi: 10.1016/j.freeradbiomed.2014.10.311