Formation and Signaling Actions of Electrophilic Fatty Acids Apply basic chemical principles in the understanding of cell signaling and the creation of new therapeutic strategies Bruce A Freeman, Ph.D. Irwin Fridovich Professor and Chair Dept. of Pharmacology and Chemical Biology University of Pittsburgh School of Medicine Nitric Oxide and Nitrite Produce Nitrogen Dioxide (.NO2) nitrates biomolecules forms electrophilic products NO2 R .NO + O2 .- ONOO.NO CO2 NO2 + MPO NO2 .NO - H+ + O2 NO2- H 2 O2 .NO 2 .NO HNO2 .NO ONO. .OH ONO. CO3.- ONOOCO2 H 2 O2 + MPO - H+ ONOO- R 2 2 Science, Nature, PNAS, J Clin Invest, J Biol Chem Mammalian NOx Cycle NO3Oral Bacteria NO2Peroxidases Acidification Deoxy-Hb XOR Nitrite-NO reaction NO2. NO. Fatty acid nitration Protein nitration Activate sGC Inhibit cytochrome oxidase Acidification SNO Protein modification NO-like bioactivity Lundberg, Weitzberg NO2-Fatty Acid Generation (Humans) 1 gm/d CLA, 8 oz/d beet root juice x 3d Normal Gastric Conditions Human Plasma NO2-CLA Basal 1.510 3 +Diet 8.010 35 3.010 Intensity, cps Intensity, cps 4.010 45 1.0 Human Urine NO2-CLA 6.010 2.010 5 4.010 3 3 1.010 5 2.010 3 0 Basal +Diet Basal 1.010 3 5.010 2 0 40 42 44 Time (min) 46 48 37 38 39 Time (min) 40 NO2-Fatty Acid Generation (Humans) [15N]O2- (nitrite, 20 mg) [15N]O3- (nitrate, 1 gm) 18:2, 3 gm @ 0, 48 hr 15NOx 15NOx 6 time points 0 pause, wash-out 24 6 time points 48 72 hr Measurements Blood pressure, heart rate Nox species – urine, blood PBMC gene expression Platelet function Mitochondrial function FDA IND, IRB PRO11120134 – Freeman, Gladwin Plasma NO2-cLA Levels Nitrite Nitrate [14N] [15N] A Key Reactivity of NO2-FA and Keto-FA Rapid, Reversible Michael Addition Reaction H RS- R' H N+ O- H O R' RS N+ H O - O- H+ H R' RS H N+ H O- O- H R' N RS H + O- O- Cysteine Reaction Rate Species K (M-1 s-1) NO 0 H2O2 ~1 15d-PGJ2 <1 NO2-oleate 190 NO2-linoleate 240 Keto-DPA, DHA ~100 Electrophilic Fatty Acids Regulate Gene Expression Human Vascular Endothelium 10-NO2-octadec-9-enoic acid 330 genes 93 genes ~425 genes total 43 genes Nrf2-regulated Inflammation Oxidant defense Metabolism DNA, protein repair Conjugation, excretion PNAS, J Biol Chem NO2 R R O Nitro-FA Keto-FA R R • Generated endogenously by both enzymatic and random redox reactions during digestion, metabolism and inflammation • Contribute to the beneficial actions of dietary unsaturated fatty acids and NO3- + NO2- (Mediterranean/Japanese diets) • Signal via Michael addition of susceptible proteins: pleitropic transcription factors, pro-inflammatory enzymes • Established metabolic pathways regulate electrophilic lipid levels COI Disclosure: Complexa, Inc. Drug Development Strategy Administer homologs of electrophilic lipid signaling mediators to increase endogenous concentrations and promote beneficial metabolic and inflammatory signaling responses Comparable Natural, Near-Natural Electrophiles * Dimethylfumarate (BG-12, Tecfidera - Biogen-Idec) * • • • • Oral drug for multiple sclerosis, FDA approval - 2012 MOA – Nrf2 activation, rapidly metabolized Not detectable in plasma after administration Recommended dosing = 240 mg, 3 x day * • • • Sulforaphane (broccoli sprouts, florets) Orally bioavailable, anti-cancer, anti-inflammatory MOA – Nrf2 activation Long track record of safety Study #1 Pre-clinical Disease Proof-of-Concept CXA-10 Protects Against Ischemic Kidney Injury (Treatment) Nitro-oleic acid protects the mouse kidney from ischemia and reperfusion injury Am J Physiol (Renal) 295:942-949, 2008 (T Yang, Univ Utah School of Medicine) CXA-10 administered 50 min and q 6H x 24 hours after ischemia CXA-10 Administration: Resulted in significant renal protection: o Decreased plasma urea and creatinine, preservation of urine output o Decreased plasma and organ indices of inflammation o Evidenced in histopathology o Decreased leucocyte recruitment No Rx No Protection + CXA-10 Protection Kidney, 24 hr post-IR Study #2 Confirmatory Kidney IR PK/PD Study (Prophylaxis) (UAB O’Brien Kidney Center, Anupam Agarwal, MD) I/R = Bilateral renal artery blocking with small clamps, 35min • Rat I/R n=6; sham n=3; CXA-10 12.5 mg/kg • Dosed 1 hr prior to ischemic insult • >45% reduction in serum creatinine * p<0.01 Basal 24 48 Time (hr) 72 Study 4 Evaluation of CXA-10 in the DOCA Salt Model of Chronic Renal Injury • CXA-10 administered to mice via chronic 4-week oral dosing, exerted significant renal-protective effects: • Significantly reduced albuminuria, fibrotic gene expression, urinary nephrin and MCP-1 excretion • Inhibited gene expression of pro-inflammatory cytokines, ECM and profibrotic factor, PAI-1 • Reduction in cardiac hypertrophy (not seen with control) • Positive impact on cholesterol metabolism • MOA - anti-inflammatory, antioxidant and anti-fibrotic effects. Control Untreated CXA-10 Histologically, untreated mice developed tubular damage and interstitial fibrosis. CXA-10 improved overall tubular appearance Representative picrosirius red stained sections (x200) Electrophilic Fatty Acids Anti-inflammatory Mediators, Drug Candidates • Naturally occurring in humans, fish, plants, insects • Levels increase during metabolic and inflammatory stress, also from diets rich in polyunsaturated fatty acids and NO2-/NO3• Mediate salutary pleitropic metabolic and inflammatory signaling • Link between metabolic/inflammatory status and adaptive, tissueprotective gene expression and enzymatic activities OA-NO2 Limits Angiotensin II-induced Hypertension p< 0.05 140 Systolic Blood Pressure (mmHg) 120 100 80 60 Mechanisms 40 20 0 Baseline OA OA-NO2 OA OA-NO2 Up • eNOS expression • HO-1 expression Angiotensin II Systolic BP decrease (mmHg) 0 1.25 2.5 5 10 20 (mg/kg) 0 -5 Down • Epoxide hydrolase • Nox expression • Nox catalytic activity * -10 -15 -20 OA OA-NO2 * * Circ Res, PNAS Remaining Challenge – ID of total pool of lipid electrophiles Metabolic Fate of Fatty Acid Nitroalkenes Protein adduction Esterification Complex lipids b-Oxidation NO2 Glucuronidation R R Excretion Signaling (PTM) Keap1/Nrf2 PPARg, HSF-1 NFkB p65, PTPs Mito: Site II-70 kDa XOR, sEH GSH reaction MDRP export Nitroalkene Reduction Prostaglandin reductase (PtGR1)