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LJ001 is a lipophilic thiazolidine derivative that inhibits the entry of numerous enveloped viruses at non-cytotoxic concentrations (IC50≤0.5 µM), and was posited to exploit the physiological difference between static viral membranes and biogenic cellular membranes. We now report on the molecular mechanism that results in LJ001's specific inhibition of virus-cell fusion.The antiviral activity of LJ001 was light-dependent, required the presence of molecular oxygen, and was reversed by singlet oxygen (1O2) quenchers, qualifying LJ001 as a type II photosensitizer. Unsaturated phospholipids were the main target modified by LJ001-generated 1O2. Hydroxylated fatty acid species were detected in model and viral membranes treated with LJ001, but not its inactive molecular analog, LJ025. 1O2-mediated allylic hydroxylation of unsaturated phospholipids leads to a trans-isomerization of the double bond and concurrent formation of a hydroxyl group in the middle of the hydrophobic lipid bilayer. LJ001-induced 1O2-mediated lipid oxidation negatively impacts on the biophysical properties of viral membranes (membrane curvature and fluidity) critical for productive virus-cell membrane fusion. LJ001 did not mediate any apparent damage on biogenic cellular membranes, likely due to multiple endogenous cytoprotection mechanisms against phospholipid hydroperoxides.Based on our understanding of LJ001's mechanism of action, we designed a new class of membrane-intercalating photosensitizers to overcome LJ001's limitations for use as an in vivo antiviral agent. Structure activity relationship (SAR) studies led to a novel class of compounds (oxazolidine-2,4-dithiones) with (1) 100-fold improved in vitro potency (IC50<10 nM), (2) red-shifted absorption spectra (for better tissue penetration), (3) increased quantum yield (efficiency of 1O2 generation), and (4) 10–100-fold improved bioavailability. Candidate compounds in our new series moderately but significantly (p≤0.01) delayed the time to death in a murine lethal challenge model of Rift Valley Fever Virus (RVFV). The viral membrane may be a viable target for broad-spectrum antivirals that target virus-cell fusion.

High density lipoprotein (HDL), its main protein, apolipoprotein A-I (apoA-I), and mimetics of apoA-I have been shown in a number of laboratories to reduce infl ammation in animal models of disease ( 1-5 ). The apoA-I mimetic peptide 4F showed great promise in a variety of mouse models of disease ( 5 ) leading to a phase I/II study in humans with a high risk of cardiovascular disease ( 6 ). In this study the 4F peptide synthesized from all D-amino acids (D-4F) was administered orally at doses that ranged from 0.43 to 7.14 mg/kg. The resulting plasma peptide levels were low [maximal plasma concentration (Cmax) 15.9 ± 6.5 ng/ml]. Despite these very low plasma levels, doses of 4.3 and 7.14 mg/kg signifi cantly improved the HDL infl ammatory index (HII), which is a measure of the ability of a test HDL to inhibit LDL-induced monocyte chemoattractant protein-1 (MCP-1) production by cultured human artery wall cells; doses of 0.43 and 1.43 mg/kg were not effective ( 6 ). A second clinical trial focused on achieving high plasma peptide levels using low doses (0.042-1.43 mg/kg) of the 4F peptide synthesized from all L-amino acids (L-4F) delivered by intravenous (IV) or subcutaneous (SQ) administration ( 7 ). Very high plasma levels were in fact achieved (e.g., Cmax 3,255 ± 630 ng/ml in the IV study), but there was no improvement in HII ( 7 ). To resolve this paradox, new studies were conducted in mice that led to the surprising discovery that the major site of action for the peptide may be in the intestine, even when the peptide is administered SQ ( 8 ). Moreover, the dose administered, not the plasma level, was the major Abstract Transgenic tomato plants were constructed with an empty vector (EV) or a vector expressing an apoA-I mimetic peptide , 6F. EV or 6F tomatoes were harvested, lyophilized, ground into powder, added to Western diet (WD) at 2.2% by weight, and fed to LDL receptor-null (LDLR ؊ / ؊ ) mice at 45 mg/kg/day 6F. After 13 weeks, the percent of the aorta with lesions was 4.1 ± 4%, 3.3 ± 2.4%, and 1.9 ± 1.4% for WD, WD + EV, and WD + 6F, respectively (WD + 6F vs. WD, P = 0.0134; WD + 6F vs. WD + EV, P = 0.0386; WD + EV vs. WD, not signifi cant). While body weight did not differ, plasma serum amyloid A (SAA), total cholesterol, triglycerides, and lysophosphatidic acid (LPA) levels were less in WD + 6F mice; P < 0.0295. HDL cholesterol and paroxonase-1 activity (PON) were higher in WD + 6F mice ( P = 0.0055 and P = 0.0254, respectively), but not in WD + EV mice. Plasma SAA, total cholesterol, triglycerides, LPA, and 15-hydroxyeicosatetraenoic acid (HETE) levels positively correlated with lesions ( P < 0.0001); HDL cholesterol and PON were inversely correlated ( P < 0.0001). After feeding WD + 6F: i ) intact 6F was detected in small intestine (but not in plasma); ii ) small intestine LPA was decreased compared with WD + EV ( P < 0.0469); and iii ) small intestine LPA 18:2 positively correlated with the percent of the aorta with lesions ( P < 0.0179). These data suggest that 6F acts in the small intestine and pr...

. Transgenic 6F tomatoes act on the small intestine to prevent systemic infl ammation and dyslipidemia caused by Western diet and intestinally derived lysophosphatidic acid. J. Lipid Res. 2013. 54: 3403-3418. Supplementary key words 6F peptide • apolipoprotein A-I mimetic peptides • atherosclerosis • lysophosphatidic acid • genetically engineered tomato plantsMimetics of apolipoprotein A-I (apoA-I) containing only 18 amino acids showed promise in animal models of disease ( 1, 2 ), and improved HDL function in humans when given orally at high doses despite achieving low plasma peptide levels ( 3 ). However, when high plasma levels were achieved with low doses of peptide given intravenously or by subcutaneous (SQ) injection, no improvement in HDL function was seen ( 4 ). Studies in mice surprisingly suggested that the major site of action for these peptides is in the Abstract We recently reported that levels of unsaturated lysophosphatidic acid (LPA) in the small intestine significantly correlated with the extent of aortic atherosclerosis in LDL receptor-null (LDLR ؊ / ؊ ) mice fed a Western diet (WD). Here we demonstrate that WD increases unsaturated (but not saturated) LPA levels in the small intestine of LDLR ؊ / ؊ mice and causes changes in small intestine gene expression. Confi rmation of microarray analysis by quantitative RT-PCR showed that adding transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F) to WD prevented many WD-mediated small intestine changes in gene expression. If instead of feeding WD, unsaturated LPA was added to chow and fed to the mice: i ) levels of LPA in the small intestine were similar to those induced by feeding WD; ii ) gene expression changes in the small intestine mimicked WD-mediated changes; and iii ) changes in plasma serum amyloid A, total cholesterol, triglycerides, HDLcholesterol levels, and the fast-performance liquid chromatography lipoprotein profi le mimicked WD-mediated changes. Adding Tg6F (but not control tomatoes) to LPA-supplemented chow prevented the LPA-induced changes. We conclude that: i ) WD-mediated systemic infl ammation and dyslipidemia may be in part due to WD-induced increases in small intestine LPA levels; and ii ) Tg6F reduces WD-mediated systemic infl ammation and dyslipidemia by preventing WDinduced increases in LPA levels in the small intestine. -Navab, M., G. Hough, G. M. Buga, F. Su, A. C. Wagner, D. Meriwether, A. Chattopadhyay, F. Gao, V. Grijalva, J. S. Danciger, B. J. Van Lenten, E. Org, A. J. Lusis, C. Pan, G. M. the Laubisch, Castera, and M. K. Grey Abbreviations: CXCL1, chemokine (CXC motif) ligand 1; EV, empty vector tomatoes (transgenic tomatoes constructed with the vector pBI121 containing the GUS gene; 6F, This work was supported in part bycontaining all L -amino acids; FPLC, fast-performance liquid chromatography; LDLR Ϫ / Ϫ , LDL receptor-null; LPA, lysophosphatidic acid; PA, phosphatidic acid; PPAR, peroxisome proliferatoractivated receptor; RT-qPCR, quantitative RT-PCR ; SAA, serum amyloid A; SQ, subcutaneous; Tg6F, t...

multiple comparisons/tests, we adjusted P values in accord with the appropriate correction. Statistical significance was set at P less than 0.05 with respect to final (adjusted) P values. Variation is reported as SEM. Additional information about Materials and Methods can be found in the Supplemental Materials.