SNARE based peptide linking as an efficient strategy to retarget botulinum neurotoxin’s enzymatic domain to specific neurons using diverse neuropeptides as targeting domains

Many disease states are caused by miss-regulated neurotransmission. A small fraction of these diseases can currently be treated with botulinum neurotoxin type A (BoNT/A). BoNT/A is composed of three functional domains – the light chain (Lc) is a zinc metalloprotease that cleaves intracellular SNAP25 which inhibits exocytosis, the translocation domain (Td) that enables the export of the light chain from the endosome to the cytosol, and the receptor binding domain (Rbd) that binds to extracellular gangliosides and synaptic vesicle glycoproteins while awaiting internalisation [1]. Current endeavours are directed towards retargeting Bont/A as well as finding safer methods of preparation and administration. Recently, our laboratory has developed a SNARE based linking strategy to recombine non-toxic BoNT/A fragments into a functional protein by simple mixing [2]. This SNARE based linking strategy permits the stepwise assembly of highly stable macromolecular complexes [2,3]. Onto these three SNARE peptides, diverse functional groups can be attached to the N- or C- terminus by direct synthesis and/or by genetic design. To enhance the therapeutic potential of BoNT/A, this method enables the rapid assembly of a large array of neuropeptide-SNAREs to their cognate LcTd-SNARE. A substitution of the Rbd with various neuropeptide sequences permits a large throughput combinatorial assay of LcTd to target new cell types. In this study, we have fused LcTd to 3 different Synaptobrevin sequences; we also use a small protein staple, and 26 different Syntaxin-neuropeptide fusions (permitting the assay of 78 new chimeric LcTd proteins with modified targeting domains). These neuropeptides such as, but not exclusively, somatostatin (SS), vasoactive intestinal peptide, substance P, opioid peptide analogues, Gonadotropin releasing hormone, and Arginine Vasopressin, which natively function through G protein coupled receptors (GPCR) can undergo agonist induced internalisation upon activation. The ability of our new constructs, once endocytosed, to inhibit neurotransmitter release was tested on different neuronal cell lines with immunoblotting of endogenous SNAP25. This cleavage by Lc reflects the ultimate readout of the enzyme’s efficacy, which incorporates the cell surface binding, internalisation kinetics, translocation of the Lc to the cytosol, and finally the enzymatic cleavage of SNAP25. Internalisation of the toxins can also be monitored with confocal microscopy and FACS by the substitution of the staple peptide for a fluorescent homologue. Figure 1 shows that whole boNT/A (upper left) can have its Rbd replaced with SNARE peptides, which will fuse together to form highly stable chimeric proteins with an altered targeting domain (right). Figure 1 also shows 4 different neuropeptide synthaxins in complex, resolved on SDS-PAGE gel (bottom left lanes 1-4, boiled 1’-4’). Fig. 1. SNARE-linked botulinum neurotoxins used for the retargeting of Bont/A. 29

Effects of Mitochondrial-Targeted Antioxidants on Real-Time Blood Nitric Oxide and Hydrogen Peroxide Release in Acute Hyperglycemia Rats

Acute hyperglycemia in non-diabetic subjects can impair vascularendothelial function, causing decreased endothelium-derived nitric oxide (NO) release and increased reactive oxygen species (ROS), such assuperoxide and hydrogen peroxide (H2O2). Hyperglycemia may induce mitochondrial dysfunction leading to ROS production and exacerbation of vascular endothelial dysfunction. We investigated whether mitochondrial-targeted antioxidants mitigate acute hyperglycemia-induced oxidative stress and reduced blood NO. To test this hypothesis, blood NO or H2O2 levels were measured simultaneously using NO or H2O2 microsensors (100 µm) which were placed into the femoral veins of anesthesized male Sprague-Dawley rats. Acute hyperglycemia was induced by infusion 20% D-glucose intravenously with or without mitochondria-targeted antioxidants (mitoquinone: mitoQ, MW=1714 g/mol, 2.3 mg/Kg; SS-31: (D-Arg)-Dmt-Lys-Phe-Amide, MW=640g/mol, 2.7 mg/Kg) for 3 hours. We found that acute hyperglycemia (200 mg/dL) significantly increased blood H2O2 by 3.0±0.5 M (n=7) and reduced blood NO by 68.0±13.5 nM (n=9) compared to the saline group at end of infusion (both p\u3c0.05). MitoQ significantly attenuated hyperglycemia– induced H2O2 levels by 2.5±0.2 M (n=7) and increased blood NO levels by 59.3±9.7 nM (n=5) (both p\u3c0.05 compared to hyperglycemia). Similarly, SS-31 significantly reduced hyperglycemia-induced blood H2O2 level by 4.0±0.6 M (n=5) and enhanced blood NO levels by 52.8±7.7 nM (n=6) at end of infusion (both p\u3c0.05 compared to hyperglycemia). In summary, acute hyperglycemia induces mitochondria-derived ROS which in turn contribute to vascular endothelial dysfunction. Therefore, mitochondria-targeted antioxidants are useful to attenuate acute hyperglycemia-induced vascular endothelial dysfunction and oxidative stress

Effects of NADPH oxidase inhibitor apocynin on real-time blood hydrogen peroxide release in femoral artery/vein ischemia and reperfusion

Background: Vascular endothelial dysfunction can initiate oxidative stress during ischemiaJreperfusion (IIR). Endothelial dysfunction is characterized by an increase in blood hydrogen peroxide (H20 ]) and a decrease in endothelial-derived nitric oxide (NO) bioavailability. Previous studies using Go 6983, a broad-spectrum protein kinase C inhibitor that can inhibit NADPH oxidase activity, has attenuated blood H20 ] levels during femoralliR in vivo. This study examines the effects of apocynin, a direct NADPH oxidase inhibitor, on real-time blood H20 ] levels in femoral I1R in vivo. H20 ] microsensors (100 Mm) were inserted into both femoral veins in anesthetized rats

Myristoylated protein kinase C beta II peptide inhibitor exerts dose-dependent inhibition of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced leukocyte superoxide release

Protein kinase C (PKC) phosphorylation of leukocyte NADPH oxidase is essential to generatesuperoxide (SO) release. Inhibition of leukocyte SO release attenuates inflammation mediated vascular injury. However, the role of PKC isoforms mediating this response has not been fully elucidated. We hypothesize that PKC beta II (βII) isoform positively regulates leukocyte NADPH oxidase, and that a cell-permeable (myr)-PKC βII peptide inhibitor (N-myr-SLNPEWNET) would dose-dependently attenuate fMLP induced leukocyte SO release. fMLP is a leukocyte chemoattractant cell membrane receptor agonist. We isolated leukocytes by peritoneal lavage from male Sprague-Dawley rats using standard methods. fMLP (1 M)-induced leukocyte SO release was measured for 120 sec spectrophotometrically by reduction of ferricytochrome c in the presence/absence of myr-PKC βII peptide inhibitor (0.2 to 20 M) in 5 x 106 leukocytes. After each assay, cell viability was determined by 0.3% trypan blue exclusion. fMLP-induced leukocyte SO release increased peak absorbance to 0.18±0.03 in controls (n=20). This response was dose-dependently inhibited by myr-PKC βII peptide inhibitor at 0.17±0.05 (0.2 M; n=9), 0.14±0.05 (0.5 M; n=11), 0.1±0.05 (1 M; n=9), 0.05±0.03 (5 M; n=9), 0.04±0.03 (10 M; n=8) and 0.05±0.03 (20 µM; n=7) and was significantly attenuated in the 5 to 20 M range compared to controls (p\u3c0.05). Moreover, cell viability was \u3e 94±1% in all study groups. These results suggest that myr-PKC βII peptide inhibitor dose-dependently inhibits fMLP-induced leukocyte SO release in the 0.2 to 5 M dose-range and these effects are attributed to inhibition of PKC βII isoform

The Effects of Protein Kinase C Beta II Peptide Modulation on Superoxide Release in Rat Polymorphonuclear Leukocytes

Phorbol 12-myristate 13-acetate (PMA; a diacylglycerol mimetic) is known to augment polymorphonuclear leukocyte (PMN) superoxide (SO) release via protein kinase C (PKC) activation. However, the role of PKC beta II (βII) mediating this response is not known. It’s known that myristic acid (myr-) conjugation facilitates intracellular delivery of the cargo sequence, and that putative PKCβII activator and inhibitor peptides work by augmenting or attenuating PKCβII translocation to cell membrane substrates (e.g. NOX-2). Therefore, we hypothesize that myr- conjugated PKCβII peptide-activator (N-myr-SVEIWD; myr-PKCβ+) would increase PMA-induced rat PMN SO release, whereas, myr-PKCβII peptide-inhibitor (N-myr-SLNPEWNET; myr-PKCβ-) would attenuate this response compared to non-drug treated controls. Rat PMNs (5x106) were incubated for 15min at 370C in the presence/absence of myr-PKCβ+/- (20 μM) or SO dismutase (SOD;10μg/mL; n=8) as positive control. PMA (100nM) induced PMN SO release was measured spectrophotometrically at 550nm via reduction of ferricytochrome c for 390 sec. PMN SO release increased absorbance to 0.39±0.04 in non-drug treated controls (n=28), and 0.49±0.05 in myr-PKCβ+(n=16). This response was significantly increased from 180 seconds to 240 seconds (p\u3c0.05). By contrast, myr-PKCβ- (0.26±0.03; n=14) significantly attenuated PMA-induced SO release compared to non-drug controls and myr-PKCβ+ (p\u3c0.05). SOD-treated samples showed \u3e90% reduction of PMA-induced SO release and was significantly different from all groups (p\u3c0.01). Cell viability ranged between 94± to 98±2% in all groups as determined by 0.2% trypan blue exclusion. Preliminary results suggest that myr-PKCβ- significantly attenuates PMA-induced SO release, whereas myr-PKCβ+ significantly augments PMA-induced SO release, albeit transiently. Additional dose response and western blot experiments are planned with myr-PKCβ+/- in PMA-induced PMN SO release assays. This research was supported by the Department of Bio-Medical Sciences and the Division of Research at PCOM and by Young Therapeutics, LLC

Mitoquinone (mitoQ) Exerts Antioxidant Effects Independent of Mitochondrial Targeted Effects in Phorbol-12-myristate-13-acetate (PMA) or N-formyl-L-methiony-L-leucyl-L-phenylalanine (fMLP) Stimulated Polymorphonuclear Leukocyte (PMN) Superoxide (SO) Release

MitoQ is a mitochondrial-targeted coenzyme Q antioxidant analog that dose-dependently restored cardiac function and reduced infarct size in isolated perfused rat hearts subjected to ischemia reperfusion (I/R). Moreover, mitoQ also dose-dependently attenuated PMA stimulated PMN superoxide (SO) release at the same concentration (10uM) as the cardioprotective dose. NADPH oxidase is the principle source of PMN SO release. We speculate that mitoQ may exert antioxidant effects independent of the mitochondria. Therefore, we hypothesized that inhibition of mitoQ on PMN-SO release will be similar as other coenzyme Q analogs: coenzyme Q1 and decylubiquinone without affecting cell viability. SO release was measured spectrophotometrically from isolated rat PMNs measured by the reduction of ferricytochrome c and were stimulated with 100nM PMA. The absorbance was measured at 550 nm up to 360sec. Positive control samples were given SO dismutase (SOD; 10ug/ml) which inhibited PMA induced SO release by \u3e90%. MitoQ significantly inhibited SO release by 56 + 3% (10uM, n=10 ,

Protein Kinase C Beta II Peptide Inhibitor Elicits Robust Effects on Attenuating Myocardial Ischemia/Reperfusion Injury

Reperfusion injury contributes to myocardial tissue damage following a heart attack partly due to the generation of reactive oxygen species (ROS) upon cardio-angioplasty. Protein kinase C beta II (PKCβII) inhibition during reperfusion with peptide inhibitor (N-myr-SLNPEWNET; PKCβII-) decreases ROS release and leukocyte infiltration in rat hind-limb and myocardial ischemia/reperfusion (I/R) studies, respectively. However, the role of activating PKCβII during reperfusion has not been previously determined. In this study, we hypothesize that myristoylated (myr)-PKCβII- will decrease infarct size and improve post-reperfused cardiac function compared to untreated controls, whereas PKCβII peptide activator (N-myr-SVEIWD; myr-PKCβII+) will show no improvement compared to control. Myristoylation of PKCβII peptides facilitate their entry into the cell in order to affect PKCβII activity by either augmenting or attenuating its translocation to cell membrane proteins, such as NOX-2. Isolated perfused rat hearts were subjected to global I(30min)/R(50min) and infused with myr-PKCβII+ (20μM; n=9), myr-PKCβII- (20µM; n=8), or plasma (control; n=9) at reperfusion. Hearts were frozen (-20oC), sectioned and stained using 1% triphenyltetrazolium chloride to differentiate necrotic tissue. The measurement of Left ventricular (LV) cardiac function was determined using a pressure transducer and infarct size was calculated as percent dead tissue vs. total heart tissue weight. Myr-PKCβII- significantly improved LV end-diastolic pressure 37±7 mmHg compared to control (58±5; p\u3c0.01) and myr-PKCβII+ (58±4; p\u3c0.01). Myr-PKCβII- significantly reduced infarct size to 14±3% compared to control (26±5%; p\u3c0.01), while myr-PKCβII+ (25±3%) showed no difference. The data indicate that myr-PKCβII- may be a putative treatment to reduce myocardial reperfusion injury when given to heart attack patients during cardio-angioplasty. Future studies are planned to determine infarct size by Image J analysis

Structural Basis for Species Selectivity in the HIV-1 gp120-CD4 Interaction: Restoring Affinity to gp120 in Murine CD4 Mimetic Peptides

The first step of HIV-1 infection involves interaction between the viral glycoprotein gp120 and the human cellular receptor CD4. Inhibition of the gp120-CD4 interaction represents an attractive strategy to block HIV-1 infection. In an attempt to explore the known lack of affinity of murine CD4 to gp120, we have investigated peptides presenting the putative gp120-binding site of murine CD4 (mCD4). Molecular modeling indicates that mCD4 protein cannot bind gp120 due to steric clashes, while the larger conformational flexibility of mCD4 peptides allows an interaction. This finding is confirmed by experimental binding assays, which also evidenced specificity of the peptide-gp120 interaction. Molecular dynamics simulations indicate that the mCD4-peptide stably interacts with gp120 via an intermolecular β-sheet, while an important salt-bridge formed by a C-terminal lysine is lost. Fixation of the C-terminus by introducing a disulfide bridge between the N- and C-termini of the peptide significantly enhanced the affinity to gp120

Effects of NOX-1 Inhibition on Real-Time Blood Nitric Oxide and Hydrogen Peroxide in Acute Hyperglycemia

Hyperglycemia has been associated with vascular endothelial dysfunction in part by a reduction in nitric oxide (NO) production and increased oxidative stress (e.g., increased superoxide (SO) and hydrogen peroxide (H2O2). Endothelial-derived NO can be significantly reduced by increased SO/H2O2 in part by the activation of NADPH oxidase during hyperglycemia. Of the 7 NADPH oxidase isoforms, NADPH oxidase isoform 1 (NOX1) is mainly expressed in the vasculature and may play a major role in hyperglycemia induced oxidative stress and vascular endothelial dysfunction. This hypothesis was tested by measuring blood NO and H2O2 levels in real time via NO and H2O2 microsensors inserted into femoral veins of rats. Hyperglycemia (e.g., 200 mg/dl) was maintained by an i.v. infusion of 30% glucose solution for 3 hours with or without a selective NOX1 inhibitor, ML171. Hyperglycemia for 3 hours resulted in significantly higher blood H2O2 levels (3.06±0.4 μM, n=9) compared to the saline infused control (P2O2 levels by 1.86±0.61 μM (