Peptides International

Louisville, KY, United States

Peptides International

Louisville, KY, United States

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Koshy S.,Baylor College of Medicine | Wu D.,Baylor College of Medicine | Hu X.,Baylor College of Medicine | Tajhya R.B.,Baylor College of Medicine | And 6 more authors.
PLoS ONE | Year: 2013

Natural killer (NK) cells are large granular lymphocytes that participate in both innate and adaptive immune responses against tumors and pathogens. They are also involved in other conditions, including organ rejection, graft-versus-host disease, recurrent spontaneous abortions, and autoimmune diseases such as multiple sclerosis. We demonstrate that human NK cells express the potassium channels Kv1.3 and KCa3.1. Expression of these channels does not vary with expression levels of maturation markers but varies between adherent and non-adherent NK cell subpopulations. Upon activation by mitogens or tumor cells, adherent NK (A-NK) cells preferentially up-regulate KCa3.1 and non-adherent (NA-NK) cells preferentially up-regulate Kv1.3. Consistent with this different phenotype, A-NK and NA-NK do not display the same sensitivity to the selective KCa3.1 blockers TRAM-34 and NS6180 and to the selective Kv1.3 blockers ShK-186 and PAP-1 in functional assays. Kv1.3 block inhibits the proliferation and degranulation of NA-NK cells with minimal effects on A-NK cells. In contrast, blocking KCa3.1 increases the degranulation and cytotoxicity of A-NK cells, but not of NA-NK cells. TRAM-34, however, does not affect their ability to form conjugates with target tumor cells, to migrate, or to express chemokine receptors. TRAM-34 and NS6180 also increase the proliferation of both A-NK and NA-NK cells. This results in a TRAM-34-induced increased ability of A-NK cells to reduce in vivo tumor growth. Taken together, our results suggest that targeting KCa3.1 on NK cells with selective blockers may be beneficial in cancer immunotherapy. © 2013 Koshy et al.


Beeton C.,Baylor College of Medicine | Pennington M.W.,Peptides International | Norton R.S.,Monash Institute of Pharmaceutical Sciences
Inflammation and Allergy - Drug Targets | Year: 2011

CCR7- effector memory T (T EM) lymphocytes are involved in autoimmune diseases such as multiple sclerosis, type 1 diabetes mellitus and rheumatoid arthritis. These cells express Kv1.3 potassium channels that play a major role in their activation. Blocking these channels preferentially inhibits the activation of CCR7- TEM cells, with little or no effects on CCR7 + naïve and central memory T cells. Blockers of lymphocyte Kv1.3 channels therefore show considerable potential as therapeutics for autoimmune diseases. ShK, a 35-residue polypeptide isolated from the Caribbean sea anemone Stichodactyla helianthus, blocks Kv1.3 channels at picomolar concentrations. Although ShK was effective in treating rats with delayed type hypersensitivity and a model of multiple sclerosis, it lacks selectivity for Kv1.3 channels over closely-related Kv1 channels. Extensive mutagenesis studies combined with elucidation of the structure of ShK led to models of ShK docked with the channel. This knowledge was valuable in the development of new ShK analogs with improved selectivity and increasing stability, which have proven efficacious in preventing and/or treating animal models of delayed type hypersensitivity, type 1 diabetes, rheumatoid arthritis, and multiple sclerosis without inducing generalized immunosuppression. They are currently undergoing further evaluation as potential immunomodulators for the treatment of autoimmune diseases. © 2011 Bentham Science Publishers.


Pennington M.W.,Peptides International | Harunur Rashid M.,University of Sydney | Tajhya R.B.,Baylor College of Medicine | Beeton C.,Baylor College of Medicine | And 2 more authors.
FEBS Letters | Year: 2012

ShK, a 35-residue peptide from a sea anemone, is a potent blocker of potassium channels. Here we describe a new ShK analogue with an additional C-terminus Lys residue and amide. ShK-K-amide is a potent blocker of Kv1.3 and, in contrast to ShK and ShK-amide, is selective for Kv1.3. To understand this selectivity, we created complexes of ShK-K-amide with Kv1.3 and Kv1.1 using docking and molecular dynamics simulations, then performed umbrella sampling simulations to construct the potential of mean force of the ligand and calculate the corresponding binding free energy for the most stable configuration. The results agree well with experimental data. © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.


Peng Y.,Sun Yat Sen University | Lu K.,Sun Yat Sen University | Li Z.,Sun Yat Sen University | Zhao Y.,Tongji University | And 8 more authors.
Neuro-Oncology | Year: 2014

Background. Tumors affecting the head, neck, and brain account for significant morbidity and mortality. The curative efficacy of radiotherapy for these tumors is well established, but radiation carries a significant risk of neurologic injury. So far, neuroprotective therapies for radiation-induced brain injury are still limited. In this study we demonstrate that Stichodactyla helianthus (ShK)-170, a specific inhibitor of the voltage-gated potassium (Kv)1.3 channel, protected mice from radiation-induced brain injury. Methods. Mice were treated with ShK-170 for 3 days immediately after brain irradiation. Radiation-induced brain injury was assessed by MRI scans and a Morris water maze. Pathophysiological change of the brain was measured by immunofluorescence. Gene and protein expressions of Kv1.3 and inflammatory factors were measured by quantitative real-time PCR, reverse transcription PCR, ELISA assay, and western blot analyses. Kv currents were recorded in the whole-cell configuration of the patch-clamp technique. Results. Radiation increased Kv1.3 mRNA and protein expression in microglia. Genetic silencing of Kv1.3 by specific short interference RNAs or pharmacological blockade with ShK-170 suppressed radiation-induced production of the proinflammatory factors interleukin-6, cyclooxygenase-2, and tumor necrosis factor-α by microglia. ShK-170 also inhibited neurotoxicity mediated by radiation-activated microglia and promoted neurogenesis by increasing the proliferation of neural progenitor cells. Conclusions. The therapeutic effect of ShK-170 is mediated by suppression of microglial activation and microglia-mediated neurotoxicity and enhanced neurorestoration by promoting proliferation of neural progenitor cells. © 2013 © The Author(s) 2013.


Chang S.C.,Monash Institute of Pharmaceutical Sciences | Huq R.,Baylor College of Medicine | Chhabra S.,Monash Institute of Pharmaceutical Sciences | Beeton C.,Baylor College of Medicine | And 3 more authors.
FEBS Journal | Year: 2015

The voltage-gated potassium channel Kv1.3 is an important target for the treatment of autoimmune diseases and asthma. Blockade of Kv1.3 by the sea anemone peptide K+-channel toxin from Stichodactyla helianthus (ShK) inhibits the proliferation of effector memory T lymphocytes and ameliorates autoimmune diseases in animal models. However, the lack of selectivity of ShK for Kv1.3 over the Kv1.1 subtype has driven a search for Kv1.3-selective analogues. In the present study, we describe N-terminally extended analogues of ShK that contain a negatively-charged Glu, designed to mimic the phosphonate adduct in earlier Kv1.3-selective analogues, and consist entirely of common protein amino acids. Molecular dynamics simulations indicated that a Trp residue at position [-3] of the tetrapeptide extension could form stable interactions with Pro377 of Kv1.3 and best discriminates between Kv1.3 and Kv1.1. This led to the development of ShK with an N-terminal Glu-Trp-Ser-Ser extension ([EWSS]ShK), which inhibits Kv1.3 with an IC50 of 34 pm and is 158-fold selective for Kv1.3 over Kv1.1. In addition, [EWSS]ShK is more than 2900-fold more selective for Kv1.3 over Kv1.2 and KCa3.1 channels. As a highly Kv1.3-selective analogue of ShK based entirely on protein amino acids, which can be produced by recombinant expression, this peptide is a valuable addition to the complement of therapeutic candidates for the treatment of autoimmune diseases. The voltage-gated potassium channel Kv1.3 is an important target for the treatment of autoimmune diseases and asthma. Amongst the most potent inhibitors of Kv1.3 is the sea anemone toxin ShK - a peptide that blocks Kv1.3, inhibiting the proliferation of effector memory T lymphocytes. In this study, Chang and colleagues used computational techniques to design N-terminally extended analogues of ShK based on their structure-activity relationships with Kv1.3. One analogue, [EWSS]ShK, inhibits Kv1.3 with an IC50 of 34 pm, is highly selective for Kv1.3 over Kv1.1, Kv1.2 and KCa3.1 channels, and is therefore a potential candidate for the treatment of autoimmune diseases. © 2015 FEBS.


Rashid M.H.,University of Sydney | Heinzelmann G.,University of Sydney | Huq R.,Baylor College of Medicine | Tajhya R.B.,Baylor College of Medicine | And 6 more authors.
PLoS ONE | Year: 2013

The voltage-gated potassium channel Kv1.3 is a well-established target for treatment of autoimmune diseases. ShK peptide from a sea anemone is one of the most potent blockers of Kv1.3 but its application as a therapeutic agent for autoimmune diseases is limited by its lack of selectivity against other Kv channels, in particular Kv1.1. Accurate models of Kv1.x-ShK complexes suggest that specific charge mutations on ShK could considerably enhance its specificity for Kv1.3. Here we evaluate the K18A mutation on ShK, and calculate the change in binding free energy associated with this mutation using the path-independent free energy perturbation and thermodynamic integration methods, with a novel implementation that avoids convergence problems. To check the accuracy of the results, the binding free energy differences were also determined from path-dependent potential of mean force calculations. The two methods yield consistent results for the K18A mutation in ShK and predict a 2 kcal/mol gain in Kv1.3/Kv1.1 selectivity free energy relative to wild-type peptide. Functional assays confirm the predicted selectivity gain for ShK[K18A] and suggest that it will be a valuable lead in the development of therapeutics for autoimmune diseases. © 2013 Rashid et al.


Jin L.,Monash Institute of Pharmaceutical Sciences | Boyd B.J.,Monash Institute of Pharmaceutical Sciences | White P.J.,Monash Institute of Pharmaceutical Sciences | Pennington M.W.,Peptides International | And 2 more authors.
Journal of Controlled Release | Year: 2015

Stichodactyla helianthus neurotoxin (ShK) is an immunomodulatory peptide currently under development for the treatment of autoimmune diseases, including multiple sclerosis and rheumatoid arthritis by parenteral administration. To overcome the low patient compliance of conventional self-injections, we have investigated the potential of the buccal mucosa as an alternative delivery route for ShK both in vitro and in vivo. After application of fluorescent 5-Fam-ShK to untreated porcine buccal mucosa, there was no detectable peptide in the receptor chamber using an in vitro Ussing chamber model. However, the addition of the surfactants sodium taurodeoxycholate hydrate or cetrimide, and formulation of ShK in a chitosan mucoadhesive gel, led to 0.05-0.13% and 1.1% of the applied dose, respectively, appearing in the receptor chamber over 5 h. Moreover, confocal microscopic studies demonstrated significantly enhanced buccal mucosal retention of the peptide (measured by mucosal fluorescence associated with 5-Fam-ShK) when enhancement strategies were employed. Administration of 5-Fam-ShK to mice (10 mg/kg in a mucoadhesive chitosan-based gel (3%, w/v) with or without cetrimide (5%, w/w)) resulted in average plasma concentrations of 2.6-16.2 nM between 2 and 6 h, which were substantially higher than the pM concentrations required for therapeutic activity. This study demonstrated that the buccal mucosa is a promising administration route for the systemic delivery of ShK for the treatment of autoimmune diseases. © 2014 Elsevier B.V.


Rashid M.H.,Monash Institute of Pharmaceutical Sciences | Rashid M.H.,University of Sydney | Huq R.,Baylor College of Medicine | Tanner M.R.,Baylor College of Medicine | And 9 more authors.
Scientific Reports | Year: 2014

HsTX1 toxin, from the scorpion Heterometrus spinnifer, is a 34-residue, C-terminally amidated peptide cross-linked by four disulfide bridges. Here we describe new HsTX1 analogues with an Ala, Phe, Val or Abu substitution at position 14. Complexes of HsTX1 with the voltage-gated potassium channels Kv1.3 and Kv1.1 were created using docking and molecular dynamics simulations, then umbrella sampling simulations were performed to construct the potential of mean force (PMF) of the ligand and calculate the corresponding binding free energy for the most stable configuration. The PMF method predicted that the R14A mutation in HsTX1 would yield a > 2â €...kcal/mol gain for the Kv1.3/Kv1.1 selectivity free energy relative to the wild-type peptide. Functional assays confirmed the predicted selectivity gain for HsTX1[R14A] and HsTX1[R14Abu], with an affinity for Kv1.3 in the low picomolar range and a selectivity of more than 2,000-fold for Kv1.3 over Kv1.1. This remarkable potency and selectivity for Kv1.3, which is significantly up-regulated in activated effector memory cells in humans, suggest that these analogues represent valuable leads in the development of therapeutics for autoimmune diseases.


PubMed | University of New South Wales, Peptides International, Monash Institute of Pharmaceutical Sciences and Purdue University
Type: Journal Article | Journal: Journal of pharmaceutical sciences | Year: 2016

HsTX1[R14A] is a potent and selective Kv1.3 channel blocker peptide with the potential to treat autoimmune diseases. Given the typically poor oral bioavailability of peptides, we evaluated pulmonary administration of HsTX1[R14A] in rats as an alternative route for systemic delivery. Plasma concentrations of HsTX1[R14A] were measured by liquid chromatography coupled with tandem mass spectrometry in rats receiving intratracheal administration of HsTX1[R14A] in solution (1-4 mg/kg) or a mannitol-based powder (1 mg/kg) and compared with plasma concentrations after intravenous administration (2 mg/kg). HsTX1[R14A] stability in rat plasma and lung tissue was also determined. HsTX1[R14A] was more stable in plasma than in lung homogenate, with more than 90% of the HsTX1[R14A] remaining intact after 5 h, compared with 40.5% remaining in lung homogenate. The terminal elimination half-life, total clearance, and volume of distribution of HsTX1[R14A] after intravenous administration were 79.6 6.5 min, 8.3 0.6 mL/min/kg, and 949.8 71.0 mL/kg, respectively (mean SD). After intratracheal administration, HsTX1[R14A] in solution and dry powder was absorbed to a similar degree, with absolute bioavailability values of 39.2 5.2% and 44.5 12.5%, respectively. This study demonstrated that pulmonary administration is a promising alternative for systemically delivering HsTX1[R14A] for treating autoimmune diseases.


PubMed | Janssen R&D L.L.C., Peptides International and University of California at San Diego
Type: | Journal: Scientific reports | Year: 2017

Pain places a devastating burden on patients and society and current pain therapeutics exhibit limitations in efficacy, unwanted side effects and the potential for drug abuse and diversion. Although genetic evidence has clearly demonstrated that the voltage-gated sodium channel, Nav1.7, is critical to pain sensation in mammals, pharmacological inhibitors of Nav1.7 have not yet fully recapitulated the dramatic analgesia observed in Nav1.7-null subjects. Using the tarantula venom-peptide ProTX-II as a scaffold, we engineered a library of over 1500 venom-derived peptides and identified JNJ63955918 as a potent, highly selective, closed-state Nav1.7 blocking peptide. Here we show that JNJ63955918 induces a pharmacological insensitivity to pain that closely recapitulates key features of the Nav1.7-null phenotype seen in mice and humans. Our findings demonstrate that a high degree of selectivity, coupled with a closed-state dependent mechanism of action is required for strong efficacy and indicate that peptides such as JNJ63955918 and other suitably optimized Nav1.7 inhibitors may represent viable non-opioid alternatives for the pharmacological treatment of severe pain.

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