Loop Peptide Research Articles

Versatile Peptide Macrocyclization with Diels-Alder Cycloadditions.

Macrocyclization can improve bioactive peptide ligands through preorganization of molecular topology, leading to improvement of pharmacologic properties like binding affinity, cell permeability, and metabolic stability. Here we demonstrate that Diels-Alder [4 + 2] cycloadditions can be harnessed for peptide macrocyclization and stabilization within a range of peptide scaffolds and chemical environments. Diels-Alder cyclization of diverse diene-dienophile reactive pairs proceeds rapidly, in high yield and with tunable stereochemical preferences on solid-phase or in aqueous solution. This reaction can be applied alone or in concert with other stabilization chemistries, such as ring-closing olefin metathesis, to stabilize loop, turn, and α-helical secondary structural motifs. NMR and molecular dynamics studies of model loop peptides confirmed preferential formation of endo cycloadduct stereochemistry, imparting significant structural rigidity to the peptide backbone that resulted in augmented protease resistance and increased biological activity of a Diels-Alder cyclized (DAC) RGD peptide. Separately, we demonstrated the stabilization of DAC α-helical peptides derived from the ERα-binding protein SRC2. We solved a 2.25 Å cocrystal structure of one DAC helical peptide bound to ERα, which unequivocally corroborated endo stereochemistry of the resulting Diels-Alder adduct, and confirmed that the unique architecture of stabilizing motifs formed with this chemistry can directly contribute to target binding. These data establish Diels-Alder cyclization as a versatile approach to stabilize diverse protein structural motifs under a range of chemical environments.

Characterization of cerium (III) ion binding to surface‐immobilized EF‐hand loop I of calmodulin

AbstractCerium has a wide range of current and emerging applications, and the binding of cerium ions to solid substrates is important for cerium recovery, or in advanced material synthesis. In this study, we investigate the affinity of a surface‐bound peptide derived from the EF‐hand loop I of calmodulin for cerium (III) ions and compare the results to a scrambled control. Results obtained via quartz crystal microbalance with dissipation are used to estimate the dissociation constant between the bound EF‐hand loop I peptide and cerium (III) ions (1.3 ± 0.1 μM), which is comparable with other dissociation constants measured for EF‐hand peptides and cerium ions in solution reported this work and in literature (0.95‐5.8 μM). Circular dichroism also suggests that the peptide binds to cerium (III) ions in solution, and undergoes a secondary structural change upon binding. Overall, this study shows that EF‐hand loop peptides are capable of binding cerium (III) ions in solution and when attached to a solid substrate.

Engineering Dynamic Surface Peptide Networks on ButyrylcholinesteraseG117H for Enhanced Organophosphosphorus Anticholinesterase Catalysis

The single residue mutation of butyrylcholinesterase (BChEG117H) hydrolyzes a number of organophosphosphorus (OP) anticholinesterases. Whereas other BChE active site/proximal mutations have been investigated, none are sufficiently active to be prophylactically useful. In a fundamentally different computer simulations driven strategy, we identified a surface peptide loop (residues 278-285) exhibiting dynamic motions during catalysis and modified it via residue insertions. We evaluated these loop mutants using computer simulations, substrate kinetics, resistance to inhibition, and enzyme reactivation assays using both the choline ester and OP substrates. A slight but significant increase in reactivation was noted with paraoxon with one of the mutants, and changes in KM and catalytic efficiency were noted in others. Simulations suggested weaker interactions between OP versus choline substrates and the active site of all engineered versions of the enzyme. The results indicate that an improvement of OP anticholinesterase hydrolysis through surface loop engineering may be a more effective strategy in an enzyme with higher intrinsic OP compound hydrolase activity.

Abstract 2248: Generation and formatting of a bispecific Affimer® biotherapeutic for the inhibition of the LAG-3 and PD-L1 pathway

Abstract Programmed Cell Death-Ligand 1 (PD-L1) binds to its receptor, Programmed Cell Death protein (PD-1) and is expressed on both a range of activated immune cells and tumor cells. Lymphocyte Activation Gene-3 (LAG-3) is a member of the Ig superfamily and is expressed on activated T cells, NK cells, DCs and B cells. Both the PD-1/PD-L1 and the LAG-3 pathway are involved in immune suppression, leading to the down-regulation of T cell activity and the generation of exhausted T cells, allowing tumor escape and metastasis. Despite the impressive success of PD-L1 targeted immunotherapy, there remains a large proportion of patients with many tumor types that fail to benefit or acquire resistance during therapy. Consequently, it is necessary to consider future combinations of immunotherapies to enhance the clinical outcomes and reduce tumor escape. LAG-3 is upregulated on T-cells following PD-1/PD-L1 pathway blockade, indicating that targeting of both pathways as a combination may lead to more durable antitumor responses. To reach the full potential of LAG-3/PD-L1 dual blockade the development of a bispecific immunotherapy may further augment T-cell activation. Affimer biotherapeutics are a new protein scaffold with great potential for the generation of biotherapeutics. Based on the human protease inhibitor Stefin A, the scaffold is small (14kDa), lacks any post-translational modifications such as disulfide bonds and expresses at high levels. Affimers against a target are generated through the use of proprietary, highly diverse phage display libraries that have been created by engineering in two nine amino acid peptide loops into the scaffold backbone. We have identified a range of potent Affimer biotherapeutic antagonists to both human PD-L1 and LAG-3. When formatted as a bispecific Fc fusion to both the N and C-terminal of a human IgG1 Fc protein the Affimers demonstrate pM to nM binding affinities and binding to both target antigens simultaneously without compromising the affinities. The bispecific Fc fusion has been shown to block the interaction of LAG-3 with its ligand, MHC class II and PD-L1 with both PD-1 and CD80 by competitive ELISA. Functionally, the LAG-3/PD-L1 bispecific enhanced T-cell activation in vitro in both SEB-driven and mixed lymphocyte reaction assays. The Fc fusion format expresses over 500 mg/L from Expi293F mammalian transient transfections and can be purified to over 95% purity. The pharmacokinetic (PK) properties showed half-life extension of the Fc fusion format in mouse by using the IgG Fc-FcRn recycling pathway, dosing at 10 mg/kg resulted in a half-life extension of over 90 hours. We have demonstrated that the Affimer scaffold protein has all the necessary attributes to be developed as a bispecific LAG-3/PD-L1 immunotherapy. The Affimer scaffold was formatted as a bispecific Fc fusion molecule with ease, had good biophysical properties and shown to be well tolerated in vivo. Citation Format: Amrik Basran, Emma Jenkins, Estelle Adam, Michele Writer, Floriane Laurent, Assa Oumie, Jyrki Sivula, Jennifer Hillman, Lisa Strong, Maureen West, Emma Stanley. Generation and formatting of a bispecific Affimer® biotherapeutic for the inhibition of the LAG-3 and PD-L1 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2248.

Abstract 4108: Preclinical evaluation of half-life extended Affimer® biotherapeutics targeting the PD-L1 pathway

Abstract Programmed Cell Death-Ligand 1 (PD-L1) binds to its receptor, Programmed Cell Death protein (PD-1) and is expressed on a range of activated immune cells and is upregulated by tumor cells and by immune cells in the tumor microenvironment. PD-L1 immunotherapies have emerged as effective treatments both as monotherapies and in combinations, providing long term responses in a subset of patients. Phage display selections have identified a range of potent Affimer biotherapeutic antagonists to both human and mouse programmed death-ligand 1 (PD-L1). We have demonstrated when formatted as bivalent molecules they have improved sub nM affinity, half-life extension in a mouse PK study, blocking activity in vitro and in vivoefficacy in syngeneic or xenograph mouse models. Affimer biotherapeutics are a 14kDa monomeric scaffold protein based on the human protease inhibitor Stefin A, lacking post-translational modifications such as disulfide bonds and glycosylation. Large diverse phage display libraries have been created by engineering in two nine amino acid peptide loops into the scaffold backbone. We have identified a range of affinity competitive binders to mouse or human PD-L1 confirmed by performing competition ELISA and SPR using Fc formatted antigen. Half-life extension of lead Affimer proteins was achieved by fusing to human IgG1Fc or as an in-line fusion (ILF) fused to serum albumin binding Affimers. We demonstrated avidity for binding to target antigen, binding to cells in vitro and blockade of the PD-1L/PD-1 interaction using Promega cell based assay. The PK properties of the formatted Affimers in mouse showed half-life extension from 40-120 hours depending on the clone and half-life extension technology used. We have demonstrated that the Affimer scaffold has the necessary properties to be developed as an immunotherapy. The anti-PD-L1 Affimer scaffold proteins were formatted as Fc fusion and in-line fusion formats which showed in vivo efficacy. These molecules will be developed further for both bispecific and drug conjugates to generate new novel biotheraputics that will give greater efficacy in the clinic. Citation Format: Amrik Basran, Emma Jenkins, Estelle Adam, Floriane Laurent, Michele Writer, Assa Oumie, Jyrki Sivula, Maureen West, Emma Stanley, Jennifer Hillman, Viviana Robles-munoz. Preclinical evaluation of half-life extended Affimer® biotherapeutics targeting the PD-L1 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4108.

Nose-to-brain delivery of hyaluronate – FG loop peptide conjugate for non-invasive hypoxic-ischemic encephalopathy therapy

The intranasal drug administration has attracted great interest as a non-invasive route allowing targeted delivery of drugs directly to the brain. However, one of the main issues in nasal drug administration is mucociliary clearance. Hyaluronate (HA) has been widely used as a mucoadhesive excipient for ocular, rectal, and vaginal delivery. Here, FG loop peptide (FGL) was conjugated to HA for improving enzymatic stability and delivery efficiency from the nose to the brain. The successful conjugation of FGL to aldehyde modified HA was confirmed by gel permeation chromatography (GPC) and 1H nuclear magnetic resonance (NMR). The outstanding enzymatic stability of HA–FGL conjugate was also corroborated by the GPC. The HA–FGL conjugate showed enhanced binding affinity onto nasal epithelial cells. In addition, in vivo nose-to-brain delivery of HA–FGL conjugate could be visualized by using an IVIS imaging system and fluorescence microscopy. Finally, in vivo therapeutic effect of HA–FGL conjugate was successfully confirmed by histological analysis, transferase-mediated uridine 5-triphosphate-biotin nick end-labeling (TUNEL) assay, immunofluorescent staining, transmission electron microscopy (TEM), and rotarod tests in hypoxic-ischemic encephalopathy model animals.

Protein engineering of GH10 family xylanases for gaining a resistance to cereal proteinaceous inhibitors

Abstract Proteinaceous inhibitors from cereals reduce the efficiency of xylanase applications as a component of animal feeds, in bread making and other technologies based on processing of cereal grains. The inhibitors of XIP-type inhibit most of fungal xylanases from the GH10 and GH11 families of glycoside hydrolases, while they have no affect on bacterial xylanases. In order to gain a resistance to XIP-type proteins, an insertion of 5 amino acid residues was introduced into the peptide loop forming the active site cleft of two GH10 xylanases (PcXylA from Penicillium canescens and TrXyn3 from Trichoderma reesei) using a protein engineering technique. Genes encoding the wild-type and engineered forms of PcXylA and TrXyn3 were cloned and expressed in Penicillium verruculosum B1-537 and P. canescens RN3-11-7 auxotrophic recipient strains, respectively. The secreted recombinant xylanases were purified for characterization. Using a method based on measuring the enzyme effect on viscosity of the native extract of rye flour and that subjected to boiling in order to inactivate its inhibitory proteins, it was shown that the 5 aa insertion leads to relieving the inhibition of PcXylA and TrXyn3 by the proteinaceous inhibitors from rye.

Mechanistic insights into the interaction of the MOG1 protein with the cardiac sodium channel Nav1.5 clarify the molecular basis of Brugada syndrome

Nav1.5 is the α-subunit of the cardiac sodium channel complex. Abnormal expression of Nav1.5 on the cell surface because of mutations that disrupt Nav1.5 trafficking causes Brugada syndrome (BrS), sick sinus syndrome (SSS), cardiac conduction disease, dilated cardiomyopathy, and sudden infant death syndrome. We and others previously reported that Ran-binding protein MOG1 (MOG1), a small protein that interacts with Nav1.5, promotes Nav1.5 intracellular trafficking to plasma membranes and that a substitution in MOG1, E83D, causes BrS. However, the molecular basis for the MOG1/Nav1.5 interaction and how the E83D substitution causes BrS remains unknown. Here, we assessed the effects of defined MOG1 deletions and alanine-scanning substitutions on MOG1's interaction with Nav1.5. Large deletion analysis mapped the MOG1 domain required for the interaction with Nav1.5 to the region spanning amino acids 146-174, and a refined deletion analysis further narrowed this domain to amino acids 146-155. Site-directed mutagenesis further revealed that Asp-148, Arg-150, and Ser-151 cluster in a peptide loop essential for binding to Nav1.5. GST pulldown and electrophysiological analyses disclosed that the substitutions E83D, D148Q, R150Q, and S151Q disrupt MOG1's interaction with Nav1.5 and significantly reduce its trafficking to the cell surface. Examination of MOG1's 3D structure revealed that Glu-83 and the loop containing Asp-148, Arg-150, and Ser-151 are spatially proximal, suggesting that these residues form a critical binding site for Nav1.5. In conclusion, our findings identify the structural elements in MOG1 that are crucial for its interaction with Nav1.5 and improve our understanding of how the E83D substitution causes BrS.

Effects of Different Cultivation Parameters on the Production of Surfactin Variants by a Bacillus subtilis Strain.

Surfactins are lipopeptide-type biosurfactants produced mainly by Bacillus species, consisting of a peptide loop of seven amino acids and a hydrophobic fatty acid chain (C12–C16). These molecules have been proven to exhibit various biological activities; thus, their therapeutic and environmental applications are considered. Within the surfactin lipopeptide family, there is a wide spectrum of different homologues and isomers; to date, more than 30 variants have been described. Since the newest members of these lipopeptides were described recently, there is no information that is available on their characteristic features, e.g., the dependence of their production from different cultivation parameters. This study examined the effects of both the different carbon sources and various metal ions on the surfactin production of a selected B. subtilis strain. Among the applied carbon sources, fructose and xylose had the highest impacts on the ratio of the different variants, regarding both the peptide sequences and the lengths of the fatty acids. Furthermore, the application of metal ions Mn2+, Cu2+ and Ni2+ in the media completely changed the surfactin variant compositions of the fermenting broths leading to the appearance of methyl esterified surfactin forms, and resulted in the appearance of novel surfactin variants with fatty acid chains containing no more than 11 carbon atoms.

Abstract 3776: Generation and formatting of an Affimer® biotherapeutic for the inhibition of the PD-L1/PD-1 pathway: Proof of concept in mouse

Abstract We have generated a range of potent Affimer antagonists to both human and mouse programmed death-ligand 1 (PD-L1) and have demonstrated that when formatted, possess the necessary affinity, pharmacokinetics (PK) and tissue penetration to modulate the immune system and demonstrate efficacy in mouse models. Affimer biotherapeutics are a new protein scaffold with great potential for the generation of biotherapeutics. Based on the human protease inhibitor Stefin A, the scaffold is small (12kDa), lacks any post translational modifications such as disulphide bonds and glycosylation. Large diverse phage display libraries have been created by engineering in two 9 amino acid peptide loops into the scaffold backbone. Using phage display, we have identified competitive binders to both mouse and human PD-L1 which are low single digit nM in affinity as determined by Biacore. By ELISA we have shown that the Affimer molecules identified compete against PD-1 and CD80 for the binding site on PD-L1 and bind to the target on a range of human cancer cell lines (e.g. lung adenocarcinoma cells), as demonstrated by FACs. Following formatting of the lead Affimer protein to the N-terminal Fc region of an IgG, we showed that we could further improve the affinity to give a KD of <40 pM as determined by Biacore. The PK properties of the Affimer-Fc fusion were determined in mouse dosed at 10 mg/kg and shown to have a terminal half-life of over 100 hours. When dosed in a tumor mouse model the Affimer-Fc fusion protein showed a statistically significant reduction in tumor volume when compared to a control antibody. We have demonstrated that the Affimer scaffold has all the necessary attributes to be developed as a therapeutic platform. The Affimer scaffold was well tolerated in vivo, demonstrated a potency and PK profile that ensured sufficient exposure in the animal to modulate the immune system to slow tumor progression in a relevant mouse model. Citation Format: Amrik Basran, Emma Stanley. Generation and formatting of an Affimer® biotherapeutic for the inhibition of the PD-L1/PD-1 pathway: Proof of concept in mouse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3776.

Mechanisms of the Aspartoacylase Catalytic Activity Regulation According to the Computer Modeling Results

The results of molecular modeling allow us to associate the catalytic activity of aspartoacylase towards the hydrolysis of N-acetylaspartate with the dynamic properties of a dimeric molecule of the enzyme. The availability of the enzyme’s active site for the substrate is controlled by the conformational dynamics of the peptide loops that form a gate to the transport channel in one of the monomers. It is shown that this model explains the results of the experimental studies according to which the point mutation K213E does not affect the catalytic function of the enzyme.

Development of a Novel Backbone Cyclic Peptide Inhibitor of the Innate Immune TLR/IL1R Signaling Protein MyD88

MyD88 is a cytoplasmic adaptor protein that plays a central role in signaling downstream of the TLRs and the IL1R superfamily. We previously demonstrated that MyD88 plays a critical role in EAE, the murine model of multiple sclerosis, and showed that the MyD88 BB-loop decoy peptide RDVLPGT ameliorates EAE. We now designed and screened a library of backbone cyclized peptides based on the linear BB loop peptide, to identify a metabolically stable inhibitor of MyD88 that retains the binding properties of the linear peptide. We identified a novel cyclic peptide protein mimetic that inhibits inflammatory responses to TLR ligands, and NFκB activation in response to IL-1 activation. The inhibitor, c(MyD 4-4), is metabolically stable in comparison to the linear peptide, blocks MyD88 in a specific manner, and inhibits MyD88 function by preventing MyD88 dimerization. Finally, treatment of mice with c(MyD 4-4) reduced the severity of clinical disease in the murine EAE model of multiple sclerosis. Thus, modulation of MyD88-dependent signaling using c(MyD 4-4) is a potential therapeutic strategy to lower innate immune inflammation in autoimmune CNS disease.

Human anti-dopamine receptor 1 monoclonal autoantibody (mAb) identifies potential mechanisms of neuronal signaling in post-infectious autoimmune-mediated neuropsychiatric disease

Abstract Neuropsychiatric and movement disorders are associated with infections and anti-neuronal autoantibodies against dopamine receptors D1R and D2R. Most characterized of these disorders is Sydenham chorea (SC), an autoimmune neurologic manifestation of streptococcal-induced rheumatic fever where autoantibodies target the basal ganglia. Pediatric autoimmune neuropsychiatric disorder associated with streptococci (PANDAS) have similar autoantibodies that react with D1R and D2R. SC-derived human mAb 24.3.1 or SC derived IgG signal D2R in transfected cells by decreasing cyclic AMP-dependent kinase (cAMP) activity, reflective of an inhibitory receptor. Anti-D2R 24.3.1 increases dopamine release from human neuronal cells. However, signaling of D1R, an activating receptor, can have different biological effects. Our PANDAS-derived anti-D1R mAb, B4C, and PANDAS sera signaled D1R by activation of cAMP in transfected cells. IgG titers against D1R were significantly elevated in SC and PANDAS sera. To further show IgG recognized the D1R receptor, peptides of the extracellular loop of D1R were constructed and a defined sequence reacted significantly with human mAb B4C and PANDAS serum IgG. Human anti-D1R mAb B4C induced cAMP activity levels comparable to dopamine in a dose-dependent manner. Combined with dopamine, mAb B4C sensitized D1R to at least 50X more responsive to dopamine. In neuropsychiatric diseases where both anti-D1R and anti-D2R autoantibodies may be in combination, anti-D1R autoantibodies could sensitize D1R making it more sensitive to excess dopamine induced by anti-D2R autoantibodies; thus, contributing to dopamine signaling abnormalities and neuropsychiatric pathogenesis.

Calcium-/calmodulin-dependent protein kinase II in occlusion-induced degenerative cartilage of rat mandibular condyle.

Activated calcium-/calmodulin-dependent protein kinaseII (CaMKII) is important to promote chondrocytes from proliferative to pre-hypertrophic state, which probably plays a role in osteoarthritis (OA), a widespread degeneration disease with enhanced aberrant chondrocyte differentiation. Our aim was to detect the role of CaMKII, and its relationship with the feedback loop of Indian hedgehog (Ihh) and Parathyroid-related peptide (PTHrP) in the temporomandibular joints (TMJs) OA. KN93, the competitive inhibitor of CaMKII, was added to the culture medium in vitro and was locally injected to rats TMJs (n=54, female) every other day for 4weeks from the beginning of the 5th and 9th week after installing of unilateral anterior crossbite (UAC), termed as 4wk+4wk and 8wk+4wk, accordingly. The RNA expression of CaMKII α (1.49±0.09), CaMKII β (3.36±0.20), Ihh (1.88±0.06) and PTHrP (1.87±0.12) was all enhanced, especially at 24dyn/cm2 in vitro (all P<.05), accompanied with downregulated expression of cartilage matrix, but upregulated markers of chondrocytes differentiation (all P<0.05). Similarity was observed in the 4wk+4wk group in vivo. In the 8wk+4wk group, UAC upregulated the RNA expression of CaMKII α (1.81±0.24), CaMKII β (1.36±0.07) and Ihh (1.70±0.21), however, down-regulated PTHrP (0.53±0.04) (all P<.05), in consonance with the protein expression. All these changes were attenuated by KN93 (all P<.05). In conclusion, CaMKII took a role, via Ihh and PTHrP pathways, in promoting biomechanically induced TMJ chondrocytes differentiation, the initiation issue of UAC stimulated osteoarthritic changes in rodent TMJs. Inhibiting CaMKII is helpful to rescue the biomechanically stimulated cartilage degradation and prospective to be a target treatment of OA.

Foldamers wave to the ribosome.

Ribosomes have now been shown to accept certain initiator tRNAs acylated with aromatic foldamer–dipeptides thereby enabling the translation of a peptide or protein with a short aromatic foldamer at the N-terminus. Some foldamer–peptide hybrids could be cyclized to generate macrocycles that present conformationally restricted peptide loops.

Crystal Structure of Cleaved Serp-1, a Myxomavirus-Derived Immune Modulating Serpin: Structural Design of Serpin Reactive Center Loop Peptides with Improved Therapeutic Function.

The Myxomavirus-derived protein Serp-1 has potent anti-inflammatory activity in models of vasculitis, lupus, viral sepsis, and transplant. Serp-1 has also been tested successfully in a Phase IIa clinical trial in unstable angina, representing a "first-in-class" therapeutic. Recently, peptides derived from the reactive center loop (RCL) have been developed as stand-alone therapeutics for reducing vasculitis and improving survival in MHV68-infected mice. However, both Serp-1 and the RCL peptides lose activity in MHV68-infected mice after antibiotic suppression of intestinal microbiota. Here, we utilize a structure-guided approach to design and test a series of next-generation RCL peptides with improved therapeutic potential that is not reduced when the peptides are combined with antibiotic treatments. The crystal structure of cleaved Serp-1 was determined to 2.5 Å resolution and reveals a classical serpin structure with potential for serpin-derived RCL peptides to bind and inhibit mammalian serpins, plasminogen activator inhibitor 1 (PAI-1), anti-thrombin III (ATIII), and α-1 antitrypsin (A1AT), and target proteases. Using in silico modeling of the Serp-1 RCL peptide, S-7, we designed several modified RCL peptides that were predicted to have stronger interactions with human serpins because of the larger number of stabilizing hydrogen bonds. Two of these peptides (MPS7-8 and -9) displayed extended activity, improving survival where activity was previously lost in antibiotic-treated MHV68-infected mice (P < 0.0001). Mass spectrometry and kinetic assays suggest interaction of the peptides with ATIII, A1AT, and target proteases in mouse and human plasma. In summary, we present the next step toward the development of a promising new class of anti-inflammatory serpin-based therapeutics.

Calmodulin Interaction with Gap Junction Intracellular Loop Peptides

Calmodulin Interaction with Gap Junction Intracellular Loop Peptides

Effects of TNF receptor blockade on in vitro cell survival and response to negative energy balance in dairy cattle

BackgroundAssociative data and some controlled studies suggest that the inflammatory cytokine tumor necrosis factor (TNF) α can induce fatty liver in dairy cattle. However, research demonstrating that TNFα is a necessary component in the etiology of bovine fatty liver is lacking. The aim of this work was to evaluate whether blocking TNFα signaling with a synthetic cyclic peptide (TNF receptor loop peptide; TRLP) would improve liver metabolic function and reduce triglyceride accumulation during feed restriction.ResultsCapability of TRLP to inhibit TNFα signaling was confirmed on primary bovine hepatocytes treated with recombinant bovine TNFα and 4 doses of TRLP (0, 1, 10, 50 μmol/L) over 24 h. Next, 4 lactating Holstein cows (parity 1.4 ± 0.5, 433 ± 131 d in milk) in an incomplete Latin rectangle design (3 × 2) were subcutaneously administered with different TRLP doses (0, 1.5, 3.0 mg/kg BW) every 4 h for 24 h, followed by an intravenous injection of TNFα (5 μg/kg BW). Before and for 2 h after TNFα injection, TRLP decreased plasma non-esterified fatty acid (NEFA) concentration (P ≤ 0.05), suggesting an altered metabolic response to inflammation. Finally, 10 non-pregnant, non-lactating Holstein cows (3.9 ± 1.1 yr of age) were randomly assigned to treatments: control (carrier: 57% DMSO in PBS) or TRLP (1.75 mg TRLP /kg BW per day). Treatments were administrated every 4 h for 7 d by subcutaneous injection to feed-restricted cows fed 30% of maintenance energy requirements. Daily blood samples were analyzed for glucose, insulin, β-hydroxybutyrate, NEFA, and haptoglobin concentrations, with no treatment effects detected. On d 7, cows completed a glucose tolerance test (GTT) by i.v. administration of a dextrose bolus (300 mg glucose/kg BW). Glucose, insulin, and NEFA responses failed to demonstrate any significant effect of treatment during the GTT. However, plasma and liver analyses were not indicative of dramatic lipolysis or hepatic lipidosis, suggesting that the feed restriction protocol failed to induce the metabolic state of interest. Injection site inflammation, assessed by a scorer blinded to treatment, was enhanced by TRLP compared to control.ConclusionsAlthough the TRLP inhibited bovine TNFα signaling and altered responses to i.v. administration of TNFα, repeated use over 7 d caused apparent local allergic responses and it failed to alter metabolism during a feed restriction-induced negative energy balance. Although responses to feed restriction seemed atypical in this study, side effects of TRLP argue against its future use as a tool for investigating the role of inflammation in metabolic impacts of negative energy balance.

Serpins: Development for Therapeutic Applications.

Serine protease inhibitors, or serpins, function as central regulators for many vital processes in the mammalian body, maintaining homeostasis for clot formation and breakdown, immune responses, lung function, and hormone or central nervous system activity, among many others. When serine protease activity or serpin-mediated regulation becomes unbalanced or dysfunctional, then severe disease states and pathogenesis can ensue. With serpinopathies, genetic mutations lead to inactive serpins or protein aggregation with loss of function. With other disorders, such as sepsis, atherosclerosis, cancer, obesity, and the metabolic syndrome, the thrombotic and thrombolytic cascades and/or inflammatory responses become unbalanced, with excess bleeding and clotting and upregulation of adverse immune responses. Returning overall balance can be engineered through introduction of a beneficial serpin replacement as a therapeutic or through blockade of serpins that are detrimental. Several drugs have been developed and are currently in use and/or in development both to replace dysfunctional serpins and to block adverse effects induced by aberrant protease or serpin actions.With this chapter, we provide a general overview of the development of a virus-derived serpin, Serp-1, and serpin reactive center loop (RCL) peptides, as therapeutics. Serp-1 is a virus-derived serpin developed as a new class of immune modulator. We will use the development of Serp-1 as a general introduction to serpin-based drug development.

Viral Serpin Reactive Center Loop (RCL) Peptides: Design and Testing.

Serpins function as a trap for serine proteases, presenting the reactive center loop (RCL) as a target for individual proteases. When the protease cleaves the RCL, the serpin and protease become covalently linked leading to a loss of function of both the protease and the serpin; this suicide inhibition is often referred to as a "mouse trap." When the RCL P1-P1' scissile bond is cut by the protease, the resulting bond between the protease and the RCL leads to insertion of the cleaved RCL into the β-sheet A and relocation of the protease to the opposite pole of the serpin, forming a suicide complex. Only a relatively small part of the serpin molecule can be removed in deletion mutations before the serpin RCL inhibitory function is lost. Serpin RCL peptides have been developed to block formation of serpin aggregates in serpinopathies, genetic serpin mutations wherein the abnormal serpins insert their RCL into adjacent serpins forming aggregates of inactive serpins.We have further posited that this natural cleavage site in the serpin RCL may form active serpin metabolites with potential to add to the serpin's inhibitory functions. We have developed RCL peptides based upon predicted serpin RCL cleavage (or metabolism) sites and tested these serpins for inhibitory function. In this chapter we describe the development of RCL-derived peptides, peptides derived based upon the RCL sequences of two myxomaviral serpins. Methods used to develop peptides are described for RCL-derived peptides from Serp-1, a thrombotic and thrombolytic serine protease inhibitor, and Serp-2, a cross class serine and cysteine protease inhibitor (Subheadings 2.1 and 3.1). Approaches to testing RCL peptide functions, in vitro by molecular assays and in vivo in models of cell migration, MHV-68 infection, and aortic allograft transplant are described (Subheadings 2.2 and 3.2).