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Objectives Autoantibodies against apolipoprotein A1 (anti‐apoA1 IgGs) and its C‐terminal region (cter apoA1) have emerged as an independent biomarker for cardiovascular disease. Cter apoA1 mimetic peptides were shown to reverse the deleterious anti‐apoA1 IgG effects in vitro. We evaluated the association of anti‐cter apoA1 IgGs with overall mortality in the general population and tested the ability of a cter apoA1 mimetic peptide to reverse the anti‐apoA1 IgG‐induced inflammatory response and mortality in vitro and in vivo, respectively. Methods Anti‐cter apoA1 IgGs were measured in serum samples of 6386 participants of the CoLaus study of which 5220 were followed for a median duration of 5.6 years. The primary outcome was overall mortality. The peptide inhibitory concentration 50% (IC50) was determined in vitro on HEK‐Blue‐4 and RAW cells. ApoE−/− mice were exposed to 16 weeks of anti‐apoA1IgG passive immunisation with and without peptide co‐incubation. Results Anti‐cter apoA1 IgGs were associated with higher interleukin 6 levels and independently predicted overall mortality; an increase of one standard deviation of anti‐cter apoA1 IgG level was associated with an 18% increase in mortality risk (hazard ratio: 1.18, 95% confidence interval: 1.04–1.33; P = 0.009). The cterApoA1 analogue reversed the antibody‐mediated inflammatory response with an IC50 of 1 µm in vitro but did not rescue the significant anti‐apoA1 IgG‐induced mortality rate in vivo (69% vs. 23%, LogRank P = 0.02). Conclusion Anti‐cter apoA1 IgG independently predicts overall mortality in the general population. Despite being effective in vitro, our cter apoA1 analogue did not reverse the anti‐apoA1 IgG‐induced mortality in mice. Our data suggest that these autoantibodies are not readily treatable through cognate peptide immunomodulation., Autoantibodies against apolipoprotein A1 (anti‐apoA1 IgGs) have emerged as an independent cardiovascular risk factor in different settings including in the general population and seem to be preferentially oriented against the its C‐terminal region (cterA1) in humans. In this translational work, we extend previous knowledge in the field by demonstrating for the first time that anti‐cterA1 IgGs are independently predictive of premature mortality in a large general population sample of 5220 individuals. Furthermore, we also report that, despite being effective in vitro, our cterA1 analogue did not reverse the significant anti‐apoA1 IgG‐induced mortality in mice.

The chemokine receptor CCR5 is a drug target to prevent transmission of HIV/AIDS. We studied four analogs of the native chemokine RANTES (CCL5) that have anti-HIV potencies of around 25 pM, which is more than four orders-of-magnitude higher than that of RANTES itself. It has been hypothesized that the ultra-high potency of the analogs is due to their ability to bind populations of receptors not accessible to native chemokines. To test this hypothesis, we developed a homogeneous dual-color fluorescence cross-correlation spectroscopy (FCCS) assay for saturation and competition binding experiments. The FCCS assay has the advantage that it does not rely on competition with radioactively labeled native chemokines used in conventional assays. We prepared site-specifically labeled fluorescent analogs using native chemical ligation of synthetic peptides, followed by bioorthogonal fluorescent labeling. We engineered a mammalian cell expression construct to provide fluorescently labeled CCR5, which was purified using a tandem immunoaffinity and size-exclusion chromatography approach to obtain monomeric fluorescent CCR5 in detergent solution. We found subnanomolar binding affinities for the two analogs 5P12-RANTES and 5P14-RANTES, and about twenty-fold reduced affinities for PSC-RANTES and 6P4-RANTES. Using homologous and heterologous competition experiments with unlabeled chemokine analogs, we conclude that the analogs all bind at the same binding site; whereas, the native chemokines (RANTES and MIP1α) fail to displace bound fluorescent analogs even at tens of micromolar concentrations. Our results can be rationalized with de novo structural models of the N-terminal tails of the synthetic chemokines that adopt a different binding mode as compared to the parent compound.

The chemokine receptor CCR5 is a drug target to prevent transmission of HIV/AIDS. We studied four analogs of the native chemokine regulated, on activation, normal T-cell-expressed, and secreted (RANTES) (CCL5) that have anti-HIV potencies of around 25 pM, which is more than four orders of magnitude higher than that of RANTES itself. It has been hypothesized that the ultrahigh potency of the analogs is due to their ability to bind populations of receptors not accessible to native chemokines. To test this hypothesis, we developed a homogeneous dual-color fluorescence cross-correlation spectroscopy assay for saturation- and competition-binding experiments. The fluorescence cross-correlation spectroscopy assay has the advantage that it does not rely on competition with radioactively labeled native chemokines used in conventional assays. We prepared site-specifically labeled fluorescent analogs using native chemical ligation of synthetic peptides, followed by bioorthogonal fluorescent labeling. We engineered a mammalian cell expression construct to provide fluorescently labeled CCR5, which was purified using a tandem immunoaffinity and size-exclusion chromatography approach to obtain monomeric fluorescent CCR5 in detergent solution. We found subnanomolar binding affinities for the two analogs 5P12-RANTES and 5P14-RANTES and about 20-fold reduced affinities for PSC-RANTES and 6P4-RANTES. Using homologous and heterologous competition experiments with unlabeled chemokine analogs, we conclude that the analogs all bind at the same binding site, whereas the native chemokines (RANTES and MIP-1α) fail to displace bound fluorescent analogs even at tens of micromolar concentrations. Our results can be rationalized with de novo structural models of the N-terminal tails of the synthetic chemokines that adopt a different binding mode as compared to the parent compound.

Cell penetrating peptides (CPPs) from the protein ZEBRA are promising candidates to exploit in therapeutic cancer vaccines, since they can transport antigenic cargos into dendritic cells and induce tumor-specific T cells. Employing CPPs for a given cancer indication will require engineering to include relevant tumor-associated epitopes, administration with an appropriate adjuvant, and testing for antitumor immunity. We assessed the importance of structural characteristics, efficiency of in vitro transduction of target cells, and choice of adjuvant in inducing the two key elements in antitumor immunity, CD4 and CD8 T cells, as well as control of tumor growth in vivo. Structural characteristics associated with CPP function varied according to CPP truncations and cargo epitope composition, and correlated with in vitro transduction efficiency. However, subsequent in vivo capacity to induce CD4 and CD8 T cells was not always predicted by in vitro results. We determined that the critical parameter for in vivo efficacy using aggressive mouse tumor models was the choice of adjuvant. Optimal pairing of a particular ZEBRA-CPP sequence and antigenic cargo together with adjuvant induced potent antitumor immunity. Our results highlight the irreplaceable role of in vivo testing of novel vaccine constructs together with adjuvants to select combinations for further development.Molecular Therapy (2016); doi:10.1038/mt.2016.134.

Endothelin-1 (ET-1) is involved in the pathogenesis of cardiac and renal diseases, and in the progression of tumour growth in cancer, but current diagnosis and treatment remain inadequate. Peptides derived from the 212 amino acid precursor preproendothelin-1 (ppET-1) may have utility as biomarkers, or cause biological effects that are unaffected by endothelin receptor antagonists. Here, we used specific immunoassays and LC-MS/MS to identify NT-proET-1 (ppET-1[18–50]), Endothelin-Like Domain Peptide (ELDP, ppET-1[93–166]) and CT-proET-1 (ppET-1[169–212]) in conditioned media from cultured endothelial cells. Synthesis of these peptides correlated with ET-1, and plasma ELDP and CT-proET-1 were elevated in patients with chronic heart failure. Clearance rates of NT-proET-1, ELDP and CT-proET-1 were determined after i.v. injection in anaesthetised rats. CT-proET-1 had the slowest systemic clearance, hence providing a biological basis for it being a better biomarker of ET-1 synthesis. ELDP contains the evolutionary conserved endothelin-like domain sequence, which potentially confers biological activity. On isolated arteries ELDP lacked direct vasoconstrictor effects. However, it enhanced ET-1 vasoconstriction and prolonged the increase in blood pressure in anaesthetised rats. ELDP may therefore contribute to disease pathogenesis by augmenting ET-1 responses.

Background: Autoantibodies to apoA-I can be considered markers and mediators of cardiovascular disease. Results: Methodologies for identification of human apoA-I epitopes are described. Two novel immunoreactive peptides were recognized by autoantibodies from cardiac patients. Conclusion: We developed a new procedure to isolate, purify, and identify apoA-I epitopes. Significance: Developing new epitope mapping approaches opens new avenues for biomarkers and therapy., Autoantibodies to apolipoprotein A-I (anti-apoA-I IgG) have been shown to be both markers and mediators of cardiovascular disease, promoting atherogenesis and unstable atherosclerotic plaque. Previous studies have shown that high levels of anti-apoA-I IgGs are independently associated with major adverse cardiovascular events in patients with myocardial infarction. Autoantibody responses to apoA-I can be polyclonal and it is likely that more than one epitope may exist. To identify the specific immunoreactive peptides in apoA-I, we have developed a set of methodologies and procedures to isolate, purify, and identify novel apoA-I endogenous epitopes. First, we generated high purity apoA-I from human plasma, using thiophilic interaction chromatography followed by enzymatic digestion specifically at lysine or arginine residues. Immunoreactivity to the different peptides generated was tested by ELISA using serum obtained from patients with acute myocardial infarction and high titers of autoantibodies to native apoA-I. The immunoreactive peptides were further sequenced by mass spectrometry. Our approach successfully identified two novel immunoreactive peptides, recognized by autoantibodies from patients suffering from myocardial infarction, who contain a high titer of anti-apoA-I IgG. The discovery of these epitopes may open innovative prognostic and therapeutic opportunities potentially suitable to improve current cardiovascular risk stratification.

BACKGROUND AND PURPOSE The µ-conopeptide family is defined by its ability to block voltage-gated sodium channels (VGSCs), a property that can be used for the development of myorelaxants and analgesics. We characterized the pharmacology of a new µ-conopeptide (µ-CnIIIC) on a range of preparations and molecular targets to assess its potential as a myorelaxant. EXPERIMENTAL APPROACH µ-CnIIIC was sequenced, synthesized and characterized by its direct block of elicited twitch tension in mouse skeletal muscle and action potentials in mouse sciatic and pike olfactory nerves. µ-CnIIIC was also studied on HEK-293 cells expressing various rodent VGSCs and also on voltage-gated potassium channels and nicotinic acetylcholine receptors (nAChRs) to assess cross-interactions. Nuclear magnetic resonance (NMR) experiments were carried out for structural data. KEY RESULTS Synthetic µ-CnIIIC decreased twitch tension in mouse hemidiaphragms (IC50= 150 nM), and displayed a higher blocking effect in mouse extensor digitorum longus muscles (IC = 46 nM), compared with µ-SIIIA, µ-SmIIIA and µ-PIIIA. µ-CnIIIC blocked NaV1.4 (IC50= 1.3 nM) and NaV1.2 channels in a long-lasting manner. Cardiac NaV1.5 and DRG-specific NaV1.8 channels were not blocked at 1 µM. µ-CnIIIC also blocked the α3β2 nAChR subtype (IC50= 450 nM) and, to a lesser extent, on the α7 and α4β2 subtypes. Structure determination of µ-CnIIIC revealed some similarities to α-conotoxins acting on nAChRs. CONCLUSION AND IMPLICATIONS µ-CnIIIC potently blocked VGSCs in skeletal muscle and nerve, and hence is applicable to myorelaxation. Its atypical pharmacological profile suggests some common structural features between VGSCs and nAChR channels.

CC chemokine receptor 5 (CCR5), the major HIV coreceptor, is a G protein-coupled receptor (GPCR) involved in cell activation and migration in response to chemokines. Blockade of CCR5 is an effective anti-HIV strategy, and potent anti-HIV chemokine analogs such as PSC-RANTES have been developed. These inhibitors act by interfering with receptor trafficking, thereby inducing prolonged intracellular sequestration of CCR5. Like many GPCRs, CCR5 is desensitized following agonist activation. The initial steps in this process are well understood, but later stages, including where CCR5 is sequestered during desensitization, and how anti-HIV chemokine analogs intervene to achieve prolonged sequestration, have yet to be elucidated in detail. In this study we demonstrate that CCR5 cycles to and from the cell surface via the endosome recycling compartment and the trans-Golgi network during desensitization, accumulating in the trans-Golgi network following internalization by both PSC-RANTES and CCL5, the native ligand from which it was derived. In addition, we show that unlike CCR5 sequestered by CCL5, CCR5 sequestered by PSC-RANTES cannot be induced to return to the cell surface by addition of the small molecule CCR5 inhibitor, TAK-779, and that association of PSC-RANTES with CCR5 is more durable than that of native CCL5 during desensitization. Our findings reconcile the previously conflicting descriptions of the location of sequestered CCR5 during desensitization, as well as providing more general insights into potential trafficking routes for endocytosed GPCRs and further elucidation of the unusual inhibitory mechanism of chemokine analogs with potent anti-HIV activity.

Phage display technology, which allows extremely rare ligands to be selected from libraries of variants according to user-defined selection criteria, has made a huge impact on the life sciences. In this chapter, we describe phage display methods for the discovery of chemokine analogs with enhanced pharmacological properties. We discuss strategies for chemokine library design and provide a recommended technique for library construction. We also describe cell-based library selection approaches that we have used to discover chemokine analogs, not only receptor antagonists but also variants with unusual effects on receptor signaling and trafficking. By providing a survey of the different phage chemokine projects that we have undertaken, we comment on the parameters most likely to affect success. Finally, we discuss how phage display-derived chemokine analogs with altered pharmacological activity represent valuable tools to better understand chemokine biology, and why certain among them have the potential to be developed as new medicines.

In the continued absence of an effective anti-HIV vaccine, approximately 2 million new HIV infections occur every year, with over 95% of these in developing countries. Calls have been made for the development of anti-HIV drugs that can be formulated for topical use to prevent HIV transmission during sexual intercourse. Because these drugs are principally destined for use in low-resource regions, achieving production costs that are as low as possible is an absolute requirement. 5P12-RANTES, an analog of the human chemokine protein RANTES/CCL5, is a highly potent HIV entry inhibitor which acts by achieving potent blockade of the principal HIV coreceptor, CCR5. Here we describe the development and optimization of a scalable low-cost production process for 5P12-RANTES based on expression in Pichia pastoris. At pilot (150 L) scale, this cGMP compliant process yielded 30 g of clinical grade 5P12-RANTES. As well as providing sufficient material for the first stage of clinical development, this process represents an important step towards achieving production of 5P12-RANTES at a cost and scale appropriate to meet needs for topical HIV prevention worldwide., Highlights • New medicines are required to prevent the spread of HIV/AIDS in low-resource regions. • 5P12-RANTES, a chemokine analog, is a promising new candidate drug. • We describe a process for producing clinical grade cGMP 5P12-RANTES in Pichia pastoris. • This is a key step to achieving production at cost and scale appropriate for use worldwide.

Background Cardiovascular disease (CVD) is the leading cause of death worldwide and new approaches for both diagnosis and treatment are required. Autoantibodies directed against apolipoprotein A-I (ApoA-I) represent promising biomarkers for use in risk stratification of CVD and may also play a direct role in pathogenesis. Methodology To characterize the anti-ApoA-I autoantibody response, we measured the immunoreactivity to engineered peptides corresponding to the different alpha-helical regions of ApoA-I, using plasma from acute chest pain cohort patients known to be positive for anti-ApoA-I autoantibodies. Principal Findings Our results indicate that the anti-ApoA-I autoantibody response is strongly biased towards the C-terminal alpha-helix of the protein, with an optimized mimetic peptide corresponding to this part of the protein recapitulating the diagnostic accuracy for an acute ischemic coronary etiology (non-ST segment elevation myocardial infarction and unstable angina) obtainable using intact endogenous ApoA-I in immunoassay. Furthermore, the optimized mimetic peptide strongly inhibits the pathology-associated capacity of anti-ApoA-I antibodies to elicit proinflammatory cytokine release from cultured human macrophages. Conclusions In addition to providing a rationale for the development of new approaches for the diagnosis and therapy of CVD, our observations may contribute to the elucidation of how anti-ApoA-I autoantibodies are elicited in individuals without autoimmune disease.

G protein-coupled receptors (GPCRs) are the largest superfamily of membrane receptors in the human genome and they are targets for a quarter of all prescription drugs. Activation of a GPCR by an agonist ligand results in G protein-mediated downstream signaling, followed by kinase action and arrestin-mediated desensitization, internalization/sequestration, and recycling. Selective manipulation of these individual steps of the GPCR activation cycle is often desired when creating drugs targeting a given receptor. We are interested in the C-C chemokine receptor CCR5 that is the major HIV coreceptor used in person-to-person transmission. Globally, the HIV/AIDS pandemic has caused nearly 30 million deaths and a similar number of people are currently infected. Certain analogues of the chemokine RANTES/CCL5 are highly potent entry inhibitors against R5-tropic HIV-1 strains, in vitro and in vivo. Three such analogues, 5P12-, 5P14-, and 6P4-RANTES, are particularly interesting because while they differ only slightly in structure they show strikingly different pharmacological profiles (G protein-linked signaling activity, stimulation of receptor internalization). We have recently developed a general, simple, and robust method for stoichiometric, site-specific fluorescence labeling of expressed GPCRs. The method is based on bioorthogonal conjugation of a fluorescent reporter group to a genetically encoded azido group introduced into expressed GPCRs using amber codon suppression.[1] We have adopted a similar strategy for fluorescent labeling of chemokines with azido groups introduced by chemical synthesis. Here we present our progress towards automated, multi-color, single-molecule fluorescence studies of the compositional and conformational dynamics of GPCR signaling complexes ("signalosomes") using fluorescently labeled chemokines and receptors in biochemically defined systems.[1] H Tian, TP Sakmar, & T Huber (2013) Site-specific labeling of genetically encoded azido groups for multi-color, single-molecule fluorescence imaging of GPCRs. Methods in Cell Biology, 117, in press.

International audience; Several gene mutations linked to intellectual disability in humans code for synaptic molecules implicated in small GTPase signaling. This is the case of the Rac/Cdc42 effector p21-activated kinase 3 (PAK3). The mechanisms responsible for the intellectual defects and the consequences of the mutation on the development and wiring of brain networks remain unknown. Here we show that expression of PAK3 mutants, suppression of PAK3, or inhibition of PAK3 function in rat hippocampal slice cultures interfere with activity-mediated spine dynamics. Inhibition of PAK3 resulted in two main alterations: (1) an increased growth of new, unstable spines, occurring in clusters, and mediated by activity; and (2) an impairment of plasticity-mediated spine stabilization interfering with the formation of persistent spines. Additionally, we find that PAK3 is specifically recruited by activity from dendrites into spines, providing a new mechanism through which PAK3 could participate in the control of both spine stabilization and local spine growth. Together, these data identify a novel function of PAK3 in regulating activity-mediated rearrangement of synaptic connectivity associated with learning and suggest that defects in spine formation and refinement during development could account for intellectual disability.