Targeting misfolding and aggregation of the amyloid-ß peptide and mutant p53 protein using multifunctional molecules.

Biomolecule misfolding and aggregation play a major role in human disease, spanning from neurodegeneration to cancer. Inhibition of these processes is of considerable interest, and due to the multifactorial nature of these diseases, the development of drugs that act on multiple pathways simultaneously is a promising approach. This Feature Article focuses on the development of multifunctional molecules designed to inhibit the misfolding and aggregation of the amyloid-ß (Aß) peptide in Alzheimer's disease (AD), and the mutant p53 protein in cancer. While for the former, the goal is to accelerate the removal of the Aß peptide and associated aggregates, for the latter, the goal is reactivation via stabilization of the active folded form of mutant p53 protein and/or aggregation inhibition. Due to the similar aggregation pathway of the Aß peptide and mutant p53 protein, a common therapeutic approach may be applicable.

An extracellular vesicular mutant KRAS-associated protein complex promotes lung inflammation and tumor growth.

Extracellular vesicles (EVs) contain more than 100 proteins. Whether there are EVs proteins that act as an 'organiser' of protein networks to generate a new or different biological effect from that identified in EV-producing cells has never been demonstrated. Here, as a proof-of-concept, we demonstrate that EV-G12D-mutant KRAS serves as a leader that forms a protein complex and promotes lung inflammation and tumour growth via the Fn1/IL-17A/FGF21 axis. Mechanistically, in contrast to cytosol derived G12D-mutant KRAS complex from EVs-producing cells, EV-G12D-mutant KRAS interacts with a group of extracellular vesicular factors via fibronectin-1 (Fn1), which drives the activation of the IL-17A/FGF21 inflammation pathway in EV recipient cells. We show that: (i), depletion of EV-Fn1 leads to a reduction of a number of inflammatory cytokines including IL-17A; (ii) induction of IL-17A promotes lung inflammation, which in turn leads to IL-17A mediated induction of FGF21 in the lung; and (iii) EV-G12D-mutant KRAS complex mediated lung inflammation is abrogated in IL-17 receptor KO mice. These findings establish a new concept in EV function with potential implications for novel therapeutic interventions in EV-mediated disease processes.

Biochemical Pathways of Cellular Mechanosensing/Mechanotransduction and Their Role in Neurodegenerative Diseases Pathogenesis.

In this review, we shed light on recent advances regarding the characterization of biochemical pathways of cellular mechanosensing and mechanotransduction with particular attention to their role in neurodegenerative disease pathogenesis. While the mechanistic components of these pathways are mostly uncovered today, the crosstalk between mechanical forces and soluble intracellular signaling is still not fully elucidated. Here, we recapitulate the general concepts of mechanobiology and the mechanisms that govern the mechanosensing and mechanotransduction processes, and we examine the crosstalk between mechanical stimuli and intracellular biochemical response, highlighting their effect on cellular organelles' homeostasis and dysfunction. In particular, we discuss the current knowledge about the translation of mechanosignaling into biochemical signaling, focusing on those diseases that encompass metabolic accumulation of mutant proteins and have as primary characteristics the formation of pathological intracellular aggregates, such as Alzheimer's Disease, Huntington's Disease, Amyotrophic Lateral Sclerosis and Parkinson's Disease. Overall, recent findings elucidate how mechanosensing and mechanotransduction pathways may be crucial to understand the pathogenic mechanisms underlying neurodegenerative diseases and emphasize the importance of these pathways for identifying potential therapeutic targets.

Truncated Pneumolysin from Streptococcus pneumoniae as a TLR4-Antagonizing New Drug for Chronic Inflammatory Conditions.

Microbial proteins have recently been found to have more benefits in clinical disease treatment because of their better-developed strategy and properties than traditional medicine. In this study, we investigated the effectiveness of a truncated peptide synthesized from the C-terminal sequence of pneumolysin, i.e., C70PLY4, in Streptococcus pneumoniae, in treating chronic inflammatory conditions. It has been shown that C70PLY4 significantly blocks the transendothelial migration of neutrophils and attenuates the formation of atherosclerotic plaque and the secretion of soluble forms of the intercellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule 1 (VCAM-1), and E-selectin in high-fat-diet/streptozotocin-induced inflammatory rats. The mechanism and the docking simulation analysis further indicated that C70PLY4 might serve as a Toll-like receptor 4 (TLR4) antagonist by competing for the binding site of MD2, an indispensable protein for lipopolysaccharide (LPS)-TLR4 interaction signaling, on the TLR4 structure. Moreover, compared to the full-length PLY, C70PLY4 seems to have no cytotoxicity in human vascular endothelial cells. Our study elucidated a possible therapeutic efficacy of C70PLY4 in reducing chronic inflammatory conditions and clarified the underlying mechanism. Thus, our findings identify a new drug candidate that, by blocking TLR4 activity, could be an effective treatment for patients with chronic inflammatory diseases.

Improved CoChR Variants Restore Visual Acuity and Contrast Sensitivity in a Mouse Model of Blindness under Ambient Light Conditions.

Severe photoreceptor cell death in retinal degenerative diseases leads to partial or complete blindness. Optogenetics is a promising strategy to treat blindness. The feasibility of this strategy has been demonstrated through the ectopic expression of microbial channelrhodopsins (ChRs) and other genetically encoded light sensors in surviving retinal neurons in animal models. A major drawback for ChR-based visual restoration is low light sensitivity. Here, we report the development of highly operational light-sensitive ChRs by optimizing the kinetics of a recently reported ChR variant, Chloromonas oogama (CoChR). In particular, we identified two CoChR mutants, CoChR-L112C and CoChR-H94E/L112C/K264T, with markedly enhanced light sensitivity. The improved light sensitivity of the CoChR mutants was confirmed by ex vivo electrophysiological recordings in the retina. Furthermore, the CoChR mutants restored the vision of a blind mouse model under ambient light conditions with remarkably good contrast sensitivity and visual acuity, as evidenced by the results of behavioral assays. The ability to restore functional vision under normal light conditions with the improved CoChR variants removed a major obstacle for ChR-based optogenetic vision restoration.

IL-8 antagonist, CXCL8(3-72)K11R/G31P coupled with probiotic exhibit variably enhanced therapeutic potential in ameliorating ulcerative colitis.

Inflammatory bowel disease (IBD) remains a major health challenge due in part to unsafe and limited treatment options, hence there is the need for alternatives. CXCL8/interleukin 8 (IL-8) is elevated in inflammation, and binds preferentially to G protein-couple receptors (GPCRs) CXCR1/2 of the CXC chemokine family to initiate cascades of downstream inflammatory signals. A mutant CXCL8 protein, CXCL8(3-72)K11R/G31P (G31P), competitively and selectively binds to CXCR1/2, making CXCL8 redundant. We explore the therapeutic potential of G31P in dextran sulfate sodium (DSS) induced ulcerative colitis (UC), and the corresponding effect if G31P treatment is augmented with Lactobacillus acidophilus (LACT). The treatment options administered significantly reduced TNF-α, IFN-γ, IL-1ß, IL-6, and IL-8, but maintained elevated levels of IL-10. CD68 and F4/80 expressions were down-regulated and showed restricted infiltration to inflamed colon, while IL-17F levels were insignificantly different from the DSS treated mice. Also, we observed up-regulation of IL-17A in G31P + LACT but not G31P treated mice if compared with Control group. The treatments ameliorated colonic fibrosis by reducing VEGF, TGF-ß, MMP-2 and MMP-9. In addition, we observed elevated levels of E-cadherin, and marginal up-regulation of occludin, suggesting the role of the treatments in regulating tight intestinal junction and adherence proteins. Mechanism-wise, G31P interferes with AKT and ERK signaling pathways. Our study suggests that G31P confers protection in IBD, particularly UC, and when G31P treatment is augmented with Lactobacillus acidophilus, the protection is variably enhanced.

Pharmacological efficacy of FGF21 analogue, liraglutide and insulin glargine in treatment of type 2 diabetes.

Fibroblast growth factor 21 (FGF21) is a promising regulator of glucose and lipid metabolism with multiple beneficial effects including hypoglycemic and lipid-lowering. Previous studies have reported that FGF21 is expected to become a new drug for treatment of diabetes. Liraglutide and insulin glargine are the two representative anti-diabetic biological drugs. In the current study, we aim to compare the long-term pharmacological efficacy of mFGF21 (an FGF21 analogue), liraglutide and insulin glargine in type 2 diabetic db/db mice. Db/db mice were initially treated with three kinds of proteins (25nmol/kg/day) by subcutaneous injection once a day for 4weeks, then subsequently be treated with once every two days for next 4weeks. After 8weeks of treatments, the blood glucose levels, body weights, glycosylated hemoglobin levels, fasting insulin levels, serum lipid profiles, hepatic biochemical parameters, oral glucose tolerance tests and hepatic mRNA expression levels of several proteins (GK, G6P, GLUT-1 and GLUT-4) associated with glucose metabolism of the experimental mice were detected. Results demonstrated that three proteins could significantly decrease the fed blood glucose levels of db/db mice. After treatment for 1week, the fed blood glucose levels of db/db mice in liraglutide group were significantly lower than those in mFGF21 and insulin glargine groups. However, after 2weeks of administration, the long-lasting hypoglycemic effect of mFGF21 was superior to liraglutide and insulin glargine up to the end of the experiments. Compared with liraglutide and insulin glargine, mFGF21 significantly reduced the glycosylated hemoglobin levels and improved the ability on glycemic control, insulin resistance, serum lipid and liver function states in db/db mice after 8weeks treatments. In addition, mFGF21 regulated glucose metabolism through increasing the mRNA expression levels of GK and GLUT-1, and decreasing the mRNA expression level of G6P. But liraglutide and insulin glargine could only up-regulate the mRNA expression of GLUT-4. In summary, as a hypoglycemic drug for long-term treatment, mFGF21 has the potential to be an ideal drug candidate for the therapy of type 2 diabetes.

Targeting mutant p53 in cancer: a long road to precision therapy.

The TP53 tumor suppressor is the most frequently mutated gene in human cancers. In recent years, a blooming of research efforts based on both cell lines and mouse models have highlighted how deeply mutant p53 proteins affect fundamental cellular pathways with cancer-promoting outcomes. Neomorphic mutant p53 activities spread over multiple levels, impinging on chromatin structure, transcriptional regulation and microRNA maturation, shaping the proteome and the cell's metabolic pathways, and also exerting cytoplasmic functions and displaying cell-extrinsic effects. These tumorigenic activities are inextricably linked with the blend of highly corrupted processes that characterize the tumor context. Recent studies indicate that successful strategies to extract core aspects of mutant p53 oncogenic potential and to identify unique tumor dependencies entail the superimposition of large-scale analyses performed in multiple experimental systems, together with a mindful use of animal models. This will hopefully soon lead to the long-awaited inclusion of mutant p53 as an actionable target of clinical antitumor therapies.

Melittin-MIL-2 fusion protein as a candidate for cancer immunotherapy.

BACKGROUND: Cytokine fusion protein that modulates the immune response holds great potential for cancer immunotherapy. IL-2 is an effective treatment against advanced cancers. However, the therapeutic efficacy of IL-2 is limited by severe systemic toxicity. Several mutants recombinant IL-2 can increase antitumor activity and minimize systemic toxicity. Melittin is an attractive anticancer candidate because of its wide-spectrum lytic properties. We previously generated a bifunctional fusion protein melittin-MIL-2, composed of melittin and a mutant IL-2. The melittin-MIL-2 inhibited the growth of human ovarian cancer SKOV3 cells in vitro and in vivo tumor growth. However, whether this antitumor effect could also be used in cancer immunotherapy was unknown. To assess its cancer immunotherapy potential, we further investigated its more effective antitumor immune response and antitumor effect against cancers of different tissue origins in vitro and in vivo. METHODS: The specific IL-2 activity of the melittin-MIL-2 fusion protein was tested on the cytokine growth dependent cell line CTLL-2. The cytolytic activity was detected by standard 4-h (51)Cr-release assays. PBMC stimulation in response to the melittin-MIL-2 was determined by IFN-γ release assay. We observed the cancer cell proliferation of different tissue origins by MTT assay. The ability of melittin-MIL-2 to inhibit tumor growth in vivo was evaluated by using human liver (SMMC-7721 cancer cells), lung (A549 cancer cells) and ovarian (SKOV3 cancer cells) cancer xenograft models. To assess the immunity within the tumor microenvironment, the level of some cytokines including IFN-γ, TNF-α, IL-12 and IL-4 was analyzed by ELISA. We injected the MDA-MB-231 cells and the melittin-MIL-2 into mice, and the anti-metastatic effect was examined by counting nodules in the lung. RESULTS: The melittin-MIL-2 was more effective in inducing T cell and NK-cell cytotoxicity. The fusion protein significantly increased IFN-γ production in PBMCs. In vitro, the melittin-MIL-2 mediated immune cells killing or directly killed the cancer cell lines of different tissue origins. In vivo, the fusion protein exhibited stronger inhibition on the growth of transplanted human tumors compared to rIL-2. The melittin-MIL-2 treatment promoted the IFN-γ secretion in tumor tissues and decreased the immunosuppressive cells in vivo. Furthermore, the fusion protein reduced lung metastasis of breast cancer. CONCLUSIONS: This study provides the evidence that the melittin-MIL-2 can produce stronger immune stimulation and antitumor effects, and the fusion protein is a potent candidate for cancer immunotherapy.

Activated protein C protects against renal ischaemia/reperfusion injury, independent of its anticoagulant properties.

Acute renal failure, a serious condition characterised by a drastic decline in renal function, often follows ischaemia/reperfusion (I/R) episodes. I/R is characterised by necrosis, inflammation and activation of coagulation, in concert causing renal tissue damage. In this context, activated protein C (APC) might be of importance in the pathogenesis of renal I/R. APC is a serine protease which has anticoagulant but also several anti-inflammatory and cytoprotective effects such as protection of endothelial barrier function. It was our objective to study the role of cytoprotective and anticoagulant functions of APC during renal I/R. C57BL/6j mice subjected to renal I/R were treated with intraperitoneally injected exogenous human APC, or two mutant forms of APC (200 µg/kg) which specifically lack anticoagulant or signalling properties. In a different experiment mice received specific monoclonal antibodies (20 mg/kg) that block the cytoprotective and/or anticoagulant properties of endogenous APC. Treatment with APC reduced tubular injury and enhanced renal function without altering the inflammatory response and did reduce renal fibrin deposition. Administration of APC mutant lacking anticoagulant properties reduced renal damage and enhanced renal function. Blocking the anticoagulant and cytoprotective functions of endogenous APC resulted in elevated tubular damage and reduced tubular cell proliferation, however, without influencing renal function or the inflammatory response. Furthermore, blocking both the anticoagulant and cytoprotective effects of APC resulted in dramatic renal interstitial haemorrhage, indicative of impaired vascular integrity. Blocking only the anticoagulant function of APC did not result in interstitial bleeding. In conclusion, the renoprotective effect of APC during I/R is independent of its anticoagulant properties.

Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging.

Signaling through the immune checkpoint programmed cell death protein-1 (PD-1) enables tumor progression by dampening antitumor immune responses. Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer. However, antibodies have inherent limitations that can curtail their efficacy in this setting, including poor tissue/tumor penetrance and detrimental Fc-effector functions that deplete immune cells. To determine if PD-1:PD-L1-directed immunotherapy could be improved with smaller, nonantibody therapeutics, we used directed evolution by yeast-surface display to engineer the PD-1 ectodomain as a high-affinity (110 pM) competitive antagonist of PD-L1. In contrast to anti-PD-L1 monoclonal antibodies, high-affinity PD-1 demonstrated superior tumor penetration without inducing depletion of peripheral effector T cells. Consistent with these advantages, in syngeneic CT26 tumor models, high-affinity PD-1 was effective in treating both small (50 mm(3)) and large tumors (150 mm(3)), whereas the activity of anti-PD-L1 antibodies was completely abrogated against large tumors. Furthermore, we found that high-affinity PD-1 could be radiolabeled and applied as a PET imaging tracer to efficiently distinguish between PD-L1-positive and PD-L1-negative tumors in living mice, providing an alternative to invasive biopsy and histological analysis. These results thus highlight the favorable pharmacology of small, nonantibody therapeutics for enhanced cancer immunotherapy and immune diagnostics.

Preclinical Assessment of Mutant Human TRIM5α as an Anti-HIV-1 Transgene.

Current HIV-1 gene therapy approaches aim at stopping the viral life cycle at its earliest steps, such as entry or immediate postentry events. Among the most widely adopted strategies are CCR5 downregulation/knockout and the use of broadly neutralizing antibodies. However, the long-term efficacy and side effects are still unclear. TRIM5α is an interferon-stimulated restriction factor that can intercept incoming retroviruses within one hour of cytosolic entry and potently inhibit the infectivity of restriction-sensitive viruses. The human TRIM5α (TRIM5αhu) generally does not efficiently target HIV-1, but point mutations in its capsid-binding domain can confer anti-HIV-1 activity. Although the mechanisms by which TRIM5αhu mutants inhibit HIV-1 are relatively well understood, their characterization as potential transgenes for gene therapy is lacking. Additionally, previous reports of general immune activation by overexpression of TRIM5α have hindered its broad adoption as a potential transgene. Here we demonstrate the ability of the R332G-R335G TRIM5αhu mutant to efficiently restrict highly divergent HIV-1 strains, including Group O, as well as clinical isolates bearing cytotoxic T lymphocyte escape mutations. R332G-R335G TRIM5αhu efficiently protected human lymphocytes against HIV-1 infection, even when expressed at relatively low levels following lentiviral transduction. Most importantly, under these conditions Rhesus macaque TRIM5α (TRIM5αRh) and TRIM5αhu (wild-type or mutated) had no major effects on the NF-κB pathway. Transgenic TRIM5α did not modulate the kinetics of IκBα, JunB, and TNFAIP3 expression following TNF-α treatment. Finally, we show that human lymphocytes expressing R332G-R335G TRIM5αhu have clear survival advantages over unmodified parental cells in the presence of pathogenic, replication-competent HIV-1. These results support the relevance of R332G-R335G and other mutants of TRIM5αhu as candidate effectors for HIV-1 gene therapy.

Hemostatic agents of broad applicability produced by selective tuning of factor Xa zymogenicity.

There is a clinical need to develop safe therapeutic strategies to mitigate bleeding. Previously, we found that a novel zymogen-like factor Xa variant (FXa-I16L) was effective in correcting the coagulation defect in hemophilic mice. Here we expand the mutational framework to tune the FX(a) zymogen-like state. Alteration of FXa zymogenicity yields variants (V17M, I16L, I16M, V17T, V17S, and I16T) with a wide range (≤1000-fold) of reduced function toward physiologic substrates and inhibitors. The extent of zymogen-like character, including resistance to antithrombin III, correlates well with plasma half-life (<2 minutes to >4 hours). Importantly, biologic function, including that of the most zymogen-like variant (FXa-I16T), was greatly enhanced when bound to FVa membranes. This resulted in improvement of clotting times and thrombin generation in hemophilic plasma. The FXa variants were remarkably effective in mouse injury models. In these systems, the data show that the more active the protease, the more difficult it is to overcome the protective mechanism of circulating inhibitors to achieve a therapeutic benefit. Depending on the treatment situation, the more zymogen-like variants (V17S and I16T) were most useful when given before injury whereas variants exhibiting greater activity but shorter half-lives (I16L and I16M) were most effective when administered after injury. This new class of FXa variants provides a useful and flexible platform for selectively bioengineering biologic function and half-life to target different clinical bleeding scenarios.

Protective and therapeutic effects of the resuscitation-promoting factor domain and its mutants against Mycobacterium tuberculosis in mice.

The resuscitation-promoting factor (Rpf), a secretory protein first reported in Micrococcus luteus, plays a critical role in mycobacterial survival and infection. There are five functionally redundant Rpf-like proteins identified in M. tuberculosis (Mtb). All these Rpfs share a conserved Rpf domain (Rpfd) composed of approximately 70 amino acids, which possesses the same biological functions as the full-length Rpf protein. Glutamic acid at position 54 in Rpfd (E54) has been implicated in mediating multiple physiological processes, and a single amino acid substitution at residue E54 can affect the protein biological activity. In order to determine the effects of different amino acid substitutions of E54 in Rpfd on its immunogenic activity, we generated three recombinant Rpfd mutants, Rpfd1 (E54K), Rpfd2 (E54A) and Rpfd3 (E54K and D48A), based on T-cell epitope prediction and tested their potential protective/therapeutic effects against Mtb in mice. Our results demonstrated that replacement of E54 by different amino acids in Rpfd distinctively influenced its resuscitation-promoting activities and Th1-type immune responses induced in mice. Administration of Rpfd2 mutant enhanced Th1-type cellular responses (IFN-γ and IL-2) in mice (P < 0.05, Rpfd2 versus control) and provided effective protection against Mtb in mice by significantly inhibiting the growth of Mtb during the initial stage of infection. Four weeks after the challenge, the slightest pathological injury in lung was observed in the Rpfd2-immunized group among all three Rfpd mutant-immunized groups. Furthermore, Rpfd2 therapy significantly decreased the bacterial load in lung and alleviated histopathological damage in Mtb-infected mice. Together, our results suggest Rpfd2 as a novel effective vaccine candidate against Mtb.

AAV liver expression of FIX-Padua prevents and eradicates FIX inhibitor without increasing thrombogenicity in hemophilia B dogs and mice.

Emerging successful clinical data on gene therapy using adeno-associated viral (AAV) vector for hemophilia B (HB) showed that the risk of cellular immune response to vector capsid is clearly dose dependent. To decrease the vector dose, we explored AAV-8 (1-3 × 10(12) vg/kg) encoding a hyperfunctional factor IX (FIX-Padua, arginine 338 to leucine) in FIX inhibitor-prone HB dogs. Two naïve HB dogs showed sustained expression of FIX-Padua with an 8- to 12-fold increased specific activity reaching 25% to 40% activity without antibody formation to FIX. A third dog with preexisting FIX inhibitors exhibited a transient anamnestic response (5 Bethesda units) at 2 weeks after vector delivery following by spontaneous eradication of the antibody to FIX by day 70. In this dog, sustained FIX expression reached ∼200% and 30% of activity and antigen levels, respectively. Immune tolerance was confirmed in all dogs after challenges with plasma-derived FIX concentrate. Shortening of the clotting times and lack of bleeding episodes support the phenotypic correction of the severe phenotype, with no clinical or laboratory evidence of risk of thrombosis. Provocative studies in mice showed that FIX-Padua exhibits similar immunogenicity and thrombogenicity compared with FIX wild type. Collectively, these data support the potential translation of gene-based strategies using FIX-Padua for HB.

Targeting ErbB-2 nuclear localization and function inhibits breast cancer growth and overcomes trastuzumab resistance.

Membrane overexpression of ErbB-2/HER2 receptor tyrosine kinase (membrane ErbB-2 (MErbB-2)) has a critical role in breast cancer (BC). We and others have also shown the role of nuclear ErbB-2 (NErbB-2) in BC, whose presence we identified as a poor prognostic factor in MErbB-2-positive tumors. Current anti-ErbB-2 therapies, as with the antibody trastuzumab (Ttzm), target only MErbB-2. Here, we found that blockade of NErbB-2 action abrogates growth of BC cells, sensitive and resistant to Ttzm, in a scenario in which ErbB-2, ErbB-3 and Akt are phosphorylated, and ErbB-2/ErbB-3 dimers are formed. Also, inhibition of NErbB-2 presence suppresses growth of a preclinical BC model resistant to Ttzm. We showed that at the cyclin D1 promoter, ErbB-2 assembles a transcriptional complex with Stat3 (signal transducer and activator of transcription 3) and ErbB-3, another member of the ErbB family, which reveals the first nuclear function of ErbB-2/ErbB-3 dimer. We identified NErbB-2 as the major proliferation driver in Ttzm-resistant BC, and demonstrated that Ttzm inability to disrupt the Stat3/ErbB-2/ErbB-3 complex underlies its failure to inhibit growth. Furthermore, our results in the clinic revealed that nuclear interaction between ErbB-2 and Stat3 correlates with poor overall survival in primary breast tumors. Our findings challenge the paradigm of anti-ErbB-2 drug design and highlight NErbB-2 as a novel target to overcome Ttzm resistance.

Novel superactive leptin antagonists and their potential therapeutic applications.

Random mutagenesis of mouse leptin antagonist (L39A/D40A/F41) followed by selection of high-affinity mutants by yeastsurface display indicated that replacing residue D23 with a non-negatively charged amino acid (most specifically with Leu) leads to dramatically enhanced affinity of leptin toward LEPR leading to development of superactive mouse, human, ovine and rat leptin antagonists (D23L/L39A/D40A/F41A). Superactive leptin antagonist mutants of mouse, human, rat or ovine leptins were developed in our laboratory, expressed in E. coli, refolded and purified to homogeneity as monomeric proteins. Pegylation of leptin antagonists resulted in potent and effective long-acting reagents suitable for in vivo studies or therapies. In the present review we explain the mechanism of leptin inhibition and summarize the possible use of leptin antagonists as possible leptin blockers in various human pathologies such as antiinflammatory and anti-autoimmune diseases, uremic cachexia, and cancer. We also suggest the use of leptin antagonists as research reagents for creation of a novel, fast and reversible model of T2DM in mice.

Incorporation of the factor IX Padua mutation into FIX-Triple improves clotting activity in vitro and in vivo.

Using gain-of-function factor IX (FIX) for replacement therapy for haemophilia B (HB) is an attractive strategy. We previously reported a high-activity FIX, FIX-Triple (FIX-V86A/E277A/R338A) as a good substitute for FIX-WT (wild-type) in protein replacement therapy, gene therapy, and cell therapy. Here we generated a new recombinant FIX-TripleL (FIX-V86A/E277A/R338L) by replacing the alanine at residue 338 of FIX-Triple with leucine as in FIX-Padua (FIX-R338L). Purified FIX-TripleL exhibited 22-fold higher specific clotting activity and 15-fold increased binding affinity to activated FVIII compared to FIX-WT. FIX-TripleL increased the therapeutic potential of FIX-Triple by nearly 100% as demonstrated with calibrated automated thrombogram and thromboelastography. FIX-TripleL demonstrated a normal clearance rate in HB mice. The clotting activity of FIX-TripleL was consistently 2- to 3-fold higher in these mice than that of FIX-Triple or FIX-R338L. Gene delivery of adeno-associated virus (AAV) in HB mice showed that FIX-TripleL had 15-fold higher specific clotting activity than FIX-WT, and this activity was significantly better than FIX-Triple (10-fold) or FIX-R338L (6-fold). At a lower viral dose, FIX-TripleL improved FIX activity from sub-therapeutic to therapeutic levels. Under physiological conditions, no signs of adverse thrombotic events were observed in long-term AAV-FIX-treated C57Bl/6 mice. Hepatocellular adenomas were observed in the high- but not the medium- or the low-dose AAV-treated mice expressing FIX-WT or FIX-Triple, indicating the advantages of using hyperfunctional FIX variants to reduce viral doses while maintaining therapeutic clotting activity. Thus, incorporation of the FIX Padua mutation significantly improves the clotting function of FIX-Triple so as to optimise protein replacement therapy and gene therapy.

Mutant HSP70 reverses autoimmune depigmentation in vitiligo.

Vitiligo is an autoimmune disease characterized by destruction of melanocytes, leaving 0.5% of the population with progressive depigmentation. Current treatments offer limited efficacy. We report that modified inducible heat shock protein 70 (HSP70i) prevents T cell-mediated depigmentation. HSP70i is the molecular link between stress and the resultant immune response. We previously showed that HSP70i induces an inflammatory dendritic cell (DC) phenotype and is necessary for depigmentation in vitiligo mouse models. Here, we observed a similar DC inflammatory phenotype in vitiligo patients. In a mouse model of depigmentation, DNA vaccination with a melanocyte antigen and the carboxyl terminus of HSP70i was sufficient to drive autoimmunity. Mutational analysis of the HSP70i substrate-binding domain established the peptide QPGVLIQVYEG as invaluable for DC activation, and mutant HSP70i could not induce depigmentation. Moreover, mutant HSP70iQ435A bound human DCs and reduced their activation, as well as induced a shift from inflammatory to tolerogenic DCs in mice. HSP70iQ435A-encoding DNA applied months before spontaneous depigmentation prevented vitiligo in mice expressing a transgenic, melanocyte-reactive T cell receptor. Furthermore, use of HSP70iQ435A therapeutically in a different, rapidly depigmenting model after loss of differentiated melanocytes resulted in 76% recovery of pigmentation. Treatment also prevented relevant T cells from populating mouse skin. In addition, ex vivo treatment of human skin averted the disease-related shift from quiescent to effector T cell phenotype. Thus, HSP70iQ435A DNA delivery may offer potent treatment opportunities for vitiligo.