The designation,synthesis, and affinity determination of affinity peptide for anthrax protective antigen

Detection of anthrax protective antigen is an effective way to diagnose anthracnose, and it plays an important part in the treatment of anthracnose. Affinity peptides, as a miniature biological recognition element, can quickly and effectively detect anthrax protective antigens. Based on computer-aided design technology (CAD), we have herein developed an affinity peptide design strategy for the detection of anthrax protective antigens. Firstly, six high-value mutation sites were determined based on the molecular docking between the template peptide and the receptor, and then the multi-site mutation of amino acids was carried out in order to establish a virtual peptide library. The library was selected by using molecular dynamics simulation and the best designed affinity peptide (code: P24) was found. The theoretical affinity with P24 peptide has increased by 19.8% compared with template peptide. Finally, the affinity with P24 peptide was measured by SPR technology to reach the nanomole level, which verified the effectiveness of the design strategy. The newly designed affinity peptide is expected to be used in the diagnosis of anthracnose.     

Antibodies against anthrax: mechanisms of action and clinical applications

B. anthracis is a bioweapon of primary importance and its pathogenicity depends on its lethal and edema toxins, which belong to the A-B model of bacterial toxins, and on its capsule. These toxins are secreted early in the course of the anthrax disease and for this reason antibiotics must be administered early, in addition to other limitations. Antibodies (Abs) may however neutralize those toxins and target this capsule to improve anthrax treatment, and many Abs have been developed in that perspective. These Abs act at various steps of the cell intoxication and their mechanisms of action are detailed in the present review, presented in correlation with structural and functional data. The potential for clinical application is discussed for Abs targeting each step of entry, with four of these molecules already advancing to clinical trials. Paradoxically, certain Abs may also enhance the lethal toxin activity and this aspect will also be presented. The unique paradigm of Abs neutralizing anthrax toxins thus exemplifies how they may act to neutralize A-B toxins and, more generally, be active against infectious diseases.     

Toll-like receptor 4 knockout protects against anthrax lethal toxin-induced cardiac contractile dysfunction: role of autophagy

BACKGROUND AND PURPOSE

Anthrax lethal toxin (LeTx) is known to induce circulatory shock and death, although the underlying mechanisms have not been elucidated. This study was designed to evaluate the role of toll-like receptor 4 (TLR4) in anthrax lethal toxin-induced cardiac contractile dysfunction.

EXPERIMENTAL APPROACH

Wild-type (WT) and TLR4 knockout (TLR^−/−) mice were challenged with lethal toxin (2 µg·g⁻¹, i.p.), and cardiac function was assessed 18 h later using echocardiography and edge detection. Small interfering RNA (siRNA) was employed to knockdown TLR4 receptor or class III PI3K in H9C2 myoblasts. GFP–LC3 puncta was used to assess autophagosome formation. Western blot analysis was performed to evaluate autophagy (LC3, Becline-1, Agt5 and Agt7) and endoplasmic reticulum (ER) stress (BiP, eIF2α and calreticulin).

KEY RESULTS

In WT mice, lethal toxin exposure induced cardiac contractile dysfunction, as evidenced by reduced fractional shortening, peak shortening, maximal velocity of shortening/re-lengthening, prolonged re-lengthening duration and intracellular Ca²⁺ derangement. These effects were significantly attenuated or absent in the TLR4 knockout mice. In addition, lethal toxin elicited autophagy in the absence of change in ER stress. Knockdown of TLR4 or class III PI3 kinase using siRNA but not the autophagy inhibitor 3-methyladenine significantly attenuated or inhibited lethal toxin-induced autophagy in H9C2 cells.

CONCLUSION AND IMPLICATIONS

Our results suggest that TLR4 may be pivotal in mediating the lethal cardiac toxicity induced by anthrax possibly through induction of autophagy. These findings suggest that compounds that negatively modulate TLR4 signalling and autophagy could be used to treat anthrax infection-induced cardiovascular complications.     

Small molecule inhibitors of histone deacetylase

This patent deals with novel histone deacetylase (HDAC) inhibitors and methods of their use for treating cell proliferative diseases or conditions, such as cancer, restenosis and psoriasis. Claimed herein are pharmaceutical compositions comprising small molecule HDAC inhibitors as the active ingredient, optionally combined with the use of an antisense oligonucleotide that inhibits HDAC expression. The claimed compounds were synthesised according to given reaction schemes and tested for their inhibition of HDAC enzymatic activity using nuclear extracts from cancer cells (pooled HDACs) and recombinant human HDAC1. An exemplified compound is stated to have caused a significant reduction in tumour weight and volume relative to saline-treated controls in BALB/c nude mice subcutaneously bearing A549 human lung carcinoma cells.     

Passive Immunotherapy Protects against Enteric Invasion and Lethal Sepsis in a Murine Model of Gastrointestinal Anthrax

The principal portal for anthrax infection in natural animal outbreaks is the digestive tract. Enteric exposure to anthrax, which is difficult to detect or prevent in a timely manner, could be exploited as an act of terror through contamination of human or animal food. Our group has developed a novel animal model of gastrointestinal (GI) anthrax for evaluation of disease pathogenesis and experimental therapeutics, utilizing vegetative Bacillus anthracis (Sterne strain) administered to A/J mice (a complement-deficient strain) by oral gavage. We hypothesized that a humanized recombinant monoclonal antibody (mAb) * that neutralizes the protective antigen (PA) component of B. anthracis lethal toxin (LT) and edema toxin (ET) could be an effective treatment. Although the efficacy of this anti-anthrax PA mAb has been shown in animal models of inhalational anthrax, its activity in GI infection had not yet been ascertained. We hereby demonstrate that passive immunotherapy with anti-anthrax PA mAb, administered at the same time as gastrointestinal exposure to B. anthracis, prevents lethal sepsis in nearly all cases (>90%), while a delay of up to forty-eight hours in treatment still greatly reduces mortality following exposure (65%). Moreover, passive immunotherapy protects against enteric invasion, associated mucosal injury and subsequent dissemination by gastrointestinal B. anthracis, indicating that it acts to prevent the initial stages of infection. * Expired raxibacumab being cycled off the Strategic National Stockpile; biological activity confirmed by in vitro assay.     

The cytotoxic effect of anthrax lethal toxin on human lung cells in vitro and the protective action of bovine antibodies to PA and LF

The excretion of protein toxins by vegetative cells of Bacillus anthracis is critical to the development of the lethal consequences of anthrax, particularly inhalational anthrax. Whilst the lung macrophages and other phagocytic cells transfer the spores from the lung cavities into the lymphatic system, and provide an initial germination site for the proliferation of the vegetative cells, it appears that much of the tissue pathology at the time of the host's death could be due to the action of the toxins, especially lethal toxin-protective antigen (PA) plus lethal factor (LF). The widespread tissue oedema and hypoxia may in part reflect a direct attack by lethal toxin on vascular endothelial cells. Also the distribution of the receptor for PA on a variety of cell types including epithelial cells as well as endothelial cells, and the involvement of the lungs in the pathology raises the question of whether lung epithelial cells are also susceptible to lethal toxin. To investigate this possibility a series of in vitro cytotoxicity experiments were carried out with human lung epithelial cells and microvascular endothelial cells. In these experiments lethal toxin (PA 500 ng ml(-1) plus 10-100 ng ml(-1) LF) was shown to cause a progressive loss of cell viability that developed slowly over at least 3 days. Affinity purified bovine colostrum antibodies for both PA and LF were equally effective in providing a 100% protection for epithelial cells from this cytotoxic action of lethal toxin. This was achieved at a 10:1 molar ratio of the particular antibody to its respective target.     

小分子PIM激酶抑制剂的研究进展

PIM激酶家族在各类肿瘤中高表达,并对肿瘤的发生发展起着重要的调节作用,闪此PIM激酶有望成为抗癌药物的新靶点,小分子PIM激酶抑制剂具有良好的应用前景。本文从PIM激酶家族蛋门结构、在肿瘤发生发展中的作用途径以及小分子PIM激酶抑制剂的研究进展j个方面进行综述。     

Small Molecule Inhibitors of Regulator of G Protein Signalling (RGS) Proteins

Recently regulators of G protein signalling (RGS) proteins have emerged as potential therapeutic targets since they provide an alternative method of modulating the activity of GPCRs, the target of so many drugs. Inhibitors of RGS proteins must block a protein-protein interaction (RGS-Gα), but also be cell and, depending on the therapeutic target, blood brain barrier permeable. A lead compound (1a) was identified as an inhibitor of RGS4 in a screening assay and this has now been optimised for activity, selectivity and solubility. The newly developed ligands (11b, 13) display substantial selectivity over the closely related RGS8 protein, lack the off-target calcium mobilisation activity of the lead 1a and have excellent aqueous solubility. They are currently being evaluated in vivo in rodent models of depression.     

Small molecule inhibitors in the treatment of cerebral ischemia

Introduction: Stroke is the world's second leading cause of death. Although recombinant tissue plasminogen activator is an effective treatment for cerebral ischemia, its limitations and ischemic stroke's complex pathophysiology dictate an increased need for the development of new therapeutic interventions. Small molecule inhibitors (SMIs) have the potential to be used as novel therapeutic modalities for stroke, since many preclinical and clinical trials have established their neuroprotective capabilities.

Areas covered: This paper provides a summary of the pathophysiology of stroke as well as clinical and preclinical evaluations of SMIs as therapeutic interventions for cerebral ischemia. Cerebral ischemia is broken down into four mechanisms in this article: thrombosis, ischemic insult, mitochondrial injury and immune response. Insight is provided into preclinical and current clinical assessments of SMIs targeting each mechanism as well as a summary of reported results.

Expert opinion: Many studies demonstrated that pre- or post-treatment with certain SMIs significantly ameliorated adverse effects from stroke. Although some of these promising SMIs moved on to clinical trials, they generally failed, possibly due to the poor translation of preclinical to clinical experiments. Yet, there are many steps being taken to improve the quality of experimental research and translation to clinical trials.     

Small molecule c-Met kinase inhibitors: a review of recent patents

Importance of the field: c-Met kinase is the receptor for hepatocyte growth factor. Primarily expressed on epithelial and mesenchymal cells its normal function is associated with wound healing, liver regeneration and embryo development. However, dysregulation of c-Met through overexpression, gene amplification, mutation or a ligand-dependent autocrine/paracrine loop is associated with tumorigenesis. c-Met dysregulation in human cancer patients is typically associated with a poor prognosis, aggressive disease, increased metastasis and shortened patient survival. Targeting the hepatocyte growth factor/c-Met signalling pathway as a means of cancer therapy has, therefore, become increasingly popular with a number of different therapeutic approaches undergoing clinical trials.

Areas covered by this review: This review covers the patent applications for small molecule c-Met kinase inhibitors since 2007, attempts to place them in context from a structural point of view and examines their potential applications in cancer therapy.

What the reader will gain: Readers will gain an overview of the structural types of c-Met inhibitors, the major players in the field and an insight into what is progressing into the clinic.

Take home message: This area is developing rapidly and the results of the various ongoing clinical trials will generate an increased understanding of the potential benefits and pitfalls of c-Met inhibitors as therapeutic agents.     

Substituted 1,4-benzodiazepine-2,5-diones as alpha-helix mimetic antagonists of the HDM2-p53 protein-protein interaction

Small molecule antagonists of protein-protein interactions represent a particular challenge for pharmaceutical discovery. One approach to finding molecules that can disrupt these interactions is to seek mimics of common protein structure motifs. We present an analysis of how molecules based on the 1,4-benzodiazepine-2,5-dione scaffold serve to mimic the side-chains presented by the hydrophobic face of two turns of an alpha-helix derived from the tumor suppressor protein p53, and thus antagonize the HDM2-p53 protein-protein binding interaction.     

Mechanism of Action of Delta-Enodtoxin from Bacillus Thuringiensis

Abstract

ABSTRACE

The crystalline protein bodies (BB) prcdud by Bacillus thuringiensis consist of a delta-endotoxin having toxicity either for lepidopteran, dipteran, lepidopteran and dipteran, or coleopteran larvae. The delta-endotoxin toxic for lepidopteran or coleopteran larvae consists of two peptides of molecular weight (mol. wt.) 6.5 kilcdaltons (kd). The dipteran-specific deltaendotoxin consists of one peptide of mol. wt. 2.8 kd.

The lepidopteran-specific delta-endotoxin acts by causing swelling of midgut cells after ingestion of B B and elevation of the K⁺ concentration and pH of the hemolymph, in vivo. The cell swelling induced by the toxin is observed only in insect cells but not mammalian cells, in vitro. A proposal for the mechanism of action of the toxin is as follows: the toxin reacts with a receptor on the susceptible cell surface, inducing a cascade of biochemical reactions resulting in stimulation of Na⁺ influx through Na⁺ channels, K⁺ efflux through K⁺-leak channels arid Na⁺,K⁺-pump by Na⁺,K⁺-ATPase in the membrane. Due to the net Na⁺ and K⁺ influx insect cells swell and burst. In contrast, thedipteran-specific delta-endotoxin has cytolytic activity for insect and mammalian cells, in vitro, and is hemolytic for mammalian erythrocytes, neurotoxic for the cockroach sixth abnoraml ganglion and highly entomccidal for mosquito larvae.     

Small molecule tyrosine kinase inhibitors: clinical development of anticancer agents

Numerous small molecule synthetic tyrosine kinase inhibitors are in clinical development for the treatment of human cancers. These fall into three broad categories: inhibitors of the epidermal growth factor receptor tyrosine kinase family (e.g., Iressa? and Tarceva?), inhibitors of the split kinase domain receptor tyrosine kinase subgroup (e.g., PTK787/ZK 222584 and SU11248) and inhibitors of tyrosine kinases from multiple subgroups (e.g., Gleevec?). In addition, agents targeting other tyrosine kinases implicated in cancer, such as Met, Tie-2 and Src, are in preclinical development. As experience is gained in the clinic, it has become clear that unleashing the full therapeutic potential of tyrosine kinase inhibitors will require patient preselection, better assays to guide dose selection, knowledge of mechanism-based side effects and ways to predict and overcome drug resistance.     

Small molecule inhibitor of apoptosis proteins antagonists: a patent review

Introduction: The family of inhibitor of apoptosis proteins (IAPs) plays a key role in the suppression of proapoptotic signaling; hence, a small molecule that disrupts the binding of IAPs with their functional partner should restore apoptotic response to proapoptotic stimuli in cells. The continued publication of new patent applications of IAP antagonists over the past 4 years is a testament to the continued interest surrounding the IAP family of proteins.

Areas covered: This review summarizes the IAP antagonist patent literature from 2010 to 2014. Monovalent and bivalent Smac mimetics will be covered as well as two new developments in the field: IAP antagonists coupled to or merged with other targeted agents and new BIR2 selective IAP antagonists.

Expert opinion: In addition to the well-explored scaffolds for monovalent and bivalent Smac-mimetics, some companies have taken more drastic approaches to explore new chemical space – for example, fragment-based approaches and macrocyclic inhibitors. Furthermore, other companies have designed compounds with alternative biological profiles – tethering to known kinase binding structures, trying to target to the mitochondria or introducing selective binding to the BIR2 domain. An overview of the status for the four small molecule IAP antagonists being evaluated in active human clinical trials is also provided.     

Small molecule inhibitors of peptidoglycan synthesis targeting the lipid II precursor

Bacterial peptidoglycan glycosyltransferases (GTs) of family 51 catalyze the polymerization of the lipid II precursor into linear peptidoglycan strands. This activity is essential to bacteria and represents a validated target for the development of new antibacterials. Application of structure-based virtual screening to the National Cancer Institute library using eHits program and the structure of the glycosyltransferase domain of the Staphylococcus aureus penicillin-binding protein 2 resulted in the identification of two small molecules analogues 5, a 2-[1-[(2-chlorophenyl)methyl]-2-methyl-5-methylsulfanylindol-3-yl]ethanamine and 5b, a 2-[1-[(3,4-dichlorophenyl)methyl]-2-methyl-5-methylsulfanylindol-3-yl]ethanamine that exhibit antibacterial activity against several Gram-positive bacteria but were less active on Gram-negative bacteria. The two compounds inhibit the activity of five GTs in the micromolar range. Investigation of the mechanism of action shows that the compounds specifically target peptidoglycan synthesis. Unexpectedly, despite the fact that the compounds were predicted to bind to the GT active site, compound 5b was found to interact with the lipid II substrate via the pyrophosphate motif. In addition, this compound showed a negatively charged phospholipid-dependent membrane depolarization and disruption activity. These small molecules are promising leads for the development of more active and specific compounds to target the essential GT step in cell wall synthesis.     

Role of site-directed mutagenesis and adjuvants in the stability and potency of anthrax protective antigen

Anthrax is a zoonotic infection caused by the gram-positive, aerobic, spore-forming bacterium Bacillus anthracis. Depending on the origin of the infection, serious health problems or mortality is possible. The virulence of B. anthracis is reliant on three pathogenic factors, which are secreted upon infection: protective antigen (PA), lethal factor (LF), and edema factor (EF). Systemic illness results from LF and EF entering cells through the formation of a complex with the heptameric form of PA, bound to the membrane of infected cells through its receptor. The currently available anthrax vaccines have multiple drawbacks, and recombinant PA is considered a promising second-generation vaccine candidate. However, the inherent chemical instability of PA through Asn deamidation at multiple sites prevents its use after long-term storage owing to loss of potency. Moreover, there is a distinct possibility of B. anthracis being used as a bioweapon; thus, the developed vaccine should remain efficacious and stable over the long-term. Second-generation anthrax vaccines with appropriate adjuvant formulations for enhanced immunogenicity and safety are desired. In this article, using protein engineering approaches, we have reviewed the stabilization of anthrax vaccine candidates that are currently licensed or under preclinical and clinical trials. We have also proposed a formulation to enhance recombinant PA vaccine potency via adjuvant formulation.     

Novel peptide ligands of RGS4 from a focused one-bead, one-compound library

Regulators of G protein signaling accelerate GTP hydrolysis by G alpha subunits and profoundly inhibit signaling by G protein-coupled receptors. The distinct expression patterns and pathophysiologic regulation of regulators of G protein signaling proteins suggest that inhibitors may have therapeutic potential. We previously reported the design, mechanistic evaluation, and structure-activity relationships of a disulfide-containing cyclic peptide inhibitor of RGS4, YJ34 (Ac-Val-Lys-c[Cys-Thr-Gly-Ile-Cys]-Glu-NH(2), S-S) (Roof et al., Chem Biol Drug Des, 67, 2006, 266). Using a focused one-bead, one-compound peptide library that contains features known to be necessary for the activity of YJ34, we now identify peptides that bind to RGS4. Six peptides showed confirmed binding to RGS4 by flow cytometry. Two analogs of peptide 2 (Gly-Thr-c[Cys-Phe-Gly-Thr-Cys]-Trp-NH(2), S-S with a free or acetylated N-terminus) inhibited RGS4-stimulated G alpha(o) GTPase activity at 25-50 microM. They selectively inhibit RGS4 but not RGS7, RGS16, and RGS19. Their inhibition of RGS4 does not depend on cysteine-modification of RGS4, as they do not lose activity when all cysteines are removed from RGS4. Peptide 2 has been modeled to fit in the same binding pocket predicted for YJ34 but in the reverse orientation.     

Synthesis and isozyme selectivity of small molecule protein kinase C inhibitors: a review of patents

Protein kinase C (PKC) is a ubiquitously expressed family of kinases that have key roles in the transduction of signals mediated by the hydrolysis of phospholipids for cell proliferation, gene expression, migration, differentiation and a host of other functions. Each of the 12 isozymes of PKC govern both the positive and negative aspects of cell signalling with opposing effects at times. The roles of different PKC isozymes in various disease conditions is being elucidated and evaluated by researchers at present. The need has emerged for the development of PKC isozyme-specific modulators that have therapeutic value and also help in discovering the myriad signalling pathways governed by these isozymes. This review attempts to give a synopsis of the recent developments in the design and synthesis of such small molecule inhibitors covered by patents during the past five years.     

Enhancement of the immune responses of mice to <Emphasis Type="Italic">Bacillus anthracis</Emphasis> protective antigen by CIA07 combined with alum

Anthrax is an acute zoonotic disease caused by infection with Bacillus anthracis. B. anthracis spores are highly resistant to environmental degradation and are used as a biological weapon. In this study, we investigated the adjuvant activity of CIA07 to anthrax protective antigen (PA). A/J mice were immunized intraperitoneally once, or twice with a 4-week interval, with recombinant PA alone or combined with alum, CpG1826, or CIA07 as adjuvant, and serum anti-PA IgG antibody responses were measured 4 weeks after each immunization. All three adjuvants significantly enhanced anti-PA IgG antibody titer 4 weeks after the priming and boosting immunizations, and alum gave the highest titer. In order to evaluate the adjuvant activity of CIA07 in the presence of alum, Balb/c mice were immunized 3 times at 1-week intervals with PA in combination with alum, CIA07 or alum plus CIA07, and the immune responses were assessed 2 weeks after the third immunization. The serum anti-PA IgG antibody titer of the CIA07-treated group was 14-fold higher than the group given PA alone, and the coadministration of CIA07 with alum further increased the titer 3.5-fold (P < 0.05). The toxin neutralizing activity of the sera from the mice given the combination of CIA07 and alum was 109-times higher than the animals given PA alone. The mice given CIA07 plus alum also showed a marked increase in the number of IFN-γ-, IL-2-, and IL-4-producing CD4⁺ T cells among their splenocytes. These data suggest the potential of CIA07 in combination with alum as an adjuvant for the development of a potent anthrax vaccine.