Sulfur(VI) Fluoride Exchange (SuFEx)-Enabled High-Throughput Medicinal Chemistry.

Optimization of small-molecule probes or drugs is a synthetically lengthy, challenging, and resource-intensive process. Lack of automation and reliance on skilled medicinal chemists is cumbersome in both academic and industrial settings. Here, we demonstrate a high-throughput hit-to-lead process based on the biocompatible sulfur(VI) fluoride exchange (SuFEx) click chemistry. A high-throughput screening hit benzyl (cyanomethyl)carbamate (Ki = 8 µM) against a bacterial cysteine protease SpeB was modified with a SuFExable iminosulfur oxydifluoride [RN═S(O)F2] motif, rapidly diversified into 460 analogs in overnight reactions, and the products were directly screened to yield drug-like inhibitors with 480-fold higher potency (Ki = 18 nM). We showed that the improved molecule is active in a bacteria-host coculture. Since this SuFEx linkage reaction succeeds on picomole scale for direct screening, we anticipate our methodology can accelerate the development of robust biological probes and drug candidates.

Targeting NLRP3 and Staphylococcal pore-forming toxin receptors in human-induced pluripotent stem cell-derived macrophages.

Staphylococcus aureus causes necrotizing pneumonia by secreting toxins such as leukocidins that target front-line immune cells. The mechanism by which leukocidins kill innate immune cells and trigger inflammation during S. aureus lung infection, however, remains unresolved. Here, we explored human-induced pluripotent stem cell-derived macrophages (hiPSC-dMs) to study the interaction of the leukocidins Panton-Valentine leukocidin (PVL) and LukAB with lung macrophages, which are the initial leukocidin targets during S. aureus lung invasion. hiPSC-dMs were susceptible to the leukocidins PVL and LukAB and both leukocidins triggered NLPR3 inflammasome activation resulting in IL-1ß secretion. hiPSC-dM cell death after LukAB exposure, however, was only temporarily dependent of NLRP3, although NLRP3 triggered marked cell death after PVL treatment. CRISPR/Cas9-mediated deletion of the PVL receptor, C5aR1, protected hiPSC-dMs from PVL cytotoxicity, despite the expression of other leukocidin receptors, such as CD45. PVL-deficient S. aureus had reduced ability to induce lung IL-1ß levels in human C5aR1 knock-in mice. Unexpectedly, inhibiting NLRP3 activity resulted in increased wild-type S. aureus lung burdens. Our findings suggest that NLRP3 induces macrophage death and IL-1ß secretion after PVL exposure and controls S. aureus lung burdens.

Receptor-Based Peptides for Inhibition of Leukotoxin Activity.

The Gram-negative bacterium Aggregatibacter actinomycetemcomitans, commonly associated with localized aggressive periodontitis (LAP), secretes an RTX (repeats-in-toxin) protein leukotoxin (LtxA) that targets human white blood cells, an interaction that is driven by its recognition of the lymphocyte function-associated antigen-1 (LFA-1) integrin. In this study, we report on the inhibition of LtxA-LFA-1 binding as an antivirulence strategy to inhibit LtxA-mediated cytotoxicity. Specifically, we designed and synthesized peptides corresponding to the reported LtxA binding domain on LFA-1 and characterized their capability to inhibit LtxA binding to LFA-1 and subsequent cytotoxic activity in human immune cells. We found that several of these peptides, corresponding to sequential ß-strands in the LtxA-binding domain of LFA-1, inhibit LtxA activity, demonstrating the effectiveness of this approach. Further investigations into the mechanism by which these peptides inhibit LtxA binding to LFA-1 reveal a correlation between toxin-peptide affinity and LtxA-mediated cytotoxicity, leading to a diminished association between LtxA and LFA-1 on the cell membrane. Our results demonstrate the possibility of using target-based peptides to inhibit LtxA activity, and we expect that a similar approach could be used to hinder the activity of other RTX toxins.

Inhibition of Cholera Toxin and Other AB Toxins by Polyphenolic Compounds.

Cholera toxin (CT) is an AB-type protein toxin that contains a catalytic A1 subunit, an A2 linker, and a cell-binding B homopentamer. The CT holotoxin is released into the extracellular environment, but CTA1 attacks a target within the cytosol of a host cell. We recently reported that grape extract confers substantial resistance to CT. Here, we used a cell culture system to identify twelve individual phenolic compounds from grape extract that inhibit CT. Additional studies determined the mechanism of inhibition for a subset of the compounds: two inhibited CT binding to the cell surface and even stripped CT from the plasma membrane of a target cell; two inhibited the enzymatic activity of CTA1; and four blocked cytosolic toxin activity without directly affecting the enzymatic function of CTA1. Individual polyphenolic compounds from grape extract could also generate cellular resistance to diphtheria toxin, exotoxin A, and ricin. We have thus identified individual toxin inhibitors from grape extract and some of their mechanisms of inhibition against CT.

Ranking Differential Drug Activities from Dose-Response Synthetic Lethality Screens.

Synthetic lethal screens are used to discover new combination treatments for cancer. In traditional high-throughput synthetic lethal screens, compounds are tested at a single dose, and hit selection is based on threshold activity values from the variance of the efficacy of the compounds tested. The limitation of the single-dose screening for synthetic lethal screens is that it does not allow for the robust detection of differential activities from compound collections with a broad range of potencies and efficacies. There is therefore a need to develop screening approaches that enable the identification of compounds with synthetic lethal effects based on changes in both potency and efficacy. Here we describe the implementation of a dose response-based synthetic lethal screen to find drugs that enhance or mitigate the cytotoxic effect of an immunotoxin protein (HA22). We developed a data analysis framework for the selection of compounds with enhancing or mitigating cytotoxic activities based on the use of dose-response parameters. The data analysis framework includes an ensemble ranking approach that allows the use of multiple dose-response parameters in a nonparametric fashion. Quantitative high-throughput screening (HTS) enables the identification of compounds with synthetic lethal activity not identified by single-dose HTS.

Inhibition of LtxA toxicity by blocking cholesterol binding with peptides.

The leukotoxin (LtxA) produced by Aggregatibacter actinomycetemcomitans kills host immune cells, allowing the bacterium to establish an ecological niche in the upper aerodigestive tract of its human host. The interaction of LtxA with human immune cells is both complex and multifaceted, involving membrane lipids as well as cell-surface proteins. In the initial encounter with the host cell, LtxA associates with lymphocyte function-associated antigen-1, a cell surface adhesion glycoprotein. However, we have also demonstrated that the toxin associates strongly with the plasma membrane lipids, specifically cholesterol. This association with cholesterol is regulated by a cholesterol recognition amino acid consensus (CRAC) motif, with a sequence of (334) LEEYSKR(340), in the N-terminal region of the toxin. Here, we have demonstrated that removal of cholesterol from the plasma membrane or mutation of the LtxA CRAC motif inhibits the activity of the toxin in THP-1 cells. To inhibit LtxA activity, we designed a short peptide corresponding to the CRAC(336) motif of LtxA (CRAC(336WT)). This peptide binds to cholesterol and thereby inhibits the toxicity of LtxA in THP-1 cells. Previously, we showed that this peptide inhibits LtxA toxicity against Jn.9 (Jurkat) cells, indicating that peptides derived from the cholesterol-binding site of LtxA may have a potential clinical applicability in controlling infections of repeats-in-toxin-producing organisms.

Inhibitory effect of totarol on exotoxin proteins hemolysin and enterotoxins secreted by Staphylococcus aureus.

Staphylococcus aureus (S. aureus) causes a wide variety of infections, which are of major concern worldwide. S. aureus produces multiple virulence factors, resulting in food infection and poisoning. These virulence factors include hyaluronidases, proteases, coagulases, lipases, deoxyribonucleases and enterotoxins. Among the extracellular proteins produced by S. aureus that contribute to pathogenicity, the exotoxins α-hemolysin, staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin B (SEB) are thought to be of major significance. Totarol, a plant extract, has been revealed to inhibit the proliferation of several pathogens effectively. However, there are no reports on the effects of totarol on the production of α-hemolysin, SEA or SEB secreted by S. aureus. The aim of this study was to evaluate the effects of totarol on these three exotoxins. Hemolysis assay, western blotting and real-time reverse transcriptase-PCR assay were performed to identify the influence of graded subinhibitory concentrations of totarol on the production of α-hemolysin and the two major enterotoxins, SEA and SEB, by S. aureus in a dose-dependent manner. Moreover, an enzyme linked immunosorbent assay showed that the TNF-α production of RAW264.7 cells stimulated by S. aureus supernatants was inhibited by subinhibitory concentrations of totarol. Form the data, we propose that totarol could potentially be used as a promising natural compound in the food and pharmaceutical industries.

Identification and Co-complex Structure of a New S. pyogenes SpeB Small Molecule Inhibitor.

The secreted Streptococcus pyogenes cysteine protease SpeB is implicated in host immune system evasion and bacterial virulence. We present a small molecule inhibitor of SpeB 2477 identified from a high-throughput screen based on the hydrolysis of a fluorogenic peptide substrate Ac-AIK-AMC. 2477 inhibits other SpeB-related proteases but not human caspase-3, suggesting that the molecule targets proteases with the papain-like structural fold. A 1.59 Å X-ray crystal structure of 2477 bound to the SpeB active site reveals the mechanism of inhibition and the essential constituents of 2477 necessary for binding. An assessment against a panel of 2477 derivatives confirms our structural findings and shows that a carbamate and nitrile on 2477 are required for SpeB inhibition, as these moieties provide an extensive network of electrostatic and hydrogen-bonding interactions with SpeB active site residues. Surprisingly, despite 2477 having a reduced inhibitory potential against papain, the majority of 2477-related compounds inhibit papain to a much greater and broader extent than SpeB. These findings indicate that SpeB is more stringently selective than papain for this panel of small molecule inhibitors. On the basis of our structural and biochemical characterization, we propose modifications to 2477 for subsequent rounds of inhibitor design that will impart specificity to SpeB over other papain-like proteases, including alterations of the compound to exploit the differences in CA protease active site pocket sizes and electrostatics.

Protective effects of bacterial osmoprotectant ectoine on bovine erythrocytes subjected to staphylococcal alpha-haemolysin.

Ectoine (ECT) is a bacterial compatible solute with documented protective action however no data are available on its effects on various cells against bacterial toxins. Therefore, we determined the in vitro influence of ECT on bovine erythrocytes subjected to staphylococcal α-haemolysin (HlyA). The cells exposed to HlyA alone showed a distinct haemolysis and reduced glutathione (GSH)/oxidised glutathione (GSSG) level, however the toxic effects were attenuated in the combinations of HlyA + ECT suggesting ECT-induced protection of erythrocytes from HlyA.

A peptide antagonist of CD28 signaling attenuates toxic shock and necrotizing soft-tissue infection induced by Streptococcus pyogenes.

Staphylococcus aureus and group A Streptococcus pyogenes (GAS) express superantigen (SAg) exotoxin proteins capable of inducing lethal shock. To induce toxicity, SAgs must bind not only to the major histocompatibility complex II molecule of antigen-presenting cells and the variable ß chain of the T-cell receptor but also to the dimer interface of the T-cell costimulatory receptor CD28. Here, we show that the CD28-mimetic peptide AB103 (originally designated "p2TA") protects mice from lethal challenge with streptococcal exotoxin A, as well as from lethal GAS bacterial infection in a murine model of necrotizing soft-tissue infection. Administration of a single dose of AB103 increased survival when given up to 5 hours after infection, reduced inflammatory cytokine expression and bacterial burden at the site of infection, and improved muscle inflammation in a dose-dependent manner, without compromising cellular and humoral immunity. Thus, AB103 merits further investigation as a potential therapeutic in SAg-mediated necrotizing soft-tissue infection.

Pretreatment with lipopolysaccharide ameliorates Pseudomonas exotoxin A-induced hepatotoxicity in rats.

CONTEXT: Liver injury can be induced by various hepatotoxicants, including Pseudomonas aeruginosa exotoxin A (PEA). Our previous study indicated that PEA-induced rat hepatotoxicity was T cells and Kupffer cells dependent. Several reports have demonstrated that non-toxic doses of bacterial lipopolysaccharide (LPS) can protect liver against the chemicals-induced toxicity such as acetaminophen and concanavalin-A. OBJECTIVE: This study aimed to investigate the protecting mechanisms of LPS on PEA-induced hepatotoxicity. RESULTS: Rats pretreated with LPS (40 µg/kg, 12 h before PEA admission) significantly decreased animal mortality, serum enzyme (ALT, AST and T-bil) activities, histopathological changes and hepatocytes apoptosis following challenge with PEA. The concentrations of tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ) and interleukin-2 (IL-2) were reduced, but IL-6 and IL-10 were increased in the serum. In addition, prior treatment of these LPS-pretreated rats with gadolinium chloride (GdCl3), a selective Kupffer cell depletion agent, markedly enhanced liver injury after PEA administration. In contrast, the pretreatment of LPS to T-cell deficient athymic nude rats still display significant attenuation of PEA-induced liver injury. This observation further confirmed our hypothesis that LPS ameliorate PEA-hepatotoxicity was through Kupffer cells but not T cells. Moreover, LPS-induced hepatoprotection ability was neutralized by co-treatment with anti-TNF-α antibodies, but not with anti-IFN-γ antibodies. Finally, replacement of LPS with RS-LPS (Rhodobacter sphaeroides LPS), a Toll like receptor-4 (TLR-4) antagonist, resulted in severe hepatotoxicity. CONCLUSION: These results suggested that Kupffer cells, TNF-α and TLR-4 play central mediator roles during the hepatoprotection against PEA-induced hepatotoxicity conferred by LPS.

3-aminopiperidine-based peptide analogues as the first selective noncovalent inhibitors of the bacterial cysteine protease IdeS.

A series of eight peptides corresponding to the amino acid sequence of the hinge region of IgG and 17 newly synthesized peptide analogues containing a piperidine moiety as a replacement of a glycine residue were tested as potential inhibitors of the bacterial IgG degrading enzyme of Streptococcus pyogenes , IdeS. None of the peptides showed any inhibitory activity of IdeS, but several piperidine-based analogues were identified as inhibitors. Two different analysis methods were used: an SDS-PAGE based assay to detect IgG cleavage products and a surface plasmon resonance spectroscopy based assay to quantify the degree of inhibition. To investigate the selectivity of the inhibitors for IdeS, all compounds were screened against two other related cysteine proteases (SpeB and papain). The selectivity results show that larger analogues that are active inhibitors of IdeS are even more potent as inhibitors of papain, whereas smaller analogues that are active inhibitors of IdeS inhibit neither SpeB nor papain. Two compounds were identified that exhibit high selectivity against IdeS and will be used for further studies.

A quinol peroxidase inhibitor prevents secretion of a leukotoxin from Aggregatibacter actinomycetemcomitans.

BACKGROUND AND OBJECTIVE: Quinol peroxidase (QPO) catalyzes peroxidase activity using quinol in the respiratory chain as a substrate. Quinol peroxidase is essential for the secretion of leukotoxin (LtxA), which destroys leukocytes and erythrocytes in humans and is one of the major virulence factors of Aggregatibacter actinomycetemcomitans, which is associated with localized aggressive periodontitis. In the present study, we aimed to find a highly potent QPO inhibitor to attenuate the virulence of A. actinomycetemcomitans. MATERIAL AND METHODS: For screening of QPO inhibitors, QPO activity was measured kinetically by SpectraMax Plus with 96-well UV plates. Three hundred compounds in the Kitasato Institute for Life Sciences Chemical Library were screened. Secretion of LtxA in the culture supernatant was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cytotoxicity against human promyelocytic leukemia cell line (HL-60) cells from the culture supernatant was measured by Trypan Blue exclusion test. RESULTS: The present study characterized ascofuranone as a highly potent inhibitor of QPO (K(i) = 9.557 +/- 0.865 nm). Ascofuranone inhibited secretion of LtxA by A. actinomycetemcomitans in a dose-dependent manner, making A. actinomycetemcomitans less pathogenic to HL-60 cells. CONCLUSION: Quinol peroxidase inhibitors are promising candidates as alternative drugs for the treatment and prevention of the onset of localized aggressive periodontitis. Using ascofuranone as a seed compound, further study of QPO inhibitors could provide novel chemotherapeutic strategies for controlling localized aggressive periodontitis.

Quantitative high-throughput screening identifies inhibitors of anthrax-induced cell death.

Here, we report the results of a quantitative high-throughput screen (qHTS) measuring the endocytosis and translocation of a beta-lactamase-fused-lethal factor and the identification of small molecules capable of obstructing the process of anthrax toxin internalization. Several small molecules protect RAW264.7 macrophages and CHO cells from anthrax lethal toxin and protected cells from an LF-Pseudomonas exotoxin fusion protein and diphtheria toxin. Further efforts demonstrated that these compounds impaired the PA heptamer pre-pore to pore conversion in cells expressing the CMG2 receptor, but not the related TEM8 receptor, indicating that these compounds likely interfere with toxin internalization.

Abrogation of streptococcal pyrogenic exotoxin B-mediated suppression of phagocytosis in U937 cells by Cordyceps sinensis mycelium via production of cytokines.

Streptococcal pyrogenic exotoxin B (SPE B) is a virulent factor in group A streptococcal infection. We previously showed that SPE B reduced phagocytosis in human monocytic U937 cells. Here we show that the mycelium extract of Cordyceps sinensis (CS), a Chinese immunomodulatory herbal medicine, increased phagocytosis in U937 cells. Neither heat nor trypsin pretreatment prevented CS extract from causing this increase. Further studies indicated that SPE B-mediated suppression of U937 cell phagocytic activity was abrogated by CS extract. Factors in the conditioned medium from CS-extract-treated U937 cells were responsible for blocking the SPE B-mediated suppression of phagocytosis. Heating the conditioned medium eliminated the increase, which suggested that the U937-cell protein products augmented phagocytosis. Analyzing cytokine mRNA expression of U937 cells revealed increases in interferon-gamma (IFN-gamma), interleukin (IL)-12 p35 and p40, and tumor necrosis factor-alpha (TNF-alpha), but not in IL-1beta, IL-6, or IL-8. Treating U937 cells with anti-IFN-gamma, IL-12, and TNF-alpha antibodies also eliminated the conditioned medium-induced increase in phagocytosis. Taken together, SPE B inhibited phagocytosis, but CS mycelium extract abrogated this inhibition by causing cytokine production.

Cisplatin inhibition of anthrax lethal toxin.

Bacillus anthracis lethal toxin (LT) produces symptoms of anthrax in mice and induces rapid lysis of macrophages derived from certain inbred strains. LT is comprised of a receptor binding component, protective antigen (PA), which delivers the enzymatic component, lethal factor (LF), into cells. We found that mouse macrophages were protected from toxin by the antitumor drug cis-diammineplatinum (II) dichloride (cisplatin). Cisplatin was shown to inhibit LT-mediated cleavage of cellular mitogen-activated protein kinases (MEKs) without inhibiting LF's in vitro proteolytic activity. Cisplatin-treated PA lost 100% of its ability to function in toxicity assays when paired with untreated LF, despite maintaining the ability to bind to cells. Cisplatin-treated PA was unable to form heptameric oligomers required for LF binding and translocation. The drug was shown to modify PA in a reversible noncovalent manner. Not surprisingly, cisplatin also blocked the actions of anthrax edema toxin and of LF-Pseudomonas aeruginosa exotoxin A fusion peptide (FP59), both of which require PA for translocation. Treatment of BALB/cJ mice or Fischer F344 rats with cisplatin at biologically relevant concentrations completely protected the animals from a coadministered lethal dose of LT. However, treatment with cisplatin 2 hours before or after animals received a lethal bolus of toxin did not protect them.

SpeB-Spi: a novel protease-inhibitor pair from Streptococcus pyogenes.

This study presents evidence for a novel protease-protease inhibitor couple, SpeB-Spi, in the human pathogen Streptococcus pyogenes. The gene for the inhibitor Spi is located directly downstream of the gene for the streptococcal cysteine protease SpeB. Spi is 37% identical and 70% similar to the sequence of the SpeB propeptide, suggesting that Spi and the SpeB propeptide might bind to SpeB in an analogous manner. Secondary structure predictions and molecular modelling suggested that Spi would adopt a structure similar to the SpeB propeptide. The spi gene was co-transcribed with speB on the 1.7 knt and 2.2 knt transcripts previously identified for speB. The Spi protein was purified by SpeB-affinity chromatography from the S. pyogenes cytoplasm. Recombinant Spi was produced and purified, and shown to bind to SpeB and to inhibit its protease activity. Although a similar genetic arrangement of protease and inhibitor is present in staphylococci, this is the first example of an inhibitor molecule that is a structural homologue of the cognate propeptide, and which is genetically linked to the protease gene. Thus, this represents a novel system whereby bacteria may control the intracellular activity of their proteases.

Selective inhibition of anthrax edema factor by adefovir, a drug for chronic hepatitis B virus infection.

Edema factor (EF), a key virulence factor in anthrax pathogenesis, has calmodulin (CaM)-activated adenylyl cyclase activity. We have found that adefovir dipivoxil, a drug approved to treat chronic infection of hepatitis B virus, effectively inhibits EF-induced cAMP accumulation and changes in cytokine production in mouse primary macrophages. Adefovir diphosphate (PMEApp), the active cellular metabolite of adefovir dipivoxil, inhibits the adenylyl cyclase activity of EF in vitro with high affinity (K(i) = 27 nM). A crystal structure of EF-CaM-PMEApp reveals that the catalytic site of EF forms better van der Waals contacts and more hydrogen bonds with PMEApp than with its endogenous substrate, ATP, providing an explanation for the approximately 10,000-fold higher affinity EF-CaM has for PMEApp versus ATP. Adefovir dipivoxil is a clinically approved drug that can block the action of an anthrax toxin. It can be used to address the role of EF in anthrax pathogenesis.

Toward the elucidation of the catalytic mechanism of the mono-ADP-ribosyltransferase activity of Pseudomonas aeruginosa exotoxin A.

The catalytic mechanism for the mono-ADP-ribosyltransferase activity of Pseudomonas aeruginosa exotoxin A was investigated by steady-state and stopped-flow kinetic analyses. The rate constants for binding of the NAD(+) substrate to the enzyme were found to be 4.7 +/- 0.4 microM(-1) s(-1) and 194 +/- 15 s(-1) for k(on) and k(off), respectively. The k(on) and k(off) rate constants for the eEF-2 substrate binding to the enzyme were 320 +/- 39 microM(-1) s(-1) and 131 +/- 22 s(-1), respectively. A potent, competitive inhibitor against the enzyme, 1,8-naphthalimide, bound the enzyme with k(on) and k(off) rates of 82 +/- 9 microM(-1) s(-1) and 51 +/- 6 s(-1), respectively. Furthermore, the binding on and off rates for the reaction products, ADP-ribose and nicotinamide, were too rapid for detection with the stopped-flow technique. Investigation of the pre-steady-state kinetics for the ADP-ribose transferase activity of the toxin-enzyme showed that there is no pre-steady-state complex formed during the catalytic cycle. Binding of NAD+ and smaller compounds representing the various parts of this substrate were investigated by the fluorescence quenching of the intrinsic toxin fluorescence. The binding data revealed a significant structural change in the enzyme upon NAD+ binding that could not be accounted for on the basis of the sum of the structural changes induced by the various NAD+ constituents. Product inhibition studies were conducted with nicotinamide and eEF-2-ADP-ribose, and the results indicate that the reaction involves a random-order ternary complex mechanism. Detailed kinetic analysis revealed that the eEF-2 substrate shows sigmoidal kinetic behavior with the enzyme, and fluorescence resonance energy transfer measurements indicated that wheat germ eEF-2 is oligomeric in solution.

Identification of peptide inhibitors of Pseudomonas aeruginosa exotoxin A function using a yeast two-hybrid approach.

The yeast two-hybrid system was used to identify peptide inhibitors of exotoxin A of Pseudomonas aeruginosa with the goal of using these to design peptide-based drugs against the toxin. A random peptide library consisting of 10(7) peptides ranging in length from 16 to 63 residues was screened for peptides that interact with the C-domain of exotoxin A. From the 10(7) transformants screened, three unique peptides of 63, 61 and 25 amino acids in length were found to specifically interact with the enzyme domain. The genes encoding these peptides were cloned and expressed as fusion proteins with the maltose-binding protein. In vitro inhibition measurements indicated that two of the peptides were modest inhibitors of toxin enzyme activity. These peptides now provide the basis for the development of more potent inhibitors, which will serve as lead inhibitors for evolution of potent peptide-based therapeutics.