Anti-Endotoxin Agents. 2. Pilot High-Throughput Screening for Novel Lipopolysaccharide-Recognizing Motifs in Small Molecules

Wood, Stewart J.; Miller, Kelly A.; David, Sunil A.

doi:10.2174/1386207043328229

Cited by 31 publications

(43 citation statements)

References 49 publications

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“…2A), and (ii) additional, appropriately positioned hydrophobic group(s) are obligatory for the interaction to manifest in neutralization of endotoxicity. Leads obtained from high-throughput screening on focused libraries (4,44,45) and from molecular modeling studies and in silico docking studies (19) suggested that extension of the spermine backbone and the introduction of an additional, appropriately positioned amine on the scaffold would allow favorable salt bridges with the lipid A phosphate groups, analogous to that observed in complexes of lipid A and PMB (2), as well as additional H-bond interactions with the 3-deoxy-D-manno-octulosonic acid (KDO) sugars (30) in the inner-core region of LPS (Fig. 2B).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Bound To Shock: Protection from Lethal Endotoxemic Shock by a Novel, Nontoxic, Alkylpolyamine Lipopolysaccharide Sequestrant

Sil

Shrestha

Kimbrell

et al. 2007

Antimicrob Agents Chemother

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Lipopolysaccharide (LPS), or endotoxin, a structural component of gram-negative bacterial outer membranes, plays a key role in the pathogenesis of septic shock, a syndrome of severe systemic inflammation which leads to multiple-system organ failure. Despite advances in antimicrobial chemotherapy, sepsis continues to be the commonest cause of death in the critically ill patient. This is attributable to the lack of therapeutic options that aim at limiting the exposure to the toxin and the prevention of subsequent downstream inflammatory processes. Polymyxin B (PMB), a peptide antibiotic, is a prototype small molecule that binds and neutralizes LPS toxicity. However, the antibiotic is too toxic for systemic use as an LPS sequestrant. Based on a nuclear magnetic resonance-derived model of polymyxin B-LPS complex, we had earlier identified the pharmacophore necessary for optimal recognition and neutralization of the toxin. Iterative cycles of pharmacophore-based ligand design and evaluation have yielded a synthetically easily accessible N 1 ,mono-alkyl-mono-homologated spermine derivative, DS-96. We have found that DS-96 binds LPS and neutralizes its toxicity with a potency indistinguishable from that of PMB in a wide range of in vitro assays, affords complete protection in a murine model of LPS-induced lethality, and is apparently nontoxic in vertebrate animal models.Endotoxin, or lipopolysaccharide (LPS), a structural component of the outer membrane of most gram-negative bacteria (31), plays a pivotal role in septic shock, a syndrome of systemic toxicity which occurs frequently as a sequel to serious systemic gram-negative infections (23). The activation by LPS of the innate immune response, mediated via toll-like receptor 4 (TLR4) (39), leads to a dysregulated production of numerous inflammatory mediators, including tumor necrosis factor alpha (TNF-␣), interleukin-1␤ (IL-1␤), and IL-6 (11), gamma interferon (IFN-␥), and IL-12, which appears to be inadequately compensated for by the production of anti-inflammatory cytokines, such as IL-10 and transforming growth factor ␤ (6). The resultant systemic inflammatory response progresses to the frequently fatal syndrome of multiple-system organ failure (3). Despite continuing advances in antimicrobial chemotherapy, the incidence of sepsis has risen almost threefold from 1979 through 2000 (25), emphasizing an urgent, unmet need to develop therapeutic options specifically targeting the pathophysiology of sepsis.The toxicity of LPS resides in its structurally highly conserved glycolipid component called lipid A (22), which is composed of a hydrophilic, bis-phosphorylated diglucosamine backbone, and a hydrophobic domain comprised of acyl chains in amide and ester linkages (14). Polymyxin B (PMB) is a membrane-active peptide antibiotic (37) known to sequester LPS and abrogate its toxicity (12, 16). The otoand nephrotoxicity of PMB limit its systemic use and have led to the development of an extracorporeal hemoperfusion cartridge based on PMB covalently immobilized on a polysty...

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Section: Resultsmentioning

confidence: 99%

“…The BC displacement assay for quantifying binding affinity has been described previously (5,43,44). Briefly, to the first column (16 wells) of a Corning Nonbinding Surface 384-well flat-bottom black fluorescence microplate 80-l aliquots of 1 mM stock solutions of DS-96 or PMB (reference compound) were added in quadruplicate and were…”

Section: Methodsmentioning

confidence: 99%

Bound To Shock: Protection from Lethal Endotoxemic Shock by a Novel, Nontoxic, Alkylpolyamine Lipopolysaccharide Sequestrant

Sil

Shrestha

Kimbrell

et al. 2007

Antimicrob Agents Chemother

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“…Binding to the lipid A region of LPS was determined using the BODIPY-TR-cadaverine displacement assay (34,35), in which the probe bound to cell-free LPS is self-quenched but fluoresces when released in solution. Stock solutions of BODIPY-TRcadaverine (500 M) and LPS from P. aeruginosa (5 mg/ml) were prepared by dissolution in Tris buffer (50 mM, pH 7.4) (the Tris buffer was supplemented with 0.5% methanol for the former).…”

Section: Methodsmentioning

confidence: 99%

New Amphiphilic Neamine Derivatives Active against Resistant Pseudomonas aeruginosa and Their Interactions with Lipopolysaccharides

Sautrey

Zimmermann

Deleu

et al. 2014

Antimicrob Agents Chemother

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The development of novel antimicrobial agents is urgently required to curb the widespread emergence of multidrug-resistant bacteria like colistin-resistant Pseudomonas aeruginosa. We previously synthesized a series of amphiphilic neamine derivatives active against bacterial membranes, among which 3=,6-di-O-[(2؆-naphthyl)propyl]neamine (3=,6-di2NP), 3=,6-di-O-[(2؆-naphthyl)butyl]neamine (3=,6-di2NB), and 3=,6-di-O-nonylneamine (3=,6-diNn) showed high levels of activity and low levels of cytotoxicity (L. Zimmermann et al., J. Med. Chem. 56:7691-7705, 2013). We have now further characterized the activity of these derivatives against colistin-resistant P. aeruginosa and studied their mode of action; specifically, we characterized their ability to interact with lipopolysaccharide (LPS) and to alter the bacterial outer membrane (OM). The three amphiphilic neamine derivatives were active against clinical colistin-resistant strains (MICs, about 2 to 8 g/ml), The most active one (3=,6-diNn) was bactericidal at its MIC and inhibited biofilm formation at 2-fold its MIC. They cooperatively bound to LPSs, increasing the outer membrane permeability. Grafting long and linear alkyl chains (nonyl) optimized binding to LPS and outer membrane permeabilization. The effects of amphiphilic neamine derivatives on LPS micelles suggest changes in the cross-bridging of lipopolysaccharides and disordering in the hydrophobic core of the micelles. The molecular shape of the 3=,6-dialkyl neamine derivatives induced by the nature of the grafted hydrophobic moieties (naphthylalkyl instead of alkyl) and the flexibility of the hydrophobic moiety are critical for their fluidifying effect and their ability to displace cations bridging LPS. Results from this work could be exploited for the development of new amphiphilic neamine derivatives active against colistin-resistant P. aeruginosa.

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“…[15] Hence, there is a great deal of A C H T U N G T R E N N U N G interest in developing therapeutic agents that can efficiently bind LPS. [16] Third, whereas the therapeutic strategy directed against viral glycans was a success, a similar approach to antibacterial therapies has not been systematically explored due to difficulties in finding molecules that can selectively bind to bacterial glycans. [17] Herein, we report our findings in screening of 10 000 random 20-mer peptide sequences printed on a glass slide with newly developed luminescent LPS glycoprobes for potential lectinomimetic activity.…”

Section: Introductionmentioning

confidence: 99%

Bacterial Glycoprofiling by Using Random Sequence Peptide Microarrays

et al. 2009

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Current analytical methods have been slow in addressing the growing need for glyco-analysis. A new generation of more empirical high-throughput (HTP) tools is needed to aid the advance of this important field. To this end, we have developed a new HTP screening platform for identification of surface-immobilized peptides that specifically bind O-antigenic glycans of bacterial lipopolysaccharides (LPS). This method involves screening of random sequence peptide libraries in addressable high-density microarray format with the newly developed luminescent LPS-quantum dot micelles. Screening of LPS fractions from O111:B4 and O55:B5 serotypes of E. coli on a microarray consisting of 10,000 20-mer peptide features revealed minor differences, while comparison of LPS from E. coli O111:B4 and P. aeruginosa produced sets of highly specific peptides. Peptides strongly binding to the E. coli LPS were highly enriched in aromatic and cationic amino acids, and most of these inhibited growth of E. coli. Flow cytometry and isothermal titration calorimetry (ITC) experiments showed that some of these peptides bind LPS in-solution with a K(d) of 1.75 microM. Peptide selections against P. aeruginosa were largely composed of hydrogen-bond forming amino acids in accordance with dramatic compositional differences in O-antigenic glycans in E. coli and P. aeruginosa. While the main value of this approach lies in the ability to rapidly differentiate bacterial and possibly other complex glycans, the peptides discovered here can potentially be used off-array as antiendotoxic and antimicrobial lead compounds, and on-array/on-bead as diagnostic and affinity reagents.

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Anti-Endotoxin Agents. 2. Pilot High-Throughput Screening for Novel Lipopolysaccharide-Recognizing Motifs in Small Molecules

Cited by 31 publications

References 49 publications

Bound To Shock: Protection from Lethal Endotoxemic Shock by a Novel, Nontoxic, Alkylpolyamine Lipopolysaccharide Sequestrant

Bound To Shock: Protection from Lethal Endotoxemic Shock by a Novel, Nontoxic, Alkylpolyamine Lipopolysaccharide Sequestrant

New Amphiphilic Neamine Derivatives Active against Resistant Pseudomonas aeruginosa and Their Interactions with Lipopolysaccharides

Bacterial Glycoprofiling by Using Random Sequence Peptide Microarrays

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