Lysine–spermine conjugates: hydrophobic polyamine amides as potent lipopolysaccharide sequestrants

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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...

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Sil

Shrestha

Kimbrell

et al. 2007

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“…LTA is a TLR2 agonist, while LPS binds to TLR4. Since we have demonstrated that several members of the acylpolyamine class bind to and neutralize LPS (5)(6)(7)35), it was of particular importance to examine in reporter gene assays the LTA-binding and -neutralizing activities of the polyamine sulfonamides which respond to TLR2 while obviating the falsepositive and spurious results that arise due to contamination with trace quantities of LPS, which is a common problem (19).…”

Section: Resultsmentioning

confidence: 99%

Structure-Activity Relationships of Antimicrobial and Lipoteichoic Acid-Sequestering Properties in Polyamine Sulfonamides

Warshakoon

Burns

David

2009

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We have recently confirmed that lipoteichoic acid (LTA), a major constituent of the gram-positive bacterial surface, is the endotoxin of gram-positive bacteria that induces proinflammatory molecules in a Toll-like receptor 2 (TLR2)-dependent manner. LTA is an anionic amphipath whose physicochemical properties are similar to those of lipopolysaccharide (LPS), which is found on the outer leaflet of the outer membranes of gram-negative organisms. Hypothesizing that compounds that sequester LPS could also bind to and inhibit LTA-induced cellular activation, we screened congeneric series of polyamine sulfonamides which we had previously shown effectively neutralized LPS both in vitro and in animal models of endotoxemia. We observed that these compounds do bind to and neutralize LTA, as reflected by the inhibition of TLR2-mediated NF-B induction in reporter gene assays. Structure-activity studies showed a clear dependence of the acyl chain length on activity against LTA in compounds with spermine and homospermine scaffolds. We then sought to examine possible correlations between the neutralizing potency toward LTA and antimicrobial activity in Staphylococcus aureus. A linear relationship between LTA sequestration activity and antimicrobial activity for compounds with a spermine backbone was observed, while all compounds with a homospermine backbone were equally active against S. aureus, regardless of their neutralizing potency toward LTA. These results suggest that the number of protonatable charges is a key determinant of the activity toward the membranes of gram-positive bacteria. The development of resistance to membrane-active antibiotics has been relatively slower than that to conventional antibiotics, and it is possible that compounds such as the acylpolyamines may be useful clinically, provided that they have an acceptable safety profile and margin of safety. A more detailed understanding of the mechanisms of interactions of these compounds with LPS and LTA, as well as the gram-negative and -positive bacterial cell surfaces, will be instructive and should allow the rational design of analogues which combine antisepsis and antibacterial properties.Sepsis and its sequel, septic shock, a consequence of systemic inflammation that leads to multiple-organ failure (32

“…Polyamines and their analogues exhibit versatile biological activities, including anticancer, antiparasitic, antiendotoxin, and antibacterial activities (32)(33)(34). Squalamine (from dogfish shark) and cinodine (from Nocardia spp.)…”

Section: Discussionmentioning

confidence: 99%

N-Terminally Modified Linear and Branched Spermine Backbone Dipeptidomimetics against Planktonic and Sessile Methicillin-Resistant Staphylococcus aureus

Dewangan

Joshi

Kumari

et al. 2014

c Toward the discovery of useful therapeutic molecules, we report the design and synthesis of a focused library of new ultrashort N-terminally modified dipeptidomimetics, with or without modifications in the spermine backbone leading to linear (series 1) or branched (series 2) tryptophans, as antimicrobial agents. Eight peptidomimetics in the library showed good antibacterial activity (MICs of 1.77 to 14.2 g/ml) against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis bacterial strains. Tryptophan fluorescence measurements on artificial bacterial or mammalian mimic membranes and assessment of the MRSA potential depolarization ability of the designed compounds revealed membrane interactions dependent on tryptophan positioning and N-terminal tagging. Among active peptidomimetics, compounds 1c and 1d were found to be nonhemolytic, displaying rapid bactericidal activity (at 4؋ MIC) against exponentially growing MRSA. Further, scanning electron microscopy of peptidomimetic 1c-and 1d-treated MRSA showed morphological changes with damage to cell walls, defining a membrane-active mode of action. Moreover, peptidomimetics 1c and 1d did not induce significant drug resistance in MRSA even after 17 passages. We also investigated the activity of these molecules against MRSA biofilms. At sub-MIC levels (ϳ2 to 4 g/ml), both peptidomimetics inhibited biofilm formation. At concentrations higher than the MIC (35 to 140 g/ml), peptidomimetics 1c and 1d significantly reduced the metabolic activity and biomass of mature (24-h) MRSA biofilms. These results were corroborated by confocal laser scanning microscopy (live/dead assay). The in vitro protease stability and lower cytotoxicity of peptidomimetics against peripheral blood mononuclear cells (PBMCs) support them being novel staphylocidal peptidomimetics. In conclusion, this study provides two peptidomimetics as potential leads for treatment of staphylococcal infections under planktonic and sessile conditions.