Bound to shock: protection from lethal endotoxemic shock by a novel, nontoxic, alkylpolyamine lipopolysaccharide sequestrant

Abstract

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.

FIG. 1.

The chemical structure of DS-96 [N¹-(3-aminopropyl)-N⁴-(3-(3-(hexadecylamino)-propylamino)propyl)butane-1,4-diamine; pentatrifluoroacetate salt].

FIG. 2.

(A) A nuclear magnetic resonance-derived model of the PMB (stick representation with van der Waals surface)-lipid A (ball-and-stick) complex (2). Bidentate salt bridges (dotted lines) are postulated to occur between each phosphate group on lipid A and two pairs of the γ-NH₂ groups of Dab residues (2). The poly-acyl domain of lipid A is shown truncated. (B) Molecular-modeling-derived geometry of the complex between LPS and DS-96. The atomic coordinates of LPS were derived from its crystal structure (14). The mono-homologated spermine backbone is predicted to form salt bridges with both phosphate groups on lipid A, as well as participating in additional ionic H bonds with the inner core KDO sugars.

FIG. 3.

(A) Binding affinities of DS-96 and PMB (reference compound) to E. coli O111:B4 LPS determined by BODIPY-cadaverine displacement assay. The ED₅₀s for the two compounds are 1.2 ± 0.16 μM and 1.31 ± 0.12 μM, respectively. (B) Inhibitory activity of NO production by DS-96 and PMB in LPS-stimulated murine J774 macrophage cells. The IC₅₀s for DS-96 and PMB are, respectively, 21 ± 3 nM and 27 ± 2 nM.

FIG. 4.

Inhibition of NF-κB reporter gene induction in HEK-293 cells stably transfected with TLR4/CD-14/MD-2/NF-κB-SEAP construct. Cells were stimulated with either LPS (10 ng/ml) or recombinant human TNF-α (100 ng/ml) and exposed to graded concentrations of test compounds. Both DS-96 and PMB inhibit LPS-stimulated NF-κB induction with identical potencies (IC₅₀, 32 ± 2 nM) but show no effect on TNF-α-stimulated cells, showing specificity of action. Shown on the left are negative and positive (LPS alone and TNF-α alone) controls.

FIG. 5.

(A) Schild-type analysis of dependence of IC₅₀ (NF-κB induction) of DS-96 and PMB on the dose of LPS used. HEK-4 cells were stimulated with escalating (1 ng/ml to 10 μg/ml) doses of E. coli O111:B4 LPS, preincubated with graded doses of either PMB or DS-96. IC₅₀s for either compound were determined at each LPS stimulus dose. (B) Inhibition of NF-κB reporter gene induction by DS-96 and PMB in HEK-293 cells stimulated with 10 ng/ml LPS isolated from a wide variety of gram-negative bacteria. A stimulus of 10 ng/ml LPS was used. Note that the IC₅₀s for both PMB and DS-96 are very similar, irrespective of the source of LPS. With serovar Abortus equi LPS as stimulus, DS-96 was observed to be more potent (IC₅₀, 82 nM) than PMB (IC₅₀, 562 nM).

FIG. 6.

(A) Inhibition of phosphorylation of p38 MAP kinase in neutrophils in whole human blood (ex vivo), stimulated with either LPS (100 ng/ml) or human TNF-α (100 ng/ml) for 15 min in the presence of graded concentrations of DS-96 or PMB. Quantification of p38 MAPK was performed using flow cytometry. (B) Forward-scatter/side-scatter profile and the gating for p38 MAPK-negative and -positive gates obtained on unstimulated cells (negative control), respectively. Back gating on the p38 MAPK-positive cells (dark-shaded peak) maps to the polymorphonuclear population. (C to E) Inhibition of LPS-induced proinflammatory cytokine production in human blood. Whole human blood was stimulated with 100 ng/ml LPS preincubated with graded concentrations of either DS-96 or PMB. Cytokine levels were quantified using a multiplexed flow-cytometric bead array system (CBA). Only TNF-α, IL-6, and IL-8 levels were quantifiable; IL-10 and IL-12p70 levels were below detection limits, and signal-to-noise ratios for IL-1β were unacceptably high.

FIG. 7.

(A) Comparison of in vivo potencies of PMB and DS-96: dose-dependent increase in survival in mice challenged with a supralethal dose of LPS (200 ng/animal). (B) Schild-type response in vivo: dose dependence of survival in mice challenged with escalating supralethal doses of LPS (200, 500, or 1,000 ng/animal). The LD₁₀₀ of LPS was determined to be 100 ng/mouse. (C) Time course (pharmacodynamics) of protection conferred by 8 mg/kg of DS-96 administered subcutaneously at various times prior to and following supralethal (200 ng/mouse) LPS challenge.

FIG. 8.

Time course of cytokine levels in mice receiving 4 mg/kg DS-96 and challenged with a 200-ng/ml dose of LPS. Cohorts of five animals each received either 4 mg/kg DS-96 or saline s.c. at t = −1 h, followed by d-galactosamine-LPS given i.p. at t = 0 h. At t = 1, 2, or 3 h, animals were bled by terminal cardiac puncture, and cytokines were assayed in plasma by cytometric bead array assays. It should be noted that only TNF-α, IL-6, and macrophage chemotactic protein 1 levels were quantifiable; IL-10, IL-12p70, and IFN-γ levels were below detection limits.