Systemic bacterial infections are associated with high mortality. The access of bacteria or constituents thereof to systemic circulation induces the massive release of immunomodulatory mediators, ultimately causing tissue hypoperfusion and multiple-organ failure despite adequate antibiotic treatment. Lipid A, the "endotoxic principle" of bacterial lipopolysaccharide (LPS), is one of the major bacterial immunostimuli. Here we demonstrate the biological efficacy of rationally designed new synthetic antilipopolysaccharide peptides (SALPs) based on the Limulus anti-LPS factor for systemic application. We show efficient inhibition of LPS-induced cytokine release and protection from lethal septic shock in vivo, whereas cytotoxicity was not observed under physiologically relevant conditions and concentrations. The molecular mechanism of LPS neutralization was elucidated by biophysical techniques. The lipid A part of LPS is converted from its "endotoxic conformation," the cubic aggregate structure, into an inactive multilamellar structure, and the binding affinity of the peptide to LPS exceeds those of known LPS-binding proteins, such as LPS-binding protein (LBP). Our results thus delineate a novel therapeutic strategy for the clinical management of patients with septic shock.The life-threatening clinical consequences of sepsis and septic shock arise from recognition of microbial immunostimulatory molecules by the hosts' professional immune cells and the release of hemodynamically active mediators. The most potent immunostimulatory constituents are part of the microbial cell envelope, such as lipopolysaccharide (LPS) or lipoproteins. They are released continuously due to cell growth and division and massively liberated as a consequence of the attack of the immune system. In the case of Gram-negative bacteria, the most potent factor is LPS, which, therefore, is also called an endotoxin. LPS concentrations in blood serum as low as 1 ng/ml are able to cause sepsis. Septic shock resulting from bacterial infection remains a frequent cause of death, particularly in intensive care units, with more than 200,000 people dying each year in the United States alone. Death by septic shock can happen despite appropriate broad-range antibiotic treatment, which may kill bacteria but is not only incapable of neutralizing immunostimulatory LPS but also may promote its release into circulation (11).The response of mammalian cells to LPS is initiated by its interaction with serum proteins such as lipopolysaccharidebinding protein (LBP) and specific receptors and/or binding proteins of immune cells such as soluble CD14 (sCD14) and membrane-bound CD14 (mCD14), which finally leads to cell activation through the Toll-like receptor 4 (TLR4)-MD-2 pathway (31). The hydrophobic moiety of LPS, lipid A, anchoring LPS to the bacterial outer membrane, constitutes the "endotoxic principle" of LPS (24). Enterobacterial lipid A consists of a diglucosamine backbone phosphorylated at positions 1 and 4Ј, to which six acyl chains are linked at positions 2,3 a...

NK-2, a membrane-acting antimicrobial peptide, was derived from the cationic core region of porcine NK-lysin and consists of 27 amino acid residues. It adopts an amphipathic, ␣-helical secondary structure and has been shown to interact specifically with membranes of negatively charged lipids. We therefore investigated the interaction of NK-2 with lipopolysaccharide (LPS), the main, highly anionic component of the outer leaflet of the outer membrane of gram-negative bacteria, by means of biophysical and biological assays. As model organisms and a source of LPS, we used Salmonella enterica strains with various lengths of the LPS carbohydrate moiety, including smooth LPS, rough LPS, and deep rough LPS (LPS Re) mutant strains. NK-2 binds to LPS Re with a high affinity and induces a change in the endotoxin-lipid A aggregate structure from a cubic or unilamellar structure to a multilamellar one. This structural change, in concert with a significant overcompensation of the negative charges of LPS, is thought to result in the neutralization of the endotoxic LPS activity in a cell culture system. Neutralization of LPS activity by NK-2 as well as its antibacterial activity against the various Salmonella strains strongly depends on the length of the sugar chains of LPS, with LPS Re being the most sensitive. This suggests that a hydrophobic peptide-LPS interaction is necessary for efficient neutralization of the biological activity of LPS and that the long carbohydrate chains, besides their function as a barrier for hydrophobic drugs, also serve as a trap for polycationic substances.

The peptide NK-2 is an effective antimicrobial agent with low hemolytic and cytotoxic activities and is thus a promising candidate for clinical applications. It comprises the ␣-helical, cationic core region of porcine NK-lysin a homolog of human granulysin and of amoebapores of pathogenic amoeba. Here we visualized the impact of NK-2 on Escherichia coli by electron microscopy and used NK-2 as a template for sequence variations to improve the peptide stability and activity and to gain insight into the structure/ function relationships. We synthesized 18 new peptides and tested their activities on seven Gram-negative and one Gram-positive bacterial strains, human erythrocytes, and HeLa cells. Although all peptides appeared unordered in buffer, those active against bacteria adopted an ␣-helical conformation in membrane-mimetic environments like trifluoroethanol and negatively charged phosphatidylglycerol (PG) liposomes that mimick the cytoplasmic membrane of bacteria. This conformation was not observed in the presence of liposomes consisting of zwitterionic phosphatidylcholine (PC) typical for the human cell plasma membrane. The interaction was paralleled by intercalation of these peptides into PG liposomes as determined by FRET spectroscopy. A comparative analysis between biological activity and the calculated peptide parameters revealed that the decisive factor for a broad spectrum activity is not the peptide overall hydrophobicity or amphipathicity, but the possession of a minimal positive net charge plus a highly amphipathic anchor point of only seven amino acid residues (two helical turns).

The paper describes the design, synthesis, and testing of inhibitors of folate-synthesizing enzymes and of whole cell cultures of Candida albicans. The target enzymes used were dihydropteroic acid synthase (SYN) and dihydrofolate reductase (DHFR). Several series of new 2,4-diaminopyrimidines were synthesized and tested as inhibitors of DHFR and compared with their activity against DHFR derived from mycobacteria and Escherichia coli. To test for selectivity, also rat DHFR was used. A series of substituted 4-aminodiphenyl sulfones was tested for inhibitory activity against SYN and the I(50) values compared to those obtained previously against Plasmodium berghei- and E. coli-derived SYN. Surprisingly, QSAR equations show very similar structural dependencies. To find an explanation for the large difference in the I(50) values observed for enzyme inhibition (SYN, DHFR) and for inhibition of cell cultures of Candida, mutant strains with overexpressed efflux pumps and strains in which such pumps are deleted were included in the study and the MICs compared. Efflux pumps were responsible for the low activity of some of the tested derivatives, others showed no increase in activity after pumps were knocked out. In this case it may be speculated that these derivatives are not able to enter the cells.

Mutations in fibrillin-1 give rise to Marfan syndrome (MFS) characterized by vascular, skeletal, and ocular abnormalities. Fibrillins form the backbone of extracellular matrix microfibrils in tissues including blood vessels, bone, and skin. They are crucial for regulating elastic fiber biogenesis and growth factor bioavailability. To compare the molecular consequences of mutations causing the severe neonatal MFS with mutations causing the milder classical MFS, we introduced representative point mutations from each group in a recombinant human fibrillin-1 fragment. Structural effects were analyzed by circular dichroism spectroscopy and analytical gel filtration chromatography. Proteolytic susceptibility was probed with non-physiological and physiological proteases, including plasmin, thrombin, matrix metalloproteinases, and cathepsins. All mutant proteins showed a similar gross secondary structure and no differences in heat stability as compared with the wild-type protein. Proteins harboring neonatal mutations were typically more susceptible to proteolytic cleavage compared with those with classical mutations and the wild-type protein. Proteolytic neo-cleavage sites were found both in close proximity and distant to the mutations, indicating small but significant structural changes exposing cryptic cleavage sites. We also report for the first time that cathepsin K and V cleave non-mutated fibrillin-1 at several domain boundaries. Compared with the classical mutations and the wild type, the group of neonatal mutations more severely affected the ability of fibrillin-1 to interact with heparin/heparan sulfate, which plays a role in microfibril assembly. These results suggest differential molecular pathogenetic concepts for neonatal and classical MFS including enhanced proteolytic susceptibility for physiologically relevant enzymes and loss of function for heparin binding.