Current Topics in Medicinal Chemistry - Volume 4, Issue 11, 2004

The initial systematic investigations of the toxic effects of putrescent material of plant and animal origin by Peter L. Panum (around 1856), led to the conclusion that the toxic principle originates from living microorganisms. Since then research on endotoxin, later defined as lipopolysaccharides (LPS), has become a major subject of immunological and immunochemical research. Great interest in LPS, the integral components of the cell wall of Gram-negative bacteria, stems from their manifold and diverse biological activities, their complex and unique physico-chemical properties, structural characteristics, serological properties and pathophysiological functions. Endotoxin is an extremely potent inducer of immune response. Already at extremely low concentrations it incites cellular responses and at higher concentrations can lead to sepsis and septic shock, a multiple organ dysfunction syndrome with high mortality that is particularly disturbing in intensive care units. However, depending upon the amount and route of introduction, LPS is also capable of producing beneficial effects in higher organisms by activating immune cells thus enhancing the capacity to cope with microbial infections and even tumors. Sepsis is a complex disease state depending on the patient history, the predisposition on the particular pathogen that a patient has been infected with, and the specific pathways activated in a patient that may lead to organ dysfunction. All these factors plus ambiguous diagnostic criteria defining sepsis make it difficult to determine the right treatment. The progress in successfully treating sepsis has been slow and costly. In recent years great strides have been made in the elucidation of the molecular basis of sepsis and thus providing new possibilities to combat sepsis and septic shock. This thematic issue of Current Topics in Medicinal Chemistry aims at presenting the state of art in endotoxin research with emphasis on the therapeutic potential promised by the basic knowledge of structures and molecular assemblies of the players in the molecular mechanisms involved in LPS recognition and neutralization of its toxic activity. The first contribution to this issue by S. H. Diks and co-workers describes the complexity of LPS signal transduction.The molecular mechanisms involved in the recognition of LPS and in the initiation of an immune response are discussed. Follows the review by K. Brandenburg and A. Wiese describing the relationships between the structure, function and activity of endotoxins. The unique structural features of LPS and the physico-chemical parameters important for the function of the outer membrane of bacterial cell wall are outlined. The inhibition of endotoxin response by synthetic TLR4 antagonists is extensively reviewed by D. P. Rossignol and coworkers. The authors discuss the evolution of LPS antagonists with emphasis on the SAR and development of E5564, an advanced and potent LPS antagonist that may prove to be of benefit in a variety of endotoxin-mediated diseases. R. Jerala and M. Porro are presenting an overview of endotoxin neutralizing peptides and recent developments in preparation of novel types of these compounds modified by lipophilic moieties and non-peptidic molecules, particularly lipopolyamines. They also present alternative applications of endotoxin binding peptides such as extracorporeal endotoxin removal and endotoxoid (the complex between Synthetic Anti-Endotoxin Peptides-SAEP and LPS) based vaccines against Neisseria meningitidis. The last contribution by P. Pristovsek and J. Kidric is concerned with the search for the molecular motifs of specific binding of LPS by anti-endotoxin proteins and peptides. The understanding of LPS mechanism of action at the atomic level is expected to provide leads for the development of new immunomodulatory compounds for the treatment of Gram-negative sepsis.

Lipopolysaccharide (LPS) is the principal initiator of septic shock and it is to a large extent responsible for post-operative mortality. The use of antibiotics is still the most successful therapy against infection that may lead to sepsis and septic shock. With the advent of antibiotic resistant strains like MRSA the usefulness of conventional antibiotics is declining and new treatment strategies for LPS-mediated septic shock are called for. In this review we discuss the molecular mechanisms that are involved in the recognition of LPS and in the initiation of an immune response. Furthermore, we also review the recent insights in the signal transduction including receptor clustering and signalosome activation. Further insight into LPS-dependent signal transduction will assist the development of novel rational therapy.

Molecules of endotoxin (lipopolysaccharides, LPS), forming a unique molecular class with peculiar physicochemical properties, impart a very important role in the formation and function of the outer membrane (OM). The latter is strictly asymmetric with the LPS monolayer forming the outer leaflet and the phospholipid (PL) monolayer forming the inner leaflet. Thus, the OM builds a functional lipid environment for the OM proteins (Omp's, porins) and the LPS layer is the first locus of interaction of the bacterial cells with components of the host's immune system, . Therefore its physical state and biochemical parameters (such as the fluidity of the lipid A acyl chains and the backbone charge density) essentially influence the defense of bacteria against the attack of the human immune systems such as the complement and antimicrobial peptides / proteins. LPS, released from the bacterial cell, is responsible for a variety of biological effects which can be ascribed to the unique structural features of LPS- the three-dimensional supramolecular structure and the intramolecular conformation - which are essential determinants of the bioactivity of endotoxins. Here, the physico-chemical parameters which are important on the one side for the function of the OM and on the other side for the activity of isolated LPS are reviewed.

Endotoxin, from the outer membrane of Gram-negative bacteria, has been implicated as the etiological agent of a variety of pathologies ranging from relatively mild (fever) to lethal (septic shock, organ failure, and death). While endotoxin (also known as lipopolysaccharide or LPS) is a complex heterogeneous molecule, the toxic portion of LPS (the lipid A portion) is relatively similar across a wide variety of pathogenic strains of bacteria, making this molecule an attractive target for the development of an LPS antagonist. Research over the past fifteen years focused on the design of various lipid A analogs including monosaccharide, acyclic and disaccharide compounds has lead to the development of E5564, an advanced, unique and highly potent LPS antagonist. E5564 is a stable, pure LPS antagonist that is selective against endotoxin-mediated activation of immune cells in vitro and in animal models. In Phase I clinical trials, we have developed an ex vivo endotoxin antagonism assay that has provided results on pharmacodynamic activity of E5564 in addition to the more typical safety and pharmacokinetic evaluations. Results from these assays have been reinforced by analysis of in vivo antagonistic activity using a human endotoxemia model. Results from all of these studies indicate that E5564 is an effective in vivo antagonist of endotoxin, and may prove to be of benefit in a variety of endotoxin-mediated diseases. This review discusses the evolution of synthetic LPS antagonists with emphasis on the SAR and development of E5564 and its precursors.

Neutralization and sequestration of bacterial lipopolysaccharide which plays a key role in gram-negative sepsis is required to block the progression of sepsis at early stages in addition to destroying bacteria. Many of the host defense peptides which have antimicrobial activity are also able to bind to and neutralize LPS, however, these two activities do not necessarily correlate. Due to its toxicity application of polymyxin B as the prototype of LPS neutralizing peptide is limited to topical applications and extracorporeal removal of endotoxin. Development of novel endotoxin neutralizing peptides without the toxicity of polymyxin B have been based on the natural host defense peptides, fragments of LPS binding proteins and engineered peptides. Neutralization of LPS can be achieved through several different peptide fold motifs, which are reviewed in this article. Endogenous host defense peptides, fragments of endotoxin-binding proteins and synthetic anti-endotoxin peptides fold into α-helical, β-hairpin, extended and compact conformations without regular secondary structure. In animal models many of the peptides have demonstrated good in vitro and in vivo endotoxin neutralizing activity but up to now none of the peptides has been approved for clinical application with an anti-endotoxin indication. Recent developments include preparation of novel types of endotoxin neutralizing compounds such as peptides modified by lipophilic moieties and non-peptidic molecules, particularly lipopolyamines and on the other hand additional medical applications such as extracorporeal endotoxin removal, targeting to inflammation sites or endotoxoid based vaccines.

Lipopolysaccharide (LPS) induced Gram-negative sepsis and septic shock remain lethal in up to 60 % of cases, and LPS antagonists that neutralize its endotoxic action are the subject of intensive research. The molecular motifs of specific binding of LPS by antiendotoxin proteins and peptides may lead to an understanding of LPS action at the atomic level and provide clues for the development of new immunomodulatory compounds for use as therapy in the treatment of Gram-negative bacterial sepsis. The interaction of LPS with its cognate binding proteins has been structurally elucidated in the single case of the X-ray crystallographic structure of LPS in complex with the integral outer membrane protein FhuA from E. coli K-12 (Ferguson et al., Science 1999, 282, 2215). This structure and other known structures of LPS binding proteins have been used to propose a common binding motif of LPS to proteins. Another independent source of structural information are solution structures of peptides in complex with LPS that can be determined using the transferred NOE effect. The molecular mechanisms of biological activity of bacterial endotoxins can additionally be probed by theoretical means. The growing structural knowledge is opening pathways to the design of peptides or peptidomimetics with improved antiendotoxin properties.