Mark D. Cunningham

A novel Escherichia coli lipid A mutant that produces an antiinflammatory lipopolysaccharide.

A unique screen was used to identify mutations in Escherichia coli lipid A biosynthesis that result in a decreased ability to stimulate E-selectin expression by human endothelial cells. A mutation was identified in the msbB gene of E. coli that resulted in lipopolysaccharide (LPS) that lacks the myristoyl fatty acid moiety of the lipid A. Unlike all previously reported lipid A mutants, the msbB mutant was not conditionally lethal for growth. Viable cells or purified LPS from an msbB mutant had a 1000-10,000-fold reduction in the ability to stimulate E-selectin production by human endothelial cells and TNF alpha production by adherent monocytes. The cloned msbB gene was able to functionally complement the msbB mutant, restoring both the LPS to its native composition and the ability of the strain to stimulate immune cells. Nonmyristoylated LPS acted as an antagonist for E-selectin expression when mixed with LPS obtained from the parental strain. These studies demonstrate a significant role for the myristate component of LPS in immune cell activation and antagonism. In addition, the msbB mutant allowed us to directly examine the crucial role that the lipid A structure plays when viable bacteria are presented to host defense cells.

Beta-lactam antibiotics potentiate magainin 2 antimicrobial activity in vitro and in vivo.

The ability of magainin 2 to augment antibiotic therapy was examined. Susceptibility to magainin 2 was determined on Escherichia coli incubated in the presence and absence of sublethal concentrations of antibiotics both in vitro and in vivo. Experiments in buffer and normal human serum revealed that E. coli exposed to sublethal amounts of cefepime, a beta-lactam antibiotic, was significantly more susceptible to the antimicrobial activity of magainin 2. Bacteria incubated with subinhibitory concentrations of other beta-lactam type antibiotics, but not amikacin (an aminoglycoside) or ciprofloxacin (a quinolone), were also more susceptible to magainin 2 in normal human serum. Bacteria were less susceptible to magainin 2 when they were examined in heat-inactivated serum. Complement was shown to be required for magainin 2 activity in serum by using C8-deficient sera. The combination of magainin 2 and cefepime was shown to be more antimicrobial in normal human serum for a variety of bacterial strains. Magainin 2 was completely inactive as a therapeutic agent when it was administered alone (2 mg per mouse) but significantly increased the survival of mice when it was administered with a low level of cefepime.

Toll-Like Receptor 9 Can Be Expressed at the Cell Surface of Distinct Populations of Tonsils and Human Peripheral Blood Mononuclear Cells

ABSTRACT Unmethlylated CpG dinucleotides induce a strong T-helper-1-like inflammatory response, presumably mediated by Toll-like receptor 9 (TLR9). However, the nature and cellular localization of TLR9 in primary human cells remain controversial. Here we demonstrate, using flow cytometry and immunofluorescence microscopy techniques, that TLR9 can be expressed at the cell surface. The primary human cell subsets that were positive for TLR9 expression were distinct depending on the tissues analyzed. Specifically, in human peripheral blood mononuclear cells (PBMC) the majority of cell surface TLR9+ cells were confined to the major histocompatibility complex (MHC) class II+ CD19− populations that express CD11c and/or CD14, whereas in tonsils the same gated population contained primarily MHC class II+ CD19+ cells. Cells positive for surface expression represented a minor fraction of the total cell populations examined, varying between 2 and 10%. In addition, we found that TLR9 expression at the surface of PBMC was up-regulated approximately fourfold following stimulation with the gram-negative bacterial cell wall component lipopolysaccharide, suggesting a potential modulatory role of TLR4 agonists on TLR9 expression. Taken together, these data validate human TLR9 expression at the surface of primary cells, in addition to the previously described intracellular localization. Further, our results suggest that human antigen-presenting cells comprise the major cell populations expressing cell surface TLR9.

Peptides related to the carboxyl terminus of human platelet factor IV with antibacterial activity.

A peptide (C13) corresponding to the last 13 amino acids of the carboxyl terminus of human platelet factor IV was found to be antibacterial. Amino acid substitutions predicted to disrupt either the amphipathic or alpha-helical nature of C13 rendered the peptide inactive. Antibacterial activity was demonstrated in normal human serum on bacteria which had been previously exposed to low levels of cefepime, a beta-lactam antibiotic. Peptide analogues were examined for more potent antibacterial activity in an antibacterial assay that employed normal human serum and low levels of cefepime. A peptide analogue (C18G) with 80-fold more antibacterial activity than C13 was identified. Studies in C8-deficient sera confirmed an essential role of human serum complement for optimal antibacterial activity. Additional studies showed low levels of cefepime, although not essential, enhanced the antibacterial activity of C18G. Animal protection experiments demonstrated that either peptide C18G or an analogue with all D amino acids (C18X) significantly increased the survival of neutropenic mice when coadministered with a low level of cefepime. This work has resulted in the identification of a new group of antibacterial peptides.

CD14 employs hydrophilic regions to "capture" lipopolysaccharides.

CD14 participates in the host innate inflammatory response to bacterial LPS obtained from Escherichia coli and other Gram-negative bacteria. Evidence from several laboratories suggests that different regions of the amino-terminal portion of the molecule may be involved in LPS binding. In this report a series of single-residue serine replacement and charge reversal mutations were generated to further elucidate the mechanism by which this protein may bind a multitude of different LPS ligands. Single-residue CD14 mutation proteins were examined for their ability to bind LPS obtained from E. coli, Porphyromonas gingivalis, and Helicobacter pylori and facilitate the activation of E-selectin from human endothelial cells. In addition, the single-residue CD14 mutation proteins were employed to perform monoclonal epitope-mapping studies with three LPS-blocking Abs that bound tertiary epitopes. Evidence that several different hydrophilic regions of the amino-terminal region of CD14 are involved in LPS binding was obtained. Epitope-mapping studies revealed that these hydrophilic regions are located on one side of the protein surface. These studies suggest that CD14 employs a charged surface in a manor similar to the macrophage scavenger receptor to “capture” LPS ligands and “present” them to other components of the innate host defense system.

An intracellular targeted NLS peptide inhibitor of karyopherin α:NF-κB interactions

The nuclear import of transcription factors involves proteins termed karyopherins. Previously, we described an intracellular targeted dual nuclear localization sequence (NLS) peptide inhibitor of processes dependent upon the transcription factor NF-kappa B. We have now developed a homogeneous solution based assay and show that NF-kappa B interacts with karyopherin alpha and that the dual NLS peptide inhibits this interaction. We also show that both L- and D-amino acid containing peptides bind well to karyopherin alpha 2, whereas, the L-amino acid peptides bind more efficiently than the D-amino acid peptide to karyopherin alpha1.

Dimethyl-diphenyl-propanamide derivatives as nonsteroidal dissociated glucocorticoid receptor agonists.

A series of 2,2-dimethyl-3,3-diphenyl-propanamides as novel glucocorticoid receptor modulators is reported. SAR exploration led to the identification of 4-hydroxyphenyl propanamide derivatives displaying good agonist activity in GR-mediated transrepression assays and reduced agonist activity in GR-mediated transactivation assays. Compounds 17 and 30 showed anti-inflammatory activity comparable to prednisolone in the rat carrageenan-induced paw edema model, with markedly decreased side effects with regard to increases in blood glucose and expression of hepatic tyrosine aminotransferase. A hypothetical binding mode accounting for the induction of the functional activity by a 4-hydroxyl group is proposed.

Azaxanthene Based Selective Glucocorticoid Receptor Modulators: Design, Synthesis, and Pharmacological Evaluation of (S)-4-(5-(1-((1,3,4-Thiadiazol-2-yl)amino)-2-methyl-1-oxopropan-2-yl)-5H-chromeno[2,3-b]pyridin-2-yl)-2-fluoro-N,N-dimethylbenzamide (BMS-776532) and Its Methylene Homologue (BMS-791826)

Structurally novel 5H-chromeno[2,3-b]pyridine (azaxanthene) selective glucocorticoid receptor (GR) modulators have been identified. A screening paradigm utilizing cellular assays of GR-mediated transrepression of proinflammatory transcription factors and transactivation of GR-dependent genes combined with three physiologically relevant assays of cytokine induction in human whole blood has allowed for the identification of high affinity, selective GR ligands that display a broad range of pharmacological profiles. Agonist efficacy in reporter assays can be tuned by halogenation of a pendent phenyl ring and correlates well with efficacy for cytokine inhibition in human whole blood. A hypothetical binding mode is proposed, invoking an expanded ligand binding pocket resembling that of arylpyrazole-bound GR structures. Two compounds of close structural similarity (35 and 37; BMS-776532 and BMS-791826, respectively) have been found to maintain distinct and consistent levels of partial agonist efficacy across several assays, displaying anti-inflammatory activity comparable to that of prednisolone 2 in suppressing cytokine production in whole blood and in rodent models of acute and chronic inflammation.

Novel Synthesis of the Hexahydroimidazo[1,5b]isoquinoline Scaffold: Application to the Synthesis of Glucocorticoid Receptor Modulators†

The first stereoselective synthesis of the hexahydroimidazo[1,5b]isoquinoline (HHII) scaffold as a surrogate for the steroidal A-B ring system is described. The structure-activity relationships of the analogs derived from this scaffold show that the basic imidazole moiety is tolerated by the glucocorticoid receptor (GR) in terms of binding affinity, although the partial agonist activity in the transrepressive assays depends on the substitution pattern on the B-ring. More importantly, most compounds in the HHII series bearing a tertiary alcohol moiety on the B-ring are either inactive or significantly less active in inducing GR-mediated transactivation, thus displaying a dissociated pharmacology in vitro.

Discovery of potent and selective nonsteroidal indazolyl amide glucocorticoid receptor agonists

Modification of a phenolic lead structure based on lessons learned from increasing the potency of steroidal glucocorticoid agonists lead to the discovery of exceptionally potent, nonsteroidal, indazole GR agonists. SAR was developed to achieve good selectivity against other nuclear hormone receptors with the ultimate goal of achieving a dissociated GR agonist as measured by human in vitro assays. The specific interactions by which this class of compounds inhibits GR was elucidated by solving an X-ray co-crystal structure.