Novel guanide-substituted compounds bind to CXCR4 and inhibit breast cancer metastasis.

CXCR4 has been shown to be overexpressed on breast cancer cells including the human MDA-MB-231 cell line. Cancer cells overexpressing the CXCR4 receptor are capable of undergoing metastasis to organs expressing high levels of CXCL12. We have synthesized numerous guanide, biguanide, phenylguanide, and naphthylguanide compounds that bind to CXCR4 at the CXCL12-binding site and thus should prevent CXCR4-facilitated cancer metastasis. The novel compounds presented here were tested for CXCR4 affinity, toxicity, receptor activation, and for their ability to prevent breast cancer metastases. Three of the compounds bound to CXCR4 at IC50 values of 0.06-0.2 µmol/l, with no associated cell toxicity or receptor activation at these concentrations. These high CXCR4 affinity compounds also showed inhibition of in-vitro wound migration. They were then tested in an in-vivo mouse breast cancer lung colony model. All of these compounds showed reductions in the number of MDA-MB-231 lung metastases compared with mock-treated control mice without evidence of cardiac, liver, or kidney toxicity in treated mice.

Improved guanide compounds which bind the CXCR4 co-receptor and inhibit HIV-1 infection.

The G-protein coupled receptor CXCR4 is a co-receptor for HIV-1 infection and is involved in signaling cell migration and proliferation. In a previous study of non-peptide, guanide-based CXCR4-binding compounds, spermine and spermidine phenylguanides inhibited HIV-1 entry at low micromolar concentrations. Subsequently, crystal structures of CXCR4 were used to dock a series of naphthylguanide derivatives of the polyamines spermidine and spermine. Synthesis and evaluation of the naphthylguanide compounds identified our best compound, spermine tris-1-naphthylguanide, which bound CXCR4 with an IC(50) of 40 nM and inhibited the infection of TZM-bl cells with X4, but not R5, strains of HIV-1 with an IC(50) of 50-100 nM.

Staphylococcus epidermidis strategies to avoid killing by human neutrophils.

Staphylococcus epidermidis is a leading nosocomial pathogen. In contrast to its more aggressive relative S. aureus, it causes chronic rather than acute infections. In highly virulent S. aureus, phenol-soluble modulins (PSMs) contribute significantly to immune evasion and aggressive virulence by their strong ability to lyse human neutrophils. Members of the PSM family are also produced by S. epidermidis, but their role in immune evasion is not known. Notably, strong cytolytic capacity of S. epidermidis PSMs would be at odds with the notion that S. epidermidis is a less aggressive pathogen than S. aureus, prompting us to examine the biological activities of S. epidermidis PSMs. Surprisingly, we found that S. epidermidis has the capacity to produce PSMδ, a potent leukocyte toxin, representing the first potent cytolysin to be identified in that pathogen. However, production of strongly cytolytic PSMs was low in S. epidermidis, explaining its low cytolytic potency. Interestingly, the different approaches of S. epidermidis and S. aureus to causing human disease are thus reflected by the adaptation of biological activities within one family of virulence determinants, the PSMs. Nevertheless, S. epidermidis has the capacity to evade neutrophil killing, a phenomenon we found is partly mediated by resistance mechanisms to antimicrobial peptides (AMPs), including the protease SepA, which degrades AMPs, and the AMP sensor/resistance regulator, Aps (GraRS). These findings establish a significant function of SepA and Aps in S. epidermidis immune evasion and explain in part why S. epidermidis may evade elimination by innate host defense despite the lack of cytolytic toxin expression. Our study shows that the strategy of S. epidermidis to evade elimination by human neutrophils is characterized by a passive defense approach and provides molecular evidence to support the notion that S. epidermidis is a less aggressive pathogen than S. aureus.

18ß-Glycyrrhetinic Acid Induces Metabolic Changes and Reduces Staphylococcus aureus Bacterial Cell-to-Cell Interactions.

The rise in bacterial resistance to common antibiotics has raised an increased need for alternative treatment strategies. The natural antibacterial product, 18ß-glycyrrhetinic acid (GRA) has shown efficacy against community-associated methicillin-resistant Staphylococcus aureus (MRSA), although its interactions against planktonic and biofilm modes of growth remain poorly understood. This investigation utilized biochemical and metabolic approaches to further elucidate the effects of GRA on MRSA. Prolonged exposure of planktonic MRSA cell cultures to GRA resulted in increased production of staphyloxanthin, a pigment known to exhibit antioxidant and membrane-stabilizing functions. Then, 1D 1H NMR analyses of intracellular metabolite extracts from MRSA treated with GRA revealed significant changes in intracellular polar metabolite profiles, including increased levels of succinate and citrate, and significant reductions in several amino acids, including branch chain amino acids. These changes reflect the MRSA response to GRA exposure, including potentially altering its membrane composition, which consumes branched chain amino acids and leads to significant energy expenditure. Although GRA itself had no significant effect of biofilm viability, it seems to be an effective biofilm disruptor. This may be related to interference with cell-cell aggregation, as treatment of planktonic MRSA cultures with GRA leads to a significant reduction in micro-aggregation. The dispersive nature of GRA on MRSA biofilms may prove valuable for treatment of such infections and could be used to increase susceptibility to complementary antibiotic therapeutics.

Novel compounds containing multiple guanide groups that bind the HIV coreceptor CXCR4.

The G-protein-coupled receptor CXCR4 acts as a coreceptor for human immunodeficiency virus type 1 (HIV-1) infection, as well as being involved in signaling cell migration and proliferation. Compounds that block CXCR4 interactions have potential uses as HIV entry inhibitors to complement drugs such as maraviroc that block the alternate coreceptor CCR5 or in cancer therapy. The peptide T140, which contains five arginine residues, is the most potent antagonist of CXCR4 developed to date. In a search for nonpeptide CXCR4 ligands that could inhibit HIV entry, three series of compounds were synthesized from 12 linear and branched polyamines with 2, 3, 4, 6, or 8 amino groups, which were substituted to produce the corresponding guanidines, biguanides, or phenylguanides. The resulting compounds were tested for their ability to compete with T140 for binding to the human CXCR4 receptor expressed on mammalian cells. The most effective compounds bound CXCR4 with a 50% inhibitory concentration of 200 nM, and all of the compounds had very low cytotoxicity. Two series of compounds were then tested for their ability to inhibit the infection of TZM-bl cells with X4 and R5 strains of HIV-1. Spermine phenylguanide and spermidine phenylguanide inhibited infection by X4 strains, but not by R5 strains, at low micromolar concentrations. These results support further investigation and development of these compounds as HIV entry inhibitors.

Exposure of Methicillin-Resistant Staphylococcus aureus to Low Levels of the Antibacterial THAM-3ΦG Generates a Small Colony Drug-Resistant Phenotype.

This study investigated resistance against trishexylaminomelamine trisphenylguanide (THAM-3ΦG), a novel antibacterial compound with selective microbicidal activity against Staphylococcus aureus. Resistance development was examined by culturing methicillin resistant S. aureus (MRSA) with sub-lethal doses of THAM-3ΦG. This quickly resulted in the formation of normal (WT) and small colonies (SC) of S. aureus exhibiting minimal inhibitory concentrations (MICs) 2× and 4× greater than the original MIC. Continuous cell passaging with increasing concentrations of THAM-3ΦG resulted in an exclusively SC phenotype with MIC >64 mg/L. Nuclear magnetic resonance (NMR)-based metabolomics and multivariate statistical analysis revealed three distinct metabolic profiles for THAM-3ΦG treated WT, untreated WT, and SC (both treated and untreated). The metabolome patterns of the SC sample groups match those reported for other small colony variants (SCV) of S. aureus. Supplementation of the SCV with menadione resulted in almost complete recovery of growth rate. This auxotrophism was corroborated by NMR analysis revealing the absence of menaquinone production in the SCV. In conclusion, MRSA rapidly acquires resistance to THAM-3ΦG through selection of a slow-growing menaquinone auxotroph. This study highlights the importance of evaluating and monitoring resistance to novel antibacterials during development.

Characterization of the antibacterial activity of Bald's eyesalve against drug resistant Staphylococcus aureus and Pseudomonas aeruginosa.

Bald's eyesalve is an Anglo-Saxon medicinal remedy that has been used through ancient times to treat eye sty infections and may represent a source of ancientbiotics. This study assessed the efficacy of Bald's eyesalve against several strains of Staphylococcus aureus and Pseudomonas aeruginosa, including a multi-drug resistant phenotype, and identified the principal compound conveying antibacterial activity. Bald's eyesalve formulations were produced by combining garlic, onion or leek, wine, bovine bile, and brass, with specific ingredient omissions in several formulations, followed by incubation at 4 °C for 9 days. Bald's eyesalve formulation ES-GBBr exhibited the greatest antibacterial activity against S. aureus and P. aeruginosa. Fractionation of ES-GBBr using molecular size exclusion and organic solvent partitioning isolated its antibacterial activity to the small molecule nonpolar fraction, and 1D 1H NMR revealed the identity of the antibacterial agent to be allicin. Depletion of allicin from this fraction by addition of exogenous cysteine established that all observable growth inhibition originated from allicin. Quantification of allicin demonstrated that its concentration was significantly greater in ES-GBBr compared to the ES-O formulation; however, this was not due to greater yield. The antibacterial activity of allicin against S. aureus was antagonized by other ingredients within Bald's eyesalve, whereas they were additive or synergistic against P. aeruginosa. These results suggest that neither leek nor onion is necessary for the antibacterial efficacy of Bald's eyesalve against S. aureus or P. aeruginosa, and while allicin was identified as the principal antibacterial agent present, its activity is influenced differentially in the presence of additional Bald's eyesalve ingredients when used against S. aureus compared to P. aeruginosa. Ancientbiotics may provide a source of promising antibacterials; however, identifying the source of activity and assessing distinct formulations for cooperative effects are essential to using ancient remedies, such as Bald's eyesalve, effectively against drug resistant pathogens.

Peptides selected from a phage display library with an HIV-neutralizing antibody elicit antibodies to HIV gp120 in rabbits, but not to the same epitope.

Monoclonal antibodies specific for the conserved CD4 binding site region of the HIV envelope protein gp120 were used to select phage from two different random peptide display libraries. Synthetic peptides were made with sequences corresponding to those displayed on the selected phage, and peptide-protein fusions were expressed that contained the selected phage-displayed peptide sequence and either the N-terminal domain of the phage pIII protein or the small heat shock protein of Methanococcus jannaschii or both. For monoclonal antibody 5145A, these constructs containing the selected peptide sequences were all capable of specifically inhibiting the binding of 5145A to HIV-1 gp120. Rabbits immunized with peptide-protein fusions produced antisera that bound to recombinant HIV-1 gp120, but did not bind to HIV-infected cells nor neutralize HIV. The antisera also did not compete with CD4 or antibodies to the CD4 binding site for binding to gp120.

Coupling Peptide Antigens to Virus-Like Particles or to Protein Carriers Influences the Th1/Th2 Polarity of the Resulting Immune Response.

We have conjugated the S9 peptide, a mimic of the group B streptococcal type III capsular polysaccharide, to different carriers in an effort to elicit an optimal immune response. As carriers, we utilized the soluble protein keyhole limpet hemocyanin and virus-like particles (VLPs) from two plant viruses, Cowpea Chlorotic Mottle Virus and Cowpea Mosaic Virus. We have found that coupling the peptide to the soluble protein elicits a Th2 immune response, as evidenced by the production of the peptide-specific IgG1 antibody and IL-4/IL-10 production in response to antigen stimulation, whereas the peptide conjugated to VLPs elicited a Th1 response (IgG2a, IFN-γ). Because the VLPs used as carriers package RNA during the assembly process, we hypothesize that this effect may result from the presence of nucleic acid in the immunogen, which affects the Th1/Th2 polarity of the response.

Antibacterial activity of THAM Trisphenylguanide against methicillin-resistant Staphylococcus aureus.

This study investigated the potential antibacterial activity of three series of compounds synthesized from 12 linear and branched polyamines with 2-8 amino groups, which were substituted to produce the corresponding guanides, biguanides, or phenylguanides, against Acinetobacter baumannii, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. Antibacterial activity was measured for each compound by determining the minimum inhibitory concentration against the bacteria, and the toxicity towards mammalian cells was determined. The most effective compound, THAM trisphenylguanide, was studied in time-to-kill and cytoplasmic leakage assays against methicillin-resistant Staphylococcus aureus (MRSA, USA300) in comparison to chlorhexidine. Preliminary toxicity and MRSA challenge studies in mice were also conducted on this compound. THAM trisphenylguanide showed significant antibacterial activity (MIC ∼1 mg/L) and selectivity against MRSA relative to all the other bacteria examined. In time-to-kill assays it showed increased antimicrobial activity against MRSA versus chlorhexidine. It induced leakage of cytoplasmic content at concentrations that did not reduce cell viability, suggesting the mechanism of action may involve membrane disruption. Using an intraperitoneal mouse model of invasive MRSA disease, THAM trisphenylguanide reduced bacterial burden locally and in deeper tissues. This study has identified a novel guanide compound with selective microbicidal activity against Staphylococcus aureus, including a methicillin-resistant (MRSA) strain.

Structure of the Fab fragment of F105, a broadly reactive anti-human immunodeficiency virus (HIV) antibody that recognizes the CD4 binding site of HIV type 1 gp120.

We have determined the crystal structure of the Fab fragment from F105, a broadly reactive human antibody with limited potency that recognizes the CD4 binding site of gp120. The structure reveals an extended CDR H3 loop with a phenylalanine residue at the apex and shows a striking pattern of serine and tyrosine residues. Modeling the interaction between gp120 and F105 suggests that the phenylalanine may recognize the binding pocket of gp120 used by Phe(43) of CD4 and that numerous tyrosine and serine residues form hydrogen bonds with the main chain atoms of gp120. A comparison of the F105 structure to that of immunoglobulin G1 b12, a much more potent and broadly neutralizing antibody with an overlapping epitope, suggests similarities that contribute to the broad recognition of human immunodeficiency virus by both antibodies. While the putative epitope for F105 shows significant overlap with that predicted for b12, it appears to differ from the b12 epitope in extending across the interface between the inner and outer domains of gp120. In contrast, the CDR loops of b12 appear to interact predominantly with the outer domain of gp120. The difference between the predicted epitopes for b12 and F105 suggests that the unique potency of b12 may arise from its ability to avoid the interface between the inner and outer domains of gp120.

Evidence for direct interaction between actin and the cystic fibrosis transmembrane conductance regulator.

Previous studies have demonstrated that actin filament organization controls the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel function. The precise molecular nature of the interaction between actin and CFTR, however, remains largely unknown. In this report, interactions between actin and purified human epithelial CFTR were directly assessed by reconstitution of the channel protein in a lipid bilayer system and by atomic force microscopy (AFM). CFTR-containing liposomes in solution were deposited on freshly cleaved mica and imaging was performed in tapping-mode AFM. CFTR function was also determined in identical preparations. Images of single CFTR molecules were obtained, and addition of monomeric actin below its critical concentration showed the formation of actin filaments associated with CFTR. The data indicate a direct interaction between actin and CFTR exists, which may explain the regulatory role of the cytoskeleton in ion channel function. This was confirmed by functional studies of CFTR single-channel currents, which were regulated by addition of various conformations of actin. The present study indicates that CFTR may directly bind actin and that this interaction helps affect the functional properties of this channel protein.