Pamela Hamill

Peptide Antimicrobial Agents

SUMMARY Antimicrobial host defense peptides are produced by all complex organisms as well as some microbes and have diverse and complex antimicrobial activities. Collectively these peptides demonstrate a broad range of antiviral and antibacterial activities and modes of action, and it is important to distinguish between direct microbicidal and indirect activities against such pathogens. The structural requirements of peptides for antiviral and antibacterial activities are evaluated in light of the diverse set of primary and secondary structures described for host defense peptides. Peptides with antifungal and antiparasitic activities are discussed in less detail, although the broad-spectrum activities of such peptides indicate that they are important host defense molecules. Knowledge regarding the relationship between peptide structure and function as well as their mechanism of action is being applied in the design of antimicrobial peptide variants as potential novel therapeutic agents.

Intracellular Receptor for Human Host Defense Peptide LL-37 in Monocytes

The human cationic host defense peptide LL-37 has a broad range of immunomodulatory, anti-infective functions. A synthetic innate defense regulator peptide, innate defense regulator 1 (IDR-1), based conceptually on LL-37, was recently shown to selectively modulate innate immunity to protect against a wide range of bacterial infections. Using advanced proteomic techniques, ELISA, and Western blotting procedures, GAPDH was identified as a direct binding partner for LL-37 in monocytes. Enzyme kinetics and mobility shift studies also indicated LL-37 and IDR-1 binding to GAPDH. The functional relevance of GAPDH in peptide-induced responses was demonstrated by using gene silencing of GAPDH with small interfering RNA (siRNA). Previous studies have established that the induction of chemokines and the anti-inflammatory cytokine IL-10 are critical immunomodulatory functions in the anti-infective properties of LL-37 and IDR-1, and these functions are modulated by the MAPK p38 pathway. Consistent with that, this study demonstrated the importance of the GAPDH interactions with these peptides since gene silencing of GAPDH resulted in impaired p38 MAPK signaling, downstream chemokine and cytokine transcriptional responses induced by LL-37 and IDR-1, and LL-37-induced cytokine production. Bioinformatic analysis, using InnateDB, of the major interacting partners of GAPDH indicated the likelihood that this protein can impact on innate immune pathways including p38 MAPK. Thus, this study has demonstrated a novel function for GAPDH as a mononuclear cell receptor for human cathelicidin LL-37 and immunomodulatory IDR-1 and conclusively demonstrated its relevance in the functioning of cationic host defense peptides.

Novel anti-infectives: is host defence the answer?

Resistance to antimicrobial agents and the limited development of novel agents are threatening to worsen the burden of infections that are already a leading cause of morbidity and mortality. This has increased interest in the development of novel strategies such as selective modulation of our natural immune defences. Innate immunity is a complex, evolutionarily conserved, multi-facetted response to defeating infection that is naturally stimulated by pathogenic organisms through pattern recognition receptors on host cells. It is amplifiable and broad spectrum but if overstimulated can lead to the potential for harmful inflammatory responses. A broad variety of therapies are already available or increasingly under development, to stimulate protective innate immunity without overtly stimulating harmful inflammation or even suppressing such damaging pro-inflammatory responses.

Systems biology evaluation of immune responses induced by human host defence peptide LL-37 in mononuclear cells

The immune system is very complex, it involves the integrated regulation and expression of hundreds of proteins. To understand in greater detail how the human host defence immunomodulatory peptide LL-37 interacts with innate immunity, a systems approach was pursued. Polychromatic flow cytometry was employed to demonstrate that within human peripheral blood mononuclear cells, CD14+ monocytes, myeloid and plasmocytoid dendritic cells and T- and B-lymphocytes, all responded to LL-37, with the differential production of intracellular cytokines. Microarray analyses with CD14+ monocytes indicated the differential expression of 475 genes in response to stimulation with LL-37. To understand this complex response, bioinformatic interrogation, using InnateDB, of the gene ontology, signalling pathways and transcription factor binding sites was undertaken. Activation of the IkappaBalpha/NFkappaB, mitogen-activated protein kinases p38, ERK1/2 and JNK, and PI3K signalling pathways in response to LL-37 was demonstrated by pathway and ontology over-representation analyses, and confirmed experimentally by inhibitor studies. Computational analysis of the predicted transcription factor binding sites upstream of the genes that were regulated by LL-37 predicted the involvement of several transcription factors including NFkappaB and five novel factors, AP-1, AP-2, SP-1, E2F1, and EGR, which were experimentally confirmed to respond to LL-37 by performing transcription factor array studies on nuclear extracts from LL-37 treated mononuclear cells. These data are discussed as reflecting the integration of several responsive signalling pathways through the involvement of transcription factor complexes in gene expression activated by LL-37 in human mononuclear cells.

Development of a red-shifted fluorescence-based assay for SARS-coronavirus 3CL protease: identification of a novel class of anti-SARS agents from the tropical marine sponge Axinella corrugata

Abstract SARS-coronavirus (SARS-CoV) encodes a main protease, 3CLpro, which plays an essential role in the viral life cycle and is currently the prime target for discovering new anti-coronavirus agents. In this article, we report our success in developing a novel red-shifted (RS) fluorescence-based assay for 3CLpro and its application for identifying small-molecule anti-SARS agents from marine organisms. We have synthesised and characterised the first generation of a red-shifted internally quenched fluorogenic substrate (RS-IQFS) for 3CLpro based on resonance energy transfer between the donor and acceptor pair CAL Fluor Red 610 and Black Hole Quencher-1 (K m and k cat values of 14 μM and 0.65 min-1). The RS-IQFS primary sequence was selected based on the results of our screening analysis of 3CLpro performed using a series of blue-shifted (BS)-IQFSs corresponding to the 3CLpro-mediated cleavage junctions of the SARS-CoV polyproteins. In contrast to BS-IQFSs, the RS-IQFS was not susceptible to fluorescence interference from coloured samples and allowed for successful screening of marine natural products and identification of a coumarin derivative, esculetin-4-carboxylic acid ethyl ester, a novel 3CLpro inhibitor (IC50=46 μM) and anti-SARS agent (EC50=112 μM; median toxic concentration >800 μM) from the tropical marine sponge Axinella corrugata.

A SARS-coronovirus 3CL protease inhibitor isolated from the marine sponge Axinella cf. corrugata: structure elucidation and synthesis

Two coumarin derivatives, esculetin-4-carboxylic acid methyl ester (1) and esculetin-4-carboxylic acid ethyl ester (2), have been isolated from the marine sponge Axinella cf. corrugata. Structure determination included analysis of spectroscopic data and total synthesis of compound 2. These are the first coumarin derivatives isolated from a marine sponge. The ethyl ester 2 was found to be an in vitro inhibitor of SARS 3CL-protease and an effective inhibitor of SARS-CoV replication in Vero cells at non-cytotoxic concentrations.

Novel Blue- and Red-Shifted Internally Quenched Fluorogenic Substrates for Continuous Monitoring of SARS-CoV 3CLpro

Introduction The SARS coronavirus (SARS-CoV)-encoded main protease, 3CL or M, plays an essential role in the viral life cycle and is currently the main focus for the development of anti-coronaviral therapies. The genome of SARS-CoV is translated in the cytoplasm of infected cells to generate several large polyproteins. For viral replication to occur, these polyproteins must be proteolytically processed into individual proteins, a process that is mediated by two viral proteases; PL2 and 3CL [1]. 3CL is considered the main viral protease as it releases the key replicative proteins of the virus, including the viral RNA polymerase and helicase proteins. Since the emergence of SARS-CoV in late 2002, the development of sensitive assays to detect recombinant 3CL activity in formats amenable to highthroughput screening (HTS) has been a priority for the generation of novel 3CL inhibitors as anti-viral therapies. Here, we report our success in developing blueand red-shifted internally quenched fluorogenic substrates (IQFSs) based on resonance energy transfer between the donor and acceptor pairs Abz/Tyr(3-NO2) [IQFS-1], and CAL Fluor Red 610 (CalRed 610)/Black Hole Quencher-2 (BHQ-2) [IQFS-2], using a decapeptide sequence corresponding to that of the cleavage site between the non-structural protein (nsp)4/nsp5 in the SARS-CoV polyprotein pp1ab.