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.

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.

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.

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Dois derivados cumarínicos, o ester metílico do ácido 4-esculetinocarboxílico (1) e o éster etílico do ácido 4-esculetinocarboxílico (2), foram isolados da esponja marinha Axinella cf. corrugata. A determinação estrutural dos compostos isolados foi realizada pela análise de seus dados espectroscópicos e pela síntese do composto 2. Estes são os primeiros derivados cumarínicos isolados de uma esponja marinha. O éster etílico 2 apresentou atividade in vitro contra a SARS 3Cl-protease e de inibição de células Vero infectadas com o SARS coronavírus, em concentrações que não provocaram citotoxicidade.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.Keywords: esculetin, marine sponge, Axinella cf. corrugata, SARS, anti-viral IntroductionIn 2002 and 2003, several cases of a life-threatening respiratory disease that was ultimately named "severe acute respiratory syndrome" (SARS) were reported from Guangdong Province in mainland China, Hong Kong, Canada, and Vietnam.1 The etiological agent responsible for SARS was quickly found to be a novel coronavirus (SARS-CoV). Among the viral gene products are several large polyproteins that must be proteolytically processed to generate the individual proteins required for viral replication to occur. Two viral proteases, PL2 pro and 3CL pro , are involved in degrading the large polyproteins. 2The viral protease 3CL pro is considered the most important of the two because it is responsible for the release of the key replicative proteins of the vírus, including the viral RNA polymerase and helicase proteins. The central role of 3CL pro in SARS-CoV replication has made it a high profile target for developing antiviral drugs to treat SARS.2 Recently, a novel fluorescence resonance energy transfer (FRET)-based assay to screen for 3CL pro inhibitors has been developed in one of our laboratories.3 As part of a screening of crude marine sponge extracts and pure compounds isolated from the marine sponges using this assay, we have found that the new natural product esculetin-4-carboxylic acid ethyl ester (2) isolated from Axinella cf. corrugata collected in Brazil is a an effective inhibitor of 3CL pro in vitro. 441 Lira et al. Vol. 18, No. 2, 2007 In our current search for new biologically active marine natural products, 4,5 we decided to investigate the MeOH crude extract of the sponge Axinella cf. corrugata, which displayed cytotoxic activity against breast MCF-7 and colon B16 cancer cell lines. Although the cytotoxic activity was lost during the crude extract fractionation, photodiode array detection-HPLC analysi...

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 (Km and kcat values of 14 microM 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 microM) and anti-SARS agent (EC50=112 microM; median toxic concentration>800 microM) from the tropical marine sponge Axinella corrugata.

Severe acute respiratory syndrome-associated coronavirus 2 is a major global health issue and is driving the need for new therapeutics. The surface spike protein, which plays a central role in virus infection, is currently the target for vaccines and neutralizing treatments. The emergence of novel variants with multiple mutations in the spike protein may reduce the effectiveness of neutralizing antibodies by altering the binding activity of the protein with angiotensin-converting enzyme 2 (ACE2). To understand the impact of spike protein mutations on the binding interactions required for virus infection and the effectiveness of neutralizing monoclonal antibody (mAb) therapies, the binding activities of the original spike protein receptor binding domain (RBD) sequence and the reported spike protein variants were investigated using surface plasmon resonance (SPR). In addition, the interactions of the ACE2 receptor, an anti-spike monoclonal antibody (mAb1), a neutralizing monoclonal antibody (mAb2), the original spike RBD sequence, and mutants D614G, N501Y, N439K, Y453F, and E484K were assessed. Compared to the original RBD, the Y453F and N501Y mutants displayed a significant increase in ACE2 binding affinity, whereas D614G had a substantial reduction in binding affinity. All mAb-RBD mutant proteins displayed a reduction in binding affinities relative to the original RBD, except for the E484K-mAb1 interaction. The potential neutralizing capability of mAb1 and mAb2 was investigated. Accordingly, mAb1 failed to inhibit the ACE2-RBD interaction and mAb2 inhibited the ACE2-RBD interactions for all RBD mutants, except mutant E484K, which only displayed partial blocking.

The hepatitis C virus (HCV) nonstructural (NS)3-NS4A serine protease heterocomplex is a prime target for development of novel HCV therapies, due to its essential role in maturation of the viral polyprotein. While the mode of substrate/inhibitor recognition of the HCV NS3/NS4A serine protease has been extensively studied in vitro, important molecular aspects of the mechanism of action for this membrane-bound multifunctional enzyme remain unresolved in vivo. In particular, what influence does membrane association exert on the specificity and catalysis of NS3-4A protease? To carry out this study, we developed a specific and sensitive protease assay using a unique internally quenched fluorogenic substrate (IQFS). Our IQFS enables for the first time the direct, specific detection of NS3-4A protease activity within membrane fractions isolated from human cells expressing NS3-4A and the determination of its steady-state kinetic parameters, which were found to be K(m) = 51 +/- 3 microM and k(cat) = 0.39 min(-1). We also show that our fluorescence-based bioassay can be used to evaluate specifically the potency and mode of action of NS3-4A directed inhibitors, such as in the case of a known NS3-4A substrate-analogue inhibitor (K(i) = 22 nM). Our results indicate that the membrane anchoring of NS3 by NS4A does not affect the substrate/inhibitor recognition by the NS3-4A protease domain. Further investigation may reveal whether membrane association could be important for regulating other enzymatic activities associated with NS3 (e.g., helicase and/or ATPase) and/or regulating the recently proposed cross-talk between the protease and helicase activities.