A hybrid ligand and structure-based virtual screening of NCI compound library identifies potential SAPT1 inhibitors

open access articleSecretory aspartate proteases (SAPs) is a key virulence factor of Candida spp. enabling adherence to and invasion of host tissues through breakdown of host proteins related to immunological and structural defenses, making them potential drug targets for drug-resistant mycoses, especially where the available therapies fail. To date, no SAP inhibitors for Candida tropicalis, one of the top five most common species isolated in Candida-related mycoses, has been reported. In this study, we report first-time identification of a set of potential C. tropicalis SAP1 (SAPT1) inhibitors through a hybrid ligand-and structure-based virtual screening of National Cancer Institute (NCI) library compounds. The NCI library was refined by filtering off non-druglike molecules. Referring to a known C. albicans SAP inhibitor, a similarity search was performed for the refined library, in addition to a pharmacophore screen using a model of the ligand-receptor interactions between a peptide substrate and SAPT1 obtained from crystallographic data. The compounds selected from these screens were subject to molecular docking to the SAPT1 active site and the top-scoring ligand-receptor complexes were further included in MM-GBSA calculations to optimize the predicted binding affinities. Finally, the selected 16 compounds, which were confirmed to make key interactions with the catalytic residues, were in silico evaluated and found eligible for certain pharmacokinetic properties. As a future prospect, obtaining these virtual hits and testing them in vitro against SAPT1 could validate the virtual screening process and yield the first small molecule inhibitors of SAPT

Structure-function analysis for the development of peptide inhibitors for a Gram-positive quorum sensing system

open access articleThe Streptococcus pneumoniae Rgg144/SHP144 regulator-peptide quorum sensing (QS) system is critical for nutrient utilization, oxidative stress response, and virulence. Here, we characterized this system by assessing the importance of each residue within the active short hydrophobic peptide (SHP) by alanine-scanning mutagenesis and testing the resulting peptides for receptor binding and activation of the receptor. Interestingly, several of the mutations had little effect on binding to Rgg144 but reduced transcriptional activation appreciably. In particular, a proline substitution (P21A) reduced transcriptional activation by 29-fold but bound with a 3-fold higher affinity than the wild-type SHP. Consistent with the function of Rgg144, the mutant peptide led to decreased utilization of mannose and increased susceptibility to superoxide generator paraquat. Pangenome comparison showed full conservation of P21 across SHP144 allelic variants. Crystallization of Rgg144 in the absence of peptide revealed a comparable structure to the DNA bound and free forms of its homologs suggesting similar mechanisms of activation. Together, these analyses identify key interactions in a critical pneumococcal QS system. Further manipulation of the SHP has the potential to facilitate the development of inhibitors that are functional across strains. The approach described here is likely to be effective across QS systems in multiple species

Inactivation of the Complement Lectin Pathway by Candida tropicalis Secreted Aspartyl Protease-1

open access articleCandida tropicalis is an opportunistic fungal pathogen and is one of the most frequently isolated non-albicans species. It can cause localised as well as invasive systemic infections particularly in immunocompromised patients. Increased resistance to common anti-fungal drugs is an emerging problem. In order to establish disseminated infections, Candida has evolved several strategies to escape the host immune system. A detailed understanding of how C. tropicalis escapes the host immune attack is needed as it can help develop novel anti-fungal therapies. Secreted aspartyl proteinases (Saps) of C. albicans have been shown to be determinants of virulence and immune evasion. However, the immune evasion properties of C. tropicalis Saps have been poorly characterised. This study investigated the immune evasion properties of C. tropicalis secreted aspartic protease 1 (Sapt1). Sapt1 was recombinantly produced using a Kluyveromyces lactis yeast expression system. A range of complement proteins and immunogloublins were screened to test if Sapt1 had any proteolytic activity. Sapt1 efficiently cleaved human mannose-binding lectin (MBL) and collectin-11, which are the initiating molecules of the lectin pathway of the complement system, but not l-ficolin. In addition, Sapt1 cleaved DC-SIGN, the receptor on antigen presenting dendritic cells. Proteolysis was prominent in acidic condition (pH 5.2), a characteristic of aspartyl protease. No proteolytic activity was detected against complement proteins C1q, C3, C3b, IgG and IgA. In view of the ability of Sapt1 to cleave MBL and collectin-11, we found that Sapt1 could prevent activation of the complement lectin pathway. RT-qPCR analysis using three different C. tropicalis clinical isolates (oral, blood and peritoneal dialysis fluid) revealed relatively higher levels of mRNA expression of Sapt1 gene when compared to a reference strain; Sapt1 protein was found to be secreted by all the tested strains. Lectin pathway and its initiating components are crucial to provide front line defence against Candida infections. For the first time, we have shown that a Candida protease can proteolytically degrade the key initiating components of lectin pathway and inhibit complement activation. Findings from this study highlight the importance of exploring Sapt1 as a potential therapeutic target. We conclude that C. tropicalis secretes Sapt1 to target the complement lectin pathway, a key pattern recognition and clearance mechanism, for its survival and pathogenesis

Engineering novel complement activity into a pulmonary surfactant protein

Complement neutralizes invading pathogens, stimulates inflammatory and adaptive immune responses, and targets non- or altered-self structures for clearance. In the classical and lectin activation pathways, it is initiated when complexes composed of separate recognition and activation subcomponents bind to a pathogen surface. Despite its apparent complexity, recognition-mediated activation has evolved independently in three separate protein families, C1q, mannose-binding lectins (MBLs), and serum ficolins. Although unrelated, all have bouquet-like architectures and associate with complement-specific serine proteases: MBLs and ficolins with MBL-associated serine protease-2 (MASP-2) and C1q with C1r and C1s. To examine the structural requirements for complement activation, we have created a number of novel recombinant rat MBLs in which the position and orientation of the MASP-binding sites have been changed. We have also engineered MASP binding into a pulmonary surfactant protein (SP-A), which has the same domain structure and architecture as MBL but lacks any intrinsic complement activity. The data reveal that complement activity is remarkably tolerant to changes in the size and orientation of the collagenous stalks of MBL, implying considerable rotational and conformational flexibility in unbound MBL. Furthermore, novel complement activity is introduced concurrently with MASP binding in SP-A but is uncontrolled and occurs even in the absence of a carbohydrate target. Thus, the active rather than the zymogen state is default in lectin·MASP complexes and must be inhibited through additional regions in circulating MBLs until triggered by pathogen recognition

Structure of the C1r-C1s interaction of the C1 complex of complement activation.

The multiprotein complex C1 initiates the classical pathway of complement activation on binding to antibody-antigen complexes, pathogen surfaces, apoptotic cells, and polyanionic structures. It is formed from the recognition subcomponent C1q and a tetramer of proteases C1r2C1s2 as a Ca2+-dependent complex. Here we have determined the structure of a complex between the CUB1-EGF-CUB2 fragments of C1r and C1s to reveal the C1r-C1s interaction that forms the core of C1. Both fragments are L-shaped and interlock to form a compact antiparallel heterodimer with a Ca2+ from each subcomponent at the interface. Contacts, involving all three domains of each protease, are more extensive than those of C1r or C1s homodimers, explaining why heterocomplexes form preferentially. The available structural and biophysical data support a model of C1r2C1s2 in which two C1r-C1s dimers are linked via the catalytic domains of C1r. They are incompatible with a recent model in which the N-terminal domains of C1r and C1s form a fixed tetramer. On binding to C1q, the proteases become more compact, with the C1r-C1s dimers at the center and the six collagenous stems of C1q arranged around the perimeter. Activation is likely driven by separation of the C1r-C1s dimer pairs when C1q binds to a surface. Considerable flexibility in C1s likely facilitates C1 complex formation, activation of C1s by C1r, and binding and activation of downstream substrates C4 and C4b-bound C2 to initiate the reaction cascade.Funding for this work was provided by the Medical Research Council (Grant G1000191/1, to R.W., P.C.E.M., and W.J.S.)

Molecular basis of sugar recognition by collectin-K1 and the effects of mutations associated with 3MC syndrome

Background Collectin-K1 (CL-K1, or CL-11) is a multifunctional Ca2+-dependent lectin with roles in innate immunity, apoptosis and embryogenesis. It binds to carbohydrates on pathogens to activate the lectin pathway of complement and together with its associated serine protease MASP-3 serves as a guidance cue for neural crest development. High serum levels are associated with disseminated intravascular coagulation, where spontaneous clotting can lead to multiple organ failure. Autosomal mutations in the CL-K1 or MASP-3 genes cause a developmental disorder called 3MC (Carnevale, Mingarelli, Malpuech and Michels) syndrome, characterised by facial, genital, renal and limb abnormalities. One of these mutations (Gly204Ser in the CL-K1 gene) is associated with undetectable levels of protein in the serum of affected individuals. Results In this study, we show that CL-K1 primarily targets a subset of high-mannose oligosaccharides present on both self- and non-self structures, and provide the structural basis for its ligand specificity. We also demonstrate that three disease-associated mutations prevent secretion of CL-K1 from mammalian cells, accounting for the protein deficiency observed in patients. Interestingly, none of the mutations prevent folding nor oligomerization of recombinant fragments containing the mutations in vitro. Instead, they prevent Ca2+ binding by the carbohydrate-recognition domains of CL-K1. We propose that failure to bind Ca2+ during biosynthesis leads to structural defects that prevent secretion of CL-K1, thus providing a molecular explanation of the genetic disorder. Conclusions We have established the sugar specificity of CL-K1 and demonstrated that it targets high-mannose oligosaccharides on self- and non-self structures via an extended binding site which recognises the terminal two mannose residues of the carbohydrate ligand. We have also shown that mutations associated with a rare developmental disorder called 3MC syndrome prevent the secretion of CL-K1, probably as a result of structural defects caused by disruption of Ca2+ binding during biosynthesis

Molecular characterization of the lectin pathway of complement activation

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Carbohydrate recognition and complement activation by rat ficolin-B

Ficolins are innate immune components that bind to PAMPs and structures on apoptotic cells. Humans produce two serum forms (L-and H-ficolin) and a leukocyte-associated form (M-ficolin), whereas rodents and most other mammals produce ficolins-A and -B, orthologues of L-and M-ficolin, respectively. All three human ficolins, together with mouse and rat ficolin-A, associate with mannan-binding lectin-associated serine proteases (MASPs) and activate the lectin pathway of complement on PAMPs. By contrast, mouse ficolin-B does not bind MASPs and cannot activate complement. Because of these striking differences together with the lack of functional information for other ficolin-B orthologues, we have characterized rat ficolin-B, and compared its physical and biochemical properties with its serum counterpart. The data show that both rat ficolins have archetypal structures consisting of oligomers of a trimeric subunit. Ficolin-B recognized mainly sialyated sugars, characteristic of exogenous and endogenous ligands, whereas ficolin-A had a surprisingly narrow specificity, binding strongly to only one of 320 structures tested: an N-acetylated trisaccharide. Surprisingly, rat ficolin-B activated MASP-2 comparable to ficolin-A. Mutagenesis data reveal that lack of activity in mouse ficolin-B is probably caused by a single amino acid change in the putative MASP-binding site that blocks the ficolin-MASP interaction

Candida Pathogenicity and Interplay with the Immune System

Candida species are opportunistic fungal pathogens that are part of the normal skin and mucosal microflora. Overgrowth of Candida can cause infections such as thrush or life-threatening invasive candidiasis in immunocompromised patients. Though Candida albicans is highly prevalent, several non-albicans species are also isolated from nosocomial infections. Candida sp. are over presented in the gut of people with Crohn’s disease and certain types of neurological disorders, with hyphal form and biofilms being the most virulent states. In addition, Candida uses several secreted and cell surface molecules such as pH related antigen 1, High affinity glucose transporter, Phosphoglycerate mutase 1 and lipases to establish pathogenicity. A strong innate immune response is elicited against Candida via dendritic cells, neutrophils and macrophages. All three complement pathways are also activated. Production of proinflammatory cytokines IL-10 and IL-12 signal differentiation of CD4+ cells into Th1 and Th2 cells, whereas IL-6, IL-17 and IL-23 induce Th17 cells. Importance of T-lymphocytes is reflected in depleted T-cell count patients being more prone to Candidiasis. Anti- Candida antibodies also play a role against candidiasis using various mechanisms such as targeting virulent enzymes and exhibiting direct candidacidal activity. However, the significance of antibody response during infection remains controversial. Furthermore, some of the Candida strains have evolved molecular strategies to evade the sophisticated host attack by proteolysis of components of immune system and interfering with immune signalling pathways. Emergence of several non-albicans species that are resistant to current antifungal agents makes treatment more difficult. Therefore, deeper insight into interactions between Candida and the host immune system is required for discovery of novel therapeutic options

Structural basis of the C1q/C1s interaction and its central role in assembly of the C1 complex of complement activation

Complement component C1, the complex that initiates the classical pathway of complement activation, is a 790-kDa assembly formed from the target-recognition subcomponent C1q and the modular proteases C1r and C1s. The proteases are elongated tetramers that become more compact when they bind to the collagen-like domains of C1q. Here, we describe a series of structures that reveal how the subcomponents associate to form C1. A complex between C1s and a collagen-like peptide containing the C1r/C1sbinding motif of C1q shows that the collagen binds to a shallow groove via a critical lysine side chain that contacts Ca2+-coordinating residues. The data explain the Ca2+-dependent binding mechanism, which is conserved in C1r and also in mannan-binding lectin-associated serine proteases, the serine proteases of the lectin pathway activation complexes. In an accompanying structure, C1s forms a compact ring-shaped tetramer featuring a unique head-to-tail interaction at its center that replicates the likely arrangement of C1r/C1s polypeptides in the C1 complex. Additional structures reveal how C1s polypeptides are positioned to enable activation by C1r and interaction with the substrate C4 inside the cage-like assembly formed by the collagenous stems of C1q. Together with previously determined structures of C1r fragments, the results reported here provide a structural basis for understanding the early steps of complement activation via the classical pathway