Current Drug Targets (v.13, #3)

Infectious diseases have been one of the most critical threats to humans throughout evolution. Antibiotics have played a major role in controlling infectious diseases and increasing life span since the 1940s. Antibiotics target molecules involved in essential bacterial functions such as DNA, RNA, protein and lipid metabolism or cell wall formation. Wide use of antibiotics generates strong positive selective pressure for bacterial stains that are resistant to antibiotics, which arose shortly after the introduction of antibiotic use [1]. Over the years, nearly all major pathogens have developed resistance to antibiotics; stains have emerged that are resistant to most available antibiotics which pose a serious challenge to public healthcare systems worldwide. The emergence of multiply resistant strains is attributed to inappropriate and excessive use of antibiotics [2]. On the other hand, the development of novel antibiotics has been lagging [3, 4]. There is a thus an urgent need to develop new antimicrobial therapy which calls for more research on the mechanisms of bacterial pathogenicity that may lead to innovative alternative therapeutic approaches. In this special issue about bacterial virulence and the development of novel antimicrobial approaches, we have compiled seven reviews dealing with topics ranging from the mechanism of bacterial infection to the current status of antibiotic development. McArthur et al. describe the role of the major virulence factor streptokinase in Streptococcus pyogenes infection and discuss potential strategies to disrupt streptokinase's function for the treatment of invasive streptococcal infection. Streptococcus pyogenes is one of the most common human pathogens, causing various diseases from pharyngitis and mild skin infection to severe life threatening invasive diseases. S. pyogenes produces streptokinase to specifically activate human plasminogen, which plays a significant role in the pathogenicity of S. pyogenes infection. Based on the critical role of streptokinase in streptococcal virulence, novel therapeutical strategies are proposed to inhibit the interaction of streptokinase with plasminogen. Thomas and Lee provide a broader picture of S. pyogenes virulence, highlighting a number of major virulence factors of S. pyogenes. Potential therapeutic approaches dealing with streptococcal infection such as antimicrobial peptides, bacteriophage therapy and utilizing pathogenomics to identify potential therapeutic and vaccine targets are described. Gamez and Hammerschmidt highlight studies on Streptococcus pneumoniae adhesins and the potential of using adhesins as vaccine candidates. This review examines the pathogenic role of pneumococcal adhesins and their interactions with host proteins to evade host immune and cellular responses. These authors suggested that pneumococcal adhesins will be effective candidates for adhesin-based vaccines that may potentially prevent pneumococcal infections. Kline et al. describe type III secret systems (T3SS) and the potential of targeting it for antimicrobial therapy in Gram negative pathogens. T3SSs are Gram negative bacterial transmembrane protein systems that translocate virulence determinants or effector proteins into host cells. T3SS are well conserved among several Gram negative bacterial pathogens, making it feasible to identify compounds inhibiting the assembly or secretion process of T3SS in a broad range of Gram negative pathogens. This article listed efforts that identified inhibitors of T3SS by whole cell based high throughput screening and by specifically targeting certain proteins in the system by either in silico design or immuno-inhibition. The advantages and drawbacks of both approaches were discussed. Whole cell based high throughput screening will identify multiple broad spectrum inhibitors capable of inhibiting function of T3SS in multiple pathogens. Substantial challenges remain to identify the targets of the inhibitors of T3SS in such complicated systems and further optimization of leads. Target based screening on the other hand may prove to be of narrow spectrum and limited potency. Despite of the limitation of these approaches, the research summarized in this article illustrates the premise of some alternative approaches to target important virulence mechanisms of pathogens to counter the rise of antibiotic resistance. Robinson et al. assess the potential of targeting DNA replication for novel therapy. Due to the advances made in last decade in genome sequencing and other high throughput techniques, it is now feasible to assess the degree of conservation of bacterial DNA replication machinery between a wide range of bacteria in order to choose the best therapeutic targets. This article described our current understanding of bacterial DNA replication, progress in development of novel inhibitors of components of DNA replication machinery and opportunity to utilize genomic and structural knowledge to aid the design of potential inhibitors. However, significant hurdles still exist with this approach. Nonetheless, with an abundance of information of protein structure and protein-protein interaction, selective inhibitors may be developed in near future. The final two reviews describe the general landscape of searching for novel antibiotics. In spite of the obstacles for developing novel antibiotics, such as lack of economic incentives, the academic and commercial research communities continue contributing great effort to antibiotic research and development. Bulter and Cooper review screening strategies used to identify potential antibiotics. The article describes various screening methods for new antibiotics. In addition to well established traditional whole cell screening, high throughput screening combining with genomic information, screening with whole organism infection models, high content screening by analysing living cells and whole organisms, and antisense technology have all been implemented to advance antibacterial drug development. An interesting area for screening for antibiotics is targeting virulence mechanisms. These potential virulence inhibitors allow for non-selective mechanisms of control by inhibiting virulence instead of cell proliferation, resulting in a decreased selective pressure for evolution of antibiotic resistance. Lahiri et al. focus on structural and biophysical approaches in screening for hits and rational design of leads based on a deeper understanding of structure-activity relationships. Detail description of applying protein crystallography as well as solution based biophysical methods in selecting, characterizing and validating promising targets as well as identifying and optimizing lead compounds that inhibit their functions were provided and a number of case studies were listed. In summary, this special issue illustrates current studies searching for antimicrobial reagents and highlights the potential of utilizing our knowledge of virulence mechanism of bacterial pathogens to explore novel strategies to treat infectious disease, which is especially relevant given rising antibiotic resistance among major pathogens. Finally, I would like to thank Dr. Mark Walker (The University of Queensland) for his kind assistance in editing this special issue.

Streptococcus pyogenes is a major human pathogen responsible for numerous diseases ranging from uncomplicated skin and throat infections to severe, life threatening invasive disease such as necrotising fasciitis and streptococcal toxic shock syndrome. These severe invasive infections progress rapidly and produce high rates of morbidity and mortality despite the implementation of aggressive treatment plans. The activation of plasminogen and the acquisition of plasmin activity at the bacterial cell surface is critical for the invasive pathogenesis of this organism. To facilitate this process, S. pyogenes secrete streptokinase, a potent plasminogen activating protein. Here, we describe the role of streptokinase in invasive pathogenesis and discuss some potentially useful strategies for disruption of streptokinase mediated plasminogen activation which could be employed to treat severe invasive S. pyogenes infections.

Group A Streptococcus (GAS) is a leading human pathogen that causes a multitude of diseases from pharyngitis, and impetigo, to more severe outcomes such as rheumatoid arthritis and necrotizing fasciitis. GAS remains a global burden as currently no vaccine exists that is completely effective. In this review we highlight recent studies on the virulence of GAS and present several approaches that have extended those findings into aims at combating GAS disease. These and other studies such as recent genome-wide efforts into host-pathogen relationships of GAS disease will likely reveal new targets of intervention. Given the recent rise in GAS strains that have acquired resistance to several types of antibiotics, it is crucial that we continue to increase our knowledge of the mechanisms underlying GAS disease.

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the upper respiratory tract in humans. However, these apparently harmless bacteria have also a high virulence potential and are known as the etiologic agent of respiratory and life-threatening invasive diseases. Dissemination of pneumococci from the nasopharynx into the lungs or bloodstream leads to community-acquired pneumonia, septicaemia and meningitis. Traditionally, pneumococcal diseases are treated with antibiotics and prevented with polysaccharide-based vaccines. However, due to the dramatic increase in antibiotic resistance and limitations of the current available vaccines, the burden of diseases remains high. Thus, combating pneumococcal transmission and infections has emphasized the need for a new generation of proteinbased vaccines. Interactions of pneumococci with soluble host proteins or cellular receptors are crucial for adherence, colonization, transmigration of host barriers and immune evasion. Therefore, surface-exposed proteins involved in these pathogenic processes and virtually expressed by all pneumococcal strains and serotypes are the prime potential targets for an immunogenic and highly protective pneumococcal-derived carrier protein of a vaccine. In this review, we will address the state of the art in deciphering, i). the conservation, distribution and pathogenic role of recently discovered pneumococcal adhesins in colonization and invasive diseases, ii). the interactions of these virulence factors with hostproteins and receptors, iii). the subversion of the host immune and cellular responses, and iv). the potential of pneumococcal adhesins as vaccine candidates.

Type III Secretion Systems (T3SSs) are highly organized multi-protein nanomachines which translocate effector proteins from the bacterial cytosol directly into host cells. These systems are required for the pathogenesis of a wide array of Gram-negative bacterial pathogens, and thus have attracted attention as potential antibacterial drug targets. A decade of research has enabled the identification of natural products, conventional small molecule drug-like structures, and proteins that inhibit T3SSs. The mechanism(s) of action and molecular target(s) of the majority of these inhibitors remain to be determined. At the same time, structural biology methods are providing an increasingly detailed picture of the functional arrangement of the T3SS component proteins. The confluence of these two research areas may ultimately identify non-classical drug targets and facilitate the development of novel therapeutics.

New antibiotics with novel modes of action are required to combat the growing threat posed by multi-drug resistant bacteria. Over the last decade, genome sequencing and other high-throughput techniques have provided tremendous insight into the molecular processes underlying cellular functions in a wide range of bacterial species. We can now use these data to assess the degree of conservation of certain aspects of bacterial physiology, to help choose the best cellular targets for development of new broad-spectrum antibacterials. DNA replication is a conserved and essential process, and the large number of proteins that interact to replicate DNA in bacteria are distinct from those in eukaryotes and archaea; yet none of the antibiotics in current clinical use acts directly on the replication machinery. Bacterial DNA synthesis thus appears to be an underexploited drug target. However, before this system can be targeted for drug design, it is important to understand which parts are conserved and which are not, as this will have implications for the spectrum of activity of any new inhibitors against bacterial species, as well as the potential for development of drug resistance. In this review we assess similarities and differences in replication components and mechanisms across the bacteria, highlight current progress towards the discovery of novel replication inhibitors, and suggest those aspects of the replication machinery that have the greatest potential as drug targets.

The emergence of multi-drug resistant bacteria is one of the most critical medical problems currently facing humankind, which will only get worse if no new antibacterial drugs are launched. This article will first review commonly used screening strategies used to identify potential new antibiotics and then discuss novel screening methods. In addition, new assays, methods, biological targets and compounds with novel modes of action undergoing pre-clinical or clinical development are briefly discussed.

New antibacterial drugs are urgently needed to combat the growing problem of multidrug resistant bacterial infections. Major advances in bacterial genomics have uncovered many unexploited targets, leading to the possibility of discovering new antibacterials with novel mechanisms that would circumvent resistance. Many of these targets are soluble enzymes that vary in their degrees of mechanistic complexity. Protein crystallography as well as solution based biophysical methods are playing an increasingly important role in selecting, characterizing and validating promising targets as well as identifying and optimizing lead compounds that inhibit their functions. Advances made in recent years in sensitivity, resolution and throughput of biophysical tools are allowing multiple approaches to screening for hits and rational design of leads based on a deeper understanding of structure-activity relationships. However, the path from a lead compound to a safe and efficacious antibacterial drug still remains challenging. Structural and biophysical approaches have had less of an impact on this later phase of discovery than on the lead generation phase.

The superfamily of proteins that contain C-type lectin-like domains (CTLDs) represents a large number of functionally diverse extracellular proteins (reviewed in [1, 2]). They are subclassified into at least 17 families, primarily according to the architecture of their often multiple domains. The canonical structure of the CTLD features a characteristic double-loop that is stabilized by highly conserved disulfide bridges and hydrophobic and polar interactions. One of these loops, referred to as the long loop region, is particularly flexible and is key for carbohydrate binding and the capacity of the CTLD to bind often simultaneously to one or more ligands. Indeed, this fold is remarkably variable and is estimated to occur with more than 1013 distinct amino acid sequences, analogous to the hypervariable region of immunoglobulins. It is therefore not surprising that the CTLD is highly adaptable, confers multiple functions to the protein, and that it binds not only to sugars, but also to many other structures, including proteins, lipids and inorganic molecules. In this minireview, we focus on the so-called Type XIV CTLD-containing proteins. These are type I transmembrane glycoproteins that have a single N-terminal CTLD that is connected sequentially via a hydrophobic stretch, to a series of 3-6 epidermal growth factor (EGF)-like repeats, a serine-threonine-rich mucin-containing domain, a short transmembrane region, and finally a 20-55 amino acid residue cytoplasmic tail. There are four members of this family, some with several descriptive names that reflect their origins of discovery. These include CD93, thrombomodulin (TM), CD248 (endosialin) and CLEC14a [3]. The latter member is a recent addition to the family, with only limited functional data, and thus will not be discussed in this review. The others represent glycoproteins that have gained wide attention due to discoveries that point to their relevance in human health and disease, particularly in the fields of innate immunity and inflammation. As with many C-type lectins, a common theme of members of the Type XIV family is their participation in modulating the response to injury, each in its own way. As will be seen in this minireview, TM, CD93 and CD248 differentially regulate innate immunity, cell proliferation and inflammation by virtue of their being comprised of additional structural motifs, having unique cellular and temporal patterns of expression, and interacting with distinct protein partners. TM is prominently expressed by endothelial cells, some myeloid cells, and dendritic cells. The CTLD of TM is the only one of the Type XIV family members in which a function has been ascribed and ligands have been identified. The CTLD clearly dampens inflammation and innate immune responses in a variety of disease models [4]. It does so in concert with three of its six EGF-like repeats, which serve primarily to protect against excess clot formation. As Morser describes in this issue of CDT [5], TM plays a central protective role in innate immunity and hemostatic control, and soluble fragments of TM are holding promise for therapies in a range of difficult-to-treat diseases. Interestingly, CD93 may have arisen from a gene duplication of the gene for TM. CD93 is similarly expressed by endothelial and myeloid cells, but also by platelets, hematopoietic stem cells, and several lymphocyte subtypes [6]. In contrast to TM, the EGF-like repeats of CD93 are not known to regulate coagulation. However, as Bohlson and colleagues report [7] CD93 also plays a key role in innate immunity by promoting phagocytosis and leukocyte adhesion. More recently revealed, CD93 participates in acquired immunity by maintaining the integrity of antibody-producing plasma cells. These properties are probably relevant in human disease, as CD93 is increasingly being shown to modulate multiple inflammatory and immune diseases and at least in some conditions, polymorphisms may portend an increased risk of inflammatory vascular disease. For a few reasons, CD248 stands somewhat apart from the rest of the family. In spite of early reports to the contrary, CD248 is not expressed by endothelial cells [8]. Rather, it is found in activated stromal and perivascular cells in tumors and inflammatory lesions and is almost undetectable in most adult tissues. It also uniquely contains a complement regulatory motif residing between the CTLD and EGF-like containing domain, the function of which remains unknown. Like TM, its gene is intronless [9]. Like CD93, it contains a C-terminal PDZ-binding motif with unknown function, but which is important for endocytosis of CD248 [10]. Generally opposing TM and CD93, CD248 appears to promote inflammation [11], possibly via intracellular signaling via its cytoplasmic tail and/or through extracellular matrix ligands that interact with the ectodomain. This minireview provides a comprehensive update on three important CTLD-containing proteins, members of a structurally related family, all of which play important yet distinct roles in innate immunity. It is intriguing that in spite of the structural similarities of their CTLDs and their overall domain architectures, TM, CD93 and CD248 do indeed display profound functional differences. Is this based on the variability within the CTLDs themselves? Or are there multiple context-dependent factors that are responsible? What common regulatory factors orchestrate the differential expression and function of the CTLDcontaining proteins to optimize the organism's response to injury and to most efficiently promote tissue repair? Are there more direct interactions between these molecules that in some situations may be expressed at the same site? Are there bioactive soluble forms of CD248, similar to those for TM and CD93 that are being exploited for diagnostic and therapeutic use? Hopefully this review will encourage further study to address some of these questions that lead to new diagnostic approaches and innovative therapeutic strategies.

CD93: Recent Advances and Implications in Disease by Mallary C. Greenlee-Wacker (411-420).
While it has been known for some time that CD93 regulates several processes involved in innate immunity and inflammation including phagocytosis and adhesion, the function of CD93 in disease progression is only now being elucidated. Recent in vivo studies in mice, and genome wide studies in mice and humans, have provided clues about its molecular function. Following a comprehensive review of CD93 expression patterns, this review will focus on recent findings over the last three years that address the putative function of CD93 in inflammation and innate immunity.

Thrombomodulin (TM) is a type 1 membrane bound glycoprotein that has a C-type lectin domain at its Nterminus, 6 copies of the epidermal growth factor-like (EGF) motif and serine/threonine rich domain carrying a glycosoaminoglycan external to the membrane. TM binds thrombin changing thrombin's substrate specificity from procoagulant and pro-inflammatory to anti-coagulant and anti-inflammatory because of the activation of protein C (PC) and thrombin-activatable fibrinolysis inhibitor (TAFI). Thrombin's anion binding site 1 binds to TM's EGF domains 5 and 6. EGF4 is required for PC activation and EGF3 and 4 for TAFI activation in addition to EGF56. The X-ray structure of thrombin bound to TM has been solved and shows few major alterations in the active site of thrombin. The lectin domain can bind high mobility group box protein 1 (HMGB1) and a sugar, Lewisy. TM's lectin domain behaves as an antagonist to HMGB1 endowing it with intrinsic anti-inflammatory activity. Treatment of dendritic cells with TM converts them from immunogenic to tolerogenic. TM is necessary for maintenance of pregnancy as well as prevention of coagulation throughout life. Soluble TM has been developed as an anticoagulant possessing favorable pharmacokinetics that has been approved for treatment of disseminated intravascular coagulation in Japan.

CD248, also known as endosialin or tumor endothelial marker-1 (TEM-1), is a C-type lectin-like domain (CTLD) containing cell surface glycoprotein that is expressed by stromal cells of proliferating tissues during embryogenesis and postnatally in tumors and inflammatory lesions. Loss-of-function studies in mice support the notion that CD248 promotes tumor growth and inflammation, observations that are stimulating interest in evaluating this molecule as a therapeutic target. In spite of these advances, the mechanisms by which CD248 modulates cancer and inflammation remain largely enigmatic. This review highlights our current understanding of CD248, its structure, pattern of expression, regulation and function in various disease processes.