Receptor mimicry as novel therapeutic treatment for biothreat agents

The specter of intentional release of pathogenic microbes and their toxins is a real threat. This article reviews the literature on adhesins of biothreat agents, their interactions with oligosaccharides and the potential for anti-adhesion compounds as an alternative to conventional therapeutics. The minimal binding structure of ricin has been well characterised and offers the best candidate for successful anti-adhesion therapy based on the Galβ1-4GlcNAc structure. The botulinum toxin serotypes A-F bind to a low number of gangliosides (GT1b, GQ1b, GD1a and GD1b) hence it should be possible to determine the minimal structure for binding. The minimal disaccharide sequence of GalNAcβ1-4Gal found in the gangliosides asialo-GM1 and asialo-GM2 is required for adhesion for many respiratory pathogens. Although a number of adhesins have been identified in bacterial biothreat agents such as Yersinia pestis, Bacillus anthracis, Francisella tularensis, Brucella species and Burkholderia pseudomallei, specific information regarding their in vivo expression during pneumonic infection is lacking. Limited oligosaccharide inhibition studies indicate the potential of GalNAcβ1-4Gal, GalNAcβ-3Gal and the hydrophobic compound, para-nitrophenol as starting points for the rational design of generic anti-adhesion compounds. A cocktail of multivalent oligosaccharides based on the minimal binding structures of identified adhesins would offer the best candidates for anti-adhesion therapy.     

Harnessing calcineurin as a novel anti-infective agent against invasive fungal infections

The number of immunocompromised patients with invasive fungal infections continues to increase and new antifungal therapies are not keeping pace with the growing incidence of these infections and their associated mortality. Calcineurin inhibition is currently used to exert effective immunosuppression following organ transplantation and in treating various other conditions. However, the calcineurin pathway is also intricately involved in the growth and pathogenesis of the three major fungal pathogens of humans, Cryptococcus neoformans, Candida albicans and Aspergillus fumigatus, and the exploitation of fungal calcineurin pathways holds great promise for the future development of novel antifungal agents. This Review summarizes our current understanding of calcineurin biology in these fungal species, and its exciting potential role in treating invasive fungal infections.     

Glyco-optimization of aminoglycosides: new aminoglycosides as novel anti-infective agents

Glyco-optimization (OPopS) of aminoglycosides has been performed by replacing the existing sugar moiety with a variety of sugar derivatives. Glycosylation of the 6-position of nebramine provided a library of novel 4,6-linked aminoglycosides (AMGs). Among them, compounds 8b,g,i,l, and 8u with 2"-amino, 2",3"-diamino, 2",4"-diamino, 3",4"-diamino, 3"-amino groups, respectively, showed significant antimicrobial activity against Gram-(+) and -(-) bacteria. Several were particularly potent against Pseudomonus aeruginosa with MICs in the 1-2 microg/mL range.     

A molecular basis for functional peptide mimicry of a carbohydrate antigen

Peptides may substitute for carbohydrate antigens in carbohydrate-specific immunological reactions. Using the recognition properties of an anti-Lewis Y (LeY) antibody, BR55-2, as a model system, we establish a molecular perspective for peptide mimicry by comparing the three-dimensional basis of BR55-2 binding to LeY with the binding of the same antibody to peptides. The peptides compete with LeY, as demonstrated by enzyme-linked immunosorbent assay and Biacore analysis. The computer program LUDI was used to epitope map the antibody-combining site, correlating peptide reactivity patterns. This approach identified amino acids interacting with the same BR55-2 functional residue groups that recognize the Fucalpha(1-3) moiety of LeY. Molecular modeling indicates that the peptides adopt an extended turn conformation within the BR55-2 combining site, serving to overlap the peptides with the LeY spatial position. Peptide binding is associated with only minor changes in BR55-2, relative to the BR55-2-LeY complex. Anti-peptide serum distinguishes the Fucalpha(1-3) from the Fucalpha(1-4) linkage, therefore differentiating difucosylated neolactoseries antigens. These results further confirm that peptides and carbohydrates can bind to the same antibody-binding site and that peptides can structurally and functionally mimic salient features of carbohydrate epitopes.     

Structural basis for norovirus inhibition and fucose mimicry by citrate

Human noroviruses bind with their capsid-protruding domains to histo-blood-group antigens (HBGAs), an interaction thought to direct their entry into cells. Although human noroviruses are the major cause of gastroenteritis outbreaks, development of antivirals has been lacking, mainly because human noroviruses cannot be cultivated. Here we use X-ray crystallography and saturation transfer difference nuclear magnetic resonance (STD NMR) to analyze the interaction of citrate with genogroup II (GII) noroviruses. Crystals of citrate in complex with the protruding domain from norovirus GII.10 Vietnam026 diffracted to 1.4 Å and showed a single citrate bound at the site of HBGA interaction. The citrate interaction was coordinated with a set of capsid interactions almost identical to that involved in recognizing the terminal HBGA fucose, the saccharide which forms the primary conserved interaction between HBGAs and GII noroviruses. Citrate and a water molecule formed a ring-like structure that mimicked the pyranoside ring of fucose. STD NMR showed the protruding domain to have weak affinity for citrate (460 μM). This affinity, however, was similar to the affinities of the protruding domain for fucose (460 μM) and H type 2 trisaccharide (390 μM), an HBGA shown previously to be specifically recognized by human noroviruses. Importantly, competition STD NMR showed that citrate could compete with HBGA for norovirus binding. Together, the results suggest that citrate and other glycomimetics have the potential to block human noroviruses from binding to HBGAs.     

Characteristics of protein-carbohydrate interactions as a basis for developing novel carbohydrate-based antirejection therapies

The relative shortage of human organs for transplantation is today the major barrier to a broader use of transplantation as a means of treating patients with end-stage organ failure. This barrier could be partly overcome by an increased use of blood group ABO-incompatible live donors, and such trials are currently underway at several transplant centres. If xenotransplantation can be used clinically in the future, the human organ shortage will, in principle, be eradicated. In both these cases, carbohydrate antigens and the corresponding anti-carbohydrate antibodies are the major primary immunological barriers to overcome. Refined carbohydrate-based therapeutics may permit an increased number of ABO-incompatible transplantations to be carried out, and may remove the initial barriers to clinical xenotransplantation. Here, we will discuss the chemical characteristics of protein-carbohydrate interactions and outline carbohydrate-based antirejection therapies as used today in experimental as well as in clinical settings. Novel mucin-based adsorbers of natural anti-carbohydrate antibodies will also be described.     

The molecular basis of melanism and mimicry in a swallowtail butterfly

Melanism in Lepidoptera, either industrial or in mimicry, is one of the most commonly cited examples of natural selection [1] [2]. Despite extensive studies of the frequency and maintenance of melanic genes in insect populations [1] [2], there has been little work on the underlying molecular mechanisms. Nowhere is butterfly melanism more striking than in the Eastern Tiger Swallowtail (Papilio glaucus) of North America [3] [4] [5]. In this species, females can be either yellow (wild type) or black (melanic). The melanic form is a Batesian mimic of the distasteful Pipevine Swallowtail (Battus philenor), which is also black in overall color. Melanism in P. glaucus is controlled by a single Y-linked (female) black gene [6]. Melanic females, therefore, always have melanic daughters. Black melanin replaces the background yellow in melanic females. Here, we show that the key enzyme involved is N-beta-alanyl-dopamine-synthase (BAS), which shunts dopamine from the melanin pathway into the production of the yellow color pigment papiliochrome and also provides products for cuticle sclerotization. In melanic females, this enzyme is suppressed, leading to abnormal melanization of a formerly yellow area, and wing scale maturation is also delayed in the same area. This raises the possibility that either reduced BAS activity itself is preventing scale sclerotization (maturation) or, in contrast, that the delay in scale maturation precludes expression of BAS at the correct stage. Together, these data show how changes in expression of a single gene product could result in multiple wing color phenotypes. The implications for the genetic control of mimicry in other Lepidoptera are discussed.     

Llama single domain antibodies as a tool for molecular mimicry

In camelids, a subset of the immunoglobulins consists of heavy-chain homodimers devoid of light chains, and are thus called heavy-chain IgGs (hcIgGs). Their variable region (VHH) is the smallest antigen-binding fragment possible, and being just one polypeptide chain it is especially suitable for engineering. In particular, camelid single domain antibodies might be very useful for molecular mimicry and anti-idiotypic vaccination. In the present work, we show that llamas immunized with an anti-DNA mouse mAb develop an important anti-Id response. Selection of VHHs by phage display, with specific elution of bound phages with the external antigenic DNA, shows that selected private anti-Id VHHs compete for binding to the external antigen and bear a functional mimicry of the DNA. These results indicate that llama anti-Id single domain antibodies would be an excellent tool for molecular mimicry studies.     

Telomerase inhibitors as novel antitumor drugs

Telomerase activity is detected in most types of human tumors, but it is almost undetectable in normal somatic cells; therefore, telomerase is a promising therapeutic target. The present review describes various approaches to telomerase inhibition, namely, antisense therapy, RNA interference, and the use of ribozymes and agents interacting with the telomeric G-quadruplex. The use of these compounds in clinical research is analyzed in the review.     

The GM2 Glycan Serves as a Functional Coreceptor for Serotype 1 Reovirus

Viral attachment to target cells is the first step in infection and also serves as a determinant of tropism. Like many viruses, mammalian reoviruses bind with low affinity to cell-surface carbohydrate receptors to initiate the infectious process. Reoviruses disseminate with serotype-specific tropism in the host, which may be explained by differential glycan utilization. Although α2,3-linked sialylated oligosaccharides serve as carbohydrate receptors for type 3 reoviruses, neither a specific glycan bound by any reovirus serotype nor the function of glycan binding in type 1 reovirus infection was known. We have identified the oligosaccharide portion of ganglioside GM2 (the GM2 glycan) as a receptor for the attachment protein σ1 of reovirus strain type 1 Lang (T1L) using glycan array screening. The interaction of T1L σ1 with GM2 in solution was confirmed using NMR spectroscopy. We established that GM2 glycan engagement is required for optimal infection of mouse embryonic fibroblasts (MEFs) by T1L. Preincubation with GM2 specifically inhibited type 1 but not type 3 reovirus infection of MEFs. To provide a structural basis for these observations, we defined the mode of receptor recognition by determining the crystal structure of T1L σ1 in complex with the GM2 glycan. GM2 binds in a shallow groove in the globular head domain of T1L σ1. Both terminal sugar moieties of the GM2 glycan, N-acetylneuraminic acid and N-acetylgalactosamine, form contacts with the protein, providing an explanation for the observed specificity for GM2. Viruses with mutations in the glycan-binding domain display diminished hemagglutination capacity, a property dependent on glycan binding, and reduced capacity to infect MEFs. Our results define a novel mode of virus-glycan engagement and provide a mechanistic explanation for the serotype-dependent differences in glycan utilization by reovirus.     

Effects of perceived patient demand on prescribing anti-infective drugs

BACKGROUND: Although patient demand is frequently cited by physicians as a reason for inappropriate prescribing, the phenomenon has not been adequately studied. The objectives of this study were to determine the prevalence of perceived patient demand in physician-patient encounters; to identify characteristics of the patient, physician and prescribing situation that are associated with perceived demand; and to determine the influence of perceived demand on physicians'' prescribing behaviour. METHODS: An observational study using 2 survey approaches was conducted in February and March 1996. Over a 2-day period 20 family physicians in the Toronto area completed a brief questionnaire for each patient encounter related to suspected infectious disease. Physicians were later asked in an interview to select and describe 1 or 2 incidents from these encounters during which perceived patient demand influenced their prescribing (critical incident technique). RESULTS: Perceived patient demand was reported in 124 (48%) of the 260 physician-patient encounters; however, in almost 80% of these encounters physicians did not think that the demand had much influence on their decision to prescribe an anti-infective. When clinical need was uncertain, 28 (82%) of 34 patients seeking an anti-infective were prescribed one, and physicians reported that they were influenced either "moderately" or "quite a bit" by perceived patient demand in over 50% of these cases. Of the 35 critical prescribing incidents identified during the interviews, anti-infectives were prescribed in 17 (49%); the reasons for prescribing in these situations were categorized. INTERPRETATION: This study provides preliminary data on the prevalence and influence of perceived patient demand in prescribing anti-infectives. Patient demand had more influence on prescribing when physicians were uncertain of the need for an anti-infective.     

Improved Method for Drawing of a Glycan Map,and the First Page of Glycan Atlas,Which Is a Compilation of Glycan Maps for a Whole Organism

Glycan Atlas is a set of glycan maps over the whole body of an organism. The glycan map that includes data of glycan structure and quantity displays micro-heterogeneity of the glycans in a tissue, an organ, or cells. The two-dimensional glycan mapping is widely used for structure analysis of N-linked oligosaccharides on glycoproteins. In this study we developed a comprehensive method for the mapping of both N- and O-glycans with and without sialic acid. The mapping data of 150 standard pyridylaminated glycans were collected. The empirical additivity rule which was proposed in former reports was able to adapt for this extended glycan map. The adapted rule is that the elution time of pyridylamino glycans on high performance liquid chromatography (HPLC) is expected to be the simple sum of the partial elution times assigned to each monosaccharide residue. The comprehensive mapping method developed in this study is a powerful tool for describing the micro-heterogeneity of the glycans. Furthermore, we prepared 42 pyridylamino (PA-) glycans from human serum and were able to draw the map of human serum N- and O-glycans as an initial step of Glycan Atlas editing.     

Structural basis of antigen mimicry in a clinically relevant melanoma antigen system

Although mimics of human tumor antigens are effective immunogens to overcome host unresponsiveness to the nominal antigen, the structural basis of this mimicry remains poorly defined. Therefore, in this study we have characterized the structural basis of the human high molecular weight-melanoma-associated antigen (HMW-MAA) mimicry by the mouse anti-idiotypic (anti-id) monoclonal antibody (mAb) MK2-23. Using x-ray crystallography, we have characterized the three-dimensional structure of the anti-id mAb MK2-23 Fab' and shown that its heavy chain complementarity-determining region (CDR3) (H3) and its light chain CDR1 (L1) are closely associated. These moieties are the source of HMW-MAA mimicry, since they display partial amino acid sequence homology along with a similar structural fold with the HMW-MAA core protein. Furthermore, a 15-residue peptide comprising the H3 loop of anti-id mAb MK2-23 demonstrates HMW-MAA-like in vitro and in vivo reactivity. This peptide in conjunction with the structural data will facilitate the characterization of the effect of the degree of antigen mimicry on the induction of a self-antigen-specific immune response by a mimic.     

Molecular mimicry between HLA-DR alleles associated with rheumatoid arthritis and Proteus mirabilis as the Aetiological basis for autoimmunity

Molecular mimicry is one of the pathological mechanisms proposed to explain the association between microorganisms and autoimmune diseases. This review deals with the association between bacteria and rheumatic diseases with a special emphasis on rheumatoid arthritis where upper urinary tract infection by Proteus mirabilis is the possible cause of this severe, arthritic condition. Prospective trials involving anti-Proteus therapy should be carried out.     

AT-rich islands in genomic DNA as a novel target for AT-specific DNA-reactive antitumor drugs

Interstrand cross-links at T(A/T)4A sites in cellular DNA are associated with hypercytotoxicity of an anticancer drug, bizelesin. Here we evaluated whether these lethal effects reflect targeting critical genomic regions. An in silico analysis of human sequences showed that T(A/T)4A motifs are on average scarce and scattered. However, significantly higher local motif densities were identified in distinct minisatellite regions (200-1000 base pairs of approximately 85-100% AT), herein referred to as "AT islands." Experimentally detected bizelesin lesions agree with these in silico predictions. Actual bizelesin adducts clustered within the model AT island naked DNA, whereas motif-poor sequences were only sparsely adducted. In cancer cells, bizelesin produced high levels of lesions (approximately 4.7-7.1 lesions/kilobase pair/microM drug) in several prominent AT islands, compared with markedly lower lesion levels in several motif-poor loci and in bulk cellular DNA (approximately 0.8-1.3 and approximately 0.9 lesions/kilobase pair/microM drug, respectively). The identified AT islands exhibit sequence attributes of matrix attachment regions (MARs), domains that organize DNA loops on the nuclear matrix. The computed "MAR potential" and propensity for supercoiling-induced duplex destabilization (both predictive of strong MARs) correlate with the total number of bizelesin binding sites. Hence, MAR-like AT-rich non-coding domains can be regarded as a novel class of critical targets for anticancer drugs.     

Resistance to anti-infective drugs and the threat to public health

Increasing resistance of pathogens to anti-infective drugs is an urgent public health problem that must be addressed through more prudent use of these drugs in human medicine and in animal husbandry, agriculture, and aquaculture.     

Insulin signal mimicry as a mechanism for the insulin-like effects of vanadium

Among several metals, vanadium has emerged as an extremely potent agent with insulin-like properties. These insulin-like properties have been demonstrated in isolated cells, tissues different animal models of type I and type II diabetes as well as a limited number of human subjects. Vanadium treatment has been found to improve abnormalities of carbohydrate and lipid metabolism and of gene expression in rodent models of diabetes. In isolated cells, it enhances glucose transport, glycogen and lipid synthesis, and inhibits gluconeogenesis and lipolysis. The molecular mechanism responsible for the insulin-like effects of vanadium compounds have been shown to involve the activation of several key components of insulin-signaling pathways that include the mitogen-activated-protein kinases (MAPKs) extracellular signal-regulated kinase 1/2 (ERK1/2) and p38MAPK, and phosphatidylinositol 3-kinase (PI3-K)/protein kinase B (PKB). It is interesting that the vanadium effect on these signaling systems is independent of insulin receptor protein tyrosine kinase activity, but it is associated with enhanced tyrosine phosphorylation of insulin receptor substrate-1. These actions seem to be secondary to vanadium-induced inhibition of protein tyrosine phosphatases. Because MAPK and PI3-K/PKB pathways are implicated in mediating the mitogenic and metabolic effects of insulin, respectively, it is plausible that mimicry of these pathways by vanadium serves as a mechanism for its insulin-like responses.     

Staphylococcal DNA as a basis for classification

The genome characterization of the typing strains for all 13 species of the genus Staphylococcus, included into the Approval List of the Names of Bacterial (1980), is presented. The nucleotide composition of DNA (28-33% of GC) did not permit the differentiation between staphylococcal species, but some of the groups of these species could be differentiated by the size of their genome (0.6-1.6 X 10(9) daltons). Differences in the degree of similarity between the nucleotide sequences of the DNA of all species (5-6% of DNA homology) made it possible to suggest raising the genus Staphylococcus to the rank of the family Staphylococcaceae fam. nov. The hypothetical classification scheme of this family is presented for discussion.     

Rutaceous alkaloids as models for the design of novel antitumor drugs

The chemical diversity of alkaloids in the Rutaceae is correlated with biosynthetic pathways involving various aromatic amino acid precursors, tyrosine, tryptophan, histidine, and anthranilic acid. The interest of rutaceous polyheteroaromatic alkaloids as models for the development of anticancer agents relies on their frequent ability to interact with DNA or with systems involved in the control of its topology, repair, and replication. Fagaronine and nitidine, from Zanthoxylum, demonstrate antileukemic activity, associated with topoisomerases inhibition. Evodiamine from Euodia rutaecarpa, displays antimetastatic properties. The pyranoacridone acronycine, from Sarcomelicope, exhibits antitumor activity against a broad spectrum of solid tumors. Development of synthetic analogues based on this latter natural product template followed the isolation of the unstable acronycine epoxide, which led to a hypothesis of bioactivation of acronycine by transformation of the 1,2-double bond into the corresponding oxirane. 1,2-Diacyloxy-1,2-dihydroacronycine derivatives exhibited antitumor properties, with a broadened spectrum of activity and an increased potency. The demonstration that acronycine interacted with DNA led to develop benzo[a], [b], and [c]acronycine analogs. Benzo[a] and [b] derivatives displayed significant antitumor activities. 1,2-Dihydroxy-1,2-dihydrobenzo[b]acronycine esters and diesters were active in human orthotopic models of cancers xenografted in nude mice. The activity of these compounds was correlated with their ability to give covalent adducts with DNA, involving reaction between the N-2 amino group of guanines and the ester group at the benzylic position of the drug. Cis-1,2-diacetoxy-1,2-dihydrobenzo[b]acronycine, currently developed under the code S23906-1, successfully underwent phase I and is currently under phase II clinical trials.