Alkaloidal Sugar Mimetics: Biological Activities and Therapeutic Applications

Abstract

Alkaloids mimicking the structures of sugars inhibit glycosidases because of a structural resemblance to the sugar moiety of the natural substrate. Glycosidases are involved in a wide range of important biological processes, such as intestinal digestion, post-translational processing of glycoproteins and the lysosomal catabolism of glycoconjugates. The realization that alkaloidal sugar mimics might have enormous therapeutic potential in many diseases such as viral infection, cancer and diabetes led to increasing interest and demand for these compounds. In this review, the structural basis of the specificity of alkaloidal sugar mimics and their current and potential applications to biomedical problems are reviewed.     

Crustacean peptide and peptide-like pheromones and kairomones

Crustacean peptide pheromones, kairomones, and substituted amino sugar kairomones are reviewed from a historical perspective. These crustacean information molecules are secondary functions of structural polymers. They are partial hydrolysis products, generated usually by the action of trypsin-like enzymes on proteins, and glycosidase enzymes on glycoproteins and proteoglycans. Structure-function studies based upon synthetic mimics of peptide information molecules show neutral amino acids with a basic carboxyl terminal are active in modifying physiological and or behavioral responses. Behaviorally active substituted amino sugar mimics are disaccharide hydrolysis products of heparin and chondroitin sulfate. Similar molecules are also used as information molecules by a variety of other marine organisms indicating they are a common biological theme.     

Principal signalling complexes in haematopoiesis: structural aspects and mimetic discovery

Blood production is a highly regulated process involving multiple inhibitory and stimulatory cytokines present in the haematopoietic stem cell niche. Small molecules mimics of these signalling molecules have substantial potential as drugs and in the development of bioreactors to generate blood products. We review the structural biology of the extracellular signalling domains of five of the most important cytokines, analyze their structure-property relationships, and summarize the progress in developing small molecule mimics using the molecular information from structural biology and mutation studies.     

Penicillium roqueforti: a multifunctional cell factory of high value‐added molecules

This is a comprehensive review, with 114 references, of the chemical diversity found in the fungus Penicillium roqueforti. Secondary metabolites of an alkaloidal nature are described, for example, ergot alkaloids such as festuclavine, isofumigaclavines A and B, and diketopiperazine alkaloids such as roquefortines A–D, which are derived from imidazole. Other metabolites are marcfortines A–C, PR‐toxin, eremofortines A–E, mycophenolic and penicillic acids, and some γ‐lactones. Also, recent developments related to the structural characteristics of botryodiplodin and andrastin are studied—the latter has anticancer properties. Finally, we discuss the enzymes of P. roqueforti, which can participate in the biotechnological production of high value‐added molecules, as well as the use of secondary metabolite profiles for taxonomic purposes.     

The merits of bipartite transition-state mimics for inhibition of uracil DNA glycosylase

The glycosidic bond hydrolysis reaction of the enzyme uracil DNA glycosylase (UDG) occurs by a two-step mechanism involving complete bond breakage to the uracil anion leaving group in the first step, formation of a discrete glycosyl cation-uracil anion intermediate, followed by water attack in a second transition-state leading to the enzyme-bound products of uracil and abasic DNA. We have synthesized and determined the binding affinities of unimolecular mimics of the substrate and first transition-state (TS1) in which the uracil base is covalently attached to the sugar, and in addition, bimolecular mimics of the second addition transition state (TS2) in which the base and sugar are detached. We find that the bipartite mimics of TS2 are superior to the TS1 mimics. These results indicate that bipartite TS2 inhibitors could be useful for inhibition of glycosylases that proceed by stepwise reaction mechanisms.     

Achievements and impacts of glycosylation reactions involved in natural product biosynthesis in prokaryotes

Bioactive natural products, such as polyketides, flavonoids, glycopeptides, and aminoglycosides, have been used as therapeutic agents. Many of them contain structurally diverse sugar moieties attached to the aglycone core structures. Glycosyltransferases (GTs) catalyze the attachment of nucleotide-activated sugar substrates to acceptor aglycones. Because these sugar moieties are usually essential for biological activity, in vivo pathway engineering in prokaryotic hosts and in vitro enzymatic approaches coupled with GT engineering are currently being used to synthesize novel glycosylated derivatives, and some of them exhibited improved biological activities compared to the parent molecules. Therefore, harnessing the potential of diverse glycosylation reactions in prokaryotes will increase the structural diversity of natural products and the possibility to generate new bioactive products.     

Squaring up to DNA: pentapeptide repeat proteins and DNA mimicry

Pentapeptide repeats are a class of proteins characterized by the presence of multiple repeating sequences five amino acids in length. The sequences fold into a right-handed β-helix with a roughly square-shaped cross section. Pentapeptide repeat proteins include a number of examples which are thought to function as structural mimics of DNA and act to competitively bind to the type II topoisomerase DNA gyrase, an important antibacterial target. DNA gyrase-targeting pentapeptide repeat proteins can both inhibit DNA gyrase—a potentially useful therapeutic property—and contribute to resistance to quinolone antibacterials (by acting to prevent them forming a lethal complex with the DNA and enzyme). Pentapeptide repeat proteins are therefore of wide interest not only because of their unusual structure, function, and potential as an antibacterial target, but also because knowledge of their mechanism of action may lead to both a greater understanding of the details of DNA gyrase function as well as being a useful template for the design of new DNA gyrase inhibitors. However, many puzzling aspects as to how these DNA mimics function and indeed even their ability to act as DNA mimics itself remains open to question. This review summarizes the current state of knowledge regarding pentapeptide repeat proteins, focusing on those that are thought to mimic DNA, and speculates on potential structure-function relationships which may account for their differing specificities.     

Secondary structures without backbone: an analysis of backbone mimicry by polar side chains in protein structures.

Backbone mimicry by the formation of closed-loop C7, C10 and C13 (mimics of gamma-, beta- and alpha-turns) conformations through side chain-main chain hydrogen bonds by polar groups is a frequent observation in protein structures. A data set of 250 non-homologous and high-resolution protein crystal structures was used to analyze these conformations for their characteristic features. Seven out of the nine polar residues (Ser, Thr, Asn, Asp, Gln, Glu and His) have hydrogen bonding groups in their side chains which can participate in such mimicry and as many as 15% of all these polar residues engage in such conformations. The distributions of dihedral angles of these mimics indicate that only certain combinations of the dihedral angles involved aid the formation of these mimics. The observed examples were categorized into various classes based on these combinations, resulting in well defined motifs. Asn and Asp residues show a very high capability to perform such backbone secondary structural mimicry. The most highly mimicked backbone structure is of the C10 conformation by the Asx residues. The mimics formed by His, Ser, Thr and Glx residues are also discussed. The role of such conformations in initiating the formation of regular secondary structures during the course of protein folding seems significant.     

Microbial synthesis of the sweet-tasting plant protein thaumatin

Thaumatin, the intensely sweet-tasting protein from the African plant, Thaumatococcus daniellii, is a potential low-caloric substitute for sugar and an attractive compound for studying the structural aspects of sweetness perception. Since thaumatin has other properties, such as flavour enhancement, it could also be used in products other than low-calorie foods. Recombinant DNA techniques could be used to synthesize this protein in quantities sufficient for such applications.     

Antibody recognition of cancer-related gangliosides and their mimics investigated using in silico site mapping

Modified gangliosides may be overexpressed in certain types of cancer, thus, they are considered a valuable target in cancer immunotherapy. Structural knowledge of their interaction with antibodies is currently limited, due to the large size and high flexibility of these ligands. In this study, we apply our previously developed site mapping technique to investigate the recognition of cancer-related gangliosides by anti-ganglioside antibodies. The results reveal a potential ganglioside-binding motif in the four antibodies studied, suggesting the possibility of structural convergence in the anti-ganglioside immune response. The structural basis of the recognition of ganglioside-mimetic peptides is also investigated using site mapping and compared to ganglioside recognition. The peptides are shown to act as structural mimics of gangliosides by interacting with many of the same binding site residues as the cognate carbohydrate epitopes. These studies provide important clues as to the structural basis of immunological mimicry of carbohydrates.     

Fmoc-protected iminosugar modified asparagine derivatives as building blocks for glycomimetics-containing peptides

CSF114(Glc) is the first synthetic Multiple Sclerosis Antigenic Probe able to identify autoantibodies in a statistically significant number of Multiple Sclerosis patients. The beta-turn conformation of this glucopeptide is fundamental for a correct presentation of the epitope Asn(Glc). To verify the influence of sugar mimics in antibody recognition in Multiple Sclerosis, we synthesized Fmoc-protected Asn derivatives containing alkaloid-type sugar mimics. The corresponding glycomimetics-containing peptide derivatives of the CSF114-type sequence were tested in competitive and solid-phase non-competitive ELISA on Multiple Sclerosis patients' sera.     

Driving engineering of novel antimicrobial peptides from simulations of peptide-micelle interactions

Simulations of antimicrobial peptides in membrane mimics can provide the high resolution, atomistic picture that is necessary to decipher which sequence and structure components are responsible for activity and toxicity. With such detailed insight, engineering new sequences that are active but non-toxic can, in principle, be rationalized. Armed with supercomputers and accurate force fields for biomolecular interactions, we can now investigate phenomena that span hundreds of nanoseconds. Although the phenomena involved in antimicrobial activity, (i.e., diffusion of peptides, interaction with lipid layers, secondary structure attainment, possible surface aggregation, possible formation of pores, and destruction of the lipid layer integrity) collectively span time scales still prohibitively long for classical mechanics simulations, it is now feasible to investigate the initial approach of single peptides and their interaction with membrane mimics. In this article, we discuss the promise and the challenges of widely used models and detail our recent work on peptide-micelle simulations as an attractive alternative to peptide-bilayer simulations. We detail our results with two large structural classes of peptides, helical and beta-sheet and demonstrate how simulations can assist in engineering of novel antimicrobials with therapeutic potential.     

The conformational restriction of synthetic peptides, including a malaria peptide, for use as immunogens

A new strategy is advanced for the conformational restriction of peptidyl immunogens. Our approach is to replace putative amide-amide hydrogen bonds with covalent hydrogen-bond mimics. Because on average every other amino acid in a protein engages in this bond, the syntheses of diversely shaped peptides can be contemplated. Synthetic methods for introducing a potential hydrogen-bond mimic into a peptide with alpha-helical potential is reported and the structural consequences are discussed. The replacement of the hydrogen bond with a chemical link will modify as well as shape the peptide. To explore the consequences of these changes, a potential synthetic vaccine for malaria, the repeating tetrapeptide Asn-Pro-Asn-Ala, was conformationally restricted. Antibodies to the shaped malarial peptide showed a strong cross reaction with Plasmodium falciparum sporozoites.     

Driving engineering of novel antimicrobial peptides from simulations of peptide-micelle interactions

Simulations of antimicrobial peptides in membrane mimics can provide the high resolution, atomistic picture that is necessary to decipher which sequence and structure components are responsible for activity and toxicity. With such detailed insight, engineering new sequences that are active but non-toxic can, in principle, be rationalized. Armed with supercomputers and accurate force fields for biomolecular interactions, we can now investigate phenomena that span hundreds of nanoseconds. Although the phenomena involved in antimicrobial activity, (i.e., diffusion of peptides, interaction with lipid layers, secondary structure attainment, possible surface aggregation, possible formation of pores, and destruction of the lipid layer integrity) collectively span time scales still prohibitively long for classical mechanics simulations, it is now feasible to investigate the initial approach of single peptides and their interaction with membrane mimics. In this article, we discuss the promise and the challenges of widely used models and detail our recent work on peptide-micelle simulations as an attractive alternative to peptide-bilayer simulations. We detail our results with two large structural classes of peptides, helical and beta-sheet and demonstrate how simulations can assist in engineering of novel antimicrobials with therapeutic potential.     

Probing DNA bulges with designed helical spirocyclic molecules

Because bulged structures (unpaired bases) in nucleic acids are of general biological significance, it has been of interest to design small molecules as specific probes of bulge function. On the basis of our earlier work with the specific DNA bulge-binding metabolite obtained from the enediyne antitumor antibiotic neocarzinostatin chromophore (NCS-chrom), we have prepared three small helical spirocyclic molecules that most closely mimic the natural product. These wedge-shaped molecules resemble the natural product in having the sugar residue attached to the same five-membered ring system. In one instance, the sugar is aminoglucose in beta-glycosidic linkage, and in the other, two enantiomers have the natural sugar N-methylfucosamine in alpha-glycosidic linkage. All three analogues were found to interfere with bulge-specific cleavage by NCS-chrom and the ability of bulged DNA to serve as a template for DNA polymerase 1 in accordance with their binding affinities for DNA containing a two-base bulge. Comparable results were obtained with the analogues for the less efficiently cleaved three-base bulge DNA structures. In each situation, the enantiomers possessing the natural sugar in alpha-glycosidic linkage are the most potent inhibitors of the cleavage reaction. In the DNA polymerase reactions, again, the closest natural product mimics were the most effective in selectively impeding nucleotide extension at the bulge site, presumably by complex formation. These results demonstrate the potential usefulness of bulge-binding compounds in modifying DNA structure and function and support efforts to design and prepare reactive species of these molecules that can covalently modify bulged DNA.     

Beta-fructofuranosidase genes of the silkworm, Bombyx mori: insights into enzymatic adaptation of B. mori to toxic alkaloids in mulberry latex

Mulberry latex contains extremely high concentrations of alkaloidal sugar mimic glycosidase inhibitors, such as 1,4-dideoxy-1,4-imino-D-arabinitol (D-AB1) and 1-deoxynojirimycin (DNJ). Although these compounds do not harm the silkworm, Bombyx mori, a mulberry specialist, they are highly toxic to insects that do not normally feed on mulberry leaves. D-AB1 and DNJ are strong inhibitors of alpha-glucosidases (EC 3.2.1.20); however, they do not affect the activity of beta-fructofuranosidases (EC 3.2.1.26). Although alpha-glucosidase genes are found in a wide range of organisms, beta-fructofuranosidase genes have not been identified in any animals so far. In this study, we report the identification and characterization of beta-fructofuranosidase genes (BmSuc1 and BmSuc2) from B. mori. The BmSuc1 gene was highly expressed in the midgut and silk gland, whereas the expression of BmSuc2 gene was not detected. BmSuc1 encodes a functional beta-fructofuranosidase, whose enzymatic activity was not inhibited by DNJ or D-AB1. We also showed that BmSUC1 protein localized within the midgut goblet cell cavities. Collectively, our data clearly demonstrated that BmSuc1 serves as a sugar-digesting enzyme in the silkworm physiology. This anomalous presence of the beta-fructofuranosidase gene in the B. mori genome may partly explain why the silkworm can circumvent the mulberry's defense system.