Dynamic tracing of sugar metabolism reveals the mechanisms of action of synthetic sugar analogs.

Synthetic sugar analogs are widely applied in metabolic oligosaccharide engineering (MOE) and as novel drugs to interfere with glycoconjugate biosynthesis. However, mechanistic insights on their exact cellular metabolism over time are mostly lacking. We combined ion-pair ultrahigh performance liquid chromatography-triple quadrupole mass spectrometry mass spectrometry using tributyl- and triethylamine buffers for sensitive analysis of sugar metabolites in cells and organisms and identified low abundant nucleotide sugars, such as UDP-arabinose in human cell lines and CMP-sialic acid (CMP-NeuNAc) in Drosophila. Furthermore, MOE revealed that propargyloxycarbonyl (Poc)-labeled ManNPoc was metabolized to both CMP-NeuNPoc and UDP-GlcNPoc. Finally, time-course analysis of the effect of antitumor compound 3Fax-NeuNAc by incubation of B16-F10 melanoma cells with N-acetyl-D-[UL-13C6]glucosamine revealed full depletion of endogenous ManNAc 6-phosphate and CMP-NeuNAc within 24 h. Thus, dynamic tracing of sugar metabolic pathways provides a general approach to reveal time-dependent insights into the metabolism of synthetic sugars, which is important for the rational design of analogs with optimized effects.

Bisubstrate Inhibitors of Nicotinamide N-Methyltransferase (NNMT) with Enhanced Activity.

Nicotinamide N-methyltransferase (NNMT) catalyzes the methylation of nicotinamide to form N-methylnicotinamide. Overexpression of NNMT is associated with a variety of diseases, including a number of cancers and metabolic disorders, suggesting a role for NNMT as a potential therapeutic target. By structural modification of a lead NNMT inhibitor previously developed in our group, we prepared a diverse library of inhibitors to probe the different regions of the enzyme's active site. This investigation revealed that incorporation of a naphthalene moiety, intended to bind the hydrophobic nicotinamide binding pocket via π-π stacking interactions, significantly increases the activity of bisubstrate-like NNMT inhibitors (half-maximal inhibitory concentration 1.41 µM). These findings are further supported by isothermal titration calorimetry binding assays as well as modeling studies. The most active NNMT inhibitor identified in the present study demonstrated a dose-dependent inhibitory effect on the cell proliferation of the HSC-2 human oral cancer cell line.

Inhibitors of nicotinamide N-methyltransferase designed to mimic the methylation reaction transition state.

Nicotinamide N-methyltransferase (NNMT) is an enzyme that catalyses the methylation of nicotinamide to form N'-methylnicotinamide. Both NNMT and its methylated product have recently been linked to a variety of diseases, suggesting a role for the enzyme as a therapeutic target beyond its previously ascribed metabolic function in detoxification. We here describe the systematic development of NNMT inhibitors derived from the structures of the substrates involved in the methylation reaction. By covalently linking fragments of the NNMT substrates a diverse library of bisubstrate-like compounds was prepared. The ability of these compounds to inhibit NNMT was evaluated providing valuable insights into the structural tolerances of the enzyme active site. These studies led to the identification of new NNMT inhibitors that mimic the transition state of the methylation reaction and inhibit the enzyme with activity on par with established methyltransferase inhibitors.

N-Guanidino Derivatives of 1,5-Dideoxy-1,5-imino-d-xylitol are Potent, Selective, and Stable Inhibitors of ß-Glucocerebrosidase.

A series of lipidated guanidino and urea derivatives of 1,5-dideoxy-1,5-imino-d-xylitol were prepared from d-xylose using a concise synthetic protocol. Inhibition assays with a panel of glycosidases revealed that the guanidino analogues display potent inhibition against human recombinant ß-glucocerebrosidase with IC50 values in the low nanomolar range. Related urea analogues of 1,5-dideoxy-1,5-imino-d-xylitol were also synthesized and evaluated in the same fashion and found to be selective for ß-galactosidase from bovine liver. No inhibition of human recombinant ß-glucocerebrosidase was observed for the urea analogues. Computational studies provided insight into the potent activity of analogues bearing the substituted guanidine moiety in the inhibition of lysosomal glucocerebrosidase (GBA).

Orthoester functionalized N-guanidino derivatives of 1,5-dideoxy-1,5-imino-d-xylitol as pH-responsive inhibitors of ß-glucocerebrosidase.

Alkylated guanidino derivatives of 1,5-dideoxy-1,5-imino-d-xylitol bearing an orthoester moiety were prepared using a concise synthetic protocol. Inhibition assays with a panel of glycosidases revealed that one of the compounds prepared displays potent inhibition against human ß-glucocerebrosidase (GBA) at pH 7.0 with IC50 values in the low nanomolar range. Notably, a significant drop in inhibitory activity is observed when the same compound is tested at pH 5.2. This pH sensitive activity is due to degradation of the orthoester functionality at lower pH accompanied by loss of the alkyl group. This approach provides a degree of control in tuning enzyme inhibition based on the local pH. Compounds like those here described may serve as tools for studying various lysosomal storage disorders such as Gaucher disease. In this regard, the most active compound was also evaluated as a potential pharmacological chaperone by assessing its effect on GBA activity in an assay employing fibroblasts from Gaucher patients.

Bicyclic isoureas derived from 1-deoxynojirimycin are potent inhibitors of ß-glucocerebrosidase.

A series of bicyclic isourea derivatives were prepared from 1-deoxynojirimycin using a concise synthetic protocol proceeding via a guanidino intermediate. Inhibition assays with a panel of glycosidases revealed that these deoxynojirimycin-derived bicyclic isoureas display very potent inhibition against human recombinant ß-glucocerebrosidase with IC50 values in the low nanomolar range.

Synthesis and evaluation of protein arginine N-methyltransferase inhibitors designed to simultaneously occupy both substrate binding sites.

The protein arginine N-methyltransferases (PRMTs) are a family of enzymes that function by specifically transferring a methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) to the guanidine group of arginine residues in target proteins. The most notable is the PRMT-mediated methylation of arginine residues that are present in histone proteins which can lead to chromatin remodelling and influence gene transcription. A growing body of evidence now implicates dysregulated PRMT activity in a number of diseases including various forms of cancer. The development of PRMT inhibitors may therefore hold potential as a means of developing new therapeutics. We here report the synthesis and evaluation of a series of small molecule PRMT inhibitors designed to simultaneously occupy the binding sites of both the guanidino substrate and AdoMet cofactor. Potent inhibition and surprising selectivity were observed when testing these compounds against a panel of methyltransferases.

Oseltamivir analogues bearing N-substituted guanidines as potent neuraminidase inhibitors.

A series of oseltamivir analogues bearing an N-substituted guanidine unit were prepared and evaluated as inhibitors of neuraminidases from four strains of influenza. The two most potent analogues identified contain relatively small N-guanidine substituents (N-methyl and N-hydroxyl) and display enhanced inhibition with IC50 values in the low nanomolar range against neuraminidases from wild-type and oseltamivir-resistant strains. Potential advantages of including the N-hydroxyguanidine moiety in neuraminidase inhibitors are also discussed.

Milk thistle's active components silybin and isosilybin: novel inhibitors of PXR-mediated CYP3A4 induction.

Because cancer is often treated with combination therapy, unexpected pharmacological effects can occur because of drug-drug interactions. Several drugs are able to cause upregulation or downregulation of drug transporters or cytochrome P450 enzymes, particularly CYP3A4. Induction of CYP3A4 may result in decreased plasma levels and therapeutic efficacy of anticancer drugs. Since the pregnane X receptor (PXR) is one of the major transcriptional regulators of CYP3A4, PXR antagonists can possibly prevent CYP3A4 induction. Currently, a limited number of PXR antagonists are available. Some of these antagonists, such as sulphoraphane and coumestrol, belong to the so-called complementary and alternative medicines (CAM). Therefore, the aim was to determine the potential of selected CAM (ß-carotene, Echinacea purpurea, garlic, Ginkgo biloba, ginseng, grape seed, green tea, milk thistle, saw palmetto, valerian, St. John's Wort, and vitamins B6, B12, and C) to inhibit PXR-mediated CYP3A4 induction at the transcriptional level, using a reporter gene assay and a real-time polymerase chain reaction assay in LS180 colon adenocarcinoma cells. Furthermore, computational molecular docking and a LanthaScreen time-resolved fluorescence resonance energy transfer (TR-FRET) PXR competitive binding assay were performed to explore whether the inhibiting CAM components interact with PXR. The results demonstrated that milk thistle is a strong inhibitor of PXR-mediated CYP3A4 induction. The components of milk thistle responsible for this effect were identified as silybin and isosilybin. Furthermore, computational molecular docking revealed a strong interaction between both silybin and isosilybin and PXR, which was confirmed in the TR-FRET PXR assay. In conclusion, silybin and isosilybin might be suitable candidates to design potent PXR antagonists to prevent drug-drug interactions via CYP3A4 in cancer patients.

Tuning the preference of thiodigalactoside- and lactosamine-based ligands to galectin-3 over galectin-1.

Inhibitors for galectin-1 and -3 were synthesized from thiodigalactoside and lactosamine by derivatization of the galactose C3. Introduction of 4-phenyl-1H-1,2,3-triazol-1-yl substituents at the thiodigalactoside C3 by CuAAC, targeting arginine-arene interactions, increased the affinity to 13 nM but yielded little selectivity. The bulkier 4-(4-phenoxyphenyl)-1H-1,2,3-triazol-1-yl substituent, however, increased the preference for galectin-3 over galectin-1 to more than 200-fold. Modeling showed more arginine-arene interactions for galectin-3 than for galectin-1. Introducing 4-phenoxyaryl groups on lactosamine had a similar effect.

Unusual binding of Grb2 protein to a bivalent polyproline-ligand immobilized on a SPR sensor: intermolecular bivalent binding.

The Grb2 adapter protein is involved in the activation of the Ras signaling pathway. It recruits the Sos protein by binding of its two SH3 domains to Sos polyproline sequences. We observed that the binding of Grb2 to a bivalent ligand, containing two Sos-derived polyproline-sequences immobilized on a SPR sensor, shows unusual kinetic behavior. SPR-kinetic analysis and supporting data from other techniques show major contributions of an intermolecular bivalent binding mode. Each of the two Grb2 SH3 domains binds to one polyproline-sequence of two different ligand molecules, facilitating binding of a second Grb2 molecule to the two remaining free polyproline binding sites. A molecular model based on the X-ray structure of the Grb2 dimer shows that Grb2 is flexible enough to allow this binding mode. The results fit with a role of Grb2 in protein aggregation, achieving specificity by multivalent interactions, despite the relatively low affinity of single SH3 interactions.

Conformation and intermolecular interactions of SA2 peptides self-assembled into vesicles.

Previously we have shown that the recombinantly produced SA2 amphiphilic oligopeptide (Ac-Ala-Ala-Val-Val-Leu-Leu-Leu-Trp-Glu-Glu-COOH) self-assembles into nanovesicles (van Hell et al. 2007). In this study, the intermolecular interactions that contribute to the formation of such peptide vesicles are examined. First, analysis of a 3-hydroxyflavone fluorescent probe inserted into the peptide assemblies demonstrated that the peptide self-assembly is based on hydrophobic clustering. The polarity of this hydrophobic microenvironment was comparable to that of negatively charged lipid bilayers. A substantial level of hydration at the hydrophilic-hydrophobic interface was detected, as was further confirmed by tryptophan fluorescence analysis. However, organic solvents such as acetonitrile, tetrahydrofuran, or ethanol could not disrupt SA2 oligopeptide vesicles, whereas these solvents fully disintegrated lipid vesicles. Instead, the SA2 assembly immediately disintegrated in hydrogen breaking solvents such dimethylsulfoxide and dimethylformamide, suggesting the involvement of additional intermolecular interactions via hydrogen bonding. Circular dichroism and Fourier transform infrared spectroscopy excluded well-defined patterns of intramolecular hydrogen bonding and indicated the polyproline type II as the dominant SA2 peptide conformation, which enables intermolecular hydrogen bonding. All-atom computational simulations were used to confirm the presence of such intermolecular hydrogen bonds and degrees of hydration. On the basis of the experimental and computational data presented, we propose a model of an interdigitated peptide assembly that involves intermolecular hydrogen bonding in addition to hydrophobic interactions that stabilize SA2 oligopeptide vesicles.

Synthesis and evaluation of new thiodigalactoside-based chemical probes to label galectin-3.

New chemical probes were synthesized to label galectin-3. They are based on the high affinity thiodigalactoside ligand. The probes were synthesized with benzophenone or acetophenone moieties as the photolabel for covalent attachment to the protein. Besides labeling the protein, these aromatic photolabels also greatly enhance the affinity of the probes towards galectin-3, due to the interaction of the photolabel with two arginine guanidinium groups of the protein. The linkage between the sugar and the photolabel was varied as an ester, an amide, and a triazole. For the amide and triazole derivatives, a versatile synthetic route towards a symmetrical 3-azido-3-deoxy-thiodigalactoside was developed. The new probes were evaluated for their binding affinity of human galectin-3. They were subsequently tested for their labeling efficiency, as well as specificity in the presence of a protein mixture and a human cancer cell lysate.

Immunodominance in mouse and human CD4+ T-cell responses specific for the Bordetella pertussis virulence factor P.69 pertactin.

P.69 pertactin (P.69 Prn), an adhesion molecule from the causative agent of pertussis, Bordetella pertussis, is present in cellular and most acellular vaccines that are currently used worldwide. Although both humoral immunity and cellular immunity directed against P.69 Prn have been implicated in protective immune mechanisms, the identities of CD4(+) T-cell epitopes on the P.69 Prn protein remain unknown. Here, a single I-A(d)-restricted B. pertussis conserved CD4(+) T-cell epitope at the N terminus of P.69 Prn was identified by using a BALB/c T-cell hybridoma. The epitope appeared immunodominant among four other minor strain-conserved P.69 Prn epitopes recognized after vaccination and B. pertussis infection, and it was capable of evoking a Th1/Th17-type cytokine response. B. pertussis P.69 Prn immune splenocytes did not cross-react with natural variants of the epitope as present in Bordetella parapertussis and Bordetella bronchiseptica. Finally, it was found that the immunodominant P.69 Prn epitope is broadly recognized in the human population by CD4(+) T cells in an HLA-DQ-restricted manner. During B. pertussis infection, the epitope was associated with a Th1-type CD4(+) T-cell response. Hence, this novel P.69 Prn epitope is involved in CD4(+) T-cell immunity after B. pertussis vaccination and infection in mice and, more importantly, in humans. Thus, it may provide a useful tool for the evaluation of the type, magnitude, and maintenance of B. pertussis-specific CD4(+) T-cell mechanisms in preclinical and clinical vaccine studies.

The role of peptide loops of the Bordetella pertussis protein P.69 pertactin in antibody recognition.

Bordetella pertussis, the etiological agent of whooping cough, is re-emerging in several countries with a traditionally high vaccine uptake. In these B. pertussis strains, polymorphisms were found in several proteins, including P.69 pertactin (P.69 Prn). P.69 Prn, an adhesin, contains two variable regions which are composed of repeats, one of which flanks the receptor binding site. Antibody titers against P.69 Prn correlate with protection and P.69 Prn is one of the components of acellular pertussis vaccines. Nevertheless, little is known about the structure and location of P.69 Prn epitopes. We used a three pronged approach to identify discontinuous epitopes that are recognized by mouse monoclonal antibodies, i.e. site-directed mutagenesis, deletion mapping and competition assays. Site-directed mutagenesis was focused on regions of P.69 Prn predicted to form loops according to the crystal structure. In this report we describe the location of several discontinuous epitopes that are also recognized by human antibodies. Our results reveal an important role of the N-terminus in immune recognition. We provide data for an indirect role of loops in immune evasion by masking of epitopes. We propose that the repeat regions have evolved to allow rapid antigenic variation to deflect the immune response from the functional domain of P.69 Prn. The results presented here provide a better understanding of the structure and function of variable loops and their role in the persistence of pathogens in immunologically primed populations.

Limited plasticity in T cell recognition of modified T cell receptor contact residues in MHC class II bound peptides.

The balance between specific and degenerate T cell recognition of MHC class II bound peptides is crucial for T cell repertoire selection, and holds important implications for protective immunity versus autoimmunity. To investigate the degree of degeneracy in T cell recognition, we applied selected modifications to T cell receptor (TCR) contact residue amino acids in the MHC class II bound epitope gpMBP72-85. By using glycosylated amino acids, as an example of a posttranslational modification, large alterations were applied. Small modifications were accomplished by exchanging an arginine residue for a citrulline or an ornithine residue. Finally, the unmodified TCR contact residue side chains were shifted one atom position to the left, using peptoid residues. Both these large and subtle changes in the wild type (WT) peptide caused lack of recognition by WT peptide specific monoclonal and polyclonal T cells. Furthermore, T cells specific for the modified peptides did not cross recognize the WT peptide. Using a set of additional compounds, we investigated the specificity of these T cell populations into detail. Our data reveal a strongly limited plasticity in T cell recognition, and a high specificity for TCR contact residue side chains.

Possibilities and limitations in the rational design of modified peptides for T cell mediated immunotherapy.

Therapeutic intervention in experimental autoimmune diseases by modulation of the T cell mediated autoimmune response has been accomplished in the past using altered peptide ligands (APLs). These peptides are usually created by applying alterations to the T cell epitope recognized by the autoaggressive T cells. In this study, we investigated whether it was possible to design APLs in a rational way, using knowledge of molecular interaction in the MHC-peptide-T cell receptor (TCR) complex, for the therapeutic intervention in experimental autoimmune encephalomyelitis (EAE). Additionally, the value of peptidomimetic modification and alterations based on posttranslational modifications for the design of APLs was examined. Based on a molecular model of the MHC-peptide complex, the T cell receptor contact residues were identified and selected alterations were applied. The designed APLs were tested for MHC binding capacity, T cell recognition, blocking of the autoreactive T cell response, immunogenicity, encephalitogenicity, and therapeutic activity. Based on the results of the in vitro assays, it was expected that some of our APLs would be able to modulate EAE. Nevertheless, none of these APLs displayed clear therapeutic activity in vivo. Thus, rational design of modified peptides for immunotherapy has to await further insights into the relationships between structure and peptide/peptidomimetic induced T cell activation, and until that, there is no possibility to take advantage of the tailor made origin of peptidomimetics.

Stabilization of peptide guinea pig myelin basic protein 72-85 by N-terminal acetylation-implications for immunological studies.

Peptide gpMBP72-85, containing amino acids 72-85 of guinea pig myelin basic protein is commonly used to induce experimental autoimmune encephalomyelitis in Lewis rats. The N-terminal glutamine in this peptide can cyclize to pyroglutamic acid, leading to loss of the first MHC anchor for binding to MHC class II. Acetylation of the peptide N-terminus prevents pyroglutamic acid formation and ensures a constant quality. An increased MHC binding affinity after N-terminal acetylation was observed. This modification also enhanced T cell proliferation of a gpMBP reactive T cell clone. The encephalitogenicity of peptide gpMBP72-85 was unaffected by acetylation. It is concluded that acetylation improves the chemical stability of gpMBP72-85, and is not detrimental but rather favorable for its biochemical and immunological, in vitro, and in vivo behavior.

Generate: a program for 3-D structure generation and conformational analysis of peptides and peptidomimetics.

The program Generate, aimed at generating 3-D structures for peptides and peptidomimetics, is presented. The algorithm is based on a build-up procedure, using a library of conformations of amino acid residues. This library is built from conformational analysis of amino acids placed in a di- or tripeptide environment to mimic the surroundings of the amino acid in a true peptide, considering different positions of the residue in the peptide chain (peptidyl fragment, NH(+)(3)-terminus or COO(-)-terminus). Cis-trans isomerism in the amide bonds is taken into account by construction of rotamer libraries for different isomers. Water solvation is included through the GB/SA model. New amino acid residues can easily be added to the libraries, making it possible to generate conformations of peptidomimetics.

Major histocompatibility complex class II binding characteristics of peptoid-peptide hybrids.

The major histocompatibility complex (MHC) class II binding requirements for solvent-exposed peptide residues were systematically studied using amino acid and peptoid substitutions. In a peptoid residue, the side chain is present on the backbone nitrogen atom as opposed to the alpha-carbon atom in an amino acid residue. To investigate the effect of this side chain shifting on MHC binding, three amino acids in the central part of the peptide sticking out of the binding groove were replaced by corresponding peptoid residues. Two peptoid-peptide hybrids showed large affinity decreases in the MHC-peptide binding assay. To investigate this affinity loss, the individual contributions to MHC binding affinity of the side chain (position), the putative hydrogen bond, and the flexibility were dissected. We conclude that the side chain position as well as the backbone nitrogen atom hydrogen bonding features of solvent-exposed residues in the peptide can be important for MHC binding affinity.