Rational synthesis of a heparan sulfate saccharide that promotes the activity of BMP2.

Heparan sulfate (HS) is a glycosaminoglycan (GAG) found throughout nature and is involved in a wide range of functions including modulation of cell signalling via sequestration of growth factors. Current consensus is that the specificity of HS motifs for protein binding are individual for each protein. Given the structural complexity of HS the synthesis of libraries of these compounds to probe this is not trivial. Herein we present the synthesis of an HS decamer, the design of which was undertaken rationally from previously published data for HS binding to the growth factor BMP-2. The biological activity of this HS decamer was assessed in vitro, showing that it had the ability to both bind BMP-2 and increase its thermal stability as well as enhancing the bioactivity of BMP-2 in vitro in C2C12 cells. At the same time no undesired anticoagulant effect was observed. This decamer was then analysed in vivo in a rabbit model where higher bone formation, bone mineral density (BMD) and trabecular thickness were observed over an empty defect or collagen implant alone. This indicated that the HS decamer was effective in promoting bone regeneration in vivo.

Isofagomine Inhibits Multiple TcdB Variants and Protects Mice from Clostridioides difficile-Induced Mortality.

Clostridioides difficile causes life-threatening diarrhea and is one of the leading causes of nosocomial infections. During infection, C. difficile releases two gut-damaging toxins, TcdA and TcdB, which are the primary determinants of disease pathogenesis and are important therapeutic targets. Once in the cytosol of mammalian cells, TcdA and TcdB use UDP-glucose to glucosylate host Rho GTPases, which leads to cytoskeletal changes that result in a loss of intestinal integrity. Isofagomine inhibits TcdA and TcdB as a mimic of the glucocation transition state of the glucosyltransferase reaction. However, sequence variants of TcdA and TcdB across the clades of infective C. difficile continue to be identified, and therefore, evaluation of isofagomine inhibition against multiple toxin variants is required. Here, we show that isofagomine inhibits the glucosyltransferase domain of multiple TcdB variants and protects TcdB-induced cell rounding of the most common full-length toxin variants. Furthermore, we demonstrate that isofagomine protects against C. difficile-induced mortality in two murine models of C. difficile infection. Isofagomine treatment of mouse C. difficile infection also permitted the recovery of the gastrointestinal microbiota, an important barrier to preventing recurring C. difficile infection. The broad specificity of isofagomine supports its potential as a prophylactic to protect against C. difficile-induced morbidity and mortality.

Combined inhibition of MTAP and MAT2a mimics synthetic lethality in tumor models via PRMT5 inhibition.

Homozygous 5'-methylthioadenosine phosphorylase (MTAP) deletions occur in approximately 15% of human cancers. Co-deletion of MTAP and methionine adenosyltransferase 2 alpha (MAT2a) induces a synthetic lethal phenotype involving protein arginine methyltransferase 5 (PRMT5) inhibition. MAT2a inhibitors are now in clinical trials for genotypic MTAP-/- cancers, however the MTAP-/- genotype represents fewer than 2% of human colorectal cancers (CRCs), limiting the utility of MAT2a inhibitors in these and other MTAP+/+ cancers. Methylthio-DADMe-immucillin-A (MTDIA) is a picomolar transition state analog inhibitor of MTAP that renders cells enzymatically MTAP-deficient to induce the MTAP-/- phenotype. Here, we demonstrate that MTDIA and MAT2a inhibitor AG-270 combination therapy mimics synthetic lethality in MTAP+/+ CRC cell lines with similar effects in mouse xenografts and without adverse histology on normal tissues. Combination treatment is synergistic with a 104-fold increase in drug potency for inhibition of CRC cell growth in culture. Combined MTDIA and AG-270 decreases S-adenosyl-L-methionine and increases 5'-methylthioadenosine in cells. The increased intracellular methylthioadenosine:S-adenosyl-L-methionine ratio inhibits PRMT5 activity, leading to cellular arrest and apoptotic cell death by causing MDM4 alternative splicing and p53 activation. Combination MTDIA and AG-270 treatment differs from direct inhibition of PRMT5 by GSK3326595 by avoiding toxicity caused by cell death in the normal gut epithelium induced by the PRMT5 inhibitor. The combination of MTAP and MAT2a inhibitors expands this synthetic lethal approach to include MTAP+/+ cancers, especially the remaining 98% of CRCs without the MTAP-/- genotype.

Isofagomine inhibits multiple TcdB variants and protects mice from Clostridioides difficile induced mortality.

Clostridioides difficile causes life-threatening diarrhea and is the leading cause of healthcare associated bacterial infections in the United States. During infection, C. difficile releases the gut-damaging toxins, TcdA and TcdB, the primary determinants of disease pathogenesis and are therefore therapeutic targets. TcdA and TcdB contain a glycosyltransferase domain that uses UDP-glucose to glycosylate host Rho GTPases, causing cytoskeletal changes that result in a loss of intestinal integrity. Isofagomine inhibits TcdA and TcdB as a mimic of the oxocarbenium ion transition state of the glycosyltransferase reaction. However, sequence variants of TcdA and TcdB across the clades of infective C. difficile continue to be identified and therefore, evaluation of isofagomine inhibition against multiple toxin variants are required. Here we show that Isofagomine inhibits the glycosyltransferase activity of multiple TcdB variants and also protects TcdB toxin-induced cell rounding of the most common full-length toxin variants. Further, isofagomine protects against C. difficile induced mortality in two murine models of C. difficile infection. Isofagomine treatment of mouse C. difficile infection permitted recovery of the gastrointestinal microbiota, an important barrier to prevent recurring C. difficile infection. The broad specificity of isofagomine supports its potential as a prophylactic to protect against C. difficile induced morbidity and mortality.

Expedient synthesis of imino-C-nucleoside fleximers featuring a one-pot procedure to prepare aryl triazoles.

Nucleoside analogues such as the antiviral agents galidesivir and ribavirin are of synthetic interest. This work reports a "one-pot" preparation of similar fleximers using a bifunctional copper catalyst that generates the aryl azide in situ, which is captured by a terminal alkyne to effect triazole formation.

Kinetic and Structural Characterization of Trypanosoma cruzi Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferases and Repurposing of Transition-State Analogue Inhibitors.

Over 70 million people are currently at risk of developing Chagas Disease (CD) infection, with more than 8 million people already infected worldwide. Current treatments are limited and innovative therapies are required. Trypanosoma cruzi, the etiological agent of CD, is a purine auxotroph that relies on phosphoribosyltransferases to salvage purine bases from their hosts for the formation of purine nucleoside monophosphates. Hypoxanthine-guanine-xanthine phosphoribosyltransferases (HGXPRTs) catalyze the salvage of 6-oxopurines and are promising targets for the treatment of CD. HGXPRTs catalyze the formation of inosine, guanosine, and xanthosine monophosphates from 5-phospho-d-ribose 1-pyrophosphate and the nucleobases hypoxanthine, guanine, and xanthine, respectively. T. cruzi possesses four HG(X)PRT isoforms. We previously reported the kinetic characterization and inhibition of two isoforms, TcHGPRTs, demonstrating their catalytic equivalence. Here, we characterize the two remaining isoforms, revealing nearly identical HGXPRT activities in vitro and identifying for the first time T. cruzi enzymes with XPRT activity, clarifying their previous annotation. TcHGXPRT follows an ordered kinetic mechanism with a postchemistry event as the rate-limiting step(s) of catalysis. Its crystallographic structures reveal implications for catalysis and substrate specificity. A set of transition-state analogue inhibitors (TSAIs) initially developed to target the malarial orthologue were re-evaluated, with the most potent compound binding to TcHGXPRT with nanomolar affinity, validating the repurposing of TSAIs to expedite the discovery of lead compounds against orthologous enzymes. We identified mechanistic and structural features that can be exploited in the optimization of inhibitors effective against TcHGPRT and TcHGXPRT concomitantly, which is an important feature when targeting essential enzymes with overlapping activities.

Residence Times for Femtomolar and Picomolar Inhibitors of MTANs.

5'-Methylthioadenosine nucleosidases (MTANs) catalyze the hydrolysis of 5'-substituted adenosines to form adenine and 5-substituted ribose. Escherichia coli MTAN (EcMTAN) and Helicobacter pylori MTAN (HpMTAN) form late and early transition states, respectively. Transition state analogues designed for the late transition state bind with fM to pM affinity to both classes of MTANs. Here, we compare the residence times (off-rates) with the equilibrium dissociation constants for HpMTAN and EcMTAN, using five 5'-substituted DADMe-ImmA transition state analogues. The inhibitors dissociate orders of magnitude slower from EcMTAN than from HpMTAN. For example, the slowest release rate was observed for the EcMTAN-HTDIA complex (t1/2 = 56 h), compared to a release rate of t1/2 = 0.3 h for the same complex with HpMTAN, despite similar structures and catalytic sites for these enzymes. Other inhibitors also reveal disconnects between residence times and equilibrium dissociation constants. Residence time is correlated with pharmacological efficacy; thus, experimental analyses of dissociation rates are useful to guide physiological function of tight-binding inhibitors. Steered molecular dynamics simulations for the dissociation of an inhibitor from both EcMTAN and HpMTAN provide atomic level mechanistic insight for the differences in dissociation kinetics and inhibitor residence times for these enzymes.

Inhibition and Mechanism of Plasmodium falciparum Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase.

Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPRT) is essential for purine salvage of hypoxanthine into parasite purine nucleotides. Transition state analogue inhibitors of PfHGXPRT are characterized by kinetic analysis, thermodynamic parameters, and X-ray crystal structures. Compound 1, 9-deazaguanine linked to an acyclic ribocation phosphonate mimic, shows a kinetic Ki of 0.5 nM. Isothermal titration calorimetry (ITC) experiments of 1 binding to PfHGXPRT reveal enthalpically driven binding with negative cooperativity for the binding of two inhibitor molecules in the tetrameric enzyme. Crystal structures of 1 bound to PfHGXPRT define the hydrogen bond and ionic contacts to complement binding thermodynamics. Dynamics of ribosyl transfer from 5-phospho-α-d-ribosyl 1-pyrophosphate (PRPP) to hypoxanthine were examined by 18O isotope exchange at the bridging phosphoryl oxygen of PRPP pyrophosphate. Rotational constraints or short transition state lifetimes prevent torsional rotation and positional isotope exchange of bridging to nonbridging oxygen in the α-pyrophosphoryl group. Thermodynamic analysis of the transition state analogue and magnesium pyrophosphate binding reveal random and cooperative binding to PfHGXPRT, unlike the obligatory ordered reaction kinetics reported earlier for substrate kinetics.

Kinetic Characterization and Inhibition of Trypanosoma cruzi Hypoxanthine-Guanine Phosphoribosyltransferases.

Chagas disease, caused by the parasitic protozoan Trypanosoma cruzi, affects over 8 million people worldwide. Current antiparasitic treatments for Chagas disease are ineffective in treating advanced, chronic stages of the disease, and are noted for their toxicity. Like most parasitic protozoa, T. cruzi is unable to synthesize purines de novo, and relies on the salvage of preformed purines from the host. Hypoxanthine-guanine phosphoribosyltransferases (HGPRTs) are enzymes that are critical for the salvage of preformed purines, catalyzing the formation of inosine monophosphate (IMP) and guanosine monophosphate (GMP) from the nucleobases hypoxanthine and guanine, respectively. Due to the central role of HGPRTs in purine salvage, these enzymes are promising targets for the development of new treatment methods for Chagas disease. In this study, we characterized two gene products in the T. cruzi CL Brener strain that encodes enzymes with functionally identical HGPRT activities in vitro: TcA (TcCLB.509693.70) and TcC (TcCLB.506457.30). The TcC isozyme was kinetically characterized to reveal mechanistic details on catalysis, including identification of the rate-limiting step(s) of catalysis. Furthermore, we identified and characterized inhibitors of T. cruzi HGPRTs originally developed as transition-state analogue inhibitors (TSAIs) of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPRT), where the most potent compound bound to T. cruzi HGPRT with low nanomolar affinity. Our results validated the repurposing of TSAIs to serve as selective inhibitors for orthologous molecular targets, where primary and secondary structures as well as putatively common chemical mechanisms are conserved.

The design of protozoan phosphoribosyltransferase inhibitors containing non-charged phosphate mimic residues.

Phosphate groups play essential roles in biological processes, including retention inside biological membranes. Phosphodiesters link nucleic acids, and the reversible transfer of phosphate groups is essential in energy metabolism and cell-signalling processes. Phosphorylated metabolic intermediates are known targets for metabolic and disease-related disorders, and the enzymes involved in these pathways recognize phosphate groups in their catalytic sites. Therapeutics that target these enzymes can require charged (ionic) entities to capture the binding energy of ionic substrates. Such compounds are not cell-permeable and require pro-drug strategies for efficacy as therapeutics. Protozoan parasites such as Plasmodium and Trypanosoma spp. are unable to synthesise purines de novo and rely on the salvage of purines from the host cell to synthesise free purine bases. Purine phosphoribosyltransfereases (PPRTases) play a crucial role for purine salvage and are potential target for drug development. Here we present attempts to design inhibitors of PPRTases that are non-ionic and show affinity for the nucleotide 5'-phosphate binding site. Inhibitor design was based on known potent ionic inhibitors, reported phosphate mimics and computational modelling studies.

Synthesis and Characterization of Transition-State Analogue Inhibitors against Human DNA Methyltransferase 1.

Hypermethylation of CpG regions by human DNA methyltransferase 1 (DNMT1) silences tumor-suppression genes, and inhibition of DNMT1 can reactivate silenced genes. The 5-azacytidines are approved inhibitors of DNMT1, but their mutagenic mechanism limits their utility. A synthon approach from the analogues of S-adenosylhomocysteine, methionine, and deoxycytidine recapitulated the chemical features of the DNMT1 transition state in the synthesis of 16 chemically stable transition-state mimics. Inhibitors causing both full and partial inhibition of purified DNMT1 were characterized. The inhibitors show modest selectivity for DNMT1 versus DNMT3b. Active-site docking predicts inhibitor interactions with S-adenosyl-l-methionine and deoxycytidine regions of the catalytic site, validated by direct binding analysis. Inhibitor action with purified DNMT1 is not reflected in cultured cells. A partial inhibitor activated cellular DNA methylation, and a full inhibitor had no effect on cellular DNA methylation. These compounds provide chemical access to a new family of noncovalent DNMT chemical scaffolds for use in DNA methyltransferases.

Inhibition of Clostridium difficile TcdA and TcdB toxins with transition state analogues.

Clostridium difficile causes life-threatening diarrhea and is the leading cause of healthcare-associated bacterial infections in the United States. TcdA and TcdB bacterial toxins are primary determinants of disease pathogenesis and are attractive therapeutic targets. TcdA and TcdB contain domains that use UDP-glucose to glucosylate and inactivate host Rho GTPases, resulting in cytoskeletal changes causing cell rounding and loss of intestinal integrity. Transition state analysis revealed glucocationic character for the TcdA and TcdB transition states. We identified transition state analogue inhibitors and characterized them by kinetic, thermodynamic and structural analysis. Iminosugars, isofagomine and noeuromycin mimic the transition state and inhibit both TcdA and TcdB by forming ternary complexes with Tcd and UDP, a product of the TcdA- and TcdB-catalyzed reactions. Both iminosugars prevent TcdA- and TcdB-induced cytotoxicity in cultured mammalian cells by preventing glucosylation of Rho GTPases. Iminosugar transition state analogues of the Tcd toxins show potential as therapeutics for C. difficile pathology.

Aminofutalosine Deaminase in the Menaquinone Pathway of Helicobacter pylori.

Helicobacter pylori is a Gram-negative bacterium that is responsible for gastric and duodenal ulcers. H. pylori uses the unusual mqn pathway with aminofutalosine (AFL) as an intermediate for menaquinone biosynthesis. Previous reports indicate that hydrolysis of AFL by 5'-methylthioadenosine nucleosidase (HpMTAN) is the direct path for producing downstream metabolites in the mqn pathway. However, genomic analysis indicates jhp0252 is a candidate for encoding AFL deaminase (AFLDA), an activity for deaminating aminofutolasine. The product, futalosine, is not a known substrate for bacterial MTANs. Recombinant jhp0252 was expressed and characterized as an AFL deaminase (HpAFLDA). Its catalytic specificity includes AFL, 5'-methylthioadenosine, 5'-deoxyadenosine, adenosine, and S-adenosylhomocysteine. The kcat/Km value for AFL is 6.8 × 104 M-1 s-1, 26-fold greater than that for adenosine. 5'-Methylthiocoformycin (MTCF) is a slow-onset inhibitor for HpAFLDA and demonstrated inhibitory effects on H. pylori growth. Supplementation with futalosine partially restored H. pylori growth under MTCF treatment, suggesting AFL deamination is significant for cell growth. The crystal structures of apo-HpAFLDA and with MTCF at the catalytic sites show a catalytic site Zn2+ or Fe2+ as the water-activating group. With bound MTCF, the metal ion is 2.0 Å from the sp3 hydroxyl group of the transition state analogue. Metabolomics analysis revealed that HpAFLDA has intracellular activity and is inhibited by MTCF. The mqn pathway in H. pylori bifurcates at aminofutalosine with HpMTAN producing adenine and depurinated futalosine and HpAFLDA producing futalosine. Inhibition of cellular HpMTAN or HpAFLDA decreased the cellular content of menaquinone-6, supporting roles for both enzymes in the pathway.

Comparison of disaccharide donors for heparan sulfate synthesis: uronic acids vs. their pyranose equivalents.

Late oxidation of hexose based building blocks or the use of uronic acid containing building blocks are two complementary strategies in the synthesis of glycosaminoglycans, the latter simplifiying the later stages of the process. Here we report the synthesis and evaluation of various disaccharide donors-uronic acids and their pyranose equivalents-for the synthesis of heparan sulfate, using an established protective group strategy. Hexose based "imidate" type donors perform well in the studied glycosylations, while their corresponding uronate esters fall short; a uronate ester thioglycoside performs equal to, if not better than, a hexose thioglycoside equivalent.

Shotgun ion mobility mass spectrometry sequencing of heparan sulfate saccharides.

Despite evident regulatory roles of heparan sulfate (HS) saccharides in numerous biological processes, definitive information on the bioactive sequences of these polymers is lacking, with only a handful of natural structures sequenced to date. Here, we develop a "Shotgun" Ion Mobility Mass Spectrometry Sequencing (SIMMS2) method in which intact HS saccharides are dissociated in an ion mobility mass spectrometer and collision cross section values of fragments measured. Matching of data for intact and fragment ions against known values for 36 fully defined HS saccharide structures (from di- to decasaccharides) permits unambiguous sequence determination of validated standards and unknown natural saccharides, notably including variants with 3O-sulfate groups. SIMMS2 analysis of two fibroblast growth factor-inhibiting hexasaccharides identified from a HS oligosaccharide library screen demonstrates that the approach allows elucidation of structure-activity relationships. SIMMS2 thus overcomes the bottleneck for decoding the informational content of functional HS motifs which is crucial for their future biomedical exploitation.

Transition State Analogues Enhanced by Fragment-Based Structural Analysis: Bacterial Methylthioadenosine Nucleosidases.

Transition state analogue inhibitor design (TSID) and fragment-based drug design (FBDD) are drug design approaches typically used independently. Methylthio-DADMe-Immucillin-A (MTDIA) is a tight-binding transition state analogue of bacterial 5'-methylthioadenosine nucleosidases (MTANs). Previously, Salmonella enterica MTAN structures were found to bind MTDIA and ethylene glycol fragments, but MTDIA modified to contain similar fragments did not enhance affinity. Seventy-five published MTAN structures were analyzed, and co-crystallization fragments were found that might enhance the binding of MTDIA to other bacterial MTANs through contacts external to MTDIA binding. The fragment-modified MTDIAs were tested with Helicobacter pylori MTAN and Staphylococcus aureus MTANs (HpMTAN and SaMTAN) as test cases to explore inhibitor optimization by potential contacts beyond the transition state contacts. Replacement of a methyl group with a 2'-ethoxyethanol group in MTDIA improved the dissociation constant 14-fold (0.09 nM vs 1.25 nM) for HpMTAN and 81-fold for SaMTAN (0.096 nM vs 7.8 nM). TSID combined with FBDD can be useful in enhancing already powerful inhibitors.

Metabolic dependency of chorismate in Plasmodium falciparum suggests an alternative source for the ubiquinone biosynthesis precursor.

The shikimate pathway, a metabolic pathway absent in humans, is responsible for the production of chorismate, a branch point metabolite. In the malaria parasite, chorismate is postulated to be a direct precursor in the synthesis of p-aminobenzoic acid (folate biosynthesis), p-hydroxybenzoic acid (ubiquinone biosynthesis), menaquinone, and aromatic amino acids. While the potential value of the shikimate pathway as a drug target is debatable, the metabolic dependency of chorismate in P. falciparum remains unclear. Current evidence suggests that the main role of chorismate is folate biosynthesis despite ubiquinone biosynthesis being active and essential in the malaria parasite. Our goal in the present work was to expand our knowledge of the ubiquinone head group biosynthesis and its potential metabolic dependency on chorismate in P. falciparum. We systematically assessed the development of both asexual and sexual stages of P. falciparum in a defined medium in the absence of an exogenous supply of chorismate end-products and present biochemical evidence suggesting that the benzoquinone ring of ubiquinones in this parasite may be synthesized through a yet unidentified route.

Selective Inhibitors of Helicobacter pylori Methylthioadenosine Nucleosidase and Human Methylthioadenosine Phosphorylase.

Bacterial 5'-methylthioadenosine/ S-adenosylhomocysteine nucleosidase (MTAN) hydrolyzes adenine from its substrates to form S-methyl-5-thioribose and S-ribosyl-l-homocysteine. MTANs are involved in quorum sensing, menaquinone synthesis, and 5'-methylthioadenosine recycling to S-adenosylmethionine. Helicobacter pylori uses MTAN in its unusual menaquinone pathway, making H. pylori MTAN a target for antibiotic development. Human 5'-methylthioadenosine phosphorylase (MTAP), a reported anticancer target, catalyzes phosphorolysis of 5'-methylthioadenosine to salvage S-adenosylmethionine. Transition-state analogues designed for HpMTAN and MTAP show significant overlap in specificity. Fifteen unique transition-state analogues are described here and are used to explore inhibitor specificity. Several analogues of HpMTAN bind in the picomolar range while inhibiting human MTAP with orders of magnitude weaker affinity. Structural analysis of HpMTAN shows inhibitors extending through a hydrophobic channel to the protein surface. The more enclosed catalytic sites of human MTAP require the inhibitors to adopt a folded structure, displacing the phosphate nucleophile from the catalytic site.

Using automated glycan assembly (AGA) for the practical synthesis of heparan sulfate oligosaccharide precursors.

Herein we report synthesis of complex heparan sulfate oligosaccharide precursors by automated glycan assembly using disaccharide donor building blocks. Rapid access to a hexasaccharide was achieved through iterative solid phase glycosylations on a photolabile resin using Glyconeer™, an automated oligosaccharide synthesiser, followed by photochemical cleavage and glycan purification using simple flash column chromatography.

Dendrimer Heparan Sulfate Glycomimetics: Potent Heparanase Inhibitors for Anticancer Therapy.

Heparanase is a mammalian endoglycosidase that cleaves heparan sulfate (HS) polysaccharides and contributes to remodelling of the extracellular matrix and regulation of HS-binding protein bioavailabilities. Heparanase is upregulated in malignant cancers and inflammation, aiding cell migration and the release of signaling molecules. It is established as a highly druggable extracellular target for anticancer therapy, but current compounds have limitations, because of cost, production complexity, or off-target effects. Here, we report the synthesis of a novel, targeted library of single-entity glycomimetic clusters capped with simple sulfated saccharides. Several dendrimer HS glycomimetics display low nM IC50 potency for heparanase inhibition equivalent to comparator compounds in clinical development, and potently inhibit metastasis and growth of human myeloma tumor cells in a mouse xenograft model. Importantly, they lack anticoagulant activity and cytotoxicity, and also inhibit angiogenesis. They provide a new candidate class for anticancer and wider therapeutic applications, which could benefit from targeted heparanase inhibition.