One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis.

Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.

Novel disaccharide inhibitors for the bacterial galactosyltransferase LgtC: Design, synthesis via Heyns rearrangement, and biochemical evaluation.

Bacterial glycosyltransferases are potential targets for the development of novel antibiotics and anti-virulence agents. We report a novel inhibitor design for the retaining α-1,4-galactosyltransferase LgtC from Neisseria meningitidis. Our design is based on the installation of an electrophilic warhead on the LgtC acceptor substrate and targeted at a non-catalytic cysteine residue in the LgtC active site. We have successfully synthesised two prototype inhibitors in four steps from lactulose. The key step in our synthesis is a Heyns rearrangement, during which we observed the formation of a hitherto unknown side product. While both lactosamine derivatives behaved as moderate inhibitors of LgtC, they also retained residual substrate activity. These results suggest that in contrast to our original design, these inhibitors do not act via a covalent mode of action, but are most likely non-covalent inhibitors.

Inhibition of bacterial growth and galactosyltransferase activity of WbwC by α, ω-bis(3-alkyl-1H-imidazolium)alkane salts: Effect of varying carbon content.

A series of compounds was designed and synthesized having two imidazolium rings separated by a polymethylene spacer and having alkyl substituents on each of the imidazolium rings. The compounds were assayed for their effects on the activity of galactosyltransferase WbwC, and also on the growth of Gram-negative and Gram-positive bacteria, as well as human cells. The inhibition observed on enzyme activities and cell growth was dependent on the total number of carbons in the spacer and the alkyl substituents on the imidazolium rings. These readily synthesized, achiral compounds have potential as antimicrobial and antiseptic agents.

The endogenous galactofuranosidase GlfH1 hydrolyzes mycobacterial arabinogalactan.

Despite impressive progress made over the past 20 years in our understanding of mycolylarabinogalactan-peptidoglycan (mAGP) biogenesis, the mechanisms by which the tubercle bacillus Mycobacterium tuberculosis adapts its cell wall structure and composition to various environmental conditions, especially during infection, remain poorly understood. Being the central portion of the mAGP complex, arabinogalactan (AG) is believed to be the constituent of the mycobacterial cell envelope that undergoes the least structural changes, but no reports exist supporting this assumption. Herein, using recombinantly expressed mycobacterial protein, bioinformatics analyses, and kinetic and biochemical assays, we demonstrate that the AG can be remodeled by a mycobacterial endogenous enzyme. In particular, we found that the mycobacterial GlfH1 (Rv3096) protein exhibits exo-ß-d-galactofuranose hydrolase activity and is capable of hydrolyzing the galactan chain of AG by recurrent cleavage of the terminal ß-(1,5) and ß-(1,6)-Galf linkages. The characterization of this galactosidase represents a first step toward understanding the remodeling of mycobacterial AG.

Potential Inhibitors of Galactofuranosyltransferase 2 (GlfT2): Molecular Docking, 3D-QSAR, and In Silico ADMETox Studies.

A strategy in the discovery of anti-tuberculosis (anti-TB) drug involves targeting the enzymes involved in the biosynthesis of Mycobacterium tuberculosis' (Mtb) cell wall. One of these enzymes is Galactofuranosyltransferase 2 (GlfT2) that catalyzes the elongation of the galactan chain of Mtb cell wall. Studies targeting GlfT2 have so far produced compounds showing minimal inhibitory activity. With the current challenge of designing potential GlfT2 inhibitors with high inhibition activity, computational methods such as molecular docking, receptor-ligand mapping, molecular dynamics, and Three-Dimensional-Quantitative Structure-Activity Relationship (3D-QSAR) were utilized to deduce the interactions of the reported compounds with the target enzyme and enabling the design of more potent GlfT2 inhibitors. Molecular docking studies showed that the synthesized compounds have binding energy values between -3.00 to -6.00 kcal mol-1. Two compounds, #27 and #31, have registered binding energy values of -8.32 ± 0.01, and -8.08 ± 0.01 kcal mol-1, respectively. These compounds were synthesized as UDP-Galactopyranose mutase (UGM) inhibitors and could possibly inhibit GlfT2. Interestingly, the analogs of the known disaccharide substrate, compounds #1-4, have binding energy range of -10.00 to -19.00 kcal mol-1. The synthesized and newly designed compounds were subjected to 3D-QSAR to further design compounds with effective interaction within the active site. Results showed improved binding energy from -6.00 to -8.00 kcal mol-1. A significant increase on the binding affinity was observed when modifying the aglycon part instead of the sugar moiety. Furthermore, these top hit compounds were subjected to in silico ADMETox evaluation. Compounds #31, #70, #71, #72, and #73 were found to pass the ADME evaluation and throughout the screening, only compound #31 passed the predicted toxicity evaluation. This work could pave the way in the design and synthesis of GlfT2 inhibitors through computer-aided drug design and can be used as an initial approach in identifying potential novel GlfT2 inhibitors with promising activity and low toxicity.

8-Hydroxyquinoline Glycoconjugates: Modifications in the Linker Structure and Their Effect on the Cytotoxicity of the Obtained Compounds.

Small molecule nitrogen heterocycles are very important structures, widely used in the design of potential pharmaceuticals. Particularly, derivatives of 8-hydroxyquinoline (8-HQ) are successfully used to design promising anti-cancer agents. Conjugating 8-HQ derivatives with sugar derivatives, molecules with better bioavailability, selectivity, and solubility are obtained. In this study, 8-HQ derivatives were functionalized at the 8-OH position and connected with sugar derivatives (D-glucose or D-galactose) substituted with different groups at the anomeric position, using copper(I)-catalyzed 1,3-dipolar azide-alkyne cycloaddition (CuAAC). Glycoconjugates were tested for inhibition of the proliferation of cancer cell lines (HCT 116 and MCF-7) and inhibition of ß-1,4-galactosyltransferase activity, which overexpression is associated with cancer progression. All glycoconjugates in protected form have a cytotoxic effect on cancer cells in the tested concentration range. The presence of additional amide groups in the linker structure improves the activity of glycoconjugates, probably due to the ability to chelate metal ions present in many types of cancers. The study of metal complexing properties confirmed that the obtained glycoconjugates are capable of chelating copper ions, which increases their anti-cancer potential.

Fragment Growing to Design Optimized Inhibitors for Human Blood Group†B Galactosyltransferase (GTB).

Human blood group B galactosyltransferase (GTB) catalyzes the galactosylation of the H antigen and is responsible for the formation of the blood group antigen of phenotype B. The ABO blood group system is well studied and routinely serotyped before transfusion and transplantation. Blood type subgroups have been repeatedly linked to an increased occurrence of diseases (e.g., a highly increased incidence rate for pancreatic cancer for individuals with blood group phenotype B). 3-Phenyl-5-(piperazin-1-yl)-1,2,4-thiadiazole 1 has previously been described to inhibit GTB with a Ki value of 800 µm. In this work, we describe a computer-guided fragment-growing approach for the optimization of this fragment that was subsequently realized by synthesizing the most promising ligands. Enlarging the phenyl moiety of fragment 1 to a naphthyl moiety resulted in ligand 3-(naphthalene-1-yl)-5-(piperazin-1-yl)-1,2,4-thiadiazole 2 a, which shows a threefold improvement in binding affinity (Ki =271 µm).

Renal globotriaosylceramide facilitates tubular albumin absorption and its inhibition protects against acute kidney injury.

To elucidate the physiologic function of renal globotriaosylceramide (Gb3/CD77), which up-to-date has been associated exclusively with Shiga toxin binding, we have analyzed renal function in Gb3-deficient mice. Gb3 synthase KO (Gb3S-/-) mice displayed an increased renal albumin and low molecular weight protein excretion compared to WT. Gb3 localized at the brush border and within vesicular structures in WT proximal tubules and has now been shown to be closely associated with the receptor complex megalin/cubilin and with albumin uptake. In two clinically relevant mouse models of acute kidney injury caused by myoglobin as seen in rhabdomyolysis and the aminoglycoside gentamicin, Gb3S-/- mice showed a preserved renal function and morphology, compared to WT. Pharmacologic inhibition of glucosylceramide-based glycosphingolipids, including Gb3, in WT mice corroborated the results of genetically Gb3-deficient mice. In conclusion, our data significantly advance the current knowledge on the physiologic and pathophysiologic role of Gb3 in proximal tubules, showing an involvement in the reabsorption of filtered albumin, myoglobin and the aminoglycoside gentamicin.

Synthesis of 8-hydroxyquinoline glycoconjugates and preliminary assay of their ß1,4-GalT inhibitory and anti-cancer properties.

8-Hydroxyquinoline scaffold is a privileged structure used in designing a new active agents with therapeutic potential. Its connections with the sugar unit is formed to improve the pharmacokinetic properties. The broad spectrum of activity of quinoline derivatives, especially glycoconjugates, is often associated with the ability to chelate metal ions or with the ability to intercalate into DNA. Simple and effective methods of synthesis glycoconjugates of 8-hydroxyquinoline and 8-hydroxyquinaldine derivatives, containing an O-glycosidic bond or a 1,2,3-triazole linker in their structure, have been developed. The obtained glycoconjugates were tested for their ability to inhibit ß-1,4-Galactosyltransferase, as well as inhibit cancer cell proliferation. It was found that used glycoconjugation strategy influenced both improvement of activity and improvement of the bioavailability of 8-HQ derivatives. Their activity depends on type of attached sugar, presence of protecting groups in sugar moiety and presence of a linker between sugar and quinolone aglycone.

Lactosylceramide synthase ß-1,4-GalT-V: A novel target for the diagnosis and therapy of human colorectal cancer.

Little is known about an oncogenic signal transducer ß-1,4-galactosyltransferase-V (ß-1,4-GalT-V), in human colorectal cancer. Using quantitative RT-PCR, immunohistochemical staining and ELISA assays, we determined that ß-1,4-GalT-V gene/protein expression is specifically increased in human colorectal cancer (CRC) tumors, compared to visibly normal tissue. Furthermore, we observed a marked increase in its enzymatic activity, and its product lactosylceramide. Moreover, we found increased dihydrosphingolipid metabolites, in particular dihydrosphingomyelin in cancer tissue compared to normal. Further, inhibition of glycosphingolipid synthesis by the synthetic ceramide analog, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), concurrently inhibited colorectal cancer cell (HCT-116) proliferation, as well as ß-1,4-GalT-V mass and several glycosphingolipid levels. We conclude that ß-1,4-GalT-V may serve as a diagnostic and therapeutic biomarker for the progression of human colorectal cancer, and consequently, inhibition of GSL synthesis may be a novel approach for the treatment of this life-threatening disease.

C1GALT1 predicts poor prognosis and is a potential therapeutic target in head and neck cancer.

Core 1 ß1,3-galactosyltransferase (C1GALT1) controls the crucial step of GalNAc-type O-glycosylation and is overexpressed in various human malignancies. However, its role in head and neck squamous cell carcinoma (HNSCC) remains unclear. Here we demonstrate that C1GALT1 expression is upregulated in HNSCC tumors and is associated with adverse clinicopathologic features. Moreover, high C1GALT1 expression predicts poor disease-free and overall survivals. C1GALT1 overexpression enhances HNSCC cell viability, migration, and invasion, which can be reversed by erlotinib. Silencing of C1GALT1 suppresses the malignant behavior both in vitro and in vivo. Mass spectrometry and lectin pull-down assays demonstrate that C1GALT1 modifies O-glycans on EGFR. Blocking O-glycan elongation on EGFR by C1GALT1 knockdown decreases EGF-EGFR binding affinity and inhibits EGFR signaling, thereby suppressing malignant phenotypes. Using molecular docking simulations, we identify itraconazole as a C1GALT1 inhibitor that directly binds C1GALT1 and promotes its proteasomal degradation, leading to significant blockade of C1GALT1-mediated effects in HNSCC cells in vitro and in vivo. Collectively, our findings demonstrate a critical role of O-glycosylation in HNSCC progression and highlight the therapeutic potential of targeting C1GALT1 in HNSCC treatment.

1-C-phosphonomethyl- and 1-C-difluorophosphonomethyl-1,4-imino-l-arabinitols as Galf transferase inhibitors: A comparison.

The convenient preparation of iminopentitol derivatives, based on a 1,4-dideoxy-1,4-imino-l-arabinitol scaffold carrying ß-phosphono(difluoromethyl) or ß-phosphonomethyl appendages, as Galf-1P mimics, is reported. The compounds were tested for their ability to inhibit GlfT2, a vital galactofuranosyltransferase involved in the cell wall biosynthesis of mycobacteria. Interestingly, the Galf-1P mimics lacking a fluorine atom (7 and 8) were very poor inhibitors, showing less than 20% inhibition of GlfT2, whereas compounds 2 and 3, which contains a difluoromethylenephosphonate moiety were more potent inhibitors. Compound 3 that is fully deprotected was the most potent showing a significant IC50 value (0.9 mm), despite the absence of the diphosphate linkage present in the parent sugar nucleotide. This study paves the way to the synthesis of more complex ß-phosphonomethyl-imino-l-arabinitol derivatives as simplified mimics of UDP-α-d-Galf.

Synthesis of Double-Modified Xyloside Analogues for Probing the ß4GalT7 Active Site.

Monosubstituted naphthoxylosides have been shown to function as substrates for, and inhibitors of, the enzyme ß4GalT7, a key enzyme in the biosynthetic pathway leading to glycosaminoglycans and proteoglycans. In this article, we explore the synthesis of 16 xyloside analogues, modified at two different positions, as well as their function as inhibitors of and/or substrates for the enzyme. Seemingly simple compounds turned out to require complex synthetic pathways. A meta-analysis of the synthetic work shows that, regardless of the abundance of methods available for carbohydrate synthesis, even simple modifications can turn out to be problematic, and double modifications present additional challenges due to conformational, steric, and stereoelectronic effects.

LC3-Dependent Autophagy in Pig 2-Cell Cloned Embryos Could Influence the Degradation of Maternal mRNA and the Regulation of Epigenetic Modification.

In this study, the distribution as well as the effect of autophagy on reprogramming in pig cloned embryos were observed immediately after somatic cell nuclear transfer. Results showed that the LC3 was at the highest level in cloned embryos at 2-cell stage, and it decreased with the development from 2-cell stage to blastocyst. Different to cloned embryos, the intensity of LC3 in parthenogenetic activation (PA) embryos was at the highest level at 4-cell stage. A markedly higher level of Bmp15, H1foo, and Dppa3 was shown in cloned embryos at 2-cell stage (p < 0.05 or p < 0.01), but a significantly lower level of LC3, Sox2, and eIF1A was observed at 4-cell stage (p < 0.05), compared with PA embryos. When the efficient interfering by the LC3 siRNA was performed on the cloned embryos (p < 0.01), not only the mRNA level of maternal Cyclin B, Bmp15, Gdf9, c-mos, H1foo, and Dppa3 was increased significantly (p < 0.05), but also the expression of Dnmt1 and Dnmt3b was obviously upregulated (p < 0.05). Although the expression of Sox2 and Oct4 is not changed, the expression of Stat3 decreased significantly (p < 0.05). Furthermore with the treatment of 200 nM rapamycin, the expression of eIF1A and Stat3 was significantly increased at 4-cell stage. In conclusion, the LC3-dependent autophagy mainly occurred in cloned embryos at 2-cell stage, but at 4-cell stage in PA embryos. In addition, the modulation of autophagy could affect genome activation by influencing the degradation of maternal mRNA and regulating the expression of DNA methyltransferase.

Synthesis and preliminary biological assay of uridine glycoconjugate derivatives containing amide and/or 1,2,3-triazole linkers.

A series of UDP-sugar analogues was synthesized and their preliminary biological activity was evaluated. Glycoconjugates of uridine 1 and 2 were synthesized by condensation of uridine-5'-carboxylic acid and 1-amino sugars derivatives of d-glucose and d-galactose, glycoconjugates 3 and 4 were synthesized by azide-alkyne 1,3-dipolar cycloaddition (CuAAC) of 1-azido sugars and propargylamide derivatives of uridine while glycoconjugates 5 and 6 were synthesized by CuAAC of propargyl ß-O-glycosides and 5'-azido uridine. Evaluation of inhibitory activity of compounds 1-6 against commercially available ß-1,4-galactosyltransferase I (ß4GalT) show that compound 5 inhibited the enzyme in µmolar range. Additionally, the antitumor activity of the obtained glycoconjugates 1-6 were tested using MTT assay.

Uncharged nucleoside inhibitors of ß-1,4-galactosyltransferase with activity in cells.

We report 5-substituted uridine derivatives as novel, uncharged inhibitors of ß-1,4-galactosyltransferase and chemical tools for cellular applications. The new inhibitors reduce P-selectin glycoprotein 1 (PSGL-1) expression in human monocytes. Our results also provide novel insights into a unique mode of glycosyltransferase inhibition.

Targeting of Proteoglycan Synthesis Pathway: A New Strategy to Counteract Excessive Matrix Proteoglycan Deposition and Transforming Growth Factor-ß1-Induced Fibrotic Phenotype in Lung Fibroblasts.

Stimulation of proteoglycan (PG) synthesis and deposition plays an important role in the pathophysiology of fibrosis and is an early and dominant feature of pulmonary fibrosis. Transforming growth factor-ß1 (TGF-ß1) is a major cytokine associated with fibrosis that induces excessive synthesis of matrix proteins, particularly PGs. Owing to the importance of PGs in matrix assembly and in mediating cytokine and growth factor signaling, a strategy based on the inhibition of PG synthesis may prevent excessive matrix PG deposition and attenuates profibrotic effects of TGF-ß1 in lung fibroblasts. Here, we showed that 4-MU4-deoxy-ß-D-xylopyranoside, a competitive inhibitor of ß4-galactosyltransferase7, inhibited PG synthesis and secretion in a dose-dependent manner by decreasing the level of both chondroitin/dermatan- and heparin-sulfate PG in primary lung fibroblasts. Importantly, 4-MU4-deoxy-xyloside was able to counteract TGF-ß1-induced synthesis of PGs, activation of fibroblast proliferation and fibroblast-myofibroblast differentiation. Mechanistically, 4-MU4-deoxy-xyloside treatment inhibited TGF-ß1-induced activation of canonical Smads2/3 signaling pathway in lung primary fibroblasts. The knockdown of ß4-galactosyltransferase7 mimicked 4-MU4-deoxy-xyloside effects, indicating selective inhibition of ß4-galactosyltransferase7 by this compound. Collectively, this study reveals the anti-fibrotic activity of 4-MU4-deoxy-xyloside and indicates that inhibition of PG synthesis represents a novel strategy for the treatment of lung fibrosis.

Prevention of cardiac hypertrophy by the use of a glycosphingolipid synthesis inhibitor in ApoE-/- mice.

ApoE-/- mice fed a high fat and high cholesterol (HFHC) diet (20% fat and 1.25% cholesterol) from 12 weeks of age to 36 weeks revealed an age-dependent increase in the left ventricular mass (LV mass) and decline in fractional shortening (FS%), which worsened with HFHC diet. These traits are indicative of maladaptive pathological cardiac hypertrophy and dysfunction. This was accompanied by loading of glycosphingolipids and increased gene expression of ANP, BNP in myocardial tissue. Masson's trichrome staining revealed a significant increase in cardiomyocyte size and fibrosis. In contrast, treatment with 5 and 10 µM D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of glucosylceramide synthase and lactosylceramide synthase, dose-dependently decreased the load of glycosphingolipids and preserved fractional shortening and maintained left ventricular mass to normal 12-week-old control levels over a 6 month treatment period. Our mechanistic studies showed that D-PDMP inhibited cardiac hypertrophy by inhibiting the phosphorylation of mitogen-activated protein kinase (MAPK). We propose that associating increased glycosphingolipid synthesis with cardiac hypertrophy could serve as a novel approach to prevent this phenotype in experimental animal models of diet -induced atherosclerotic heart disease.

Generation of α1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs.

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs.

[How independent pharmacological screenings in plants and humans led to the discovery of a new family of lipid metabolism inhibitors]. / Identification par deux criblages simultanés indépendants d'une famille d'inhibiteurs du métabolisme des glycérolipides.

In eukaryotic cells, phosphatidic acid (PA) and diacylglycerol (DAG), are at the origin of all membrane glycerolipids. Their interconversion is achieved by dephosphorylation of PA and phosphorylation of DAG: they form therefore a metabolic hub. PA and DAG are also known to be versatile signaling molecules. Two independent pharmacological screenings conducted on plant and human targets, led to the discovery of a new family of compounds acting on enzymes binding to either PA or DAG, in biological contexts that seemed initially independent. On the one hand, in plants, monogalactosyldiacylglycerol synthases (MGDG synthases or MGD) are responsible for the synthesis of MGDG, which is the most profuse lipid of photosynthetic membranes, and thus essential for metabolism and development. MGD use DAG as substrate. On the other hand, in mammals, phospholipases D (PLD), that produce PA, are involved in a variety of signaling cascades that control a broad spectrum of cellular functions, and play a role in the development of cancers. The two independent pharmacological screenings described in this review aimed to identify inhibitory molecules of either MGD of the plant model Arabidopsis, or human PLD. In both cases, the obtained molecules are piperidinyl-benzimidazolone derivatives, thereby allowing to propose this family of molecules as a novel source of inspiration for the search of compounds interfering with glycerolipid metabolism, that could be useful for other biological and therapeutics contexts.