Constructing a human complex type N-linked glycosylation pathway in Kluyveromyces marxianus.

Glycosylation can affect various protein properties such as stability, biological activity, and immunogenicity. To produce human therapeutic proteins, a host that can produce glycoproteins with correct glycan structures is required. Microbial expression systems offer economical, rapid and serum-free production and are more amenable to genetic manipulation. In this study, we developed a protocol for CRISPR/Cas9 multiple gene knockouts and knockins in Kluyveromyces marxianus, a probiotic yeast with a rapid growth rate. As hyper-mannosylation is a common problem in yeast, we first knocked out the α-1,3-mannosyltransferase (ALG3) and α-1,6-mannosyltransferase (OCH1) genes to reduce mannosylation. We also knocked out the subunit of the telomeric Ku domain (KU70) to increase the homologous recombination efficiency of K. marxianus. In addition, we knocked in the MdsI (α-1,2-mannosidase) gene to reduce mannosylation and the GnTI (ß-1,2-N-acetylglucosaminyltransferase I) and GnTII genes to produce human N-glycan structures. We finally obtained two strains that can produce low amounts of the core N-glycan Man3GlcNAc2 and the human complex N-glycan Man3GlcNAc4, where Man is mannose and GlcNAc is N-acetylglucosamine. This study lays a cornerstone of glycosylation engineering in K. marxianus toward producing human glycoproteins.

A new small-molecule KRE2 inhibitor against invasive Candida parapsilosis infection.

AIM: To investigate the antifungal activity of MOL3, a small molecule that was selected by virtual screening, against Candida spp. MATERIALS & METHODS: The antifungal activity of MOL3 was evaluated using standard strains and clinical isolates. Activity was evaluated in both in vitro tests and animal models. RESULTS: The minimum fungicidal concentration of MOL3 against Candida spp. ranged from 16 to 128 mg/l. MOL3 at the sub-minimum fungicidal concentration inhibited hyphal elongation. The remaining yeast cells presented morphological changes and were metabolically inactive. MOL3 was toxicologically inert both in vitro and in the animal model. MOL3 also reduced experimental systemic infection by C. parapsilosis in mice. CONCLUSION: The selection of MOL3 by virtual screening was successful, revealing a promising antifungal candidate.

Cellular Consequences of Diminished Protein O-Mannosyltransferase Activity in Baker's Yeast.

O-Mannosylation is a type of protein glycosylation initiated in the endoplasmic reticulum (ER) by the protein O-mannosyltransferase (PMT) family. Despite the vital role of O-mannosylation, its molecular functions and regulation are not fully characterized. To further explore the cellular impact of protein O-mannosylation, we performed a genome-wide screen to identify Saccharomyces cerevisiae mutants with increased sensitivity towards the PMT-specific inhibitor compound R3A-5a. We identified the cell wall and the ER as the cell compartments affected most upon PMT inhibition. Especially mutants with defects in N-glycosylation, biosynthesis of glycosylphosphatidylinositol-anchored proteins and cell wall ß-1,6-glucan showed impaired growth when O-mannosylation became limiting. Signaling pathways that counteract cell wall defects and unbalanced ER homeostasis, namely the cell wall integrity pathway and the unfolded protein response, were highly crucial for the cell growth. Moreover, among the most affected mutants, we identified Ost3, one of two homologous subunits of the oligosaccharyltransferase complexes involved in N-glycosylation, suggesting a functional link between the two pathways. Indeed, we identified Pmt2 as a substrate for Ost3 suggesting that the reduced function of Pmt2 in the absence of N-glycosylation promoted sensitivity to the drug. Interestingly, even though S. cerevisiae Pmt1 and Pmt2 proteins are highly similar on the sequence, as well as the structural level and act as a complex, we identified only Pmt2, but not Pmt1, as an Ost3-specific substrate protein.

Evaluation of monovalent and multivalent iminosugars to modulate Candida albicans ß-1,2-mannosyltransferase activities.

ß-1,2-Linked oligomannosides substitute the cell wall of numerous yeast species. Several of those including Candida albicans may cause severe infections associated with high rates of morbidity and mortality, especially in immunocompromised patients. ß-1,2-Mannosides are known to be involved in the pathogenic process and to elicit an immune response from the host. In C. albicans, the synthesis of ß-mannosides is under the control of a family of nine genes coding for putative ß-mannosyltransferases. Two of them, CaBmt1 and CaBmt3, have been shown to initiate and prime the elongation of the ß-mannosides on the cell-wall mannan core. In the present study, we have assessed the modulating activities of monovalent and multivalent iminosugar analogs on these enzymes in order to control the enzymatic bio-synthesis of ß-mannosides. We have identified a monovalent deoxynojirimycin (DNJ) derivative that inhibits the CaBmt1-catalyzed initiating activity, and mono-, tetra- and polyvalent deoxymannojirimycin (DMJ) that modulate the CaBmt1 activity toward the formation of a single major product. Analysis of the aggregating properties of the multivalent iminosugars showed their ability to elicit clusterization of both CaBmt1 and CaBmt3, without affecting their activity. These results suggest promising roles for multivalent iminosugars as controlling agents for the biosynthesis of ß-1,2 mannosides and for monovalent DNJ derivative as a first target for the design of future ß-mannosyltransferase inhibitors.

Lead selection and characterization of antitubercular compounds using the Nested Chemical Library.

Discovering new drugs to treat tuberculosis more efficiently and to overcome multidrug resistance is a world health priority. To find novel antitubercular agents several approaches have been used in various institutions worldwide, including target-based approaches against several validated mycobacterial enzymes and phenotypic screens. We screened more than 17,000 compounds from Vichem's Nested Chemical Library™ using an integrated strategy involving whole cell-based assays with Corynebacterium glutamicum and Mycobacterium tuberculosis, and target-based assays with protein kinases PknA, PknB and PknG as well as other targets such as PimA and bacterial topoisomerases simultaneously. With the help of the target-based approach we have found very potent hits inhibiting the selected target enzymes, but good minimal inhibitory concentrations (MIC) against M. tuberculosis were not achieved. Focussing on the whole cell-based approach several potent hits were found which displayed minimal inhibitory concentrations (MIC) against M. tuberculosis below 10 µM and were non-mutagenic, non-cytotoxic and the targets of some of the hits were also identified. The most active hits represented various scaffolds. Medicinal chemistry-based lead optimization was performed applying various strategies and, as a consequence, a series of novel potent compounds were synthesized. These efforts resulted in some effective potential antitubercular lead compounds which were confirmed in phenotypic assays.

Discovery of genetic biomarkers contributing to variation in drug response of cytidine analogues using human lymphoblastoid cell lines.

BACKGROUND: Two cytidine analogues, gemcitabine and cytosine arabinoside (AraC), are widely used in the treatment of a variety of cancers with a large individual variation in response. To identify potential genetic biomarkers associated with response to these two drugs, we used a human lymphoblastoid cell line (LCL) model system with extensive genomic data, including 1.3 million SNPs and 54,000 basal expression probesets to perform genome-wide association studies (GWAS) with gemcitabine and AraC IC50 values. RESULTS: We identified 11 and 27 SNP loci significantly associated with gemcitabine and AraC IC50 values, respectively. Eleven candidate genes were functionally validated using siRNA knockdown approach in multiple cancer cell lines. We also characterized the potential mechanisms of genes by determining their influence on the activity of 10 cancer-related signaling pathways using reporter gene assays. Most SNPs regulated gene expression in a trans manner, except 7 SNPs in the PIGB gene that were significantly associated with both the expression of PIGB and gemcitabine cytotoxicity. CONCLUSION: These results suggest that genetic variation might contribute to drug response via either cis- or trans- regulation of gene expression. GWAS analysis followed by functional pharmacogenomics studies might help identify novel biomarkers contributing to variation in response to these two drugs and enhance our understanding of underlying mechanisms of drug action.

A phenylalanine to serine substitution within an O-protein mannosyltransferase led to strong resistance to PMT-inhibitors in Pichia pastoris.

Protein O-mannosyltransferases (PMTs) catalyze the initial reaction of protein O-mannosylation by transferring the first mannose unit onto serine and threonine residues of a nascent polypeptide being synthesized in the endoplasmic reticulum (ER). The PMTs are well conserved in eukaryotic organisms, and in vivo defects of these enzymes result in cell death in yeast and congenital diseases in humans. A group of rhodanine-3-acetic acid derivatives (PMTi) specifically inhibits PMT activity both in vitro and in vivo. As such, these chemical compounds have been effectively used to minimize the extent of O-mannosylation on heterologously produced proteins from different yeast expression hosts. However, very little is known about how these PMT-inhibitors interact with the PMT enzyme, or what structural features of the PMTs are required for inhibitor-protein interactions. To better understand the inhibitor-enzyme interactions, and to gain potential insights for developing more effective PMT-inhibitors, we isolated PMTi-resistant mutants in Pichia pastoris. In this study, we report the identification and characterization of a point mutation within the PpPMT2 gene. We demonstrate that this F664S point mutation resulted in a near complete loss of PMTi sensitivity, both in terms of growth-inhibition and reduction in O-mannosylglycan site occupancy. Our results provide genetic evidence demonstrating that the F664 residue plays a critical role in mediating the inhibitory effects of these PMTi compounds. Our data also indicate that the main target of these PMT-inhibitors in P. pastoris is Pmt2p, and that the F664 residue most likely interacts directly with the PMTi-compounds.

Novel small molecules for the treatment of infections caused by Candida albicans: a patent review (2002-2010).

INTRODUCTION: The fungal pathogen Candida albicans is one of the leading causes of infections affecting immunodeficient individuals, including those HIV-infected and patients undergoing cancer therapy. Emerging problems in terms of therapeutic efficacy and drug resistance have highlighted the need to consider new therapeutic approaches, based on the exploitation of virulence factors as alternatives to conventional drug targets. AREAS COVERED: Advances in the development of anti-Candida drugs are examined in this review, as reflected by the patent literature since 2002 along with selected peer-reviewed publications. Taking into account a total of 26 patents, the discussion encompasses several therapeutic approaches, including azoles as ergosterol biosynthesis inhibitors, glucan and chitin synthase inhibitors, and secreted aspartyl protease inhibitors. EXPERT OPINION: New analogs of existing drugs are being developed as broad spectrum antifungals to improve efficacy and circumvent drug resistance. Also, candidate drugs targeting new virulence factors are promising to overcome limitations due to poor efficacy and the rising of drug resistance observed for several available drugs. Efforts for the discovery and development of antifungal agents should be equivalent to other therapeutic areas, and advances in the generation of therapeutic agents with fungus-specific mechanisms of action are of highest priority.

Modular approach to triazole-linked 1,6-α-D-oligomannosides to the discovery of inhibitors of Mycobacterium tuberculosis cell wall synthetase.

Aiming at developing inhibitors of mannosyltransferases, the enzymes that participate in the biosynthesis of the cell envelope of Mycobacterium tuberculosis, the synthesis of a range of designed triazole-linked 1,6-oligomannosides up to a hexadecamer has been accomplished by a modular approach centered on the Cu(I)-catalyzed azide-alkyne cycloaddition as key process. The efficiency and fidelity of the cycloaddition are substantiated by high yields (76-96%) and exclusive formation of the expected 1,4-disubstituted triazole ring in all oligomer assembling reactions. Key features of oligomers thus prepared are the anomeric carbon-carbon bond of all mannoside residues and the 6-deoxymannoside capping residue. Suitable bioassays with dimer, tetramer, hexamer, octamer, decamer, and hexadecamer showed variable inhibitor activity against mycobacterial α-(1,6)-mannosyltransferases, the highest activity (IC(50) = 0.14-0.22 mM) being registered with the hexamannoside and octamannoside.

Rationally designed squaryldiamides - a novel class of sugar-nucleotide mimics?

Sugar-nucleotides such as GDP-mannose, GDP-fucose and UDP-glucose are important biomolecules with a central role in carbohydrate and glycoconjugate biosynthesis, metabolism and cell signalling. Analogues and mimics of naturally occurring sugar-nucleotides are sought after as chemical tools and inhibitor candidates for sugar-nucleotide-dependent enzymes including glycosyltransferases. Many sugar-nucleotides bind to their target glycosyltransferases via coordination of the diphosphate group to a divalent metal cofactor in the active site. The identification of uncharged, chemically stable surrogates for the diphosphate group, with the ability to coordinate to a divalent metal, is therefore an important design criteria for the development of sugar-nucleotide mimics. Here, we describe the rational design and synthesis of a novel class of sugar-nucleotide mimics based on a squaryldiamide scaffold, an uncharged phosphate isostere. We demonstrate by comprehensive NMR titration experiments that the new sugar-nucleotide mimics coordinate efficiently to Mg(2+), and provide results from biological studies with a therapeutically relevant mannosyltransferase from Trypanosoma brucei. Our findings suggest that squaryldiamides are a promising template for the development of sugar-nucleotide mimics, and illustrate the considerable potential of the squarylamide group as a fragment for inhibitor design.

Synthesis of alkyl and cycloalkyl alpha-d-mannopyranosides and derivatives thereof and their evaluation in the mycobacterial mannosyltransferase assay.

The synthesis of a series of alkyl (having from C6 to C20 aglycones), cyclohexyl, and cyclohexylalkyl alpha-d-mannopyranosides, 6-deoxygenated analogs, thioglycosides, and sulfones derived thereof, is reported. Here, under the in vitro assay conditions used, none of the 15 tested compounds acted as an inhibitor of the mannose transfer catalyzed by the enzymes present in mycobacterial membrane and cell wall fractions. Mannopyranosides comprising shorter aliphatic, up to 8 carbon atoms long linear, or cyclic aglycone served as the acceptor substrates in the mycobacterial mannosyltransferase reaction. The thioglycosides exhibited similar behavior, in contrast to the sulfones, which were essentially not recognized by the mycobacterial enzymes. 6-Deoxygenated glycosides were not processed by the enzymes, suggesting that the mannose transfer occurs at position 6 of the acceptors.

Epimeric and amino disaccharide analogs as probes of an alpha-(1-->6)-mannosyltransferase involved in mycobacterial lipoarabinomannan biosynthesis.

Mycobacterial lipoarabinomannan (LAM) is an important, immunologically active glycan found in the cell wall of mycobacteria, including the human pathogen Mycobacterium tuberculosis. At the core of LAM is a mannan domain comprised of alpha-(1-->6)-linked-mannopyranose (Manp) residues. Previously, we and others have demonstrated that alpha-Manp-(1-->6)-alpha-Manp disaccharides (e.g., Manp-(1-->6)-alpha-ManpOctyl, ) are the minimum acceptor substrates for enzymes involved in the assembly of the LAM mannan core. We report here the synthesis five epimeric and three amino analogs of , and their subsequent biochemical evaluation against an alpha-(1-->6)-ManT activity present in a membrane preparation from M. smegmatis. Changing the manno- configuration of either residue of to talo- or gluco- led to a reduction or loss of activity, thus confirming earlier work showing that the C-2 and C-4 hydroxyl groups of each monosaccharide were important for enzymatic recognition. Characterization of the products formed from these analogs was done using a combination of mass spectrometry and glycosidase digestion, and full substrate kinetics were also performed. The analogs in which the acceptor hydroxyl group had been replaced with an amino group were, as expected, not substrates for the enzyme, but were weak inhibitors.

New insights into the early steps of phosphatidylinositol mannoside biosynthesis in mycobacteria: PimB' is an essential enzyme of Mycobacterium smegmatis.

Phosphatidyl-myo-inositol mannosides (PIMs) are key glycolipids of the mycobacterial cell envelope. They are considered not only essential structural components of the cell but also important molecules implicated in host-pathogen interactions. Although their chemical structures are well established, knowledge of the enzymes and sequential events leading to their biosynthesis is still incomplete. Here we show for the first time that although both mannosyltransferases PimA and PimB' (MSMEG_4253) recognize phosphatidyl-myo-inositol (PI) as a lipid acceptor, PimA specifically catalyzes the transfer of a Manp residue to the 2-position of the myo-inositol ring of PI, whereas PimB' exclusively transfers to the 6-position. Moreover, whereas PimB' can catalyze the transfer of a Manp residue onto the PI-monomannoside (PIM1) product of PimA, PimA is unable in vitro to transfer Manp onto the PIM1 product of PimB'. Further assays using membranes from Mycobacterium smegmatis and purified PimA and PimB' indicated that the acylation of the Manp residue transferred by PimA preferentially occurs after the second Manp residue has been added by PimB'. Importantly, genetic evidence is provided that pimB' is an essential gene of M. smegmatis. Altogether, our results support a model wherein Ac1PIM2, a major form of PIMs produced by mycobacteria, arises from the consecutive action of PimA, followed by PimB', and finally the acyltransferase MSMEG_2934. The essentiality of these three enzymes emphasizes the interest of novel anti-tuberculosis drugs targeting the initial steps of PIM biosynthesis.

Inactivation of Mycobacterium tuberculosis mannosyltransferase pimB reduces the cell wall lipoarabinomannan and lipomannan content and increases the rate of bacterial-induced human macrophage cell death.

The Mycobacterium tuberculosis (M.tb) cell wall contains an important group of structurally related mannosylated lipoglycans called phosphatidyl-myo-inositol mannosides (PIMs), lipomannan (LM), and mannose-capped lipoarabinomannan (ManLAM), where the terminal alpha-[1-->2] mannosyl structures on higher order PIMs and ManLAM have been shown to engage C-type lectins such as the macrophage mannose receptor directing M.tb phagosome maturation arrest. An important gene described in the biosynthesis of these molecules is the mannosyltransferase pimB (Rv0557). Here, we disrupted pimB in a virulent strain of M.tb. We demonstrate that the inactivation of pimB in M.tb does not abolish the production of any of its cell wall mannosylated lipoglycans; however, it results in a quantitative decrease in the ManLAM and LM content without affecting higher order PIMs. This finding indicates gene redundancy or the possibility of an alternative biosynthetic pathway that may compensate for the PimB deficiency. Furthermore, infection of human macrophages by the pimB mutant leads to an alteration in macrophage phenotype concomitant with a significant increase in the rate of macrophage death.

First small molecular inhibitors of T. brucei dolicholphosphate mannose synthase (DPMS), a validated drug target in African sleeping sickness.

Drug-like molecules with activity against Trypanosoma brucei are urgently required as potential therapeutics for the treatment of African sleeping sickness. Starting from known inhibitors of other glycosyltransferases, we have developed the first small molecular inhibitors of dolicholphosphate mannose synthase (DPMS), a mannosyltransferase critically involved in glycoconjugate biosynthesis in T. brucei. We show that these DPMS inhibitors prevent the biosynthesis of glycosylphosphatidylinositol (GPI) anchors, and possess trypanocidal activity against live trypanosomes.

Designing sleeping sickness control.

Control of African trypanosomiasis caused by the protozoan parasite Trypanosoma brucei is an important issue in medicine, veterinary medicine, and agricultural economy. Because vaccine development is unlikely, development of safer and more effective chemotherapeutics is critical. The biosynthetic pathway of glycosylphosphatidylinositol (GPI), which acts as membrane anchors of coat proteins, variant surface glycoproteins, and transferrin receptors, is a validated target of drug development. An article in this issue reports the first chemically synthesized inhibitor of the third mannosyltransferase from the GPI pathway, stimulating further investigation toward practical and useful compounds.

Probing enzymes late in the trypanosomal glycosylphosphatidylinositol biosynthetic pathway with synthetic glycosylphosphatidylinositol analogues.

Glycosylphosphatidylinositol (GPI)-anchored proteins are abundant in the protozoan parasite Trypanosoma brucei, the causative agent of African sleeping sickness in humans and the related disease Nagana in cattle, and disruption of GPI biosynthesis is genetically and chemically validated as a drug target. Here, we examine the ability of enzymes of the trypanosomal GPI biosynthetic pathway to recognize and process a series of synthetic dimannosyl-glucosaminylphosphatidylinositol analogues containing systematic modifications on the mannose residues. The data reveal which portions of the natural substrate are important for recognition, explain why mannosylation occurs prior to inositol acylation in the trypanosomal pathway, and identify the first inhibitor of the third alpha-mannosyltransferase of the GPI biosynthetic pathway.

Efficient antibody production upon suppression of O mannosylation in the yeast Ogataea minuta.

When antibodies were expressed in the methylotrophic yeast Ogataea minuta, we found that abnormal O mannosylation occurred in the secreted antibody. Yeast-specific O mannosylation is initiated by the addition of mannose at serine (Ser) or threonine (Thr) residues in the endoplasmic reticulum via protein O mannosyltransferase (Pmt) activity. To suppress the addition of O-linked sugar chains on antibodies, we examined the possibility of inhibiting Pmt activity by the addition of a Pmt inhibitor during cultivation. The Pmt inhibitor was found to partially suppress the O mannosylation on the antibodies. Surprisingly, the suppression of O mannosylation was associated with an increased amount of assembled antibody (H2L2) and enhanced the antigen-binding activity of the secreted antibody. In this study, we demonstrated the expression of human antibody in O. minuta and elucidated the relationship between O mannosylation and antibody production in yeast.

Transcriptional and physiological adaptation to defective protein-O-mannosylation in Candida albicans.

Five Pmt isoforms O-mannosylate secretory proteins in Candida albicans. Comparisons of genome-wide transcript patterns of each pmt mutant revealed commonly downregulated genes involved in glycolysis and glycerol production. Increased phosphorylation of the Cek1p- but not the Mkc1p-MAP kinase, as well as increased transcript levels for some stress-related genes were detected in the pmt1 strain but not in the other pmt mutants. The transcriptomal pattern after short-term inhibition of Pmt1p activity confirmed stress responses, but did not indicate an alteration of glycolytic flow. Short- but not long-term adaptation to Pmt1p inhibition required signalling components Cek1p, Mkc1p, Efg1p and Tpk1p. Cna1p (calcineurin) but not its downstream effectors Crz1p and Crz2p was generally essential to allow growth during Pmt1p inhibition; accordingly, cyclosporin A strongly inhibited growth of the pmt1 mutant. The lack of Pmt isoforms influenced transcript levels for the remaining isoforms both positively and negatively, suggesting complex cross-regulation among PMT genes. These results confirm individual functions of Pmt isoforms but suggest a common biphasic adaptation response to Pmt deficiency. While known signalling pathways modulate adaptation for a short-term, long-term adaptation requires calcineurin, adjustments of remaining Pmt activities and of glycolytic flow.

Galactose-derived phosphonate analogues as potential inhibitors of phosphatidylinositol biosynthesis in mycobacteria.

Galactose-based phosphonate analogues of myo-inositol-1-phosphate and phosphatidylinositol have been synthesized from methyl beta-d-galactopyranoside. Michaelis-Arbuzov reaction of isopropyl diphenyl phosphite or triisopropyl phosphite with a 6-iodo-3,4-isopropylidene galactoside afforded the corresponding phosphonates. Deprotection of the diphenyl phosphonate afforded methyl beta-d-galactoside 6-phosphonate, an analogue of myo-inositol-1-phosphate. The diisopropyl esters of the diisopropyl phosphonate were selectively deprotected and the corresponding anion was coupled with 1,2-dipalmitoyl-sn-glycerol using dicyclohexylcarbodiimide. Deprotection afforded a methyl beta-d-galactoside-derived analogue of phosphatidylinositol. The galactose-derived analogues of phosphatidylinositol and myo-inositol-1-phosphate were not substrates for mycobacterial mannosyltransferases (at concentrations up to 1 mM) involved in phosphatidylinositol mannoside biosynthesis in a cell-free extract of Mycobacterium smegmatis. The galactose-derived phosphonate analogue of phosphatidylinositol was shown to be an inhibitor at 0.01 mM of PimA mannosyltransferase involved in the synthesis of phosphatidylinositol mannoside from phosphatidylinositol, and a weaker inhibitor of the next mannosyltransferase(s), which catalyzes the mannosylation of phosphatidylinositol mannoside.