Mannose and phosphomannose isomerase regulate energy metabolism under glucose starvation in leukemia.

Diverse metabolic changes are induced by various driver oncogenes during the onset and progression of leukemia. By upregulating glycolysis, cancer cells acquire a proliferative advantage over normal hematopoietic cells; in addition, these changes in energy metabolism contribute to anticancer drug resistance. Because leukemia cells proliferate by consuming glucose as an energy source, an alternative nutrient source is essential when glucose levels in bone marrow are insufficient. We profiled sugar metabolism in leukemia cells and found that mannose is an energy source for glycolysis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway. Leukemia cells express high levels of phosphomannose isomerase (PMI), which mobilizes mannose to glycolysis; consequently, even mannose in the blood can be used as an energy source for glycolysis. Conversely, suppression of PMI expression or a mannose load exceeding the processing capacity of PMI inhibited transcription of genes related to mitochondrial metabolism and the TCA cycle, therefore suppressing the growth of leukemia cells. High PMI expression was also a poor prognostic factor for acute myeloid leukemia. Our findings reveal a new mechanism for glucose starvation resistance in leukemia. Furthermore, the combination of PMI suppression and mannose loading has potential as a novel treatment for driver oncogene-independent leukemia.

MPI-based bioinformatic analysis and co-inhibitory therapy with mannose for oral squamous cell carcinoma.

Mannose induces tumor cell apoptosis and inhibits glucose metabolism by accumulating intracellularly as mannose 6-phosphate while the drug sensitivity of tumors is negatively correlated with mannose phosphate isomerase gene (MPI) expression. In this study, we performed a first attempt to explore the relationship between the targeted gene MPI and immune infiltration and genetic and clinical characteristics of head and neck squamous carcinoma (HNSC) using computational algorithms and bioinformatic analysis, and further to verify the co-inhibition effects of mannose with genotoxicity, immune responses, and microbes dysbiosis in oral squamous cell carcinoma (OSCC) in vitro and in vivo. Our results found that patients with lower MPI expression had higher survival rate. The enhancement of MPI expression was in response to DNA damage gene, and ATM inhibitor was verified as a potential drug with a synergistic effect with mannose on HSC-3. In the HNSC, infiltrated immunocytes CD8+ T cell and B cell were the significantly reduced risk cells, while IL-22 and IFN-γ showed negative correlation with MPI. Finally, mannose could reverse immunophenotyping caused by antibiotics in mice, resulting in the decrease of CD8+ T cells and increase of myeloid-derived suppressor cells (MDSCs). In conclusion, the MPI gene showed a significant correlation with immune infiltration and genetic and clinical characteristics of HNSC. The treatment of ATM inhibitor, immune regulating cells of CD8+ T cells and MDSCs, and oral microbiomes in combination with mannose could exhibit co-inhibitory therapeutic effect for OSCC.

Synthesis and evaluation of malonate-based inhibitors of phosphosugar-metabolizing enzymes: class II fructose-1,6-bis-phosphate aldolases, type I phosphomannose isomerase, and phosphoglucose isomerase.

In the design of inhibitors of phosphosugar metabolizing enzymes and receptors with therapeutic interest, malonate has been reported in a number of cases as a good and hydrolytically-stable surrogate of the phosphate group, since both functions are dianionic at physiological pH and of comparable size. We have investigated a series of malonate-based mimics of the best known phosphate inhibitors of class II (zinc) fructose-1,6-bis-phosphate aldolases (FBAs) (e.g., from Mycobacterium tuberculosis), type I (zinc) phosphomannose isomerase (PMI) from Escherichia coli, and phosphoglucose isomerase (PGI) from yeast. In the case of FBAs, replacement of one phosphate by one malonate on a bis-phosphorylated inhibitor (1) led to a new compound (4) still showing a strong inhibition (K(i) in the nM range) and class II versus class I selectivity (up to 8×10(4)). Replacement of the other phosphate however strongly affected binding efficiency and selectivity. In the case of PGI and PMI, 5-deoxy-5-malonate-D-arabinonohydroxamic acid (8) yielded a strong decrease in binding affinities when compared to its phosphorylated parent compound 5-phospho-D-arabinonohydroxamic acid (2). Analysis of the deposited 3D structures of the kinetically evaluated enzymes complexed to the phosphate-based inhibitors indicate that malonate could be a good phosphate surrogate only if phosphate is not tightly bound at the enzyme active site, such as in position 7 of compound 1 for FBAs. These observations are of importance for further design of inhibitors of phosphorylated-compounds metabolizing enzymes with therapeutic interest.

Preliminary studies on the inhibition of D-sorbitol-6-phosphate 2-dehydrogenase from Escherichia coli with substrate analogues.

D-Sorbitol-6-phosphate 2-dehydrogenase catalyzes the NADH-dependent conversion of D-fructose 6-phosphate to D-sorbitol 6-phosphate and improved production and purification of the enzyme from Escherichia coli is reported. Preliminary inhibition studies of the enzyme revealed 5-phospho-D-arabinonohydroxamic acid and 5-phospho-D-arabinonate as new substrate analogue inhibitors of the F6P catalyzed reduction with IC50 values of (40 +/- 1) microM and (48 +/- 3) microM and corresponding Km/IC50 ratio values of 14 and 12, respectively. Furthermore, we report here the phosphomannose isomerase substrate D-mannose 6-phosphate as the best inhibitor of E. coli D-sorbitol-6-phosphate 2-dehydrogenase yet reported with an IC50 = 7.5 +/- 0.4 microM and corresponding Km/IC50 ratio = about 76.

Inhibition of type I and type II phosphomannose isomerases by the reaction intermediate analogue 5-phospho-D-arabinonohydroxamic acid supports a catalytic role for the metal cofactor.

The phosphomannose isomerases (PMI) comprise three families of proteins: type I, type II, and type III PMIs. Members of all three families catalyze the reversible isomerization of D-mannose 6-phosphate (M6P) and D-fructose 6-phosphate (F6P) but share little or no sequence identity. Because (1) PMIs are essential for the survival of several microorganisms, including yeasts and bacteria, and (2) the PMI enzymes from several pathogens do not share significant sequence identity to the human protein, PMIs have been considered as potential therapeutic targets. Elucidation of the catalytic and regulatory mechanisms of the different types of PMIs is strongly needed for rational species-specific drug design. To date, inhibition and crystallographic studies of all PMIs are still largely unexplored. As part of our research program on aldose-ketose isomerases, we report in this paper the evaluation of two new inhibitors of type I and type II PMIs from baker's yeast and Pseudomonas aeruginosa, respectively. We found that 5-phospho-D-arabinonohydroxamic acid (5PAH), which is the most potent inhibitor of phosphoglucose isomerase (PGI), is by far the best inhibitor ever reported of both type I and type II PMI-catalyzed isomerization of M6P to F6P. 5PAH, which has an inhibition constant at least 3 orders of magnitude smaller than that of previously reported PMI inhibitors, may be the first high-energy intermediate analogue inhibitor of the enzymes. We also tested the related molecule 5-phospho-D-arabinonate (5PAA), which is a strong competitive inhibitor of PGI, and found that it does not inhibit either PMI. All together, our results are consistent with a catalytic role for the metal cofactor in PMI activity.

Mechanism of irreversible inactivation of phosphomannose isomerases by silver ions and flamazine.

Silver ions and silver-containing compounds have been used as topical antimicrobial agents in a variety of clinical situations. We have previously shown that the enzyme phosphomannose isomerase (PMI) is essential for the biosynthesis of Candida albicans cell walls. In this study, we find that PMI can be inhibited by silver ions. This process is shown to be irreversible, and is a two-step process, involving an intermediate complex with a dissociation constant, Ki, of 59 +/- 8 microM, and a maximum rate of inactivation of 0.25 +/- 0.04 min-1 in 50 mM Hepes buffer, pH 8.0 at 37 degrees C. The enzyme can be protected against this inactivation by the substrate mannose 6-phosphate, with a dissociation constant of 0.31 +/- 0.04 mM, close to its Km value. Flamazine (silver sulfadiazine) is a silver-containing antibiotic which is used clinically as a topical antimicrobial and antifungal agent. We compared the ability of silver sulfadiazine and two other silver-containing compounds to irreversibly inactivate C. albicans PMI. The addition of the organic moiety increased the affinity of the compounds, with silver sulfadiazine showing a Ki of 190 +/- 30 nM. In all cases, the maximum inhibition rate was similar, implying a similar rate-determining step. Silver sulfadiazine does not inhibit Escherichia coli PMI, and this suggests a role of the only free cysteine, Cys-150, in the inactivation process. To confirm this, we mutated this residue to alanine in C. albicans PMI. The resultant Cys150 --> Ala mutant protein showed similar Vm and Km values to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of phosphomannose isomerase by mercury ions.

Mercury ions can inhibit Candida albicans phosphomannose isomerase (PMI) by two different processes at sub-micromolar concentrations. Kinetic studies show that mercury ions are in rapid equilibrium with the enzyme and cause a clear partial noncompetitive inhibition when mannose 6-phosphate is used as the substrate. The inhibition constants at 37 degrees C in 50 mM Hepes buffer, pH 8.0, are 35 and 57 nM for Kii and Kis, respectively. In addition to this inhibition at rapid equilibrium, mercury ions also inactivate C. albicans PMI by a much slower process, involving an irreversible mechanism. This is shown to be a two-step process, proceeding via an intermediate complex with a dissociation constant of 5.6 microM, with a maximum rate of inactivation of 0.15 min-1. The rate of irreversible inactivation can be slowed by the addition of the substrate, mannose 6-phosphate. Incubation of the enzyme with [203Hg]Cl2 causes the formation of a stable adduct with one atom of mercury incorporated into each enzyme molecule during the inactivation. Since cysteine-150 is the only iodoacetate-modifiable cysteine in the protein, we propose that this is where the mercury ion reacts during the irreversible inactivation process. In the Escherichia coli enzyme this cysteine is replaced by an asparagine, and the enzyme cannot be irreversibly inactivated by mercury ions.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of Cys-150 in the active site of phosphomannose isomerase from Candida albicans.

Candida albicans phosphomannose isomerase (PMI) (EC 5.3.1.8) has been recently cloned and overexpressed in Escherichia coli. The enzyme can be irreversibly inactivated by iodoacetate in 50 mM borate buffer, pH 9.0, in a time-dependent manner at a rate of 4.2 +/- 0.03 min-1 M-1. This inhibition can be prevented by the substrate mannose 6-phosphate with a Ks of 0.22 +/- 0.05 mM, slightly lower than its Km value. However, metals such as zinc and cadmium, which are reversible, competitive inhibitors for PMI, do not protect the enzyme against modification. The protein has been labeled by using [2-14C]iodoacetate, in the presence or absence of substrate, and the protein is fully inactivated when 1.0 thiol group is modified per molecule of enzyme. Tryptic maps of the modified protein have been produced. The protected peptide has been identified and sequenced, and the phenylthiohydantoin amino acids have been collected. The modified amino acid is Cys-150. This cysteine residue is conserved in mammalian and yeast phosphomannose isomerases, but not in bacterial species where it is replaced with asparagine. We therefore purified PMI from E. coli and showed that this enzyme is not sensitive to inactivation by iodoacetate. The iodoacetate is presumably inhibiting PMI by sterically blocking the mannose 6-phosphate binding site. Multiple sequence alignment procedures were used to try to identify potential ligands of the zinc atom that is essential for enzyme activity and thus to delineate the active site region.(ABSTRACT TRUNCATED AT 250 WORDS)