Discovery of ebselen as an inhibitor of Cryptosporidium parvum glucose-6-phosphate isomerase (CpGPI) by high-throughput screening of existing drugs.

Cryptosporidium parvum is a water-borne and food-borne apicomplexan pathogen. It is one of the top four diarrheal-causing pathogens in children under the age of five in developing countries, and an opportunistic pathogen in immunocompromised individuals. Unlike other apicomplexans, C. parvum lacks Kreb's cycle and cytochrome-based respiration, thus relying mainly on glycolysis to produce ATP. In this study, we characterized the primary biochemical features of the C. parvum glucose-6-phosphate isomerase (CpGPI) and determined its Michaelis constant towards fructose-6-phosphate (Km = 0.309 mM, Vmax = 31.72 nmol/µg/min). We also discovered that ebselen, an organoselenium drug, was a selective inhibitor of CpGPI by high-throughput screening of 1200 known drugs. Ebselen acted on CpGPI as an allosteric noncompetitive inhibitor (IC50 = 8.33 µM; Ki = 36.33 µM), while complete inhibition of CpGPI activity was not achieved. Ebselen could also inhibit the growth of C. parvum in vitro (EC50 = 165 µM) at concentrations nontoxic to host cells, albeit with a relatively small in vitro safety window of 4.2 (cytotoxicity TC50 on HCT-8 cells = 700 µM). Additionally, ebselen might also target other enzymes in the parasite, leading to the parasite growth reduction. Therefore, although ebselen is useful in studying the inhibition of CpGPI enzyme activity, further proof is needed to chemically and/or genetically validate CpGPI as a drug target.

Eimeria tenella glucose-6-phosphate isomerase: molecular characterization and assessment as a target for anti-coccidial control.

Limitations with current chemotherapeutic and vaccinal control of coccidiosis caused by Eimeria species continue to prompt development of novel controls, including the identification of new drug targets. Glucose-6-phosphate isomerase (G6-PI) has been proposed as a valid drug target for many protozoa, although polymorphism revealed by electrophoretic enzyme mobility has raised doubts for Eimeria. In this study we identified and sequenced the Eimeria tenella G6-PI orthologue (EtG6-PI) from the reference Houghton strain and confirmed its position within the prevailing taxonomic hierarchy, branching with the Apicomplexa and Plantae, distinct from the Animalia including the host, Gallus gallus. Comparison of the deduced 1647 bp EtG6-PI coding sequence with the 9016 bp genomic locus revealed 15 exons, all of which obey the intron-AG-/exon/-GT-intron splicing rule. Comparison with the Weybridge and Wisconsin strains revealed the presence of 33 single nucleotide polymorphisms (SNPs) and 14 insertion/deletion sites. Three SNPs were exonic and all yielded non-synonymous substitutions. Preliminary structural predictions suggest little association between the coding SNPs and key G6-PI catalytic residues or residues thought to be involved in the coordination of the G6-PI's substrate phosphate group. Thus, the significant polymorphism from its host orthologue and minimal intra-specific polymorphism suggest G6-PI remains a valid anti-coccidial drug target.

Impaired enzyme-to-enzyme channelling between hexokinase isoenzyme(s) and phosphoglucoisomerase in rat pancreatic islets incubated at a low concentration of D-glucose.

It was recently proposed that in rat pancreatic islets exposed to 8.3 mM D-glucose, alpha-D-glucose-6-phosphate undergoes enzyme-to-enzyme channelling between hexokinase isoenzyme(s) and phosphoglucoisomerase. To explore the identity of the hexokinase isoenzyme(s) involved in such a tunnelling process, the generation of 3HOH from the alpha- and beta-anomers of either D-[2-3H]glucose or D-[5-3H]glucose was now measured over 60 min incubation at 4 degrees C in pancreatic islets exposed only to 2.8 mM D-glucose, in order to decrease the relative contribution of glucokinase to the phosphorylation of the hexose. Under these experimental conditions, the ratio for 3HOH production from D-[2-3H]glucose/D-[5-3H]glucose at anomeric equilibrium (39.7 +/- 11.6%) and the beta/alpha ratios for the generation of 3HOH from either the D-[2-3H]glucose anomers (70.9 +/- 12.6%) or the D-[5-3H]glucose anomers (59.6 +/- 12.4%) indicated that a much greater fraction of alpha-D-glucose-6-phosphate escapes from the process of enzyme-to-enzyme channelling in the islets exposed to 2.8 mM, rather than 8.3 mM D-glucose. These findings suggest, therefore, that the postulated process of enzyme-to-enzyme channelling involves mainly glucokinase.

Suramin disrupts insulin-like growth factor-II (IGF-II) mediated autocrine growth in human SH-SY5Y neuroblastoma cells.

Suramin, traditionally used in the treatment of trypanosomiasis, is under investigation in the treatment of cancer. One side effect that limits its use is the onset of a sensorimotor polyneuropathy. In order to investigate the mechanism by which suramin induces polyneuropathy, we examined its effects on SH-SY5Y human neuroblastoma cells, an in vitro model of neuronal growth and differentiation. Addition of 50-400 micrograms/ml suramin to SH-SY5Y cells grown in 0.6% CS inhibited [3H]thymidine ([3H]TdR) incorporation and cell growth. Upon removal of suramin, [3H]TdR incorporation increased, demonstrating that levels of suramin used were cytostatic and not cytotoxic. Analysis of suramin-treated SH-SY5Y cells by flow cytometry revealed growth arrest in the G1/G0 phase of the cell cycle. IGF-II-induced SH-SY5Y growth is mediated by the type I IGF receptor (IGF-IR). Therefore, we examined its effect on IGF-IR tyrosine phosphorylation. Suramin prevented IGF-II-stimulated IGF-IR tyrosine phosphorylation. These results indicate that in SH-SY5Y cells, suramin acts as a cytostatic agent and can block IGF-II-dependent cell growth by preventing IGF-IR activation. Thus, suramin toxicity in the peripheral nervous system may be due, in part, to preventing IGF and other growth factors from activating their receptors.

Molecular analysis of glucose phosphate isomerase deficiency associated with hereditary hemolytic anemia.

We report here two new cases of glucose phosphate isomerase (GPI) deficiency associated with hemolytic anemia and present the results of molecular analysis of the five Japanese GPI variants. A Japanese girl (GPI Fukuoka) had an episode of prolonged neonatal jaundice and at 3 years of age was admitted due to acute hemolytic crisis occurring with upper respiratory tract infection. Red blood cell (RBC) GPI activity was decreased to 11.8% of normal and the reduced glutathione (GSH) level of RBCs was slightly decreased. A 54-year-old Japanese man (GPI Iwate) was hospitalized due to chronic active hepatitis, and compensated hemolysis was noted. RBC GPI activity of the proband was decreased to 18.8%, and the GSH content was about half of the normal mean value. Sequencing of the reticulocyte GPIcDNA showed homozygous missense mutations 1028CAG-->CGG (343Gln-->Arg), 14ACC-->A7C (5Thr-->lle), 671ACG-->A7G (224Thr-->Met), and 1615GAC-->AAC (539Asp-->Asn) in GPI Narita, GPI Matsumoto, GPI Iwate, and GPI Fukuoka, respectively. We also identified GPI Kinki as a compound heterozygote of 1124ACA-->AGA(375Thr-->Arg)/ 1615GAC-->AAC(539Asp-->Asn). Our findings, together with the previous results of other investigators, showed that the GPI gene mutations so far identified were heterogeneous, although most GPI variants had common biochemical characteristics such as heat instability and normal kinetics. Several amino acid substitutions were identified in the proximity of the catalytically important amino acid residues such as Ser/Asp 159/160, Asp341, and Lys518, which have been identified in the structural analysis of the pig GPI. The molecular characterization of human GPI variants, therefore, may provide new insights into the genotype-phenotype correlation of GPI deficiency as well as the structure-function relationship of this enzyme.

Antiproliferative effect of multiple autocrine loop blockade in human malignant glioma cell lines.

The effects of specific antibodies against growth factors and receptors on deoxyribonucleic acid (DNA) synthesis in two established human glioma cell lines, A172 and TM-1, were examined. Anti-platelet-derived growth factor (PDGF), anti-basic fibroblast growth factor (bFGF), and anti-epidermal growth factor receptor (EGF-R) antibodies inhibited thymidine incorporation by both cell lines in serum-free medium. Antibody specific to transforming growth factor-alpha only slightly suppressed DNA synthesis by both cell lines. Although the antiproliferative effects of anti-PDGF and anti-bFGF antibodies decreased in serum-supplemented medium, the effect of anti-EGF-R antibody was little changed. The combination of anti-PDGF, anti-bFGF, and anti-EGF-R antibodies significantly inhibited thymidine incorporation by the two cell lines even in serum-supplemented medium. This preliminary study suggests that simultaneous blockade of multiple autocrine loops may provide a new approach to the treatment of human malignant gliomas.

Effect of Plumbagin on some glucose metabolising enzymes studied in rats in experimental hepatoma.

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) isolated from Plumbago zeylanica Linn, when administered orally, at a dosage of 4 mg/kg body weight induces tumour regression in 3-methyl-4-dimethyl aminoazobenzene (3MeDAB) induced hepatoma in Wistar male rats. The purpose of this investigation was to identify the changes in the rate of glycolysis and gluconeogenesis in tumour-bearing rats and the effects of treatment with Plumbagin. The levels of certain glycolytic enzymes, namely, hexokinase; phosphoglucoisomerase; and aldolase levels increased (p < 0.001) in hepatoma bearing rats, whereas they decreased in Plumbagin administered rats to near normal levels. Certain gluconeogenic enzymes, namely, glucose-6-phosphatase and fructose-1,6-diphosphatase decreased (p < 0.001) in tumour hosts, whereas Plumbagin administration increased the gluconeogenic enzyme levels in the treated animals. These investigations indicate the molecular basis of the different biological behaviour of 3MeDAB induced hepatoma and the anticarcinogenic property of Plumbagin against hepatoma studied in rats.

Comparative study of the effects of beta-sitosterol, estradiol and progesterone on selected biochemical parameters of the uterus of ovariectomised rats.

A comparative study was made of the effects of beta-sitosterol, estradiol-17 beta and progesterone, individually and in combinations, on certain biochemical parameters important to carbohydrate metabolism in the uteri of adult ovariectomised rats. beta-Sitosterol (SITO), estradiol (E2) and combined treatment (SITO + E2) induced significant increases in glycogen concentration and the activities of glucose-6-phosphate dehydrogenase (G6PDH), phosphohexose isomerase (PHI) and total lactate dehydrogenase (LDH). Progesterone (P) administration however, raised only the uterine PHI and LDH activities. Co-administration of P with beta-sitosterol (P + SITO) suppressed the SITO-induced increase in glycogen concentration and G6PDH activity. On the other hand, combined treatment (P + SITO) augmented total LDH activity.

Response of hamster to the antifertility effect of gossypol.

One hundred and five sexually mature male hamsters were divided in different groups. In the first experiment hamsters were administered gossypol, 10 mg/kg and 20 mg/kg/body weight/day, for twenty and thirty days. In the second experiment hamsters were administered gossypol, 5, 10 and 20 mg/kg/body weight/day, for sixty days. In the third experiment, hamsters were administered gossypol 5 mg, 10 mg, 20 mg and 40 mg/kg body weight/day for 45 days. Animals in all the groups were given gossypol by oral intubation every day. No significant effect on the body weight of hamsters following gossypol treatment was observed. At low doses the weights of testis and accessory sex organs were not statistically different from those of the controls. A significant decrease in testis and epididymis weight was however observed following high doses of gossypol. Low doses of gossypol treatment did not affect the motility of the vas deferens spermatozoa. The vas deferens spermatozoa were however immotile after 40 mg/kg/day gossypol treatment. Gossypol treatment induced a series of histological changes in the seminiferous epithelium of the hamster testis. The earliest sign of drug effect was seen in spermatids and with the increase in doses the effects became more pronounced and extended to the spermatocytes. At 40 mg/kg dose an almost complete arrest of spermatogenesis was observed. Quantitatively, the ratio of pachytene spermatocytes: resting spermatocytes and step 7 spermatids: pachytene spermatocytes decreased significantly. The step 7 spermatids did not mature to step 19 spermatids at all. Histochemically activities of ATPase, SDH and LDH decreased with the increasing doses of gossypol, the activity of 3B hydroxysteroid dehydrogenase was not affected by gossypol treatment. In testis the glucose-6-phosphatase activity was not affected significantly but the activities of fructose 1, 6-diphosphatase and glucose-6-phosphate isomerase decreased significantly with the increasing doses of gossypol. Amylase activity rose significantly at higher doses. Marked changes in LDH and LDH-X were however observed with the increase in gossypol dose. In liver the activity of glucose-6-phosphatase increased significantly while the activities of fructose 1, 6-diphosphatase, glucose-6-phosphate isomerase and amylase were not affected following gossypol treatment. The glycogen contents however increased significantly following high doses of gossypol. No changes in testosterone production and plasma levels of testosterone were observed following gossypol treatment.

Effect of gossypol on the fertility of male rats.

Thirty male rats were grouped into 5 groups of 6 animals each. Animals in groups II-V were given gossypol at a dose of 5 mg/kg, 10 mg/kg, 20 mg/kg and 40 mg/kg body weight per day for 45 days respectively. Animals of group I served as control. A significant decrease in body weight after administration of 40 mg/kg body weight of gossypol was observed; low doses of gossypol, however did not affect the body weight. Testis, epididymis, prostate and seminal vesicles weights decreased gradually with the increasing doses of gossypol. With the increasing doses of gossypol, a marked decrease in the vas deferens sperm motility was observed. At 40 mg/kg dose there was a total inhibition of sperm motility. Histological studies after 5 mg/kg revealed no apparent sign of degeneration, while after 10 mg/kg dose the changes in the individual cell types were accompanied by overall disorganisation of the germinal epithelium involving displacement of the spermatocytes. The rats treated with 20-40 mg/kg gossypol showed a pronounced deleterious effect on the histological structure of the testis. The drug effect was dose dependent developing sequentially; from the uppermost layer of elongated spermatids affecting round spermatids and finally spermatocytes. Quantitatively the ratios of pachytene spermatocytes: resting spermatocytes, stage 7 spermatids: pachytene spermatocytes, and stage 19 spermatids: stage 7 spermatids and tubular diameter and germinal height decreased significantly. The activities of glucose-6-phosphatase, fructose 1, 6-diphosphatase, glucose-6-phosphate isomerase in testis decreased significantly at high dose (40 mg/kg), while the activity of amylase and glycogen content increased significantly with the increasing doses of gossypol.(ABSTRACT TRUNCATED AT 250 WORDS)