Inosine-5''-monophosphate analogues as inhibitors of human IMP cyclohydrolase and cellular growth

The novel 5-lipoxygenase (5-LO) inhibitor, ABT-761, was investigated for its effect on exercise-induced bronchoconstriction in asthmatic subjects. The relationship between 5-LO inhibition and effects on the response of the airways to exercise was examined. In a double-blind, randomized, crossover clinical trial, 10 patients with mild to moderate persistent asthma (who exhibited a fall in forced expiratory volume in one second (FEV1) > or = 20% following standardized exercise challenge) received 200 mg ABT-761 or matched placebo, orally, 5 h prior to exercise on two study days, 7-10 days apart. Lung function, urinary leukotriene E4 (LTE4) and ex vivo calcium ionophore-stimulated LTB4 release in whole blood were measured prior to dosing, prior to exercise and at various time points up to 4 h post-exercise. The mean (SD) maximal percentage fall in FEV1 after exercise was 27.1 (12)% on placebo and 19.9 (10)% on ABT-761 days, respectively (p<0.05). Post-exercise fall in FEV1 was significantly attenuated at 5, 10, 15 and 30 min after exercise and the mean area under curve, representing the overall effect of exercise from 0-45 min post-challenge, was also significantly attenuated by ABT-761 (p<0.001). Ex vivo LTB4 release was inhibited by more than 80% throughout the 4 h post-exercise period, indicating that 5-LO was extensively inhibited at all time points. Urinary LTE4 in the post-exercise period was significantly lower after ABT-761 day than after placebo (40.1 (17.6) versus 89.8 (58.2) pg x mg creatinine(-1); p<0.05). Inhibition of LTB4 release in ABT-761-treated patients correlated positively with the attenuation of post-exercise FEV1 decline (r=0.711; p<0.05). We conclude that ABT-761 is effective in suppressing exercise-induced bronchoconstriction and that this protection is related quantitatively to the degree of 5-lipoxygenase inhibition.     

Insights into the mechanism of domain closure and substrate specificity of glutamate dehydrogenase from Clostridium symbiosum

Comparisons of the structures of glutamate dehydrogenase (GluDH) and leucine dehydrogenase (LeuDH) have suggested that two substitutions, deep within the amino acid binding pockets of these homologous enzymes, from hydrophilic residues to hydrophobic ones are critical components of their differential substrate specificity. When one of these residues, K89, which hydrogen-bonds to the gamma-carboxyl group of the substrate l-glutamate in GluDH, was altered by site-directed mutagenesis to a leucine residue, the mutant enzyme showed increased substrate activity for methionine and norleucine but negligible activity with either glutamate or leucine. In order to understand the molecular basis of this shift in specificity we have determined the crystal structure of the K89L mutant of GluDH from Clostridium symbiosum. Analysis of the structure suggests that further subtle differences in the binding pocket prevent the mutant from using a branched hydrophobic substrate but permit the straight-chain amino acids to be used as substrates. The three-dimensional crystal structure of the GluDH from C. symbiosum has been previously determined in two distinct forms in the presence and absence of its substrate glutamate. A comparison of these two structures has revealed that the enzyme can adopt different conformations by flexing about the cleft between its two domains, providing a motion which is critical for orienting the partners involved in the hydride transfer reaction. It has previously been proposed that this conformational change is triggered by substrate binding. However, analysis of the K89L mutant shows that it adopts an almost identical conformation with that of the wild-type enzyme in the presence of substrate. Comparison of the mutant structure with both the wild-type open and closed forms has enabled us to separate conformational changes associated with substrate binding and domain motion and suggests that the domain closure may well be a property of the wild-type enzyme even in the absence of substrate.     

Pharmacokinetics of cladribine in plasma and its 5''-monophosphate and 5''-triphosphate in leukemic cells of patients with chronic lymphocytic leukemia

PURPOSE: To determine whether microtubule- and actin-altering drugs, which have been shown to increase aqueous humor outflow, cause cellular contraction in human trabecular meshwork (HTM) cells. METHODS: HTM cells were plated in culture dishes containing a polymerized deformable silicone substrate. After 48 hours, the dishes were placed on an inverted microscope and treated with ethacrynic acid, colchicine, vinblastine, cytochalasin B, or 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and then recorded on videotape for 15 minutes. An increase in silicone substrate wrinkle size and/or number indicated a contraction. Sham controls were used. RESULTS: Cellular contraction was observed with ethacrynic acid, colchicine, and vinblastine in the 10(-5) to 10(-4) M dosage range. Pretreatment with H-7 blocked these effects. Cytochalasin B did not produce cellular contraction. CONCLUSIONS: Microtubule disruption causes cellular contraction in HTM cells, and this effect depends on an intact actin cytoskeleton network. Contraction of trabecular meshwork cells in response to various stimuli is an attractive hypothesis for possible homeostatic mechanisms in the outflow pathway, and this may serve as a focus for novel glaucoma drug development.     

Lactate transport by cortical synaptosomes from adult rat brain: characterization of kinetics and inhibitor specificity

Since lactate released by glial cells may be a key substrate for energy in neurons, the kinetics for the uptake of L-[U-14C]lactate by cortical synaptic terminals from 7- to 8-week-old rat brain were determined. Lactate uptake was temperature-dependent, and increased by 64.9% at pH 6.2, and decreased by 43.4% at pH 8.2 relative to uptake at pH 7.3. Uptake of monocarboxylic acids was saturable with increasing substrate concentration. Eadie-Hofstee plots of the data gave evidence of two carrier-mediated uptake mechanisms with a high-affinity Km of 0.66 mM and Vmax of 3.66 mM for pyruvate, and a low-affinity system with a Km of 9.9 mM for both lactate and pyruvate and Vmax values of 16.6 and 23.1 nmol/30 s/mg protein for lactate and pyruvate, respectively. Saturable uptake was seen in the presence of 10 mM alpha-cyano-4-hydroxycinnamate. Lactate transport by synaptic terminals was much more sensitive to inhibition by sulfhydryl reagents than transport in astrocytes. Addition of 0.5 and 2 mM mersalyl decreased the uptake of 1 mM lactate by synaptic terminals by 59.3 and 66.37%, respectively. Pyruvate moderately decreased lactate transport, whereas 3-hydroxybutyrate had little effect. Quercetin, an inhibitor of lactate release, had little effect on the content of 14C lactate in synaptic terminals, supporting the concept that the majority of lactate produced within brain is from glial cells. Oxidation of L-[U-14C]lactate by synaptosomes was saturable, and yielded a Km of 1.23 mM and a Vmax of 116 nmol/h/mg protein. Overall the studies show that synaptic terminals from adult brain have a high capacity for transport and oxidation of lactate, consistent with the proposed role for this compound in metabolic trafficking in brain. Furthermore, the data provide kinetic evidence of two carrier-mediated mechanisms for monocarboxylic acid transport by synaptosomes and demonstrate that uptake of lactate by synaptic terminals is regulated differently than transport by astrocytes. Uptake of lactate by synaptic terminals also has differences from the systems described for neurons.     

Synthesis, structure, and antiproliferative activity of selenophenfurin, an inosine 5'-monophosphate dehydrogenase inhibitor analogue of selenazofurin

The synthesis and biological activity of selenophenfurin (5-beta-D-ribofuranosylselenophene-3-carboxamide, 1), the selenophene analogue of selenazofurin, are described. Glycosylation of ethyl selenophene-3-carboxylate (6) under stannic chloride-catalyzed conditions gave 2- and 5-glycosylated regioisomers, as a mixture of alpha- and beta-anomers, and the beta-2,5-diglycosylated derivative. Deprotected ethyl 5-beta-D-ribofuranosylselenophene-3-carboxylate (12 beta) was converted into selenophenfurin by ammonolysis. The structure of 12 beta was determined by 1H- and 13C-NMR, crystallographic, and computational studies. Selenophenfurin proved to be antiproliferative against a number of leukemia, lymphoma, and solid tumor cell lines at concentrations similar to those of selenazofurin but was more potent than the thiophene and thiazole analogues thiophenfurin and tiazofurin. Incubation of K562 cells with selenophenfurin resulted in inhibition of IMP dehydrogenase (IMPDH) (76%) and an increase in IMP pools (14.5-fold) with a concurrent decrease in GTP levels (58%). The results obtained confirm the hypothesis that the presence of heteroatoms such as S or Se in the heterocycle in position 2 with respect to the glycosidic bond is essential for both cytotoxicity and IMP dehydrogenase inhibitory activity in this type of C-nucleosides.     

Substrate and inhibitor specificity of the lactate carrier of human neutrophils

Comitin (p24) was first identified in Dictyostelium discoideum as a membrane-associated protein which binds in gel overlay assays to G and F actin. To analyze its actin-binding properties we used purified, bacterially expressed comitin and found that it binds to F actin in spin down experiments and increases the viscosity of F actin solutions even under high-salt conditions. Immunofluorescence studies, cell fractionation experiments and EM studies of vesicles precipitated with comitin-specific monoclonal antibodies showed that comitin was present in D. discoideum on: (a) a perinuclear structure with tubular or fibrillary extensions; and (b) on vesicles distributed throughout the cell. In immunofluorescence experiments using comitin antibodies NIH 3T3 fibroblasts showed a similar staining pattern as D. discoideum cells. Using bona fide Golgi markers the perinuclear structure was identified as the Golgi apparatus. The results were supported by an electron microscopic study using cryosections. Based on these data we propose that also in Dictyostelium the stained perinuclear structure is the Golgi apparatus. In vivo the perinuclear structure was found to be attached to the actin and the microtubule network. Alteration of the actin network or depolymerization of the microtubules led to its dispersal into vesicles distributed throughout the cell. These results suggest that the Golgi apparatus in D. discoideum is connected to the actin network by comitin. This protein seems also to be present in mammalian cells.     

Expression, purification, and characterization of inosine 5'-monophosphate dehydrogenase from Borrelia burgdorferi

Inosine 5'-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in de novo guanine nucleotide biosynthesis. IMPDH converts IMP to xanthosine 5'-monophosphate with concomitant conversion of NAD+ to NADH. All IMPDHs characterized to date contain a 130-residue "subdomain" that extends from an N-terminal loop of the alpha/beta barrel domain. The role of this subdomain is unknown. An IMPDH homolog has been cloned from Borrelia burgdorferi, the causative agent of Lyme disease (Margolis, N., Hogan, D., Tilly, K., and Rosa, P. A. (1994) J. Bacteriol. 176, 6427-6432). This homolog has replaced the subdomain with a 50-residue segment of unrelated sequence. We have expressed and characterized the B. burgdorferi IMPDH homolog. This protein has IMPDH activity, which unequivocally demonstrates that the subdomain is not required for catalytic activity. The monovalent cation and dinucleotide binding sites of B. burgdorferi IMPDH are significantly different from those of human IMPDH. Therefore, these sites are targets for the design of specific inhibitors for B. burgdorferi IMPDH. Such inhibitors might be new treatments for Lyme disease.     

An RNA splicing enhancer-like sequence is a component of a splicing inhibitor element from Rous sarcoma virus

The accumulation in infected cells of large amounts of unspliced viral RNA for use as mRNA and genomic RNA is a hallmark of retrovirus replication. The negative regulator of splicing (NRS) is a long cis-acting RNA element in Rous sarcoma virus that contributes to unspliced RNA accumulation through splicing inhibition. One of two critical sequences located in the NRS 3' region resembles a minor class 5' splice site and is required for U11 small nuclear ribonucleoprotein (snRNP) binding to the NRS. The second is a purine-rich region in the 5' half that interacts with the splicing factor SF2/ASF. In this study we investigated the possibility that this purine-rich region provides an RNA splicing enhancer function required for splicing inhibition. In vitro, the NRS acted as a potent, orientation-dependent enhancer of Drosophila doublesex pre-mRNA splicing, and enhancer activity mapped to the purine-rich domain. Analysis of a number of site-directed and deletion mutants indicated that enhancer activity was diffusely located throughout a 60-nucleotide area but only the activity associated with a short region previously shown to bind SF2/ASF correlated with efficient splicing inhibition. The significance of the enhancer activity to splicing inhibition was demonstrated by using chimeras in which two authentic enhancers (ASLV and FP) were substituted for the native NRS purine region. In each case, splicing inhibition in transfected cells was restored to levels approaching that observed for the NRS. The observation that a nonfunctional version of the FP enhancer (FPD) that does not bind SF2/ASF also fails to block splicing when paired with the NRS 3' region supports the notion that SF2/ASF binding to the NRS is relevant, but other SR proteins may substitute if an appropriate binding site is supplied. Our results are consistent with a role for the purine region in facilitated snRNP binding to the NRS via SF2/ASF.     

Antitumour activity and schedule dependency of 8-chloroadenosine-3'',5''-monophosphate (8-ClcAMP) against human tumour xenografts

We have mapped the chromosomal locations of three human nuclear genes for putative components of the apparatus of mitochondrial gene expression, using a combination of in situ hybridization and interspecies hybrid mapping. The genes RPMS12 (mitoribosomal protein S12, a conserved protein component of the mitoribosomal accuracy center), TUFM (mitochondrial elongation factor EF-Tu), and AFG3L1 (similar to the yeast genes Afg3 and Rca1 involved in the turnover of mistranslated or misfolded mtDNA-encoded polypeptides) were initially characterized by a combination of database sequence analysis, PCR, cloning, and DNA sequencing. RPMS12 maps to chromosome 19q13.1, close to the previously mapped gene for autosomal dominant hearing loss DFNA4. The TUFM gene is located on chromosome 16p11.2, with a putative pseudogene or variant (TUFML) located very close to the centromere of chromosome 17. AFG3L1 is located on chromosome 16q24, very close to the telomere. By virtue of their inferred functions in mitochondria, these genes should be regarded as candidates of disorders sharing features with mitochondrial disease syndromes, such as sensorineural deafness, diabetes, and retinopathy.     

Purification of a 5-HT uptake inhibitor from the venom of Cerastes vipera

Heart rate (HR) and heart rate variability (HRV) are risk markers in cardiac disease. HRV is also an index of the sympathovagal modulation of heart rate. Their relations have been rarely analyzed. We aimed to study such relations in normal adult conscious rats by using a novel bradycardic agent, a sinus node inhibitor, S-16257. Placebo-drug crossover designs were used while monitoring HR with telemetry and analyzing HRV in both time and frequency domains. S-16257 (2 mg/kg; n = 10) decreased HR by 29% and markedly increased HRV in parallel. By using various combinations of S-16257, atropine (2 mg/kg), and propranolol (4 mg/kg), a positive relation was shown between RR interval and various indexes of HRV: the slower the HR, the greater the HRV. Nevertheless, there is one exception to this correlation. When S-16257 was associated with both atropine and propranolol, the deep bradycardia was accompanied by a reduction of HRV, which indicates that the physiologic negative correlation between HR and HRV is not an intrinsic property of the pacemaker but is highly dependent on the two components of the autonomic system.     

Change of antigenic and neutralizing specificity in substitutional epitope peptides of hemophilia B inhibitor

We have previously described the epitope mapping and functional neutralization of three factor IX inhibitors in hemophilia B (HB-1, 3, and 7) by synthetic peptides (13). However, A concentration of synthetic peptide of about 1000 times the concentration of factor IX in plasma was essential to neutralize the purified antibodies. We now report that substitutional synthetic peptides of epitope are able to neutralize the factor IX inhibitor with a lower concentration. Using two major epitope peptides, 156Val-Asn-Ser-Thr-Glu-Ala-Glu-Thr-Ile164 and 168Ile-Thr-Gln-Ser-Thr-Gln-Ser-Phe-Asn176, we designed changes of antigenicity using the systematic substitution of different amino acids at each residue of the epitope peptides and neutralization rate of factor IX inhibitors by a lower concentration of substitutional synthetic peptides conjugated with bovine serum albumin (BSA). Twenty-five substitutional peptides for HB-1, twenty substitutional peptides for HB-3 and forty-four substitutional peptides for HB-7 reacted stronger than the native sequences. One of the peptides, 1.0 microM of 156Val-Asn-Ser-Thr-Glu-Tyr-Glu-Thr-Ile164 conjugated with BSA, neutralized 26.5% of inhibitor in HB-1's plasma maximally. Our data show that high antigenicity peptides conjugated with BSA ranging in concentration from 0.1 microM to 1.0 microM are able to neutralize factor IX inhibitors in plasma and there is a possibility of peptide neutralization inhibitor therapy.     

Steady-state kinetic mechanism, stereospecificity, substrate and inhibitor specificity of Enterobacter cloacae nitroreductase

Enterobacter cloacae nitroreductase (NR) is a flavoprotein which catalyzes the pyridine nucleotide-dependent reduction of nitroaromatics. Initial velocity and inhibition studies have been performed which establish unambiguously a ping-pong kinetic mechanism. NADH oxidation proceeds stereospecifically with the transfer of the pro-R hydrogen to the enzyme and the amide moiety of the nicotinamide appears to be the principal mediator of the interaction between NR and NADH. 2,4-Dinitrotoluene is the most efficient oxidizing substrate examined, with a kcat/KM an order of magnitude higher than those of p-nitrobenzoate, FMN, FAD or riboflavin. Dicoumarol is a potent inhibitor competitive vs. NADH with a Ki of 62 nM. Several compounds containing a carboxyl group are also competitive inhibitors vs. NADH. Yonetani-Theorell analysis of dicoumarol and acetate inhibition indicates that their binding is mutually exclusive, which suggests that the two inhibitors bind to the same site on the enzyme. NAD+ does not exhibit product inhibition and in the absence of an electron acceptor, no isotope exchange between NADH and 32P-NAD+ could be detected. NR catalyzes the 4-electron reduction of nitrobenzene to hydroxylaminobenzene with no optically detectable net formation of the putative two-electron intermediate nitrosobenzene.