Novel amide-based inhibitors of inosine 5'-monophosphate dehydrogenase

A series of novel amide-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are described.     

Pneumocystis carinii f. sp. carinii synthesizes de novo four homologs of ubiquinone

Ubiquinone, coenzyme Q, plays a pivotal role in electron transport and is a target for chemotherapy against a number of eukaryotic infectious agents, including Pneumocystis carinii. Coenzyme Q10 was previously identified as the major ubiquinone homolog in P. carinii isolated and purified from rat lungs; CoQ9 was also present. In contrast, CoQ9 and CoQ8 (but not CoQ10) were detected in the lungs of uninfected rat controls. These observations suggested that the pathogen synthesizes CoQ10, and perhaps CoQ9 as well. In the present study, CoQ biosynthesis in P. carinii was examined in greater detail. Radiolabeled mevalonate, a precursor of the CoQ polyprenyl chain, was incorporated in vitro into P. carinii ubiquinones. Incorporation of radiolabeled mevalonate into P. carinii CoQ was not enhanced by treating cells with lovastatin, suggesting that the cells did not transport the drug, or that a lovastatin-insensitive pathway for de novo synthesis of isoprenoids may also function in this organism. Radiolabeled precursors of the ring moiety, including shikimic acid, p-hydroxybenzoic acid, and tyrosine were also incorporated into P. carinii CoQ. Unexpectedly, it was found that not only CoQ9 and CoQ10, but also CoQ7, and CoQ8, were metabolically radiolabeled by all the precursors tested, indicating that the organism synthesizes CoQ7, CoQ8, CoQ9, and CoQ10. Metabolic radiolabeling of ubiquinones in rat lung controls was not detected in experiments using either radioactive mevalonate or p-hydroxybenzoate. Thus the incorporations measured using purified P. carinii preparations were due to the enzymes of the organism.     

Biochemical analysis of the modular enzyme inosine 5'-monophosphate dehydrogenase.

Two prominent domains have been identified in the X-ray crystal structure of inosine-5'-monophosphate dehydrogenase (IMPDH), a core domain consisting of an alpha/beta barrel which contains the active site and an inserted subdomain whose structure is less well defined. The core domain encompassing amino acids 1-108 and 244-514 of wild-type human IMPDH (II) connected by the tetrapeptide linker Ile-Arg-Thr-Gly was expressed. The subdomain including amino acids 99-244 of human wild-type IMPDH (II) was expressed as a His-tagged fusion protein, where the His-tag was removable by enterokinase cleavage. These two proteins as well as wild-type human IMPDH (II), all proteins expressed in Escherichia coli, have been purified to apparent homogeneity. Both the wild-type and core domain proteins are tetrameric and have very similar enzymatic activities. In contrast, the subdomain migrates as a monomer or dimer on a gel filtration column and lacks enzymatic activity. Circular dichroism spectropolarimetry indicates that the core domain retains secondary structure very similar to full-length IMPDH, with 30% alpha-helix and 30% beta-sheet vs 33% alpha-helix and 29% beta-sheet for wild-type protein. Again, the subdomain protein is distinguished from both wild-type and core domain proteins by its content of secondary structure, with only 15% each of alpha-helix and beta-sheet. These studies demonstrate that the core domain of IMPDH expressed separately is both structurally intact and enzymatically active. The availability of the modules of IMPDH will aid in dissecting the architecture of this enzyme of the de novo purine nucleotide biosynthetic pathway, which is an important target for immunosuppressive and antiviral drugs.     

Kinetic mechanism of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase

IMP dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with conversion of NAD+ to NADH. This reaction is the rate-limiting step in de novo guanine nucleotide biosynthesis. IMPDH is a target for antitumor, antiviral, and immunosuppressive chemotherapy. We have determined the complete kinetic mechanism for IMPDH from Tritrichomonas foetus using ligand binding, isotope effect, pre-steady-state kinetic, and rapid quench kinetic experiments. Both substrates bind to the free enzyme, which suggests a random mechanism. IMP binds to the enzyme in two steps. Two steps are also involved when IMP binds to a mutant IMPDH in which the active site Cys is substituted with a Ser. This observation suggests that this second step may be a conformational change of the enzyme. No Vm isotope effect is observed when [2-2H]IMP is the substrate which indicates that hydride transfer is not rate-limiting. This result is confirmed by the observation of a pre-steady-state burst of NADH production when monitored by absorbance. However, when NADH production was monitored by fluorescence, the rate constant for the exponential phase is 5-10-fold lower than when measured by absorbance. This observation suggests that the fluorescence of enzyme-bound NADH is quenched and that this transient represents NADH release from the enzyme. The time-dependent formation and decay of [14C]E-XMP intermediates was monitored using rapid quench kinetics. These experiments indicate that both NADH release and E-XMP hydrolysis are rate-limiting and suggest that NADH release precedes hydrolysis of E-XMP.     

Direct assay method for inosine 5'-monophosphate dehydrogenase activity

A rapid microassay method for the accurate measurement of the activity of inosine 5'-monophosphate dehydrogenase in crude tissue extracts was described. [8-14C]IMP and the radioactive products were separated by high-voltage electrophoresis in 0.1 M potassium phosphate buffer, pH 7.0, for 45 min. This separation method provides an analysis of the possible interfering reactions such as the metabolic conversion of the substrate IMP to inosine and adenylosuccinate, and the loss of the product XMP to xanthosine or GMP and to other metabolites. Low blank values were consistently obtained with this method because the XMP spot moves faster than the IMP spot. The major advantages of this assay method are direct measurement of IMP dehydrogenase activity in crude extracts, high sensitivity (with a limit of detection of 5 pmol of XMP production), high reproducibility (less than +/- 3.6%), low blank values (60-80 cpm), speed (2 h per 30 assays), and capability to measure activity in small amounts of tissue (10-50 mg wet wt).     

Species-specific inhibition of inosine 5'-monophosphate dehydrogenase by mycophenolic acid

IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+) to NADH. This reaction is the rate-limiting step in de novo guanine nucleotide biosynthesis. Mycophenolic acid (MPA) is a potent inhibitor of mammalian IMPDHs but a poor inhibitor of microbial IMPDHs. MPA inhibits IMPDH by binding in the nicotinamide half of the dinucleotide site and trapping the covalent intermediate E-XMP. The MPA binding site of resistant IMPDH from the parasite Tritrichomonas foetuscontains two residues that differ from human IMPDH. Lys310 and Glu431 of T. foetus IMPDH are replaced by Arg and Gln, respectively, in the human type 2 enzyme. We characterized three mutants of T. foetusIMPDH: Lys310Arg, Glu431Gln, and Lys310Arg/Glu431Gln in order to determine if these substitutions account for the species selectivity of MPA. The mutation of Lys310Arg causes a 10-fold decrease in the K(i) for MPA inhibition and a 8-13-fold increase in the K(m) values for IMP and NAD(+). The mutation of Glu431Gln causes a 6-fold decrease in the K(i) for MPA. The double mutant displays a 20-fold increase in sensitivity to MPA. Pre-steady-state kinetics were performed to obtain rates of hydride transfer, NADH release, and hydrolysis of E-XMP for the mutant IMPDHs. The Lys310Arg mutation results in a 3-fold increase in the accumulation level of E-XMP, while the Glu431Gln mutation has only a minimal effect on the kinetic mechanism. These experiments show that 20 of the 450-fold difference in sensitivity between the T. foetus and human IMPDHs derive from the residues in the MPA binding site. Of this, 3-fold can be attributed to a change in kinetic mechanism. In addition, we measured MPA binding to enzyme adducts with 6-Cl-IMP and EICARMP. Neither of these adducts proved to be a good model for E-XMP.     

Fine analysis of the Pneumocystis carinii f. sp. carinii genome by two-dimensional pulsed-field gel electrophoresis

Pneumocystis carinii is a general designation for a group of unusual unicellular fungal parasites responsible of pneumopathy in animal hosts. Divided into several subgroups termed the 'special forms', P. carinii is prone to an extensive karyotype variation. In previous studies, the nuclear genome of these organisms has been considered to be haploid and a set of 16 chromosomes has been assigned to P. carinii f. sp. carinii, a special form known to infect rats. We report the analysis of the genome of an isolate representative of the karyotype 1 of this special form, using two-dimensional pulsed-field gel electrophoresis procedures. The 'karyotype and restriction display' (KARD) fingerprints indicated the presence of 17 different chromosomes. The haploid genome size was estimated to be 8.4 Mbp. Some homologous chromosomes were distinguished on the basis of a single restriction fragment length polymorphism, which raises the possibility of a diploid nucleus. A restriction map of the chromosome 15, characterized by two homologues with a size difference of 7 kb, was constructed. Hybridization data indicated that insertion/deletion events may have occurred within subtelomeric regions which carry genes encoding the major surface glycoprotein (MSG) of Pneumocystis.     

Monovalent cation activation in Escherichia coli inosine 5'-monophosphate dehydrogenase

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate with the concomitant reduction of NAD to NADH. Escherichia coli IMPDH is activated by K(+), Rb(+), NH(+)(4), and Cs(+). K(+) activation is inhibited by Li(+), Na(+), Ca(2+), and Mg(2+). This inhibition is competitive versus K(+) at high K(+) concentrations, noncompetitive versus IMP, and competitive versus NAD. Thus monovalent cation activation is linked to the NAD site. K(+) increases the rate constant for the pre-steady-state burst of NADH production, possibly by increasing the affinity of NAD. Three mutant IMPDHs have been identified which increase the value of K(m) for K(+): Asp13Ala, Asp50Ala, and Glu469Ala. In contrast to wild type, both Asp13Ala and Glu469Ala are activated by all cations tested. Thus these mutations eliminate cation selectivity. Both Asp13 and Glu469 appear to interact with the K(+) binding site identified in Chinese hamster IMPDH. Like wild-type IMPDH, K(+) activation of Asp50Ala is inhibited by Li(+), Na(+), Ca(2+), and Mg(2+). However, this inhibition is noncompetitive with respect to K(+) and competitive with respect to both IMP and NAD. Asp50 interacts with residues that form a rigid wall in the IMP site; disruption of this wall would be expected to decrease IMP binding, and the defect could propagate to the proposed K(+) site. Alternatively, this mutation could uncover a second monovalent cation binding site.     

The CBS subdomain of inosine 5'-monophosphate dehydrogenase regulates purine nucleotide turnover

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyses the rate-limiting step in guanine nucleotide biosynthesis. IMPDH has an evolutionary conserved CBS subdomain of unknown function. The subdomain can be deleted without impairing the in vitro IMPDH catalytic activity and is the site for mutations associated with human retinitis pigmentosa. A guanine-prototrophic Escherichia coli strain, MP101, was constructed with the subdomain sequence deleted from the chromosomal gene for IMPDH. The ATP content was substantially elevated in MP101 whereas the GTP content was slighty reduced. The activities of IMPDH, adenylosuccinate synthetase and GMP reductase were two to threefold lower in MP101 crude extracts compared with the BW25113 wild-type strain. Guanine induced a threefold reduction in the MP101 ATP pool and a fourfold increase in the GTP pool within 10 min of addition to growing cells; this response does not result from the reduced IMPDH activity or starvation for guanylates. In vivo kinetic analysis using 14-C tracers and 33-P pulse-chasing revealed mutation-associated changes in purine nucleotide fluxes and turnover rates. We conclude that the CBS subdomain of IMPDH may coordinate the activities of the enzymes of purine nucleotide metabolism and is essential for maintaining the normal ATP and GTP pool sizes in E. coli .