Different Characteristics and Nucleotide Binding Properties of Inosine Monophosphate Dehydrogenase (IMPDH) Isoforms

We recently reported that Inosine Monophosphate Dehydrogenase (IMPDH), a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, clustered into macrostructures in response to decreased nucleotide levels and that there were differences between the IMPDH isoforms, IMPDH1 and IMPDH2. We hypothesised that the Bateman domains, which are present in both isoforms and serve as energy-sensing/allosteric modules in unrelated proteins, would contribute to isoform-specific differences and that mutations situated in and around this domain in IMPDH1 which give rise to retinitis pigmentosa (RP) would compromise regulation. We employed immuno-electron microscopy to investigate the ultrastructure of IMPDH macrostructures and live-cell imaging to follow clustering of an IMPDH2-GFP chimera in real-time. Using a series of IMPDH1/IMPDH2 chimera we demonstrated that the propensity to cluster was conferred by the N-terminal 244 amino acids, which includes the Bateman domain. A protease protection assay suggested isoform-specific purine nucleotide binding characteristics, with ATP protecting IMPDH1 and AMP protecting IMPDH2, via a mechanism involving conformational changes upon nucleotide binding to the Bateman domain without affecting IMPDH catalytic activity. ATP binding to IMPDH1 was confirmed in a nucleotide binding assay. The RP-causing mutation, R224P, abolished ATP binding and nucleotide protection and this correlated with an altered propensity to cluster. Collectively these data demonstrate that (i) the isoforms are differentially regulated by AMP and ATP by a mechanism involving the Bateman domain, (ii) communication occurs between the Bateman and catalytic domains and (iii) the RP-causing mutations compromise such regulation. These findings support the idea that the IMPDH isoforms are subject to distinct regulation and that regulatory defects contribute to human disease.     

Antitumor Potential of Acyclic Nucleoside Phosphonates

Abstract

Acyclic nucleoside phosphonates such as HPMPC (cidofovir) and PMEA (adefovir) have been identified as broad-spectrum antiviral agents that are effective against herpes-, retro- and hepadnavirus infections (PMEA) and herpes-, pox-, adeno-, polyoma-, and papillomavirus infections (HPMPC). Here we show that HPMPC and PMEA also offer great potential as antitumor agents, through the induction of tumor cell differentiation (PMEA), inhibition of angiogenesis (HPMPC) and induction of apoptosis (HPMPC). In vivo tumor regressions have been noted for choriocarcinoma (PMEA) in rats, hemangioma (HPMPC) in rats and papillomatous lesions (HPMPC) in humans. Acyclic nucleoside phosphonates can be considered as a new dimension to the discipline of chemotherapy. They have a unique mode of action that is targeted at (viral or tumoral) DNA synthesis. They exhibit a pronounced and prolonged anti-viral and/or tumoral activity that can persist for days or weeks after a single administration. Most importantly, they have a uniquely broad spectrum of indications for clinical use, encompassing both DNA- and retrovirus infections, as well as various forms of cancer of both viral and non-viral origin.     

HPLC/Tandem Ion Trap Mass Detector Methods for Determination of Inosine Monophosphate Dehydrogenase (IMPDH) and Thiopurine Methyltransferase (TPMT)

The efficiency of Mycophenolate mofetil (MMF) and Azathioprine (AZA) as immunosuppressive agents depends on the activity of 2 enzymes, inosine monophosphate dehydrogenase (IMPDH) and thiopurine methyltransferase (TPMT) respectively. We present preliminary evaluation of nonradioactive methods that apply HPLC with ion-trap mass detection to measure the activities of IMPDH in peripheral blood mononuclear cells (PBMC) and TPMT in the erythrocytes (RBC). We found IMPDH activity of 0.9 ± 0.2 nmol/hour/10⁶ PBMC and TPMT activity of 19.9 ± 4.7 nmol/hour/ml RBC in healthy subjects. These methods, following its further validation, could be useful for monitoring the activity in a clinical and experimental setting.     

Differential Sensitivities of Fast- and Slow-Cycling Cancer Cells to Inosine Monophosphate Dehydrogenase 2 Inhibition by Mycophenolic Acid

As uncontrolled cell proliferation requires nucleotide biosynthesis, inhibiting enzymes that mediate nucleotide biosynthesis constitutes a rational approach to the management of oncological diseases. In practice, however, results of this strategy are mixed and thus elucidation of the mechanisms by which cancer cells evade the effect of nucleotide biosynthesis restriction is urgently needed. Here we explored the notion that intrinsic differences in cancer cell cycle velocity are important in the resistance toward inhibition of inosine monophosphate dehydrogenase (IMPDH) by mycophenolic acid (MPA). In short-term experiments, MPA treatment of fast-growing cancer cells effectively elicited G0/G1 arrest and provoked apoptosis, thus inhibiting cell proliferation and colony formation. Forced expression of a mutated IMPDH2, lacking a binding site for MPA but retaining enzymatic activity, resulted in complete resistance of cancer cells to MPA. In nude mice subcutaneously engrafted with HeLa cells, MPA moderately delayed tumor formation by inhibiting cell proliferation and inducing apoptosis. Importantly, we developed a lentiviral vector–based Tet-on label-retaining system that enables to identify, isolate and functionally characterize slow-cycling or so-called label-retaining cells (LRCs) in vitro and in vivo. We surprisingly found the presence of LRCs in fast-growing tumors. LRCs were superior in colony formation, tumor initiation and resistance to MPA as compared with fast-cycling cells. Thus, the slow-cycling compartment of cancer seems predominantly responsible for resistance to MPA.     

Targets for the Antiviral and Antitumor Activities of Nucleoside,Nucleotide and Oligonucleotide Analogues

Abstract

The following targets can be considered in the development of antiviral agents: (i) DNA polymerase via dThd kinase, (ii) S-adenosylhomocysteine hydrolase; and in the development of antitumor agents: (iii) dTMP synthetase and (iv) protein synthesis via the 2–5A pathway.     

New Arylazo-Based (Chromene-Thiazole) Hybrids as Potential MRSA Inhibitors

A three-component protocol was established to efficiently synthesize (chromene-thiazole) and related arylazo analogs in good to excellent yields. The desired products were prepared by reacting the appropriate salicylaldehydes, 2-cyanothioacetamide, and chloroacetone or hydrazonyl chlorides. Using piperidine as a mediator in ethanol at 80 °C for 4–6 h, the three-component protocol produce the target hybrids in 87–96 % yields. The newly synthesized products showed a broad range of antibacterial activity. The addition of an arylazo unit at the chromene-C6 position significantly improved the antibacterial activity, while the impact of adding an arylazo group at the thiazole-C5 position varied based on the electronic characteristics of the para-substituted arene unit. Generally, series that is linked to two arylazo units, one at chromene-C6 and the other at thiazole-C5, showed the best activity. Some new hybrids showed effective antibacterial activity than ciprofloxacin with MIC/MBC values up to 1.9/3.9 μM against S. aureus and E. coli. Additionally, they demonstrated better effectiveness against MRSA ATCC:33591 and ATCC:43300 compared to linezolid, with MIC/MBC values up to 4.0/16.1 and 3.9/15.6 μM, respectively. The data predicted for the physicochemical properties, lipophilicity, pharmacokinetics, and drug-likeness of new arylazo-based chromene-thiazole hybrids evaluated by SwissADME. As a result of the above, products that are linked to two arylazo units can be considered drug-like scaffolds.     

Synthesis of New Nucleoside Analogues Comprising a Geminal Difluorocyclopropane Moiety as Potential Antiviral/Antitumor Agents

Abstract

Geminal difluorocyclopropane analogues of nucleosides 7a–7e were synthesized. Compounds 7a and 7c–7e were obtained by alkylation of nucleic acid bases or their appropriate precursors with (cis)-1-benzyloxymethyl-2-bromomethyl-3,3-difluorocyclopropane (8). Analogue 7b was prepared by hydrolysis of 2-amino-6-chloropurine derivative 7e. Compounds 7a–7d did not exhibit any antiviral activity against HCMV, HSV-1, HSV-2, EBV, VZV, HBV and HIV-1 or antitumor effects against murine leukemia L1210, mouse tumors PO3 or C38 and human tumor H15.     

HPLC and Synthesis Strategy in Nucleoside and (Oligo)Nucleotide Chemistry

Abstract

A judicious use of HPLC allows to simplify the synthetic approach of an (oligo)nucleotide. As an example, is reported a preparation of an antiviral (3′-5′)dinucleotide with simultaneous isolation of its (3′-3′) isomer.     

A New Subfamily of Polyphosphate Kinase 2 (Class III PPK2) Catalyzes both Nucleoside Monophosphate Phosphorylation and Nucleoside Diphosphate Phosphorylation

Inorganic polyphosphate (polyP) is a linear polymer of tens to hundreds of phosphate (P_i) residues linked by “high-energy” phosphoanhydride bonds as in ATP. PolyP kinases, responsible for the synthesis and utilization of polyP, are divided into two families (PPK1 and PPK2) due to differences in amino acid sequence and kinetic properties. PPK2 catalyzes preferentially polyP-driven nucleotide phosphorylation (utilization of polyP), which is important for the survival of microbial cells under conditions of stress or pathogenesis. Phylogenetic analysis suggested that the PPK2 family could be divided into three subfamilies (classes I, II, and III). Class I and II PPK2s catalyze nucleoside diphosphate and nucleoside monophosphate phosphorylation, respectively. Here, we demonstrated that class III PPK2 catalyzes both nucleoside monophosphate and nucleoside diphosphate phosphorylation, thereby enabling us to synthesize ATP from AMP by a single enzyme. Moreover, class III PPK2 showed broad substrate specificity over purine and pyrimidine bases. This is the first demonstration that class III PPK2 possesses both class I and II activities.     

GTP Concentrations are Elevated in Erythrocytes of Renal Transplant Recipients when Conventional Immunosuppression is Replaced by the Inosine Monophosphate Dehydrogenase Inhibitor Mycophenolic Acid Mofetil (MMF)

We show that GTP concentrations rise in the erythrocytes of renal transplant recipients receiving the immunosuppressant MMF, and demonstrate that this effect is not caused by poor renal function after engraftment. We propose a model that is consistent with our observations.     

Effective Inhibitors of Tyrosyl-DNA Phosphodiesterase 1 Based on Monoterpenoids as Potential Agents for Antitumor Therapy

Russian Journal of Bioorganic Chemistry - Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is one of the important DNA repair enzymes responsible for the repair of DNA damage caused by anticancer drugs, such...     

Association and Haplotype Analysis of purH Gene with Inosine Monophosphate Content in Chickens

The current study was designed to investigate the effects of the purH gene on chicken muscle inosine monophosphate (IMP) content. Muscle IMP content was measured in five chicken breeds. Single nucleotide polymorphisms (SNPs) were detected by PCR-SSCP and DNA sequencing. Two SNPs were detected, A/T substitution at position 8023 in exon 9, and T/C substitution at position 17446 in exon 16. The results indicated that only T17446C polymorphism was associated with IMP content. The haplotype effect was higher than the single genotype effect. We tentatively conclude that purH gene is a candidate locus or linked to a major gene that affects muscle IMP content. Haplotypes are superior to single genotypes as potential molecular markers for meat quality traits in chicken.     

Inhibitors of Renin as Potential Therapeutic Agents

Abstract

The reaction of usual (U) and atypical (A) cholinesterase phenotypes was studied with six organophosphorus compounds, two pyridinium oximes (HI-6 and PAM-2) and with 4–4-bipyridine (4,4-BP). No difference in the inhibition rate constants for the two phenotypes was found with the progressive inhibitors tabun, sarin, paraoxon and soman. The other two progressive inhibitors, VX and the positively charged phosphostigmine, inhibited the U phenotype more strongly than the A phenotype.

The positively charged reversible inhibitor HI-6 showed a higher affinity for the U than for the A phenotype, while PAM-2 and the non-charged 4,4′-BP did not show a significant difference in their affinity towards the two enzymes.

Both phenotypes phosphylated by VX or sarin were reactivatable by HI-6 and PAM-2, and the A phenotype was always reactivated more slowly than the U phenotype. The paraoxon-inhibited phenotypes were reactivated at equal rates with PAM-2 but were not reactivated with HI-6. The phosphylated phenotypes did not reactivate spontaneously during one hour.

The effect of reversible inhibitors upon the rate of phosphylation (protection) was tested with HI-6 (for inhibition by soman, tabun and paraoxon) and with 4,4′-BP (for inhibition by soman). By applying the concentrations of the protectors equal to their enzyme/inhibitor dissociation constants, a better protection of the U than of the A phenotype was achieved by HI-6, but equal protection was given by 4,4′-BP.     

Therapeutic Potential of HPMPC (Cidofovir), PMEA (Adefovir) and Related Acyclic Nucleoside Phosphonate Analogues as Broad-Spectrum Anttviral Agents

Abstract

This article reviews the antiviral features of the acyclic nucleoside phosphonate (ANP) analogues, with a special focus on the most recent findings concerning the biochemistry and clinical efficacy of HPMPC [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine; cidofovir; Vistide®] and PMEA [9-(2-phosphonyl-methoxyethyl)adenine; adefovir].     

Synthesis of Some New Nucleoside Analogues as Potential Antiviral Agents

Abstract

A novel series of pyrimidine nucleoside analogues was synthesized. 2,3-Dideoxy-2,3-anhydro-β-D-lyxofuranose was opened by sodium azide to give the corresponding azido compound, which was reduced by lithium aluminium hydride to lead to 2,3-dideoxy-2,3-epimino-β-D-ribofuranose. Pyrimidine bases were glycosylated with this synthon to give potential antiviral molecules: 1-(2,3-dideoxy-2,3-epimino-β-D-ribofuranosyl)pyrimidines.     

Structural Basis for the Specificity of Human NUDT16 and Its Regulation by Inosine Monophosphate

Human NUDT16 is a member of the NUDIX hydrolase superfamily. After having been initially described as an mRNA decapping enzyme, recent studies conferred it a role as an “housecleaning” enzyme specialized in the removal of hazardous (deoxy)inosine diphosphate from the nucleotide pool. Here we present the crystal structure of human NUDT16 both in its apo-form and in complex with its product inosine monophosphate (IMP). NUDT16 appears as a dimer whose formation generates a positively charged trench to accommodate substrate-binding. Complementation of the structural data with detailed enzymatic and biophysical studies revealed the determinants of substrate recognition and particularly the importance of the substituents in position 2 and 6 on the purine ring. The affinity for the IMP product, harboring a carbonyl in position 6 on the base, compared to purine monophosphates lacking a H-bond acceptor in this position, implies a catalytic cycle whose rate is primarily regulated by the product-release step. Finally, we have also characterized a phenomenon of inhibition by the product of the reaction, IMP, which might exclude non-deleterious nucleotides from NUDT16-mediated hydrolysis regardless of their cellular concentration. Taken together, this study details structural and regulatory mechanisms explaining how substrates are selected for hydrolysis by human NUDT16.     

Synthesis of a Nucleoside Bioconjugate System as a Potential Anti-HIV Agent

Abstract

Synthesis and anti-HIV data for a bioconjugate molecule incorporating a HIV protease inhibitor (A74704) and a HIV RT inhibitor (d4T) are presented.