Structure of vaccinia virus thymidine kinase in complex with dTTP: insights for drug design

BACKGROUND: Development of countermeasures to bioterrorist threats such as those posed by the smallpox virus (variola), include vaccination and drug development. Selective activation of nucleoside analogues by virus-encoded thymidine (dThd) kinases (TK) represents one of the most successful strategies for antiviral chemotherapy as demonstrated for anti-herpes drugs. Vaccinia virus TK is a close orthologue of variola TK but also shares a relatively high sequence identity to human type 2 TK (hTK), thus achieving drug selectivity relative to the host enzyme is challenging. RESULTS: In order to identify any differences compared to hTK that may be exploitable in drug design, we have determined the crystal structure of VVTK, in complex with thymidine 5'-triphosphate (dTTP). Although most of the active site residues are conserved between hTK and VVTK, we observe a difference in conformation of residues Asp-43 and Arg-45. The equivalent residues in hTK hydrogen bond to dTTP, whereas in subunit D of VVTK, Asp-43 and Arg-45 adopt a different conformation preventing interaction with this nucleotide. Asp-43 and Arg-45 are present in a flexible loop, which is disordered in subunits A, B and C. The observed difference in conformation and flexibility may also explain the ability of VVTK to phosphorylate (South)-methanocarbathymine whereas, in contrast, no substrate activity with hTK is reported for this compound. CONCLUSION: The difference in conformation for Asp-43 and Arg-45 could thus be used in drug design to generate VVTK/Variola TK-selective nucleoside analogue substrates and/or inhibitors that have lower affinity for hTK

3'-[4-Aryl-(1,2,3-triazol-1-yl)]-3'-deoxythymidine analogues as potent and selective inhibitors of human mitochondrial thymidine kinase

In an effort to increase the potency and selectivity of earlier identified substrate-based inhibitors of mitochondrial thymidine kinase 2 (TK-2), we now describe the synthesis of new thymidine analogues containing a 4- or 5-substituted 1,2,3-triazol-1-yl substituent at the 3'-position of the 2'-deoxyribofuranosyl ring. These analogues were prepared by Cu- and Ru-catalyzed cycloadditions of 3'-azido-3'-deoxythymidine and the appropriate alkynes, which produced the 1,4- and 1,5-triazoles, respectively. Selected analogues showed nanomolar inhibitory activity for TK-2, while virtually not affecting the TK-1 counterpart. Enzyme kinetics indicated a competitive and uncompetitive inhibition profile against thymidine and the cosubstrate ATP, respectively. This behavior is rationalized by suggesting that the inhibitors occupy the substrate-binding site in a TK-2 ATP complex that maintains the enzyme's active site in a closed conformation through the stabilization of a small lid domain

Design and synthesis of phosphonoacetic acid (PPA) ester and amide bioisosters of ribofuranosylnucleoside diphosphates as potential ribonucleotide reductase inhibitors and evaluation of their enzyme inhibitory, cytostatic and antiviral activity.

Continuing our investigations on inhibitors of ribonucleotide reductase (RNR), the crucial enzyme that catalyses the reduction of ribonu-cleotides to deoxyribonucleotides, we have now prepared and evaluated 5′-phosphonoacetic acid, amide and ester analogues of adenosine, uridine and cytidine with the aim to verify both substrate specificity and contribution to biological activity of diphosphate mimic moieties. A molecular modelling study has been conducted on the RNR R1 subunit, in order to verify the possible interaction of the proposed bioisosteric moieties. The study compounds were finally tested on the recombinant murine RNR showing a degree of inhibition that ranged from 350 μM for the UDP analogue 5′-deoxy-5′- N-(phosphon-acetyl)uridine sodium salt (amide) to 600 μM for the CDP analogue 5′- O-[(diethyl-phosphon)acetyl]cytidine (ester). None of the tested compounds displayed noteworthy cytostatic activity at 100–500 μM concentrations, whereas ADP analogue 5′- N-[(diethyl-phosphon) acetyl]adenosine (amide) and 5′-deoxy-5′- N-(phos-phon-acetyl)adenosine sodium salt (amide) showed a moderate inhibitory activity (EC50: 48 μM) against HSV-2 and a modest inhibitory activity (EC50: 110 μM) against HIV-1, respectively

Investigation of antiviral and anticancer nucleoside analog substrate recognition of drosophila melanogaster and herpes virus deoxyribonucleoside kinases

The deoxyribonucleoside kinase of the fruit fly Drosophila melanogaster (Dm-dNK) is a multisubstrate enzyme that phosphorylates pyrimidine and purine deoxyribonucleosides as well as several anticancer and antiviral nucleoside analogs. Dm-dNK is sequence related to the human deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK) and thymidine kinase 2 (M), as well as to the herpes simplex virus type-1 thymidine kinase (Hsv-1 TK). The human and viral deoxyribonucleoside kinases can phosphorylate multiple deoxyribonucleosides, whereas Dm-dNK has the ability to phosphorylate all naturally occurring deoxyribonucleosides required for DNA replication. In addition to its broad substrate specificity, Dm-dNK also exhibits higher catalytic rates for nucleoside and nucleoside analog phosphorylation compared to other nucleoside kinases. Nucleoside kinases are being investigated for possible use as suicide genes in combined gene/chemotherapy of cancer. The most commonly studied nucleoside kinase suicide gene is the HSV-1 TK gene used in combination with the guanosine nucleoside analog ganciclovir. The suicide nucleoside kinase is ratelimiting in the pharmacological activation of the cytotoxic nucleoside analogs, and mutants of HSV-1 TK with improved biochemical properties for nucleoside analog phosphorylation are more efficient suicide genes. The broad substrate specificity of Dm-dNK and its high catalytic rate makes it an interesting candidate gene for suicide gene therapy. The possible use of Dm-dNK as a suicide gene has been evaluated and it has been shown that over-expression of Dm-dNK enhances the sensitivity of cancer cells to several cytotoxic nucleoside analogs. Although Dm-dNK phosphorylates both purine and pyrimidine nucleosides, the enzyme has a preference for pyrimidine nucleosides. The maximal catalytic rate of purine and pyrimidine nucleoside phosphorylation is similar, but the enzyme exhibits higher affinity for pyrimidine nucleosides and nucleoside analogs. For suicide gene therapy application, purine nucleoside analogs may be preferred because these compounds appear to induce a higher bystander cell killing, i.e. killing of untransduced neighboring cells by transfer of phosphorylated nucleoside analogs via gap junctions. The solved structures of Dm-dNK, dGK and HSV-1 TK, reveal a common folding of these enzymes and in particular the amino acid residues involved in substrate interactions are highly conserved. However, the substrate binding site also exhibits some major differences between Dm-dNK and HSV-1 TK. Based on this structural information we performed site directed mutagenesis of the residues Asn28, I1e29, Phe1 14 and G1n81 in order to understand the determinants of the substrate specificity of the enzyme and to find Dm-dNK mutants with improved kinetic properties for application in suicide gene therapy. It has also been shown that Dm-dNK with 20 amino acid C-terminal deletion has even higher catalytic rates for deoxyribonucleosides compared to wild-type, and it was also reported that the mutagenesis of a few amino acids allows to change the substrate specificity from pyrimidines to purines. Based on these previous studies we constructed the reported mutated enzymes and designed new mutations, with and without the 20 amino acid C-terminal deletion. We measured the ability to phosphorylate ganciclovir (GCV). We have finally selected the most efficient enzymes phosphorylating GCV and expressed in an osteosarcoma TK cell line and determined the sensitivity to nucleoside analogs. The cells expressing the Met88Arg mutant enzyme showed the highest increased sensitivity to purine nucleoside analogs with 8 to 80-fold decreased IC50 compared to untransduced control cells or cells expressing the wild-type nucleoside kinase. We have also created a Dm-dNK protein targeted to the mitochondrial matrix by fusing a mitochondrial targeting signal to the N-terminus of the protein. We showed that the mitochondrial Dm-dNK was enzymatically active and that overexpression of the enzyme in an osteosarcoma TK‾deficient cell line resulted in an increased sensitivity to some nucleoside analogs such as 1-â-D-arabinofuranosylthymine (araT), (E)-5-(2-bromovinyl)-2''-deoxyuridine (BVDU), 5-bromo-2'' eoxyuridine (5-Br-dUrd) and 5fluoro2''deoxyuridine (S-F-dUrd). Labeling studies using [3H]-dThd also showed that mitochondrial expression of Dm-dNK, compared to nuclear expression, resulted in a higher specific [3H]-dTTP activity in the total MP pool and as a result a higher rate of [3H]-dTTP incorporation into nuclear DNA. We conducted structural studies on HSV-1 TK. In particular we performed site-directed mutagenesis on A1a167 and A1a168 of the HSV-1 TK. The mutated A1a168His and A1a167Phe enzymes turned out to have knocked-out the dThd activity while retaining full GCV phosphorylation ability

The characterization of human adenylate kinases 7 and 8 demonstrates differences in kinetic parameters and structural organization among the family of adenylate kinase isoenzymes

Differences in expression profiles, substrate specificities, kinetic properties and subcellular localization among the adenylate kinase (AK) isoenzymes, have been shown to be important for maintaining a proper adenine nucleotide composition for many different cell functions. In the present study human AK7 was characterized and its substrate specificity, kinetic properties and subcellular localization determined. In addition, a novel member of the human adenylate kinase family, with two functional domains, was identified and characterized and it was assigned the name AK8. AK8 is the second known human AK with two complete and active AK domains within its polypeptide chain, a feature that previously was shown also for AK5. The full length AK8, as well as its two domains AK8p1 and AK8p2, all showed similar AK enzyme activity. AK7, full length AK8, AK8p1 and AK8p2 phosphorylated AMP, CMP, dAMP and dCMP with ATP as phosphate donor and also AMP, CMP and dCMP with GTP as phosphate donor. Both AK7 and the full length AK8 showed highest affinity for AMP with ATP as phosphate donor and proved to be more efficient in AMP phosphorylation as compared to the major cytosolic isoform AK1. Expression of the proteins fused with the green fluorescent protein demonstrated a cytosolic localization for both AK7 and AK8

Retained sensitivity to cytotoxic pyrimidine nucleoside analogs in thymidine kinase 2 deficient human fibroblasts

Thymidine kinase 2 (TK2) is a mitochondrial deoxyribonucleoside kinase that phosphorylates several nucleoside analogs used in anti-viral and anti-cancer therapy. A fibroblast cell line with decreased TK2 activity was investigated in order to obtain insights in the effects of TK2 deficiency on nucleotide metabolism. The role of TK2 for the sensitivity against cytotoxic nucleoside analogs was also investigated. The TK2 deficient cells retained their sensitivity against all pyrimidine nucleoside analogs tested. This study suggests that nucleoside analog phosphorylation mediated by TK2 may be less important, compared to other deoxyribonucleoside kinases, for the cytotoxic effects of these compounds.status: publishe

Design, synthesis and binding at cloned muscarinic receptors of N-[5-(1'-substituted-acetoxymethyl)-3-oxadiazolyl] and N-[4-(1'-substituted-acetoxymethyl)-2-dioxolanyl] dialkyl amines.

none7Few muscarinic antagonists differentiate between the M4 and M2 muscarinic receptors. In a structure activity study, aimed at discovering leads for the development of a M4 muscarinic receptor-selective antagonist, we have synthesized and tested at cloned muscarinic receptors the binding of a group of dioxolane- or oxadiazole-dialkyl amines, and compared them to our compound 1, which contains the furan nucleus. Although none of these agents were particularly potent at M4 receptors (Kd values were typically 30-70 nM), furan derivatives (-)1 and (+)1 were significantly more potent at M4 receptors than at M2 receptors (approximately 3- and 4-fold, respectively). The dioxolane derivatives 12b and 12c were more than 10-fold selective for the M4 versus the M2 receptors, while the dioxolane derivative 12e was 15-fold more potent at M4 receptors than for M2 receptors. However, these agents bound to M3 receptors with potencies like that for the M4 receptor, so they are not M4-selective. The M4/M2 relative selectivities of some of our compounds are similar to the better hexahydrosiladifenidol derivatives, and may provide some important structural clues for the development of potent and selective M4 antagonists.noneS. Manfredini; I. Lampronti; S. Vertuani; N. Solaroli; M. Recanatini; D. Bryan; M. McKinneyManfredini, Stefano; Lampronti, Ilaria; Vertuani, Silvia; Solaroli, Nicola; M., Recanatini; D., Bryan; M., Mckinne