Giardia intestinalis thymidine kinase is a high-affinity enzyme crucial for DNA synthesis and an exploitable target for drug discovery.

Giardiasis is a diarrheal disease caused by the unicellular parasite Giardia intestinalis, for which metronidazole is the main treatment option. The parasite is dependent on exogenous deoxyribonucleosides for DNA replication and thus is also potentially vulnerable to deoxyribonucleoside analogs. Here, we characterized the G. intestinalis thymidine kinase, a divergent member of the thymidine kinase 1 family that consists of two weakly homologous parts within one polypeptide. We found that the recombinantly expressed enzyme is monomeric, with 100-fold higher catalytic efficiency for thymidine compared to its second-best substrate, deoxyuridine, and is furthermore subject to feedback inhibition by dTTP. This efficient substrate discrimination is in line with the lack of thymidylate synthase and dUTPase in the parasite, which makes deoxy-UMP a dead-end product that is potentially harmful if converted to deoxy-UTP. We also found that the antiretroviral drug azidothymidine (AZT) was an equally good substrate as thymidine and was active against WT as well as metronidazole-resistant G. intestinalis trophozoites. This drug inhibited DNA synthesis in the parasite and efficiently decreased cyst production in vitro, which suggests that it could reduce infectivity. AZT also showed a good effect in G. intestinalis-infected gerbils, reducing both the number of trophozoites in the small intestine and the number of viable cysts in the stool. Taken together, these results suggest that the absolute dependency of the parasite on thymidine kinase for its DNA synthesis can be exploited by AZT, which has promise as a future medication effective against metronidazole-refractory giardiasis.

Identification of novel potential inhibitors of varicella-zoster virus thymidine kinase from ethnopharmacologic relevant plants through an in-silico approach.

Although Varicella or chickenpox infection which is caused by the varicella-zoster virus (VZV) has significantly been managed through vaccination, it remains an infection that poses threats to the nearest future due to therapeutic drawbacks. The focus of this research was geared towards in silico screening for the identification of novel compounds in plants of ethnopharmacological relevance in the treatment of chicken pox in West Africa. The work evaluated 65 compounds reported to be present in Achillea millefolium, Psidium guajava and Vitex doniana sweet to identify potential inhibitors of thymidine kinase, the primary drug target of varicella zoster virus. Out of the 65 compounds docked, 42 of these compounds were observed to possess binding energies lower than -7.0 kcal/mol, however only 20 were observed to form hydrogen bond interactions with the protein. These interactions were elucidated using LigPlot+ and MM-PBSA analysis with residue Ala134 predicted as critical for binding. Pharmacological profiling predicted three potential lead compounds comprising myricetin, apigenin- 4' -glucoside and Abyssinone V to possess good pharmacodynamics properties and negligibly toxic. The molecules were predicted as antivirals including anti-herpes and involved in mechanisms comprising inhibition of polymerase, ATPase and membrane integrity, which were corroborated previously in other viruses. These drug-like compounds are plausible biotherapeutic moieties for further biochemical and cell-based assaying to discover their potential for use against chickenpox. Communicated by Ramaswamy H. Sarma.

Neural differentiation of glioblastoma cell lines via a herpes simplex virus thymidine kinase/ganciclovir system driven by a glial fibrillary acidic protein promoter.

Glioblastoma is a malignant brain tumor with poor prognosis that rapidly acquires resistance to available clinical treatments. The herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) system produces the selective elimination of HSVtk-positive cells and is a candidate for preclinical testing against glioblastoma via its ability to regulate proliferation and differentiation. Therefore, in this study, we aimed to establish a plasmid encoding the HSVtk/GCV system driven by a glial fibrillary acidic protein (GFAP) promoter and verify its possibility of neural differentiation of glioblastoma cell line under the GCV challenge. Four stable clones-N2A-pCMV-HSVtk, N2A-pGFAP-HSVtk, U251-pCMV-HSVtk, and U251-pGFAP-HSVtk-were established from neuronal N2A and glioblastoma U251 cell lines. In vitro GCV sensitivity was assessed by MTT assay for monitoring time- and dosage-dependent cytotoxicity. The capability for neural differentiation in stable glioblastoma clones during GCV treatment was assessed by performing immunocytochemistry for nestin, GFAP, and ßIII-tubulin. Under GFAP promoter control, the U251 stable clone exhibited GCV sensitivity, while the neuronal N2A clones were nonreactive. During GCV treatment, cells underwent apoptosis on day 3 and dying cells were identified after day 5. Nestin was increasingly expressed in surviving cells, indicating that the population of neural stem-like cells was enriched. Lower levels of GFAP expression were detected in surviving cells. Furthermore, ßIII-tubulin-positive neuron-like cells were identified after GCV treatment. This study established pGFAP-HSVtk-P2A-EGFP plasmids that successfully ablated GFAP-positive glioblastoma cells, but left neuronal N2A cells intact. These data suggest that the neural differentiation of glioblastoma cells can be promoted by treatment with the HSVtk/GCV system.

UL23, UL30, and UL5 characterization of HSV1 clinical strains isolated from hematology department patients.

Genotypic diagnosis of HSV drug resistance can be performed routinely in a clinically relevant time. Nevertheless, data about HSV mutations (polymorphism or resistance) is not exhaustive which hinders the interpretation of such tests. The UL23, UL30, and UL5 genes are of greatest interest as these encode, respectively, thymidine kinase, DNA polymerase, and helicase, which, if mutated may affect the effectiveness of acyclovir, foscarnet, cidofovir, and helicase-primase inhibitors. The present study aimed to extensively characterize UL23, UL30, and UL5 genes. A total of 239 clinical HSV1 recovered from patients admitted to the hematology departments of the Lyon teaching hospitals were included in this single-center retrospective study. Drug resistance was evaluated using the neutral red dye-uptake assay, and sequencing using the Sanger method. Additional information on HSV1 natural polymorphism and resistance is now available. Twenty-two amino acid substitutions related to polymorphism were described on UL23 (E43A, L50M, L68R, Q109K, A133V, A136N, S150L, D258N, S263L, P280S, N301S, A316S, M322L, I326V, D330A, D338H, Q342H, T344I, Q349R, V352L, R370W, E371D), and 6 amino acid substitutions on UL30 (G641R, G645D, E649G, G679D, R681L, I966M). Moreover, the UL23 substitution L242P was added to ACV resistance-related mutations. There were 12 substitutions on UL23 (A37S, V70M, S74L, H151N, P154S, P155Q, L159R, E225L, Y248H, Q270R, N303Y, M372I), and 8 on UL5 (L49I, L138V, S173L, A280T, A575V, V600A, A602T, D862N) that remain of unclear significance with regards to drug resistance. The mean (±standard deviation, SD) number of natural polymorphisms in UL23 was 2.53 (±2.55), in UL30 it was 0.83 (±1.02), and in UL5 it was 5.00 (±1.59) There was no association between HSV1 phenotype and the frequency of substitutions. The results reported herein provide valuable new information concerning HSV1 mutations that will assist the interpretation of genotypic assays.

Schedule-dependent synergistic effects of 5-fluorouracil and selumetinib in KRAS or BRAF mutant colon cancer models.

Combination of MEK inhibitor and 5-FU had showed limited efficacy in clinical trials. We previously reported that acquired resistance to 5-FU was related with continued activation of salvage pathway. Here we investigated whether combination of 5-FU and a MEK inhibitor had treatment sequence-dependent synergistic effects in KRAS or BRAF mutant colon cancer models. Treatment with 5-FU followed by selumetinib (FS) induced highest cell death and synergy compared with reverse (SF) and concomitant (cFS) treatment in six cell lines. SF or cFS combination induced synergy in 1 or 2 cell lines, respectively, in which the synergy was less than that by FS combination. FS enhanced apoptosis and decreased anchorage-independent growth. Induction of thymidine kinase 1, a rate-limiting enzyme in salvage pathway, by 5-FU was abrogated by subsequent treatment with selumetinib, and ERK reactivation after selumetinib was prohibited by pretreatment with 5-FU. FS altered mRNA expression in groups of genes distinct from SF. Administration of 5-FU (10 or 30 mg/kg/day) for 7 days, followed by selumetinib (10 or 25 mg/kg/day) for another 7 days, in colo205 and HCT8 xenograft models significantly decreased tumor growth compared with a single agent. However, co-administration in the reverse sequence did not show the difference in tumor size compared with the treatment of single agent. Decreased expression of Ki67 was observed in tumors from mice treated with FS. Our results suggest that sequential administration of 5-FU plus selumetinib would be a promising strategy for patients having KRAS or BRAF mutant colon cancers.

Prodrugs of herpes simplex thymidine kinase inhibitors.

BACKGROUND: Because guanine-based herpes simplex virus thymidine kinase inhibitors are not orally available, we synthesized various 6-deoxy prodrugs of these compounds and evaluated them with regard to solubility in water, oral bioavailability, and efficacy to prevent herpes simplex virus-1 reactivation from latency in a mouse model. METHODS: Organic synthesis was used to prepare compounds, High Performance Liquid Chromatography (HPLC) to analyze hydrolytic conversion, Mass Spectrometry (MS) to measure oral bioavailability, and mouse latent infection and induced reactivation to evaluate the efficacy of a specific prodrug. RESULTS: Aqueous solubilities of prodrugs were improved, oxidation of prodrugs by animal cytosols occurred in vitro, and oral absorption of the optimal prodrug sacrovir™ (6-deoxy-mCF3PG) in the presence of the aqueous adjuvant Soluplus® and conversion to active compound N(2)-[3-(trifluoromethyl)pheny])guanine (mCF3PG) were accomplished in mice. Treatment of herpes simplex virus-1 latent mice with sacrovir™ in 1% Soluplus in drinking water significantly suppressed herpes simplex virus-1 reactivation and viral genomic replication. CONCLUSIONS: Ad libitum oral delivery of sacrovir™ was effective in suppressing herpes simplex virus-1 reactivation in ocularly infected latent mice as measured by the numbers of mice shedding infectious virus at the ocular surface, numbers of trigeminal ganglia positive for infectious virus, number of corneas that had detectable infectious virus, and herpes simplex virus-1 genome copy numbers in trigeminal ganglia following reactivation. These results demonstrate the statistically significant effect of the prodrug on suppressing herpes simplex virus-1 reactivation in vivo.

Synthesis and evaluation of thymidine kinase 1-targeting carboranyl pyrimidine nucleoside analogs for boron neutron capture therapy of cancer.

A library of sixteen 2nd generation amino- and amido-substituted carboranyl pyrimidine nucleoside analogs, designed as substrates and inhibitors of thymidine kinase 1 (TK1) for potential use in boron neutron capture therapy (BNCT) of cancer, was synthesized and evaluated in enzyme kinetic-, enzyme inhibition-, metabolomic-, and biodistribution studies. One of these 2nd generation carboranyl pyrimidine nucleoside analogs (YB18A [3]), having an amino group directly attached to a meta-carborane cage tethered via ethylene spacer to the 3-position of thymidine, was approximately 3-4 times superior as a substrate and inhibitor of hTK1 than N5-2OH (2), a 1st generation carboranyl pyrimidine nucleoside analog. Both 2 and 3 appeared to be 5'-monophosphorylated in TK1(+) RG2 cells, both in vitro and in vivo. Biodistribution studies in rats bearing intracerebral RG2 glioma resulted in selective tumor uptake of 3 with an intratumoral concentration that was approximately 4 times higher than that of 2. The obtained results significantly advance the understanding of the binding interactions between TK1 and carboranyl pyrimidine nucleoside analogs and will profoundly impact future design strategies for these agents.

Testing the sensitivities of noncognate inhibitors to varicella zoster virus thymidine kinase: implications for postherpetic neuralgia therapy with existing agents.

Varicella zoster virus (VZV), a member of the human herpesvirus family, affects peripheral or cranial nerves and can reactivate years after the primary infection. Thymidine kinase (TK) is essential for VZV replication, and its active site is highly conserved in the herpesvirus family. A number of small-molecule inhibitors have already been successfully developed that target the TK of herpes simplex virus type 1 (HSV-1), which is one of the most prevalent sexually transmitted infections worldwide. In the present study, we attempted to test the sensitivities of HSV-1 TK inhibitors to their noncognate VZV TK by integrating in silico modeling and an in vitro assay. We tested nine representative HSV-1 TK inhibitors, including three FDA-approved drugs and six compounds that are under clinical development. The structures of the complexes of these inhibitor ligands with HSV-1 TK and noncognate VZV TK had been solved previously by X-ray crystallography or were modeled in the present work using a template-based approach. Subsequently, a rigorous quantum mechanics/molecular mechanics (QM/MM) nonbonded analysis that accounted for the Poisson-Boltzmann/surface area (PB/SA) solvent effect was employed to refine the complex structures and, on this basis, to evaluate the binding potencies of these complexes. As might be expected, the QM/MM-PB/SA-derived free energy was shown to be highly correlated with the HSV-1 TK inhibitory activities of the nine inhibitors. Further, it was found that the HSV-1 TK inhibitors exhibit strong binding affinities for their noncognate VZV TK, although they are still more selective for HSV-1 TK than for VZV TK. In order to test the theoretical results obtained from the computational analysis, we performed an in vitro kinase assay to determine the inhibitory potencies of three commercially available antiviral agents, namely penciclovir, ganciclovir, and aciclovir, against their noncognate target VZV TK, resulting in IC50 values of 86, 127, and 150 µM respectively, which are modestly weaker than the corresponding values obtained for HSV-1 TK. In addition, visual structure examination and virtual mutation/deletion analysis suggested that the residue Arg222 is present at the active site of HSV-1 TK but not at the active site of VZV TK, which is the reason for the difference in inhibitor selectivity between HSV-1 TK and VZV TK.

Tomato thymidine kinase is subject to inefficient TTP feedback regulation.

A promising suicide gene therapy system to treat gliomas has been reported: the thymidine kinase 1 from tomato (toTK1) combined with the nucleoside analog pro-drug zidovudine (azidothymidine, AZT), which is known to penetrate the blood-brain barrier. Transduction with toTK1 has been found to efficiently increase the sensitivity of human glioblastoma cells to AZT, and nude rats with intracranial glioblastoma grafts have shown significantly improved survival when treated with the toTK1/AZT system. We show in our paper that the strong suicidal effect of AZT together with toTK1 may be explained by reduced TTP-mediated feedback inhibition of the AZT phosphorylation.

Long-term follow-up of HIV-infected patients once diagnosed with acyclovir-resistant herpes simplex virus infection.

Acyclovir-resistant herpes simplex virus (HSV) infection is common in immunocompromised patients, but the course of such infection is little known. We describe the long-term follow-up of HIV-infected patients diagnosed once with acyclovir-resistant HSV infections. We retrospectively studied all HIV-infected patients between 2000 and 2010 diagnosed with virologically confirmed acyclovir-resistant HSV infection. Patients' socio-demographic and immunovirological characteristics were described. Response to foscarnet or cidofovir and recurrences were reported. Among 5295 HIV-infected patients, 13 (0.2%) were once diagnosed with an acyclovir-resistant HSV infection. Twelve patients were men, nine patients were of African origin. All patients reported previous acyclovir exposure and median CD4 count was 183 cells/mm(3) Ten patients presented exclusively with cutaneous lesions. Initially, 11 patients were treated with foscarnet and two with cidofovir. The median follow-up was 67 months (6-145). All patients recurred, 10 presenting at least one acyclovir-resistant HSV recurrence. The median number of acyclovir-resistant HSV recurrences per patient was 2 (0 - 5). Regarding the first and second recurrences, 7/13 (54%) and 5/11 (45%) HSV clinical isolates exhibited resistance to acyclovir, respectively. Acyclovir-resistant HSV infection prevalence was low in our cohort. The rate of acyclovir-resistant HSV episodes averaged 50% during the two first recurrences.

2,3-Dihydroxy-quinoxaline induces ATPase activity of Herpes Simplex Virus thymidine kinase.

2,3-Dihydroxy-quinoxaline, a small molecule that promotes ATPase catalytic activity of Herpes Simplex Virus thymidine kinase (HSV-TK), was identified by virtual screening. This compound competitively inhibited HSV-TK catalyzed phosphorylation of acyclovir with Ki=250 µM (95% CI: 106-405 µM) and dose-dependently increased the rate of the ATP hydrolysis with KM=112 µM (95% CI: 28-195 µM). The kinetic scheme consistent with this experimental data is proposed.

Hairy cell leukemia-new genes, new targets.

Hairy cell leukemia (HCL), a B cell malignancy comprising 2 % of all leukemias, has become quite exciting recently with regard to the development of new targets for therapy. This review will focus on advancements made within the past 1-2 years in targeted therapy for this disease. These advances may be grouped into two very difference categories, namely targeting of CD22 with the recombinant immunotoxin moxetumomab pasudotox, and targeting of the mutated BRAF component of the MAP kinase pathway. Moxetumomab pasudotox in phase I testing was recently reported to be associated with an overall response rate of 86 % and a complete remission (CR) rate of 46 % in 28 patients with relapsed and refractory HCL. Many of the CRs are without minimal residual disease (MRD). Severe or dose limiting toxicity was not observed on this trial, but a completely reversible and largely asymptomatic form of grade 2 hemolytic uremic syndrome occurred in two patients during retreatment. This agent has commenced phase III multicenter testing to validate its phase I results. An extensive number of studies have documented the V600E mutation in nearly all HCL patients, but not in similar hematologic malignancies. The thymidine kinase inhibitor vemurafenib, which inhibits the V600E mutant of BRAF, was reported to induce a CR in multiply relapsed and refractory HCL, with nearly complete clearing of MRD. One additional partial and one additional complete remission were subsequently reported.

Synthesis of N3-substituted carboranyl thymidine bioconjugates and their evaluation as substrates of recombinant human thymidine kinase 1.

Four different libraries of overall twenty three N3-substituted thymidine (dThd) analogues, including eleven 3-carboranyl thymidine analogues (3CTAs), were synthesized. The latter are potential agents for Boron Neutron Capture Therapy (BNCT) of cancer. Linker between the dThd scaffold and the m-carborane cluster at the N3-position of the 3CTAs contained amidinyl-(3e and 3f), guanidyl-(7e-7g), tetrazolylmethyl-(9b1/2-9d1/2), or tetrazolyl groups (11b1/2-11d1/2) to improve human thymidine kinase 1 (hTK1) substrate characteristics and water solubilities compared with 1st generation 3CTAs, such as N5 and N5-2OH. The amidinyl- and guanidyl-type N3-substitued dThd analogues (3a-3f and 7a-7g) had hTK1 phosphorylation rates of <30% relative to that of dThd, the endogenous hTK1 substrate, whereas the tetrazolyl-type N3-substitued dThd analogues (9a, 9b1/2-9d1/2 and 11a, 11b1/2-11d1/2) had relative phosphorylation rates (rPRs) of >40%. Compounds 9a, 9b1/2-9d1/2 and 11a, 11b1/2-11d1/2 were subjected to in-depth enzyme kinetics studies and the obtained rk(cat)/K(m) (k(cat)/K(m) relative to that of dThd) ranged from 2.5 to 26%. The tetrazolyl-type N3-substitued dThd analogues 9b1/2 and 11d1/2 were the best substrates of hTK1 with rPRs of 52.4% and 42.5% and rk(cat)/K(m) values of 14.9% and 19.7% respectively. In comparison, the rPR and rk(cat)/K(m) values of N5-2OH in this specific study were 41.5% and 10.8%, respectively. Compounds 3e and 3f were >1900 and >1500 times, respectively, better soluble in PBS (pH 7.4) than N5-2OH whereas solubilities for 9b1/2-9d1/2 and 11b1/2-11d1/2 were only 1.3-13 times better.

Recent advances in thymidine kinase 2 (TK2) inhibitors and new perspectives for potential applications.

Thymidine kinase 2 (TK2), encoded on chromosome 16q22 of the human genome, is a deoxynucleoside kinase (dNK) that catalyzes the phosphorylation of the pyrimidine deoxynucleosides 2'-deoxythymidine (dThd), 2'-deoxyuridine (dUrd) and 2'- deoxycytidine (dCyd) to the corresponding deoxynucleoside 5'-monophosphate derivatives. In contrast to the S-phase-specific thymidine kinase 1 (TK1), TK2 is constitutively expressed in the mitochondria and plays an important role in providing dNTPs for the replication and maintenance of mitochondrial DNA (mtDNA). The severe mitochondrial DNA depletion syndrome (MDS) has been associated with mutations in TK2, resulting in mtDNA depletion, isolated skeletal myopathy, and death of the individual at an early stage of life. Some antiviral nucleoside analogs, such as 3'-azido-dThd (AZT) that is targeting the human immunodeficiency virus (HIV)-encoded reverse transcriptase, are substrates for TK2 and it has been proposed that the mitochondrial toxicity observed after prolonged treatment with such drugs could be due to their interaction with TK2. Therefore, the design of specific TK2 inhibitors may be useful to investigate the role of TK2 in the maintenance and homeostasis of mitochondrial dNTP pools and its contribution to the mitochondrial toxicity of several antiviral and anticancer drugs. Since 2000, several potent and selective TK2 inhibitors have been described. Besides bringing together previously reported inhibitors, special attention will be paid in this review to the new families of TK2 inhibitors more recently described, together with modeling studies and biological assays. Moreover, the last section will be focused on several recent investigations that suggest that depletion of mtDNA can take place both in tumorigenesis and during cancer treatment with certain nucleoside analogues.

Study of [18F]FLT and [123I]IaraU for cellular imaging in HSV1 tk-transfected murine fibrosarcoma cells: evaluation of the tracer uptake using 5-fluoro, 5-iodo and 5-iodovinyl arabinosyl uridines as competitive probes.

As one of the most intensively studied probes for imaging of the cellular proliferation, [(18)F]FLT was investigated whether the targeting specificity of thymidine kinase 1 (TK1) dependency could be enhanced through a synergistic effect mediated by herpes simplex type 1 virus (HSV1) tk gene in terms of the TK1 or TK2 expression. 5-[(123)I]Iodo arabinosyl uridine ([(123)I]IaraU) was prepared in a radiochemical yield of 8% and specific activity of 21 GBq/µmol, respectively. Inhibition of the cellular uptake of these two tracers was compared by using the arabinosyl uridine analogs such as 5-iodo, 5-fluoro and 5-(E)-iodovinyl arabinosyl uridine along with 2'-fluoro-5-iodo arabinosyl uridine (FIAU). Due to potential instability of the iodo group, accumulation index of 1.6 for [(123)I]IaraU by HSV1-TK vs. control cells could virtually be achieved at 1.5 h, but dropped to 0.2 compared to 2.0 for [(18)F]FLT at 5 h. The results from competitive inhibition by these nucleosides against the accumulation of [(18)F]FLT implied that FLT exerted a mixed TK1- and TK2-dependent inhibition with HSV1-tk gene transfection because of the shifting of thymidine kinase status. Taken together, the combination of [(18)F]FLT and HSV1-TK provides a synergistic imaging potency.

[Cloning, expression, isolation and properties of thymidine kinase herpes simplex virus, strain L2].

A thymidine kinase UL23 gene (EC 2.7.1.145) from an acyclovir-sensitive strain L2 of herpes simplex virus type 1 was cloned and expressed in E. coli. Enzyme was purified by chromatography to a homogeneous state controlled by PAG electrophoresis. The Michaelis constants for the reactions with thymidine and an acyclovir were determined. It was found that enzyme phosphorilate some modified nucleosides such as d2T, d4T, d2C, 3TC, FLT, BVDU, GCV. A comparison of the purified enzyme properties and properties ofthymidine kinase of other strains of herpes simplex virus, previously published was carried out. It is shown that enzyme is inhibited by acyclovir H-phosphonate.

Effect of the tyrosine kinase inhibitor lapatinib on CUB-domain containing protein (CDCP1)-mediated breast cancer cell survival and migration.

The surface receptor CUB domain-containing protein 1 (CDCP1) is highly expressed in several adenocarcinomas and speculated to participate in anchorage-independent cell survival and cell motility. Tyrosine kinase phosphorylation seems to be crucial for intracellular signaling of CDCP1. Lapatinib, a tyrosine kinase inhibitor (TKI), is approved for treatment of HER-2/neu overexpressing metastatic breast cancer and functions by preventing autophosphorylation following HER-2/neu receptor activation. This study aimed to investigate the effect of CDCP1 expression on anchorage-independent growth and cell motility of breast cancer cells. Moreover, studies were performed to examine if lapatinib provided any beneficial effect on HER-2/neu((+)/-)/CDCP1(+) breast cancer cell lines. In our studies, we affirmed that CDCP1 prevents cells from undergoing apoptosis when cultured in the absence of cell-substratum anchorage and that migratory and invasive properties of these cells were decreased when CDCP1 was down-regulated. However, only HER-2/neu(+), but not HER-2/neu((+)/-) cells showed decreased proliferation and invasion and an enhanced level of apoptosis towards loss of anchorage when treated with lapatinib. Therefore, we conclude that CDCP1 might be involved in regulating adhesion and motility of breast cancer cells but that lapatinib has no effect on tyrosine kinases regulating CDCP1. Nonetheless, other TKIs might offer therapeutic approaches for CDCP1-targeted breast cancer therapy and should be studied considering this aspect.

Novel 1,2,4-triazole and purine acyclic cyclopropane nucleoside analogues: synthesis, antiviral and cytostatic activity evaluations.

BACKGROUND: Several published studies indicate that the acyclic guanine nucleoside analogues possessing bis(1,2-hydroxymethyl) substituted cyclopropane rings mimicking the sugar moiety are potent inhibitors of replication of several herpes viruses. METHODS: Established synthetic methods and antiviral and cytostatic activity assays were used for the evaluation of new 1,2,4-triazole and purine acyclic nucleoside analogues. RESULTS: The synthesis of new types of acyclic nucleoside analogues which incorporate 1,2,4-triazole or purine moiety bound via flexible methylenic spacer to the bis(1,2-hydroxymethyl) cyclopropane ring. None of the new compounds showed pronounced antiviral activities at subtoxic concentrations on a broad panel of DNA and RNA viruses. Evaluation of their affinity for herpes simplex type 1 (HSV-1) and varicella-zoster virus-encoded thymidine kinases (VZV TK) also showed that none of the compounds was able to significantly inhibit 1 µM deoxythymidine phosphorylation by HSV-1 and VZV TK at 500 µM concentrations. The in vitro cytostatic activity evaluation results indicated a weak antiproliferative activity for all tested compounds. Only 6-pyrrolylpurine derivative bearing a carboxylic group substituted cyclopropane ring produced a rather slight inhibitory effect at higher micromolar concentrations on a breast carcinoma cell line (MCF-7) and no cytotoxic effect on human normal fibroblasts (WI 38). CONCLUSIONS: The lack of antiherpetic activity may be due to poor, if any, recognition of the compounds by virus-induced nucleoside kinases as an alternative substrate to become metabolically activated.

Combining small interfering RNAs targeting thymidylate synthase and thymidine kinase 1 or 2 sensitizes human tumor cells to 5-fluorodeoxyuridine and pemetrexed.

Thymidylate synthase (TS) is the only de novo source of thymidylate (dTMP) for DNA synthesis and repair. Drugs targeting TS protein are a mainstay in cancer treatment, but off-target effects and toxicity limit their use. Cytosolic thymidine kinase (TK1) and mitochondrial thymidine kinase (TK2) contribute to an alternative dTMP-producing pathway, by salvaging thymidine from the tumor milieu, and may modulate resistance to TS-targeting drugs. Combined down-regulation of these enzymes is an attractive strategy to enhance cancer therapy. We have shown previously that antisense-targeting TS enhanced tumor cell sensitivity to TS-targeting drugs in vitro and in vivo. Because both TS and TKs contribute to increased cellular dTMP, we hypothesized that TKs mediate resistance to the capacity of TS small interfering RNA (siRNA) to sensitize tumor cells to TS-targeting anticancer drugs. We assessed the effects of targeting TK1 or TK2 with siRNA alone and in combination with siRNA targeting TS and/or TS-protein targeting drugs on tumor cell proliferation. Down-regulation of TK with siRNA enhanced the capacity of TS siRNA to sensitize tumor cells to traditional TS protein-targeting drugs [5-fluorodeoxyuridine (5FUdR) and pemetrexed]. The sensitization was greater than that observed in response to any siRNA used alone and was specific to drugs targeting TS. Up-regulation of TK1 in response to combined 5FUdR and TS siRNA suggests that TK knockdown may be therapeutically useful in combination with these agents. TKs may be useful targets for cancer therapy when combined with molecules targeting TS mRNA and TS protein.

The cytostatic activity of NUC-3073, a phosphoramidate prodrug of 5-fluoro-2'-deoxyuridine, is independent of activation by thymidine kinase and insensitive to degradation by phosphorolytic enzymes.

A novel phosphoramidate nucleotide prodrug of the anticancer nucleoside analogue 5-fluoro-2'-deoxyuridine (5-FdUrd) was synthesized and evaluated for its cytostatic activity. Whereas 5-FdUrd substantially lost its cytostatic potential in thymidine kinase (TK)-deficient murine leukaemia L1210 and human lymphocyte CEM cell cultures, NUC-3073 markedly kept its antiproliferative activity in TK-deficient tumour cells, and thus is largely independent of intracellular TK activity to exert its cytostatic action. NUC-3073 was found to inhibit thymidylate synthase (TS) in the TK-deficient and wild-type cell lines at drug concentrations that correlated well with its cytostatic activity in these cells. NUC-3073 does not seem to be susceptible to inactivation by catabolic enzymes such as thymidine phosphorylase (TP) and uridine phosphorylase (UP). These findings are in line with our observations that 5-FdUrd, but not NUC-3073, substantially loses its cytostatic potential in the presence of TP-expressing mycoplasmas in the tumour cell cultures. Therefore, we propose NUC-3073 as a novel 5-FdUrd phosphoramidate prodrug that (i) may circumvent potential resistance mechanisms of tumour cells (e.g. decreased TK activity) and (ii) is not degraded by catabolic enzymes such as TP which is often upregulated in tumour cells or expressed in mycoplasma-infected tumour tissue.