Inhibition Of Thymidine Kinase Research Articles

Identification of novel potential inhibitors of monkeypox virus thymidine kinase using molecular docking, molecular dynamics simulation and MM/PBSA methods.

The monkeypox spread has been announced a public health emergency of international concern (PHEIC) by the World Health Organization (WHO). Both monkeypox and smallpox viruses are placed in the genus Orthopoxvirus. Despite recommendations for the administration of smallpox drugs versus monkeypox, no specific drug for monkeypox has yet been introduced. A reliable and effective method against this outbreak can be the use of natural products. This study aimed for identification of natural flavonoid derivatives as potential thymidine kinase inhibitors, the main drug target of monkeypox virus. Thymidine kinase protein structure was predicted by homology modeling and the quality of generated model was evaluated. Then, the interaction between natural flavonoids and the modeled thymidine kinase was explored by molecular docking. Based on docking results, more than half of the flavonoids with higher docking scores compared to reference drug (ganciclovir) were exhibited better binding affinities toward the protein. In addition, stability of the top flavonoids including eupatorin, fisetin, rhamnetin and scutellarein, was confirmed by MD simulations and binding free energy calculations using MM/PBSA analysis. These selected compounds were also shown acceptable results for drug likeness and ADMET analysis. Therefore, the results of the study showed that these flavonoids could be considered as potential thymidine kinase inhibitors for use against monkeypox virus.

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

Herpès chronique périnéal résistant à l’aciclovir révélateur d’une leucémie lymphoïde chronique

Chronic HSV infection is a cause of chronic perineal ulcerations. We report a case of a chronic and refractory HSV infection revealing chronic lymphoid leukaemia. An 85-year-old woman with an 8-month history of chronic perineal ulcerations was referred to our dermatology department. She had no previous medical history of herpes infection. Skin biopsies ruled out carcinoma but were consistent with HSV infection. A local swab was positive for HSV2. Treatment with valaciclovir and intravenous acyclovir (ACV) at the recommended doses was ineffective. Laboratory tests revealed type-B chronic lymphoid leukaemia. Molecular biology studies confirmed the presence of ACV-resistant HSV via decreased thymidine kinase activity (stop codon: M183stop). Foscarnet was administered for a period of 3weeks with almost complete healing of the ulcerations. Treatment was stopped prematurely due to acute renal insufficiency and the remaining lesions were treated using imiquimod cream. Valaciclovir was prescribed to prevent further episodes. The condition recurred a mere 11months later. The prevalence of ACV-resistant HSV is 0.32% in immunocompetent patients and 3.5% in immunocompromised patients. Insufficient dosing regimens or prolonged treatment with TK inhibitors result in the local selection of pre-existing mutant HSV viruses. Foscarnet, a DNA polymerase inhibitor, is the treatment of choice in HSV-resistant infections. ACV-resistant HSV is less virulent and replicates less, with reactivations being mainly due to wild-type HSV latent in the neural ganglia. Valaciclovir can be used as a preventive treatment. To our knowledge, this is the first case of ACV-resistant HSV infection revealing chronic lymphoid leukaemia. Chronic perineal ulcerations can be the first manifestation of immunodeficiency seen for example with haematological diseases. In the event of clinical resistance of an HSV infection to recommended thymidine kinase inhibitor regimens, the use of foscarnet should be considered.

Abstract 1431: Incidence and natural history of cardiovascular toxicity associated with biologic and targeted therapies used in hematologic malignancies

Abstract INTRODUCTION: Unanticipated cardiac toxicity has been reported with anti cancer targeted and biological treatments. In this retrospective study, we aim at identifying the number of cardiac toxicity cases among patients with hematologic malignancies (HM) who were treated with targeted therapies over 10-year period with emphasis on natural history and outcomes. METHODS: With the help of the Faculty Practice Decision Support (FPDS) team, we used 114 diagnosis codes for HM and 17 codes for cardiac diseases in order to mine our electronic medical records (EPIC) and identify patients with HM and specific cardiac problems, then sorted them according to whether they received known biologic agents such as thymidine kinase inhibitors (TKIs), proteasome inhibitors, monoclonal antibodies, hypomethylating agents, and immunomodulatory drugs. RESULTS: FPDS team provided us with a file that contained medical records of 820 patients that had both HM and cardiac abnormalities. We pulled out 53 patients who received the drugs of interest. We were able to confirm cardiac toxicity defined mainly by left ventricular ejection fraction (LVEF) of < 50%, arrhythmias, or ischemic cardiovascular event in 44 patients. Ten patients were excluded because of pre-existing cardiac disease. Thus the final analysis was performed on the remaining 34 patients. The median follow-up was 9 mo (range, 1-78). The median age of this group was 66 years (range, 27-81), male/female 19/15, 26 Caucasians, and 15 were still alive. The median time from exposure to development of cardiac events was 120 days (range, <1-300). The diagnosis distribution was as follows: multiple myeloma 16, B-cell non-Hodgkin's lymphoma (NHL) 10, follicular NHL 4, Philadelphia positive acute lymphoblastic leukemia 3, MDS 1. Number of patients (n) who received suspected drugs contributing to the cardiac toxicity: bortezomib (8), carfilzomib (4), imatinib (2), dasatinib (4), Rituximab (13), and lenalidomide (3). Twenty one patients had no prior cardiac disease, while 11 had known coronary artery disease and 2 had known arrhythmias, while only 9 had no chronic co-morbidities. Fourteen patients did not receive anthracyclines including 2 who received rituximab. Nine patients had elevation in troponin and were diagnosed with NSTE myocardial infarction at the time of cardiac toxicity. Among the 19 dead patients, only 6 died of cardiac causes, while the rest died of their cancers. Repeat echocardiograms showed worsening of LVEF in 5 patients and improvement in 15 patients. CONCLUSIONS: About 4.1% of patients with HM experience unanticipated cardiac toxicity with related mortality of 17.6%. Most patients do well with stable compensated cardiac function with objective improvement in LVEF. Whether Rituximab can cause cardiac toxicity is controversial and we will present specific arguments. Citation Format: Jan S. Moreb, Haneen Mohammad, Lindsay McCullough, Anita Szady, Leslie Pettiford, Alexandra Lucas. Incidence and natural history of cardiovascular toxicity associated with biologic and targeted therapies used in hematologic malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1431.

Protein synthesis inhibitors of natural origin for CML therapy: semisynthetic homoharringtonine (Omacetaxine mepesuccinate).

Omacetaxine mepesuccinate is a drug approved in 2014 by FDA for the use in CML therapy in patients resistant to at least two thymidine kinase inhibitors (TKIs). It possesses unique mechanism of anticancer activity that is principally different from mechanism of activity of TKIs. Omacetaxine mepesuccinate inhibits protein translation through prevention of the initial elongation step of protein synthesis and its use benefits CML patients possessing the BCR-ABL oncogene. Because of the superior activity of Omacetaxine in patients who became resistant to therapy with TKIs, FDA decided on the accelerated approval of this drug taking its consideration not only its activity as such but also a favorable benefit-to-risk profile in patients included into clinical studies.

A comparative study of replicative properties of antitumor recombinant vaccinia viruses on human glioblastoma cell culture U87 and monkey kidney cell culture CV-1

In the world of today, virotherapy is one of the rapidly developing areas in the treatment of cancer, and its advantage is selective destruction of cancer cells with minimizing the destructive effect on normal cells of the body. A promising basis for the creation of oncolytic drugs is orthopoxviruses, which have a number of advantages over other viral vectors, and one of these advantages is a large capacity of the genome, which allows genes encoding proteins with antitumor properties to be cloned into their genome. In this study, we compared the replicative properties of ten variants of vaccinia virus (the strain LIVP of VACV) using human glioblastoma cell culture; some of these viruses have additional genes, such as the gene encoding granulocyte-macrophage colony stimulating factor, gene encoding apoptosis-inducing protein TRAIL and gene encoding green fluorescent protein. Furthermore, the virus with five virulence genes deleted (genes encoding hemagglutinin, γ-interferonbinding protein, thymidine kinase, complementbinding protein and Bcl2-like inhibitor of apoptosis), which has significantly lower reactogenicity and neurovirulence compared to the original strain LIVP of VACV, was studied. These data suggest that variants of vaccinia virus with a defective gene encoding thymidine kinase most actively replicate in glioblastoma cell culture.

Highly immunogenic variant of attenuated vaccinia virus.

The LIVPΔ6 strain of vaccinia virus (VACV) was created by genetic engineering on the basis of previously obtained attenuated 1421ABJCN strain by target deletion of the A35R gene encoding an inhibitor of antigen presentation by the major histocompatibility complex class II. 1421ABJCN is the LIVP strain of VACV with five inactivated virulence genes encoding hemagglutinin (A56R), γ-interferon-binding protein (B8R), thymidine kinase (J2R), complement-binding protein (C3L), and Bcl2-like inhibitor of apoptosis (N1L). The highly immunogenic LIVPΔ6 strain could be an efficient fourth-generation attenuated vaccine against smallpox and other orthopoxvirus infections.

Inhibiting cyprinid herpesvirus-3 replication with CRISPR/Cas9.

The potential of CRISPR/Cas9 gene editing to repress CyHV-3 was tested in vitro. By targeting two basic target genes necessary for the early transcription of CyHV-3, we show that virus transcription and particle release were significantly decreased by CRISPR/Cas9, as measured by quantitative real-time PCR and virus titration experiments, respectively. (A) The effectiveness is confirmed of the CRISPR/Cas9 system at repressing exogenous genes, including large viral genomic DNA, by introducing site-specific mutations in vitro. (B) The CyHV-3 virus replicates poorly in Cas9-positive cells. (C) The inhibition of thymidine kinase alone cannot block viral particle release.

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.

Prodrugs of herpes simplex thymidine kinase inhibitors

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. 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. Aqueous solubilities of prodrugs were improved, oxidation of prodrugs by animal cytosols occurred invitro, 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. 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 invivo.

Abstract B56: High sensitivity characterization of KRAS mutant subpopulations in normal lung tissue and adenocarcinomas of the lung

Abstract KRAS tumor mutational status predicts response to therapies that target the epidermal growth factor receptor (EGFR) and is being used to make treatment decisions for individual colon cancer patients. There is evidence showing that undetected KRAS mutant tumor subpopulations can subvert durable responses, lead to drug resistance, and drive relapse in colon cancer patients treated with EGFR-targeted therapies. Patients with adenocarcinomas of the lung are treated with EGFR thymidine kinase inhibitors and, theoretically, the efficacy of these treatments could be influenced by KRAS mutational status, even though KRAS mutational status is not currently evaluated prior to treatment of lung cancer patients. In order to assess the potential for undetected KRAS mutant subpopulations to impact response in lung adenocarcinoma patients treated with EGFR thymidine kinase inhibitors, the sensitive allele-specific competitive blocker polymerase chain reaction (ACB-PCR) method was used to quantify KRAS codon 12 GGT to GAT and GGT to GTT mutant fraction (MF) in 19 normal lung samples and 21 primary lung adenocarcinomas. ACB-PCR is a type of allele-specific amplification that can measure 3 mutant alleles in a background of 300,000 normal alleles and gives a quantitative readout, rather than reporting the presence/absence of mutation. ACB-PCR was used to quantify KRAS mutation in normal lung and lung tumor tissue in order to empirically define pathological levels of KRAS mutation as those above the upper 95% confidence interval of that present in normal lung. Measurable levels (≥10-5) of KRAS codon 12 GTT and/or GAT mutations were observed in 17/19 (89%) of normal lung samples. The KRAS codon 12 GAT and GTT geometric mean MFs in normal lung were 3.25 x 10-5 and 3.15 x 10-5, respectively. Normal colonic mucosa and normal lung tissue contain similar levels of KRAS codon 12 GAT mutation, however normal lung tissue has a significantly higher level of the KRAS codon 12 GTT mutation than colonic mucosa. All lung adenocarcinomas had measurable levels of both mutations. Adenocarcinomas from male smokers had significantly greater KRAS codon 12 GTT MFs than adenocarcinomas from female smokers. The highest observed KRAS codon 12 GAT MFs were associated with tumors characterized as having relatively low necrosis and tumor cell content. Importantly, 47.6% and 57.1% of lung adenocarcinomas had KRAS codon 12 GAT and GTT MFs greater than the upper 95% confidence interval for MF in normal lung (1.02 x 10-4 and 9.16 x 10-5, respectively). In addition, 28% of lung adenocarcinomas had abnormally-high levels of both mutations. Extrapolation of these results to include additional KRAS hotspot point mutations leads to the conclusion that most, if not all, lung adenocarcinomas possess greater KRAS mutant cell content than normal lung tissue. These results suggest there is a strong possibility that treatment of lung adenocarcinoma patients with therapies that target EGFR may select for the outgrowth of KRAS mutant subpopulations and contribute to the development of drug resistance and relapse. Citation Format: Meagan B. Myers, Karen L. McKim, Fanxue Meng, Barbara L. Parsons. High sensitivity characterization of KRAS mutant subpopulations in normal lung tissue and adenocarcinomas of the lung. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B56. doi: 10.1158/1557-3125.RASONC14-B56

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.

Abstract 4227: Simple identification of specific mutations using allele-specific PCR primers and micro capillary electrophoresis (WAVE MCE).

Abstract Transgenomic, Inc. has developed a size-based analysis for the detection of all EGFR Exon 19 deletions (including complex mutation) greater than 6 base pairs (ACE™ Kit EGFR Exon 19). Currently there are over 70 known EGFR Exon 19 deletions in the region important for response of non-small cell lung cancer (NSCLC) to thymidine kinase inhibitors (TKIs). The majority of these deletions are rare, but the patients may benefit from TKI treatment if the deletion is present. In most current testing paradigms, a NSCLC patient with one of these rare mutations would be considered EGFR mutation negative and not receive TKI therapy. We have designed an easy, size-based PCR assay to detect any of the deletions present in any sample after a single PCR reaction. The assay is based on the separation of PCR fragments of different sizes using microcapillary electrophyoresis (MCE). Transgenomic's WAVE® MCE platform can discriminate the deletions from the wild-type sequence in EGFR Exon 19, however the exact nature of the mutation cannot be determined. If the exact nature of the mutation is required, the PCR fragment can be sequenced. To confirm that the platform can detect common and rare mutations, plasmids containing various EGFR Exon 19 deletions were synthesized, mixed with EGFR wild-type plasmids and analyzed on the WAVE® MCE platform. The Limit of detection for the plasmid mixing experiments is between 0.5 and 1% for all plasmid constructs tested. These plasmids include 6, 9, 15, 21, 24, and 25 bp deletions . As all samples tested have some wild-type sequence present, 6 base pair deletions cannot be resolved from the wild-type fragment. Several of the deletion constructs contain those not detected by any allele-specific kit, however patients with these deletions, as determined by Sanger sequence analysis, have benefitted from TKI therapy. In addition cell line DNA and DNA isolated from FFPE sections have been analyzed for the detection of EGFR Exon 19 deletions and similar limits of detection (0.5-1.0%) were seen. This simple, size-based analysis for EGFR Exon deletions will result in NSCLC patients with both common and rare EGFR Exon 19 deletions being able to receive the treatment option best suited for their molecular profile. Citation Format: Katherine A. Richardson, Grant Wu, Rui Lin, Yanggu Shi, Phil Eastlake, Benjamin Legendre. Simple identification of specific mutations using allele-specific PCR primers and micro capillary electrophoresis (WAVE MCE). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4227. doi:10.1158/1538-7445.AM2013-4227

Text-based similarity searching for hit- and lead-candidate identification

The Pharmacophore Alignment Search Tool (PhAST) is a string-based approach to virtual screening. Molecules are represented by linear sequences which describe their respective pattern of interaction possibilities. The problem of molecule linearization is tackled by applying Minimum Volume Embedding in combination with a Diffusion Kernel to the molecular graph [1,2]. Linear representations are compared using global pairwise sequence alignment [3]. PhAST exhibited enrichment capabilities comparable or superior to most common virtual screening approaches. Compound rankings were proven to be dissimilar to those of other virtual screening techniques. It was shown that emphasis on key interactions through the application of position specific weights in the alignment process significantly increases enrichment. Significance of chemical similarity was determined in form of p-values of global alignment scores, calculated in an approach that was adapted from its original application to local sequence alignments of protein sequences utilizing Marcov chain Monte Carlo simulation [4]. Bonferroni correction was used to correct p-values with respect to the size of the screening library [5]. PhAST was employed in two prospective applications: A screening for non-nucleoside analogue inhibitors of bacterial thymidine kinase yielded a hit with a distinct structural framework but only weak activity. Screenings for drugs that are not members of the NSAID (non-steroidal anti-inflammatory drug) class as modulators of gamma secretase resulted in a potent modulator with clear structural distinction from the reference compound.

Herpes Simplex Virus Thymidine Kinase Inhibitor GLS122E and Its 6-Deoxy Prodrug GLS361B (Sacrovir™)—Potential for Preventing Viral Disease Recurrence In Vivo

Herpes Simplex Virus Thymidine Kinase Inhibitor GLS122E and Its 6-Deoxy Prodrug GLS361B (Sacrovir™)—Potential for Preventing Viral Disease Recurrence In Vivo

Effect of 3‘‐Azido‐3’‐deoxythymidine on 5‐Fluorouracil Metabolism in Human Promyelocyte Leukaemia HL‐60 Cells*

It was shown earlier that combined exposure of 5-fluorouracil and azidothymidine (AZT) causes synergistic cytotoxicity in various human tumour cell lines. The influence of AZT, a competitive inhibitor of thymidine kinase, on the metabolism of 5-fluorouracil was investigated in this study. Sequential combination treatment with 100 μM AZT and 5-fluorouracil decreased the 50% growth inhibitory concentration of 5-fluorouracil (IC50) from 5.98 μM in untreated control cells to 3.96 μM in cells pretreated with AZT. AZT did not alter the intracellular levels of 5-fluorouracil but significantly increased intracellular fluorodeoxyuridine levels as determined by high performance-liquid chromatography (1.97 ± 0.64 vs 3.69 ± 0.67 pmol/50 times 106 cells). Intracellular levels of fluorouridine increased slightly but not significantly from 6.43 ± 1.99 to 7.49 ± 1.54. However, fluorodeoxyuridine monophosphate dropped in cells preincubated with AZT from 8.34 ± 1.04 to 5.91 ± 5.91 pmol/50 times 106 cells as well as in the culture supernatant (4.37 ± 0.87 vs. 2 44 ± 0.43 pmol/50 times 106 cells). These results indicate that the inhibition of thymidine kinase by AZT might increase intracellular levels of fluorodeoxyuridine and fluorouridine, thus causing more pronounced cytotoxic effects.

Synthesis, modeling and evaluation of 3′-(1-aryl-1H-tetrazol-5-ylamino)-substituted 3′-deoxythymidine derivatives as potent and selective human mitochondrial thymidine kinase inhibitors

Based on the presumed binding mode of an earlier identified inhibitor, we herein report new 3'-modified nucleosides as potent and selective inhibitors of mitochondrial thymidine kinase (TK2). A series of thirteen 3'-amino-, 3'-guanidino- and 3'-tetrazole-containing nucleosides were synthesized and evaluated for their TK2 inhibitory activity. Within the tetrazole series, compounds with nanomolar inhibitory activity were identified. A homology model of TK2 allowed to elucidate the observed activities. Introduction of a 2-bromovinyl group on C-5 of the pyrimidine base of the most promising 3'-derivative further improved the inhibitory activity, and caused a significant increase in the selectivity for TK2 versus TK1. Interestingly, for the current series of analogues, a strong correlation was observed between TK2 and Drosophila melanogaster dNK inhibition, further substantiating the phylogenetic relationship between these two nucleoside kinases.

Multistep Virtual Screening for Rapid and Efficient Identification of Non‐Nucleoside Bacterial Thymidine Kinase Inhibitors

Antimicrobial activity of trimethoprim/sulfamethoxazole (SXT) against Staphylococcus aureus (S. aureus) is antagonized by thymidine, which is abundant in infected or inflamed human tissue. To restore the antimicrobial activity of SXT in the presence of thymidine, we screened for small-molecule inhibitors of S. aureus thymidine kinase with non-nucleoside scaffolds. We present the successful application of an adaptive virtual screening protocol for novel antibiotics using a combination of ligand- and structure-based approaches. Two consecutive rounds of virtual screening and in vitro testing were performed that resulted in several non-nucleoside hits. The most potent compound exhibits substantial antimicrobial activity against both methicillin-resistant S. aureus strain ATCC 700699 and nonresistant strain ATCC 29213, when combined with SXT in the presence of thymidine. This study demonstrates how virtual screening can be used to guide hit finding in antibacterial screening campaigns with minimal experimental effort.

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.

Effect of combinations of antiviral drugs on herpes simplex encephalitis

2-Phenylamino-6-oxo-9-(4-hydroxybutyl)purine (HBPG) is a thymidine kinase inhibitor that prevents encephalitic death in mice caused by herpes simplex virus (HSV) types 1 and 2, although its potency is somewhat less than that of acyclovir (ACV). The present study was undertaken to determine the effect of combinations of HBPG and either ACV, phosphonoformate (PFA), or cidofovir (CDF) against HSV encephalitis. BALB/c mice were given ocular infections with HSV-1 or HSV-2, and treated twice daily intraperitoneally for five days with HBPG, alone or in combination with ACV, PFA, or CDF. Animals were observed daily for up to 30 days, and the day of death of each was recorded. All of the combinations showed additivity, and the combination of HBPG + ACV appeared to be synergistic, ie, protected more mice against HSV-1 encephalitis compared with each drug given alone. Delay of treatment with HBPG for up to two days was still effective in preventing HSV-2 encephalitis. The combination of the thymidine kinase inhibitor HBPG and the antiherpes drug ACV may have synergistic activity against HSV encephalitis. The development of a potent and safe combination therapy for the prevention and/or treatment of HSV infection of the central nervous system can improve the outcome of this infection in humans.