Search Results (50)

Herpes simplex virus 2 (HSV-2) remains a significant cause of morbidity and mortality in immunocompromised individuals, despite the availability of effective antivirals. Infections caused by drug-resistant isolates are an emerging concern among these patients. Understanding evolutionary aspects of HSV-2 resistance is crucial for designing improved therapeutic strategies. Here, we characterized 11 HSV-2 isolates recovered from various body sites of a single immunocompromised patient suffering from a primary HSV-2 infection unresponsive to acyclovir and foscarnet. The isolates were analyzed phenotypically and genotypically (Sanger sequencing of viral thymidine kinase and DNA polymerase genes). Viral clone isolations, deep sequencing, viral growth kinetics, and dual infection competition assays were performed retrospectively to assess viral heterogeneity and fitness. Sanger sequencing identified mixed populations of DNA polymerase mutant variants. Viral clones were plaque-purified and genotyped, revealing 17 DNA polymerase mutations (K533E, A606V, C625R, R628C, A724V, S725G, S729N, I731F, Q732R, M789T/K, Y823C, V842M, R847C, F923L, T934A, and R964H) associated with acyclovir and foscarnet resistance. Deep-sequencing of the DNA polymerase detected drug-resistant variants ranging between 1 and 95%, although the first two isolates had a wild-type DNA polymerase. Some mutants showed reduced fitness, evidenced by (i) the frequency of variants identified by deep-sequencing not correlating with the proportion of mutants found by plaque-purification, (ii) loss of the variants upon passaging in cell culture, or (iii) reduced frequencies in competition assays. This study reveals the rapid evolution of heterogeneous drug-resistant HSV-2 populations under antiviral therapy, highlighting the need for alternative treatment options and resistance surveillance, especially in severe infections. Full article

Marine tunicates are a very attractive and abundant source of secondary metabolites with chemical diversity and biological activity. Fractionation and purification of the organic extract of the Red Sea tunicate Didemnum species resulted in the isolation and identification of three new compounds, didemnosides A and B (1 and 2) and 1,1′,3,3′-bisuracil (3), together with thymidine (4), 2′-deoxyuridine (5), homarine (6), and acetamide (7). Planar structures of the compounds were explained through analyses of their 1D (¹H and ¹³C) and 2D (¹H–¹H COSY, HSQC, and HMBC) NMR spectra and high-resolution mass spectral determinations. Compound 1 exhibited the highest growth inhibition toward the MCF-7 cancer cell line with IC₅₀ values of 0.597 μM, while other compounds were inactive (≥50 μM) against this cell line. On the other hand, compounds 1, 2, and 4–7 moderately inhibited SW-1222 and PC-3 cells with IC₅₀ values ranging between 5.25 and 9.36 μM. Molecular docking analyses of the top three active compounds on each tested cell line exposed stable interactions into the active pockets of estrogen receptor alpha (ESR1), human topoisomerase II alpha (TOP2A), and cyclin-dependent kinase 5 (CDK5) which are contemplated as essential targets in cancer treatments. Thus, compound 1 represents a scaffold for the development of more effective anticancer drugs. Full article

Background/Objectives: Oral squamous cell carcinoma (OSCC) is the most prevalent malignancy of the oral cavity and is frequently diagnosed at an advanced stage, resulting in poor prognosis and limited treatment options. Identifying reliable biomarkers that can predict tumor progression and serve as therapeutic targets remains an urgent clinical need. Methods: To identify key molecular drivers in OSCC, we performed an integrative bioinformatics analysis of five OSCC-related microarray datasets from the Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) were identified and subjected to functional enrichment, protein–protein interaction (PPI) network construction, and hub gene ranking using Cytoscape. Candidate genes were further validated using TCGA, UALCAN, and the Human Protein Atlas. In vitro functional assays were performed to evaluate the effect of TK1 knockdown on cell migration. Results: A total of 138 common DEGs were identified across datasets. GO enrichment revealed that these genes were associated with cell proliferation, extracellular matrix organization, and metastasis-related processes. Thymidine kinase 1 (TK1) was identified as a key hub gene and found to be consistently overexpressed in OSCC tissues. Kaplan–Meier analysis showed that high TK1 expression correlated with poor overall survival in head and neck cancer. TK1 knockdown in OSCC cell lines significantly impaired cell migration and wound-healing ability. Conclusions: Our findings suggest that TK1 plays an active role in promoting OSCC progression and may serve as a prognostic biomarker and potential therapeutic target for metastatic OSCC. Full article

Protein glycosylation plays a crucial role in cancer biology, influencing essential cellular processes such as cell signalling, immune recognition, and tumour metastasis. Therefore, this study highlights the therapeutic potential of targeting glycosylation in cancer treatment, as modulating these modifications could disrupt the fundamental mechanisms driving cancer progression and improve therapeutic outcomes. Recently, Tunicamycin C, a well-known glycosylation inhibitor, has shown promise in breast cancer treatment but remains unexplored in colorectal cancer (CRC). Thus, in this study, we aimed to understand the potential action of Tunicamycin C in CRC using in silico studies to identify possible drug targets for Tunicamycin C. First, we identified two target proteins using the HTDocking algorithm followed by GO and KEGG pathway searches: thymidine kinase 1 (TK1) and cAMP-dependent protein kinase catalytic subunit alpha (PKAc). Following this, molecular dynamics modelling revealed that Tunicamycin C binding induced a conformational perturbation in the 3D structures of TK1 and PKAc, inhibiting their activities. This interaction led to a stable design, promoting optimal binding of Tunicamycin C in the hydrophobic pockets of TK1 and PKAc. Serial validation studies highlighted the role of active site residues in binding stabilisation. Tunicamycin C exhibited high binding affinity with TK1 and PKAc. This study provides a way to explore and repurpose novel inhibitors of TK1 and PKAc and identify new therapeutic targets, which may block glycosylation in cancer treatment. Full article

Herpes simplex virus type 1 (HSV-1) acyclovir (ACV) resistance is acquired by mutations in the viral thymidine kinase (TK) or DNA polymerase (DNApol) genes. We previously obtained an ACV-resistant clone (HSV-1_VZV_TK_clone α) by sequential passages of HSV-1_VZV-TK, a recombinant virus which lacked its endogenous TK activity and instead expressed the varicella-zoster virus (VZV) TK ectopically. HSV-1_VZV_TK_clone α had been generated using an HSV-1_BAC in the presence of increasing concentrations of ACV. The ACV-resistant clone bore normal TK and DNApol genes. Here, we deployed next-generation full-genome sequencing of HSV-1_VZV_TK_clone α and identified a single nucleotide substitution, resulting in a P597L missense mutation in the UL29 gene product, the ICP8 protein. Recombinant HSV-1 encoding a P597L ICP8 protein was generated, and its properties and ability to confer drug resistance were analyzed. No difference in virus growth and UL29 expression was observed between the mutant recombinant, the wild type, and a revertant mutant viral strain, and susceptibility tests of these strains to ACV and other drugs using Vero, HEL, and ARPE19 cells identified that the recombinant UL29 mutant virus was resistant only to ACV. These results indicate that ICP8 may be involved in the anti-herpesvirus drugs’ mechanism of action on HSV-1. Full article

Pancreatic cancer presents formidable challenges due to rapid progression and resistance to conventional treatments. Oncolytic viruses (OVs) selectively infect cancer cells and cause cancer cells to lyse, releasing molecules that can be identified by the host’s immune system. Moreover, OV can carry immune-stimulatory payloads such as interleukin-12, which when delivered locally can enhance immune system-mediated tumor killing. OVs are very well tolerated by cancer patients due to their ability to selectively target tumors without affecting surrounding normal tissues. OVs have recently been combined with other therapies, including chemotherapy and immunotherapy, to improve clinical outcomes. Several OVs including adenovirus, herpes simplex viruses (HSVs), vaccinia virus, parvovirus, reovirus, and measles virus have been evaluated in preclinical and clinical settings for the treatment of pancreatic cancer. We evaluated the safety and tolerability of a replication-competent oncolytic adenoviral vector carrying two suicide genes (thymidine kinase, TK; and cytosine deaminase, CD) and human interleukin-12 (hIL12) in metastatic pancreatic cancer patients in a phase 1 trial. This vector was found to be safe and well-tolerated at the highest doses tested without causing any significant adverse events (SAEs). Moreover, long-term follow-up studies indicated an increase in the overall survival (OS) in subjects receiving the highest dose of the OV. Our encouraging long-term survival data provide hope for patients with advanced pancreatic cancer, a disease that has not seen a meaningful increase in OS in the last five decades. In this review article, we highlight several preclinical and clinical studies and discuss future directions for optimizing OV therapy in pancreatic cancer. We envision OV-based gene therapy to be a game changer in the near future with the advent of newer generation OVs that have higher specificity and selectivity combined with personalized treatment plans developed under AI guidance. Full article

Non-small cell lung cancer (NSCLC) represents 80% of all lung cancer cases and is characterized by low survival rates due to chemotherapy and radiation resistance. Novel treatment strategies for NSCLC are urgently needed. Liver kinase B1 (LKB1), a tumor suppressor prevalently mutated in NSCLC, activates AMP-activated protein kinase (AMPK) which in turn inhibits mammalian target of rapamycin complex 1 (mTORC1) and activates unc-51 like autophagy activating kinase 1 (ULK1) to promote autophagy. Sestrin-2 is a stress-induced protein that enhances LKB1-dependent activation of AMPK, functioning as a tumor suppressor in NSCLC. In previous studies, rosemary (Rosmarinus officinalis) extract (RE) activated the AMPK pathway while inhibiting mTORC1 to suppress proliferation, survival, and migration, leading to the apoptosis of NSCLC cells. In the present study, we investigated the anticancer potential of carnosic acid (CA), a bioactive polyphenolic diterpene compound found in RE. The treatment of H1299 and H460 NSCLC cells with CA resulted in concentration and time-dependent inhibition of cell proliferation assessed with crystal violet staining and ³H-thymidine incorporation, and concentration-dependent inhibition of survival, assessed using a colony formation assay. Additionally, CA induced apoptosis of H1299 cells as indicated by decreased B-cell lymphoma 2 (Bcl-2) levels, increased cleaved caspase-3, -7, poly (ADP-ribose) polymerase (PARP), Bcl-2-associated X protein (BAX) levels, and increased nuclear condensation. These antiproliferative and proapoptotic effects coincided with the upregulation of sestrin-2 and the phosphorylation/activation of LKB1 and AMPK. Downstream of AMPK signaling, CA increased levels of autophagy marker light chain 3 (LC3), an established marker of autophagy; inhibiting autophagy with 3-methyladenine (3MA) blocked the antiproliferative effect of CA. Overall, these data indicate that CA can inhibit NSCLC cell viability and that the underlying mechanism of action of CA involves the induction of autophagy through a Sestrin-2/LKB1/AMPK signaling cascade. Future experiments will use siRNA and small molecule inhibitors to better elucidate the role of these signaling molecules in the mechanism of action of CA as well as tumor xenograft models to assess the anticancer properties of CA in vivo. Full article

Purpose: To search for new predictive breast cancer biomarkers. We analyzed the serum concentrations of biomarkers involved in carcinogenesis, which can also be targeted by therapy. Methods: In a single-center prospective study, the serum levels of Aurora A, thymidine kinase 1, and human epidermal growth factor receptor type 3 (HER3) were determined in 119 women with BC before neoadjuvant treatment using ELISA kits. Results: The following clinical data were analyzed: age; TNM; the expression of ER, PGR, HER2, and Ki67; histological grade (G); and the response to neoadjuvant treatment (NAT) in the residual tumor burden classification (RCB). A complete pathological response (pCR) was achieved after NAT in 41 patients (34%). The highest proportion of the patients with a confirmed pCR was found for triple negative breast cancer (TNBC) (62.5%); non-luminal HER2-positive (52.6%) cancer subtypes (p = 0.0003); and in the G3 group (50%; p = 0.0078). The patients with higher levels of Aurora A were more likely to achieve pCR (p = 0.039). In the multivariate analysis, the serum Aurora A levels ≥ 4.75 ng/mL correlated with a higher rate of pCR (OR: 3.5; 95% CI: 1.2–10.1; p = 0.023). Conclusions: We showed that in a biologically heterogeneous group of BC patients, the pretreatment serum Aurora A levels were of significant value in predicting the response to NAT. Full article

The thymidine kinase (TK) and DNA polymerase (pol) genes of the herpes simplex viruses type 1 (HSV-1) and type 2 (HSV-2) are two important genes involved in antiviral resistance. We investigated the genetic polymorphisms of the HSV-TK and pol genes in clinical isolates from Korean HSV-infected patients using next-generation sequencing (NGS) for the first time in Korea. A total of 81 HSV-1 and 47 HSV-2 isolates were examined. NGS was used to amplify and sequence the TK and pol genes. Among the 81 HSV-1 isolates, 12 and 17 natural polymorphisms and 9 and 23 polymorphisms of unknown significance in TK and pol were found, respectively. Two HSV-1 isolates (2.5%) exhibited the E257K amino acid substitution in TK, associated with antiviral resistance. Out of 47 HSV-2 isolates, 8 natural polymorphisms were identified in TK, and 9 in pol, with 13 polymorphisms of unknown significance in TK and 10 in pol. No known resistance-related mutations were observed in HSV-2. These findings contribute to our understanding of the genetic variants associated with antiviral resistance in HSV-1 and HSV-2 in Korea, with frequencies of known antiviral resistance-related mutations of 2.5% and 0% in HSV-1 and HSV-2, respectively. Full article

Thymidylate synthase (TS) remains a major target for cancer therapy. TS inhibition elicits increases in DNA salvage pathway activity, detected as a transient compensatory “flare” in 3′-deoxy-3′-[¹⁸F]fluorothymidine positron emission tomography (¹⁸F-FLT PET). We determined the magnitude of the ¹⁸F-FLT flare in non-small cell lung cancer (NSCLC) patients treated with the antifolate pemetrexed in relation to clinical outcome. Method: Twenty-one patients with advanced/metastatic non-small cell lung cancer (NSCLC) scheduled to receive palliative pemetrexed ± platinum-based chemotherapy underwent ¹⁸F-FLT PET at baseline and 4 h after initiating single-agent pemetrexed. Plasma deoxyuridine (dUrd) levels and thymidine kinase 1 (TK1) activity were measured before each scan. Patients were then treated with the combination therapy. The ¹⁸F-FLT PET variables were compared to RECIST 1.1 and overall survival (OS). Results: Nineteen patients had evaluable PET scans at both time points. A total of 32% (6/19) of patients showed ¹⁸F-FLT flares (>20% change in SUVmax-wsum). At the lesion level, only one patient had an FLT flare in all the lesions above (test–retest borders). The remaining had varied uptake. An ¹⁸F-FLT flare occurred in all lesions in 1 patient, while another patient had an ¹⁸F-FLT reduction in all lesions; 17 patients showed varied lesion uptake. All patients showed global TS inhibition reflected in plasma dUrd levels (p < 0.001) and ¹⁸F-FLT flares of TS-responsive normal tissues including small bowel and bone marrow (p = 0.004 each). Notably, 83% (5/6) of patients who exhibited ¹⁸F-FLT flares were also RECIST responders with a median OS of 31 m, unlike patients who did not exhibit ¹⁸F-FLT flares (15 m). Baseline plasma TK1 was prognostic of survival but its activity remained unchanged following treatment. Conclusions: The better radiological response and longer survival observed in patients with an ¹⁸F-FLT flare suggest the efficacy of the tracer as an indicator of the early therapeutic response to pemetrexed in NSCLC. Full article

Machaeriols and machaeridiols are unique hexahydrodibenzopyran-type aralkyl phytocannabinoids isolated from Machaerium Pers. Earlier studies of machaeriol A (1) and B (2) did not show any affinity for cannabinoid receptor 1 (CB1 or CNR1), although they are structural analogs of psychoactive hexahydrocannabinol. This study comprehensively reports on the affinities of isolated Machaerium Pers. compounds, namely machaeriol A–D (1–4) and machaeridiol A–C (5–7), against cannabinoid (CB1 and CB2) and opioid (κ, δ and µ) receptors. Among the isolated compounds, machaeriol D (4) and machaeridiol A–C (5–7) showed some selective binding affinity for the CB2 receptor, using a radioligand binding assay, with K_i values of >1.3, >1.77, >2.18 and >1.1 μM, respectively. On the other hand, none of the compounds showed any binding to the CB1 receptor. Due to recent reports on the anticancer potential of the endocannabinoid system, compounds 1–7 were tested against a battery of luciferase reporter gene vectors that assess the activity of many cancer-related signaling pathways, including Stat3, Smad2/3, AP-1, NF-κB, E2F, Myc, Ets, Notch, FoxO, Wnt, Hedgehog and pTK in HeLa and T98G glioblastoma cells. Complete dose–response curves have been determined for each compound in both of these cell lines, which revealed that machaeridiol 6 displayed activities (IC₅₀ in µM in HeLa and T98G cells) towards Stat3 (4.7, 1.4), Smad2/3 (1.2, 3.0), AP-1 (5.9, 4.2), NF-κB (0.5, 4.0), E2F (5.7, 0.7), Myc (5.3, 2.0), ETS (inactive, 5.9), Notch (5.3, 4.6), Wnt (4.2, inactive) and Hedgehog (inactive, 5.0). Furthermore, a combination study between machaeriol C (3) and machaeridiol B (6) displayed additive effects for E2F, ETS, Wnt and Hedgehog pathways, where these compounds individually were either minimally active or inactive. None of the compounds inhibited luciferase expression driven by the minimal thymidine kinase promoter (pTK), indicating the lack of general cytotoxicity for luciferase enzyme inhibition at the 50 µM concentration in both of these cell lines. The significance of the inhibition of these signaling pathways via machaeridiol 5–7 and their cross-talk potential has been discussed. Full article

Thymidine kinase 1 (TK1) is an intracellular enzyme involved in DNA-precursor synthesis. Increased serum TK1 levels are used as a biomarker in various malignancies. We combined serum TK1 with PSA and evaluated its capacity to predict overall survival (OS) in 175 men with prostate cancer (PCa), detected by screening in 1988–1989 (n = 52) and during follow-up (median 22.6 years) (n = 123). TK1 was measured in frozen serum, age was stratified into four groups, and dates of PCa diagnosis and dates of death were obtained from Swedish population-based registries. The median concentration of TK1 and PSA was 0.25 and 3.8 ng/ml. TK1 was an independent variable of OS. In the multivariate analysis, PSA was not statistically significant in combination with age whereas the significance remained for TK1 + PSA. Measured once, TK1 + PSA predicted a difference of up to 10 years (depending on patient subgroup) in OS at a median of 9 years before PCa diagnosis. The TK1 concentration in 193 controls without malignancies did not differ from that of the PCa patients, hence TK1 was likely not released from incidental PCa. Thus, TK1 in the blood circulation may indicate the release of TK1 from sources other than cancers, nonetheless associated with OS. Full article

Background: The early detection of ovarian cancer is presently not effective, and it is crucial to establish biomarkers for the early diagnosis of ovarian cancer to improve the survival of patients. Materials and methods: The aim of this study was to investigate the role of thymidine kinase 1 (TK1) in combination with CA 125 or HE4 to serve as a potential diagnostic biomarkers for ovarian cancer. In this study, a set of 198 serum samples consisting of 134 ovarian tumor patients and 64 healthy age-matched controls were analyzed. The TK1 protein levels in serum samples were determined using the AroCell TK 210 ELISA. Results: A combination of TK1 protein with CA 125 or HE4 showed better performance than either of them alone in the differentiation of early stage ovarian cancer from the healthy control group, but also a significantly better performance than the ROMA index. However, this was not observed using a TK1 activity test in combination with the other markers. Furthermore, the combination of TK1 protein and CA 125 or HE4 could differentiate early stage disease (stage I, II) more efficiently from advanced-stage (stage III, IV) disease (p < 0.0001). Conclusions: The combination of TK1 protein with CA 125 or HE4 increased the potential of detecting ovarian cancer at early stages. Full article

Pseudorabies (also called Aujeszky’s disease) is a highly infectious viral disease caused by the pseudorabies virus (PRV, or Suid herpesvirus 1). Although the disease has been controlled by immunization with the PRV-attenuated vaccine, the emerging PRV variants can escape the immune surveillance in the vaccinated pig, resulting in recent outbreaks. Furthermore, the virus has been detected in other animals and humans, indicating cross-transmission of PRV. However, the mechanism of PRV cross-species transmission needs further study. In this study, we compared the amino acid sequences of glycoproteins (gD), gL, and thymidine kinase (TK) of PRV strains, human PRV hSD-1 2019 strain, and the attenuated strain Bartha-K61, followed by predication of their spatial conformation. In addition, the interactions between the viral gD protein and host nectin-1, nectin-2, and HS were also evaluated via molecular docking. The results showed that the amino acid sequence homology of the gD, gL, and TK proteins of hSD-1 2019 and JL-CC was 97.5%, 94.4%, and 99.1%, respectively. Moreover, there were mutations in the amino acid sequences of gD, gL, and TK proteins of hSD-1 2019 and JL-CC compared with the corresponding reference sequences of the Bartha strain. The mutations of gD, gL, and TK might not affect the spatial conformation of the protein domain but may affect the recognition of antibodies and antigen epitopes. Moreover, the gD protein of JL-CC, isolated previously, can bind to human nectin-1, nectin-2, and HS, suggesting the virus may be highly infectious and pathogenic to human beings. Full article

The enantioselective preparation of the two isomers of 4-hydroxy-2-cyclohexanone derivatives 1a,b was achieved, starting from a common cyclohexenone, through asymmetric transfer hydrogenation (ATH) reactions using bifunctional ruthenium catalysts. From these versatile intermediates, a stereoselective route to a cytosine analogue built on a bicyclo [4.1.0]heptane scaffold is described. Nucleoside kinase activity assays with this cyclopropyl-fused cyclohexane nucleoside, together with other related nucleosides (2a–e), were performed, showing that thymine- and guanine- containing compounds have affinity for herpes simplex virus Type 1 (HSV-1) thymidine kinase (TK) but not for human cytosolic TK-1, thus pointing to their selectivity for herpetic TKs but not cellular TKs. Full article