Thymidine Phosphorylase Research Articles

OniLon: Phase II trial of trifluridine/tipiracil (TAS-102) and nanoliposomal irinotecan (nal-IRI) in advanced colorectal cancer.

3580 Background: TAS-102 (FTD/TPI) is a combination of a nucleoside analogue and a thymidine phosphorylase inhibitor, and has showed activity in 5FU-resistant CRC. Nal-IRI achieves higher intra-tumor concentrations than irinotecan (Iri; 142-fold) and its major metabolite, SN-38 (9-fold). This resulted in superior anti-tumor activity compared to free Iri in multiple tumor xenografts. Furthermore, multiple clinical trials have established the activity of nal-IRI in combination with 5FU in pancreaticobiliary cancers. The combination of nal-IRI with the more potent nucleoside analogue TAS-102 may thus result in a more effective systemic therapy. We conducted this study to define the safety and efficacy of the combination of TAS-102 and nal-IRI in advanced CRC. Methods: Eligible patients (pts) on this investigator-initiated phase II study had advanced CRC and must have had disease progression on at least one prior therapy. Additional inclusion criteria - measurable disease, ECOG PS 0-1 & adequate organ function. Based on previously published dose escalation phase I data, the pts were treated at the recommended phase II dose of TAS-102 (given orally at 35mg/m2 bid on days 1-5) and nal-IRI (60mg/m2 IV on day 1) in 14-day cycles. The primary endpoint was objective response rate (ORR) = complete (CR) + partial (PR) response per RECIST v1.1. Multiple secondary endpoints included safety/tolerability, progression-free survival (PFS), overall survival (OS), disease control rate (DCR) and duration of response (DoR). Results: Twenty-two pts were enrolled, of whom 20 were evaluable for response. Male - 50%; ECOG PS 1 – 75%; African Americans – 38%; median age - 58yrs (interquartile range [IQR] 49-67). Median line of prior therapy was 1 (IQR 1-2) and 13.6% (3 pts enrolled during the dose escalation phase) had received prior Iri. 47.6% received concurrent Bevacizumab. ORR was 15% (3 PR, 0 CR). 60% had stable disease (SD) as best response; DCR of 75%. mPFS - 9.7 mos (95% CI: 5.6, 14.2); mOS - 10.1 mos (95% CI: 7.3, 15.9). 12-month PFS and OS rates were 34.6% and 39.5% respectively. At a median follow up of 11.9 mos, DoR was yet to be determined and 50% of the pts had maintained response for 12 mos. Median duration of progression free among pts with SD was 7.6 mos (95% CI: 4.6, 13.2), with a 12-month survival rate of 47.7% (95% CI: 15.5%, 74.5%). Median time to response (TTR) was 4.8mos. Most common treatment related toxicities included fatigue and neutropenia (G3; 27.3% each); anemia and leucopenia (G3; 18.2% each). Other G3 AEs included febrile neutropenia, diarrhea, hypokalemia, lymphopenia and nausea (4.5% each). Conclusions: We demonstrated that the combination of TAS-102 and nal-IRI had an acceptable safety profile, and showed antitumor activity in patients with advanced CRC. Additional biomarker analyses such as survival correlation with UGT1A1 phenotype and RAS mutational status are ongoing. (NCT03368963). Clinical trial information: NCT03368963 .

Integrating metabolomics and network pharmacology to assess the effects of quercetin on lung inflammatory injury induced by human respiratory syncytial virus

Quercetin (QR) has significant anti-respiratory syncytial virus (RSV) effects. However, its therapeutic mechanism has not been thoroughly explored. In this study, a lung inflammatory injury model caused by RSV was established in mice. Untargeted lung tissue metabolomics was used to identify differential metabolites and metabolic pathways. Network pharmacology was used to predict potential therapeutic targets of QR and analyze biological functions and pathways modulated by QR. By overlapping the results of the metabolomics and the network pharmacology analyses, the common targets of QR that were likely to be involved in the amelioration of RSV-induced lung inflammatory injury by QR were identified. Metabolomics analysis identified 52 differential metabolites and 244 corresponding targets, while network pharmacology analysis identified 126 potential targets of QR. By intersecting these 244 targets with the 126 targets, hypoxanthine–guanine phosphoribosyltransferase (HPRT1), thymidine phosphorylase (TYMP), lactoperoxidase (LPO), myeloperoxidase (MPO), and cytochrome P450 19A1 (CYP19A1) were identified as the common targets. The key targets, HPRT1, TYMP, LPO, and MPO, were components of purine metabolic pathways. The present study demonstrated that QR effectively ameliorated RSV-induced lung inflammatory injury in the established mouse model. Combining metabolomics and network pharmacology showed that the anti-RSV effect of QR was closely associated with purine metabolism pathways.

Curative anti-typhoid effect of Detarium microcarpum Guill. & Perr. (Leguminosae) hydroethanolic extract root bark based-on in vivo and molecular docking analyses.

Detarium microcarpum is used to treat typhoid fever, a major public health problem, by indigenous population in Africa. Though its preventive activities have been documented, the curative effect is still to be confirmed. This study aimed at evaluating the curative effects of the hydroethanolic extract of Detarium microcarpum root bark on Salmonella typhimurium-induced typhoid in rat and exploring the in-silico inhibition of some bacterial key enzymes. In vitro antioxydant, in vivo antisalmonella of the extract and in silico molecular docking assay on the isolated compounds were carried out to explore the anti-salmonella effects of Detarium microcarpum. The in vitro antioxidant properties of the extract were evaluated using DPPH, ABTS and FRAP tests. The anti-salmonella activity of the extract was assessed through feacal sample from Salmonella typhimurium-infected rat cultured in Salmonella-Shigella agar (SS agar) medium. The affinity of isolated compounds (Rhinocerotinoic acid and Microcarposide) from the extract were performed on four key enzymes (Adenylosuccinate lyase, Acetyl coenzyme A synthetase, Thymidine phosphorylase and LuxS-Quorum sensor) using molecular docking simulation to elucidate the molecular level inhibition mechanism. Crude extract of D. microcarpum root bark showed variable activities on DPPH (RSa50: 6.09±1.04μg/mL), ABTS (RSa50: 24.46±0.27), and FRAP (RSa50: 23.30±0.23). The extract at all the doses exhibited significant healing effect of infected rats, with the complete clearance. The extract restored hematological, biochemical and histological parameters closed to the normal control. The molecular docking results indicates that rhinocerotinoic acid and microcarposide present more affinity to the LuxS-Quorum sensor and Acetyl coenzyme A synthetase protein as compared to the others. These results demonstrate potent anti-typhoid activities of the hydroethanolic of Detarium microcarpum root bark extract through antioxidant properties and high inhibitory affinity of its compounds on some bacterial key enzymes that justify its use as traditional medicine to typhoid fever.

Thymidine phosphorylase facilitates retinoic acid inducible gene-I induced endothelial dysfunction

Activation of nucleic acid sensors in endothelial cells (ECs) has been shown to drive inflammation across pathologies including cancer, atherosclerosis and obesity. We previously showed that enhancing cytosolic DNA sensing by inhibiting three prime exonuclease 1 (TREX1) in ECs led to EC dysfunction and impaired angiogenesis. Here we show that activation of a cytosolic RNA sensor, Retinoic acid Induced Gene 1 (RIG-I) diminishes EC survival, angiogenesis and triggers tissue specific gene expression programs. We discovered a RIG-I dependent 7 gene signature that affects angiogenesis, inflammation and coagulation. Among these, we identified the thymidine phosphorylase TYMP as a key mediator of RIG-I induced EC dysfunction via its regulation of a subset of interferon stimulated genes. Our RIG-I induced gene signature was also conserved in the context of human diseases – in lung cancer vasculature and herpesvirus infection of lung endothelial cells. Pharmacological or genetic inhibition of TYMP rescues RIG-I induced EC death, migration arrest and restores sprouting angiogenesis. Interestingly, using RNAseq we identified a gene expression program that was RIG-I induced but TYMP dependent. Analysis of this dataset indicated that IRF1 and IRF8 dependent transcription is diminished in RIG-I activated cells when TYMP is inhibited. Functional RNAi screen of our TYMP dependent EC genes, we found that a group of 5 genes - Flot1, Ccl5, Vars2, Samd9l and Ube2l6 are critical for endothelial cell death mediated by RIG-I activation. Our observations identify mechanisms by which RIG-I drives EC dysfunction and define pathways that can be pharmacologically targeted to ameliorate RIG-I induced vascular inflammation.

RETRO-TAS, a Retrospective Observational Study of Trifluridine/Tipiracil in Chemorefractory Metastatic Colorectal Cancer.

Trifluridine/tipiracil (FTD/TPI) is an oral antimetabolite agent comprised of trifluridine, a thymidine-based nucleoside analogue that inhibits cell proliferation following its incorporation into DNA, and tipiracil that helps maintain the blood concentration of trifluridine by inhibiting the enzyme thymidine phosphorylase which inactivates trifluridine. It is approved as a third-line treatment option for patients with metastatic colorectal cancer (mCRC) and is administered at 35 mg/m2 two times daily from day 1 to 5 and from day 8 to 12 every 28 days. The aim of this investigator-initiated retrospective study (RETRO-TAS; NCT04965870) was to document real-world data on the clinical efficacy of FTD/TPI in patients with chemorefractory mCRC. The clinical characteristics of patients with mCRC treated with FTD/TPI in 8 Cancer Centres were collected to assess physician's choice in the third or beyond line of treatment as well as the duration of treatment, dose modification, and toxicity. In addition, other important prognostic features related to mCRC such as molecular profile, performance status (PS), and primary site were analyzed. Statistical analysis for progression-free survival (PFS), overall survival (OS), 6-/8-month PFS rate and disease control rate (DCR) along with Cox regression model, Kaplan-Meier curves, and log-rank tests were carried out by using Stata/MP 16.0 for Windows. From October 2018 to October 2021, a total of 200 patients with mCRC and a median age of 67.0 (IQR 58.0, 75.0) years were treated with FTD/TPI. Τhe median follow-up time was 14 months (IQR 7, 23), 158 PDs and 106 deaths were reported at the time of this analysis. Of all the patients, 58% were males and 58% had mCRC at diagnosis. The molecular analysis identified mutations in KRAS (52%), NRAS (5%), HER2 (3.5%), BRAF (3.5%), and MSI (9%). Previous treatments included radical surgery in 51.5% and adjuvant chemotherapy in 39.5% of patients. FTD/TPI was administered in the third- (70.5%), fourth- (17.0%), or fifth-line (12.5%) treatment setting. Serious adverse events related to FTD/TPI included neutropenia (2%), anaemia (1%), thrombocytopenia (0.5%), diarrhoea (0.5%), nausea (0.5%), and fatigue (4%). A reduction of FTD/TPI dose, delay of next cycle initiation, and shorter duration were reported in 25%, 31%, and 14.5% of patients, respectively. Of all the patients 71.5% received FTD/TPI as monotherapy, 24.5% in combination with bevacizumab, and 4.0% with an anti-EGFR agent. The median FTD/TPI treatment duration was 119.5 days and 81% of patients discontinued treatment due to progressive disease. The DCR recorded by investigators' assessment was 45.5%. The median PFS was 4.8 and the median OS was 11.4 months. The 6- and the 8-month PFS rate was 41.4% and 31.5%, respectively. In the multivariate analysis, PS > 1 and presence of liver and lung metastasis were adversely associated with PFS and OS whereas mutational status and tumor sidedness were not. RETRO-TAS is a real-world observational study that confirms and adds on the findings of the pivotal RECOURSE Phase III study in relation to the efficacy of FTD/TPI in the third-line setting and in all subgroups of patients regardless of mutational status and sidedness.

Dihydropyrimidone Derivatives as Thymidine Phosphorylase Inhibitors: Inhibition Kinetics, Cytotoxicity, and Molecular Docking.

Overexpression of the thymidine phosphorylase (TP) enzyme induces angiogenesis, which eventually leads to metastasis and tumor growth. The crucial role of TP in cancer development makes it an important target for anticancer drug discovery. Currently, there is only one US-FDA-approved drug, i.e., Lonsurf, a combination of trifluridine and tipiracil, for the treatment of metastatic colorectal cancer. Unfortunately, numerous adverse effects are associated with its use, such as myelosuppression, anemia, and neutropenia. Since the last few decades, the discovery of new, safe, and effective TP inhibitory agents has been rigorously pursued. In the present study, we evaluated a series of previously synthesized dihydropyrimidone derivatives 1-40 for their TP inhibitory potential. Compounds 1, 12, and 33 showed a good activity with IC50 = 314.0 ± 0.90, 303.5 ± 0.40, and 322.6 ± 1.60 µM, respectively. The results of mechanistic studies revealed that compounds 1, 12, and 33 were the non-competitive inhibitors. These compounds were also evaluated for cytotoxicity against 3T3 (mouse fibroblast) cells and were found to be non-cytotoxic. Finally, the molecular docking suggested the plausible mechanism of non-competitive inhibition of TP. The current study thus identifies some dihydropyrimidone derivatives as potential inhibitors of TP, which can be further optimized as leads for cancer treatment.

Abstract 5510: TAS102 synergizes with regorafenib against colorectal and gastric cancer cells

Abstract TAS-102 is an oral formulation consisting of trifluridine (FTD) and thymidine phosphorylase inhibitor tipiracil hydrochloride (TPI). Regorafenib is a multi-targeted tyrosine kinase inhibitor, it inhibits the activities of VEGFR2 and 3, Ret, Kit, PDGFR and Raf kinases and results in the inhibition of tumor angiogenesis and cell proliferation. TAS102 or regorafenib monotherapy can lead to an overall survival benefit in metastatic colorectal cancer (mCRC) patient previously treated with conventional chemotherapy and target therapy (Arnold et al., 2018). TAS102 in combination with bevacizumab, a monoclonal antibody against vascular endothelial growth factor A (VEGF-A), is associated with a significant and clinically relevant improvement in progression-free survival in colorectal cancer (C-TASK FORCE)(Kuboki et al., 2017; Pfeiffer et al., 2020). FTD can synergize with nintedanib to inhibit the growth of colorectal cancer xenografts(Suzuki, Nakagawa, Matsuoka, & Takechi, 2016). We previously reported that regorafenib combined with a fluoropyrimidine can delay disease progression in case reports of multidrug-resistant mCRC patients(Marks et al., 2015). Therefore, we hypothesized that the combination of TAS102 and regorafenib may be active in CRC and other gastrointestinal (GI) cancers, and may in the future provide a treatment option for patients with advanced GI cancer. We investigated the therapeutic effect of TAS102 in combination with regorafenib in preclinical studies employing cell culture, colonosphere assays that enrich for cancer stem cells, and in vivo. We found that TAS102 in combination with regorafenib has synergistic activity against multiple GI cancers in vitro including colorectal and gastric cancer, but not liver cancer cells. TAS102 inhibits colonosphere formation and this effect is potentiated by regorafenib. In vivo anti-tumor effects of TAS102 plus regorafenib appear to be due to anti-proliferative effects, necrosis and angiogenesis inhibition. Growth inhibition by TAS102 plus regorafenib occurs in xenografted tumors regardless of p53, KRAS or BRAF mutations, although more potent tumor suppression was observed with wild-type p53. Regorafenib significantly inhibits TAS102-induced angiogenesis and microvessel density in xenografted tumors, and inhibits TAS102-induced ERK1/2 activation regardless of RAS or BRAF status in vivo. Collectively, TAS102 plus regorafenib is a synergistic drug combination in preclinical models of GI cancer, with regorafenib suppressing TAS102-induced increase in microvessel density and p-ERK as contributing mechanisms. The drug combination may be further tested in gastric and other GI cancers. Citation Format: Jun Zhang, Lanlan Zhou, Shuai Zhao, Wafik S. El-Deiry. TAS102 synergizes with regorafenib against colorectal and gastric cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5510.

Thymidine phosphorylase as a molecular target for quercetin-incorporated collagen matrix in the prevention of hand-foot syndrome induced by capecitabine in rats.

Hand-foot syndrome (HFS) is a common adverse effect of capecitabine affecting the quality of life of cancer patients. To enhance the tolerability of capecitabine, this work evaluated the incorporation of quercetin into topical collagen matrix formula to target thymidine phosphorylase enzyme, oxidative stress, and apoptosis underlying HFS. Forty Sprague Dawley rats were allocated to four equal groups. The control group received distilled water orally. HFS was induced by oral capecitabine (200 mg/kg/day) for 21 days. The untreated HFS group received no treatment. In the treated groups, topical collagen and quercetin-incorporated collagen matrix formula were administered concomitantly with the HFS induction protocol. Treatment with quercetin-incorporated collagen matrix showed a significant decrease in thymidine phosphorylase level compared with the untreated and collagen-treated groups. Treatment with quercetin-incorporated collagen matrix showed a significant decrease in malondialdehyde and caspase-3 levels, and a significant increase in the total antioxidant capacity of the skin and B cell lymphoma/leukemia 2 levels compared with the untreated group. Additionally, a significant improvement in the gross picture and histopathological score of HFS was observed. In conclusion, the quercetin-incorporated collagen matrix is a promising formula for the prevention of HFS, due to the targeted effect on thymidine phosphorylase and subsequent antioxidant and antiapoptotic effects.

Thymidine Phosphorylase Deficiency or Inhibition Preserves Cardiac Function in Mice With Acute Myocardial Infarction.

Background Ischemic cardiovascular disease is the leading cause of death worldwide. Current pharmacologic therapy has multiple limitations, and patients remain symptomatic despite maximal medical therapies. Deficiency or inhibition of thymidine phosphorylase (TYMP) in mice reduces thrombosis, suggesting that TYMP could be a novel therapeutic target for patients with acute myocardial infarction (AMI). Methods and Results A mouse AMI model was established by ligation of the left anterior descending coronary artery in C57BL/6J wild-type and TYMP-deficient (Tymp-/-) mice. Cardiac function was monitored by echocardiography or Langendorff assay. TYMP-deficient hearts had lower baseline contractility. However, cardiac function, systolic left ventricle anterior wall thickness, and diastolic wall strain were significantly greater 4 weeks after AMI compared with wild-type hearts. TYMP deficiency reduced microthrombus formation after AMI. TYMP deficiency did not affect angiogenesis in either normal or infarcted myocardium but increased arteriogenesis post-AMI. TYMP deficiency enhanced the mobilization of bone marrow stem cells and promoted mesenchymal stem cell (MSC) proliferation, migration, and resistance to inflammation and hypoxia. TYMP deficiency increased the number of larger MSCs and decreased matrix metalloproteinase-2 expression, resulting in a high homing capability. TYMP deficiency induced constitutive AKT phosphorylation in MSCs but reduced expression of genes associated with retinoid-interferon-induced mortality-19, a molecule that enhances cell death. Inhibition of TYMP with its selective inhibitor, tipiracil, phenocopied TYMP deficiency, improved post-AMI cardiac function and systolic left ventricle anterior wall thickness, attenuated diastolic stiffness, and reduced infarct size. Conclusions This study demonstrated that TYMP plays an adverse role after AMI. Targeting TYMP may be a novel therapy for patients with AMI.

Synthesis, in vitro thymidine phosphorylase inhibitory activity and molecular docking study of novel pyridine-derived bis-oxadiazole bearing bis-schiff base derivatives

The current study has afforded twelve analogs (4a-l) of pyridine-derived bis-oxadiazole containing bis-schiff base and subsequently evaluated for their potential to inhibit thymidine phosphorylase(in vitro). All the synthesized analogs were structurally elucidated using various spectroscopic tools including NMR and HREIMS. All synthesized scaffolds showed varied range of inhibitory potential with IC50values ranging from 5.19 ± 1.10 to 36.18 ± 4.60 μM in comparison to 7-deazaxanthine (IC50 = 30.28 ± 2.10 μM) as a standard drug. All analogs (except analog 4 l which displayed less potency than standard drug) showed improved potency having IC50 values of 19.73 ± 2.30, 16.14 ± 1.20, 18.93 ± 1.60, 22.78 ± 1.80, 30.47 ± 3.70, 5.19 ± 1.10, 23.13 ± 1.90, 21.56 ± 2.50, 4.88 ± 1.10, 26.63 ± 2.90 and 6.67 ± 1.10 respectively.Results obatined were compared to standard 7-deazxanthine drug with IC50 values of 30.28 ± 2.10 μM. Structure-activity relationship (SAR) studies revealed that analogs bearing –NO2, -CF3, –OH and –Cl moieties at various position of aryl part showed many folds more potency than standard 7-deazaxathine standard drug. In order to determine the potential mode of interactions with thymidine phosphorylase active sites, the most active analogs 4f (bearing 3-CF3& 5-NO2), 4i (bearing 3-OH & 5-NO2), and 4 k (bearing 2-OH &5-NO2) were further subjected to molecular docking study. The results confirmed that these active analogs adopted numerous important interactions including hyrognen bonding, pi-donor hydrogen bond, pi-pi T shaped, pi-pi stacking, pi-alkyl, pi-anion, pi-sigma, halogen (flourine) and numerous Vander Waals interactions with the amino acid of enzyme being targeted.

Rs11479 in Thymidine Phosphorylase Associated with Prognosis of Patients with Colorectal Cancer Who Received Capecitabine-Based Adjuvant Chemotherapy.

Thymidine Phosphorylase (TYMP) gene was of potential significance in the process of colorectal cancer (CRC) development and played an important role in capecitabine metabolism. This study was to identify the association between TYMP polymorphism and prognosis of postoperative patients with CRC who received capecitabine-based adjuvant chemotherapy. A total of 218 patients with CRC who were treated with surgical resection and capecitabine-based adjuvant chemotherapy were included in this study retrospectively. Peripheral blood and peripheral blood mononuclear cell (PBMC) specimen of the patients were collected for the genotyping of TYMP polymorphism and TYMP mRNA expression, respectively. Univariate analysis of genotypes and prognosis was carried out by Kaplan-Meier survival analysis, Cox regression analysis was adopted in multivariate analysis. The mRNA expression of TYMP according to genotype status was analyzed using non-parameter test. Prevalence of rs11479 in TYMP among the 218 patients exhibited that minor allele frequency of rs11479 was 0.20 (GG 141 cases, GA 68 cases and AA 9 cases), which was in accordance with Hardy-Weinberg equilibrium (P=0.825). Association analysis suggested that the median disease-free survival (DFS) of patients with GG genotype and GA/AA genotype was 3.1 and 6.1 years, respectively (P=0.004). Furthermore, the median overall survival of patients with GG genotype and GA/AA genotype was 5.0 and 7.0 years, respectively (P=0.033). Multivariate Cox regression analysis exhibited that rs11479 polymorphism was an independent factor for DFS (HR = 1.64, P=0.009). Additionally, of the 65 PBMC specimens, mRNA expression results indicated that patients with GA/AA genotypes conferred significantly higher mRNA expression of TYMP than that of patients with GG genotype (P<0.001). Polymorphism rs11479 in TYMP gene might predict the prognosis of patients with CRC who received capecitabine-based adjuvant chemotherapy through mediation of the mRNA expression of TYMP. The conclusion of this study should be validated in prospective clinical trials subsequently.

Abstract P2-25-01: TRPS1 Regulates angiogenesis via JAG1 and TYMP in Triple-Negative Breast Cancer

Abstract Background: Triple-negative breast cancer (TNBC), the most difficult subtype to treat, is defined as estrogen receptor (ER), HER2, and progesterone receptor (PR) negative and comprises roughly 15-20% of all breast cancers. Patients with a TNBC diagnosis have worse outcomes and poorer survival rates when compared to women with other breast cancers, despite adjuvant chemotherapy. One of the factors contributing to its metastatic ability is the angiogenic process it undergoes. Clinical trials with anti-angiogenic therapeutics showed to be inadequate, with moderate response rates and insignificant survival gains for patients. TRPS1, a GATA and GATA-like transcription factor, has been proven to play a role in the epithelial to mesenchymal transition (EMT) which ultimately promotes tumor growth and metastasis. It has previously been shown that TRPS1 regulates angiogenesis via the expression of VEGF proteins in breast cancer. Our purpose in this study was to further investigate the functional role of TRPS1-regulated genes involved in TNBC angiogenic pathways. Methods: Sleeping-Beauty (SB) transposons help detect cancer progression genes which participate in tumorigenesis and metastasis, not easily identified with current sequencing technology. Using the SB transposon methodology, several candidate trunk drivers were elucidated using Pten mutant mice. Validation studies further showed TRPS1 as one of the eight TNBC tumor suppressor genes discovered by this technology. Then, ChIP-seq array studies were performed which showed that TRPS1 regulates expression of genes involved in the angiogenesis pathway. To corroborate the functional role of TRPS1 in angiogenesis, tube formation and sprouting assays were performed using overexpression and inactivation of TRPS1 in MDA-MD-231 and HCC70 cells, respectively. Furthermore, we performed a ChIP qPCR human angiogenesis array to determine TRPS1-regulated angiogenic genes. A luciferase reporter assay was performed to determine whether TRPS1 is a direct transcriptional regulator of these genes. Results: Inactivation of TRPS1 expression allowed tube formation and cell branching in the sprouting assays. Tumor xenografts overexpressing TRPS1 were analyzed by immunohistochemistry, showed a significant reduction of angiogenic vasculature when stained with CD31; and a substantial increase in blood vessels when TRPS1 was shRNA inactivated. Furthermore, human ChIP qPCR angiogenesis array identified 10 top candidate genes potentially regulated by TRPS1 transcription factor. Of these candidates, JAG1 and TYMP showed to have a higher fold enrichment compared to other angiogenic related genes. Finally, we validated its direct functional binding by using luciferase reporter assay, such demonstrated that TRPS1 is a direct transcriptional regulator of JAG1 and TYMP. Discussion: It has been previously established that the NOTCH ligand, JAG1 and the thymidine phosphorylase enzyme, TYMP are powerful genes participating in angiogenesis. Furthermore, elevated expression of JAG1 mRNA and protein has been associated with poor patient outcomes in breast cancer. Similarly, TYMP activity has been shown to be higher in triple-negative breast tumors, leading to poor outcomes as well. Although several anti-angiogenic drugs have been approved, tumor-acquired resistance to therapy limit their effectiveness. An increased understanding of tumor vessel mechanisms involved in tumorigenesis and metastasis is necessary to overcome the obstacles that prevent successful control of the angiogenic response in tumors. This would require as many paths as can be unraveled regarding the angiogenic mechanisms driving TNBC. Understanding the relationship between the function of TRPS1 in regulating the expression of important genes such as JAG1 and TYMP lays a better foundation for new drugs and drug targets that control and inhibit tumor angiogenesis. Citation Format: Liliana Guzman, Camila Ayerbe, Roberto Rangel, Jenny Chang, Roberto Rosato. TRPS1 Regulates angiogenesis via JAG1 and TYMP in Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-25-01.

Depletion of plasma thymidine results in growth retardation and mitochondrial myopathy in mice overexpressing human thymidine phosphorylase

Plasma thymidine levels in rodents are higher than in other mammals including humans, possibly due to a different pattern and lower level of thymidine phosphorylase expression. Here, we generated a novel knock-in (KI) mouse line with high systemic expression of human thymidine phosphorylase to investigate this difference in nucleotide metabolism in rodents. The KI mice showed growth retardation around weaning and died by 4weeks of age with a decrease in plasma thymidine level compared with the litter-control WT mice. These phenotypes were completely or partially rescued by administration of the thymidine phosphorylase inhibitor 5-chloro-6-(2-iminopyrrolidin-1-yl) methyl-2,4(1H,3H)-pyrimidinedione hydrochloride or thymidine, respectively. Interestingly, when thymidine phosphorylase inhibitor administration was discontinued in adult animals, KI mice showed deteriorated grip strength and locomotor activity, decreased bodyweight, and subsequent hind-limb paralysis. Upon histological analyses, we observed axonal degeneration in the spinal cord, muscular atrophy with morphologically abnormal mitochondria in quadriceps, retinal degeneration, and abnormality in the exocrine pancreas. Moreover, we detected mitochondrial DNA depletion in multiple tissues of KI mice. These results indicate that the KI mouse represents a new animal model for mitochondrial diseases and should be applicable for the study of differences in nucleotide metabolism between humans and mice.

An Understanding of Mechanism-Based Approaches for 1,3,4-Oxadiazole Scaffolds as Cytotoxic Agents and Enzyme Inhibitors.

The world's health system is plagued by cancer and a worldwide effort is underway to find new drugs to treat cancer. There has been a significant improvement in understanding the pathogenesis of cancer, but it remains one of the leading causes of death. The imperative 1,3,4-oxadiazole scaffold possesses a wide variety of biological activities, particularly for cancer treatment. In the development of novel 1,3,4-oxadiazole-based drugs, structural modifications are important to ensure high cytotoxicity towards malignant cells. These structural modification strategies have shown promising results when combined with outstanding oxadiazole scaffolds, which selectively interact with nucleic acids, enzymes, and globular proteins. A variety of mechanisms, such as the inhibition of growth factors, enzymes, and kinases, contribute to their antiproliferative effects. The activity of different 1,3,4-oxadiazole conjugates were tested on the different cell lines of different types of cancer. It is demonstrated that 1,3,4-oxadiazole hybridization with other anticancer pharmacophores have different mechanisms of action by targeting various enzymes (thymidylate synthase, HDAC, topoisomerase II, telomerase, thymidine phosphorylase) and many of the proteins that contribute to cancer cell proliferation. The focus of this review is to highlight the anticancer potential, molecular docking, and SAR studies of 1,3,4-oxadiazole derivatives by inhibiting specific cancer biological targets, such as inhibiting telomerase activity, HDAC, thymidylate synthase, and the thymidine phosphorylase enzyme. The purpose of this review is to summarize recent developments and discoveries in the field of anticancer drugs using 1,3,4-oxadiazoles.

QSAR Analysis, Molecular Docking and ADME Studies of Thiobarbituric Acid Derivatives as Thymidine Phosphorylase Inhibitors: A Rational Approach to Anticancer Drug Design by in silico Modelling

Background: Thymidine Phosphorylase (TP) is an imperative target for cancer researchers. In the current research, quantitative structure-activity relationship (QSAR) models were demonstrated to identify new TP inhibitors. Objective: The main objective is to perform a QSAR study on a series of 19 derivatives of thiobarbituric acid and new molecules designed and dock to check potency and efficacy for anticancer activity. Methods: Multiple linear regression analysis (MLR) was used to establish a two-dimensional quantitative structure-activity relationship (2D-QSAR) with regression coefficient values of 0.9781, 0.9513, and 0.9819 for the training set (r2), leave-one-out (LOO) dependent internal regression (q2), and external test set regression (r2 _pred), respectively. Three-dimensional quantitative structure-activity relationship (3DQSAR) model, obtained by using the simulated annealing k nearest neighbour (SA-KNN) method (q2 = 0.7880). Newly designed molecules were subjected to docking studies with 7-deazaxanthine taken as standard. Results: Molecular modelling, structure-based drug design and docking study analysis were performed. The new chemical entities (NCE’s) designed, docked towards targeted receptor and show good results as compared to the standard 7-deazaxanthine. It was found that these molecules bind similar amino acid pocket regions as that of standard. Molecules bind at the active site of TP enzyme involving H bond interactions with shorter distances showed greater affinity. At last, the oral bioavailability and toxic effect were evaluated as absorption, distribution, metabolism, and elimination (ADME) studies by computational means of the Qikprop tool of Schrodinger. Conclusion: One of the most successful and fast-increasing methodologies is molecular modelling. It not only aids in the prediction of specific target compounds but also aids in the cost reduction of valuable substances. QSAR and docking study was performed, and most of the molecules have shown good dock scores. Based on these results, NCE’s for anticancer activity were successfully designed and analysed in this research work which will be helpful for effective drug synthesis with less toxicity in the future. Others: 2D QSAR model was generated by three methods, and the best one was selected for further study. NCEs were planned based on descriptors such as topological, electrostatic, steric, and hydrophobic substitutions around the core.

Effects of gene expression in 5-FU metabolic pathways in a phase III trial evaluating adjuvant S-1 therapy compared to surgery alone following curative resection for biliary tract cancer (JCOG1202A1).

732 Background: S-1, an oral fluoropyrimidine derivative, is standard adjuvant therapy in Japanese patients with resected biliary tract cancer (BTC), based on the results of JCOG1202, a phase III trial evaluating adjuvant S-1 following curative resection for BTC compared to surgery alone. The efficacy of 5-FU-based therapy is related to the expression of enzymes involved in 5-FU metabolic pathways, such as thymidylate synthase (TS), thymidine phosphorylase (TP), orotate phosphoribosyltransferase (OPRT) and dihydropyrimidine dehydrogenase (DPD). The aim of this study was to evaluate the impact of messenger RNA (mRNA) levels of the four 5-FU metabolic pathway genes on the outcomes of patients enrolled in JCOG1202. Methods: Tumor cell RNA was isolated from formalin-fixed paraffin-embedded primary BTC specimens resected in 264 of 440 randomized patients in JCOG1202. The four 5-FU metabolic pathway genes were measured in 183 patients (surgery alone: n = 94; adjuvant S-1: n = 89) who were randomly divided to training (n = 96) or validation set (n = 87). The endpoints of interest were the predictive values of the four genes for the efficacy of adjuvant S-1 on overall survival and relapse-free survival (RFS), and we here report the results regarding RFS. Cut-off levels for mRNA expression were selected in the training set which minimized the bootstrap p-values (2,000 samples) of an interaction term of treatment (surgery alone or S-1) and mRNA expression in a Cox regression model. Results: There were no obvious differences in each mRNA level and clinical characteristics between surgery alone and adjuvant S-1 groups. RFS tended to be better with adjuvant S-1 (hazard ratio [HR] = 0.790, [95% confidence interval: 0.524-1.192]) compared to surgery alone, which was maintained in the low DPD population (HR = 0.440 [0.216-0.898] in training set and 0.748 [0.334-1.675] in validation set), in the low TP population (HR = 0.709 [0.388-1.296] and 0.602 [0.287-1.262]), and in the high OPRT population (HR = 0.520 [0.152-1.779] and 0.609 [0.161-2.304]). Conclusions: The efficacy of adjuvant S-1 on RFS was representative in BTC patients with intratumoral gene expressions of low DPD, low TP, and high OPRT, which was disappeared in the population with high DPD, high TP, and low OPRT. These biomarkers might be useful to predict therapeutic benefits with S-1 containing chemotherapy for BTC.

A Rational Approach to Anticancer Drug Design: 2D and 3D- QSAR, Molecular Docking and Prediction of ADME Properties using Silico Studies of Thymidine Phosphorylase Inhibitors

Background: Cancer is the most prevalent disease seen nowadays. Thymidine phosphorylase (TP) is an angiogenic enzyme that is overexpressed in many solid tumors. Over the years, Thymidine phosphorylase has emerged as a novel target for anticancer drug development as an inhibitor. Objective: To design novel oxadiazole-isatin pharmacophore-containing molecules and explore their structural requirements related to the anticancer activity. Methods: Pharmacophore optimisation was carried out for oxadiazole-isatin hybrid molecules using molecular modeling studies (2D and 3D QSAR). Further, the new chemical entities were designed using the combilib tool of V life software. To have a better understanding of the binding interactions, the newly designed molecules were docked. To achieve a drug-like pharmacokinetic profile, molecules were also tested for ADME prediction. Results: Two-Dimensional Quantitative Structure-Activity Relationship (2D-QSAR) model was generated using the multiple regression method with r2 = 0.84 and q2 = 0.76. Three-Dimensional Quantitative Structure-Activity Relationship (3D-QSAR) model was obtained by simulated annealing k nearest near (SA kNN) method with q2 = 0.8099. Molecular docking studies showed promising results. Compound 5 was found to be with the best dock score and the best fit to the active site pocket of the thymidylate phosphorylase enzyme. The compounds have notable absorption, distribution, metabolism, and excretion (ADME) properties that can be predicted to assure a drug-like pharmacokinetic profile. Conclusion: One of the most successful and fast-increasing methodologies is molecular modeling. It not only aids in the prediction of specific target compounds but also aids in the cost reduction of valuable substances. The successful use of molecular modeling was done in this study, with caution taken to avoid any chance co-relation. Optimised pharmacophore was obtained and new chemical entities were designed. Docking studies revealed that Compound 5 has shown better H-bond interaction with Lys 221 and Thr 151 with bond distances 2.0 Α° and 1.8 Α° which is the most active molecule. ADME tests discovered that the majority of the newly designed compounds were within a reasonable range as required in a druglike pharmacokinetic profile. Molecules 2, 4, 5, 6 can be considered as a lead for future synthesis and biological screening.

Phase I study of trifluridine/tipiracil in combination with gemcitabine (gem) and nab-paclitaxel (nab-P) in patients (pts) with advanced pancreatic ductal adenocarcinoma (PDAC).

731 Background: The incidence of PDAC is on the rise and it is predicted to be the 2nd leading cause of cancer related mortality in the next decade. Most patients present with advanced disease at diagnoses with limited systemic treatment options. Fluoropyrimidines are active in PDAC. Lonsurf (L) is an orally administered combination of a thymidine-based nucleic acid analogue, trifluridine, and a thymidine phosphorylase inhibitor, tipiracil hydrochloride. Preclinical data demonstrate Lonsurf may have activity in 5-FU resistant malignancies. This phase I study combines Gem, nab-P, and L. Methods: Gem and nab-P are dosed on days 1 and 15 IV on a 28 day cycle. L (20-30mg/m2) is dosed twice daily on days 2-6 and 16-20 (table 1). Dose escalation is by 3+3 design. Key eligibility include pts with untreated locally advanced or metastatic PDAC, ECOG 0-1, and adequate hepatic and bone marrow function. Results: 14 pts (median age 62 yrs [range 43-74]) have been enrolled. Dose was initiated at DL1. The first 3 pts were treated without DLT. DL2 exceeded the MTD with 1 patient experiencing grade 3 infection (cholangitis). Dose expansion to 7 patients was completed at DL 1 with no further DLTs. The RP2D is Gem 800mg/m2, Nab-P 100mg/m2, and L 25mg/m2. Of the 10 patients with evaluable disease, 2 (20%) had PR and 7 (70%) had SD. Pts were on study a median of 14 months (range 4 -31+). Most common grade 3/4 AEs include fatigue (46%,) neutropenia (38%), anemia (31%) anorexia (15%), nausea (15%), vomiting (15%), abdominal pain (15%), hyperglycemia (15%). No grade 5 events occurred. Conclusions: The RP2D is Gem 800mg/m2, Nab-P 100mg/m2, and L 25mg/m2. This combination was well tolerated with expected toxicities of myelosuppression with prolonged responses seen. Clinical trial information: NCT04046887 . [Table: see text]

Thymidine phosphorylase as predictors for progression free survival in patient with ovarian cancer

Thymidine phosphorylase as predictors for progression free survival in patient with ovarian cancer

Druggable Biomarkers Altered in Clear Cell Renal Cell Carcinoma: Strategy for the Development of Mechanism-Based Combination Therapy.

Targeted therapeutics made significant advances in the treatment of patients with advanced clear cell renal cell carcinoma (ccRCC). Resistance and serious adverse events associated with standard therapy of patients with advanced ccRCC highlight the need to identify alternative 'druggable' targets to those currently under clinical development. Although the Von Hippel-Lindau (VHL) and Polybromo1 (PBRM1) tumor-suppressor genes are the two most frequently mutated genes and represent the hallmark of the ccRCC phenotype, stable expression of hypoxia-inducible factor-1α/2α (HIFs), microRNAs-210 and -155 (miRS), transforming growth factor-beta (TGF-ß), nuclear factor erythroid 2-related factor 2 (Nrf2), and thymidine phosphorylase (TP) are targets overexpressed in the majority of ccRCC tumors. Collectively, these altered biomarkers are highly interactive and are considered master regulators of processes implicated in increased tumor angiogenesis, metastasis, drug resistance, and immune evasion. In recognition of the therapeutic potential of the indicated biomarkers, considerable efforts are underway to develop therapeutically effective and selective inhibitors of individual targets. It was demonstrated that HIFS, miRS, Nrf2, and TGF-ß are targeted by a defined dose and schedule of a specific type of selenium-containing molecules, seleno-L-methionine (SLM) and methylselenocystein (MSC). Collectively, the demonstrated pleiotropic effects of selenium were associated with the normalization of tumor vasculature, and enhanced drug delivery and distribution to tumor tissue, resulting in enhanced efficacy of multiple chemotherapeutic drugs and biologically targeted molecules. Higher selenium doses than those used in clinical prevention trials inhibit multiple targets altered in ccRCC tumors, which could offer the potential for the development of a new and novel therapeutic modality for cancer patients with similar selenium target expression. Better understanding of the underlying mechanisms of selenium modulation of specific targets altered in ccRCC could potentially have a significant impact on the development of a more efficacious and selective mechanism-based combination for the treatment of patients with cancer.