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David J Harrison - One of the best experts on this subject based on the ideXlab platform.
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Abstract C122: Nuc-7738, a novel ProTide modification of 3’-deoxyadenosine, activates AMPK and kills renal cancer cells in vitro
Molecular Cancer Therapeutics, 2020Co-Authors: Mary Kudsy, Michelle Myers, In Hwa Um, Mustafa Elshani, Sarah Puthur, Grant D Stewart, Maeve A Rahilly, Alex Chapman, David J HarrisonAbstract:Background: Cordycepin or 3′-deoxyadenosine inhibits the growth of cancer cells by several possible mechanisms. The ProTide Nuc-7738, a phosphoramidate transformation of cordycepin, is designed to overcome cancer resistance mechanisms through direct release of 3’-deoxyadenosine monophosphate in cells. We hypothesized that it might activate AMP-activated protein kinase (AMPK), the key cellular energy sensor, and thus disrupt metabolic homeostasis in cancer cells. Clear cell renal cell carcinoma (ccRCC) is characterized by lipid accumulation due to dysregulation of the genes fundamental in metabolic pathways and so this disease might be a promising target for metabolic treatment. Phosphorylation of AMPK (pAMPK) is associated with downregulation of mTOR signalling, highlighting the potential of Nuc-7738 to regulate this important cancer pathway. Methods: The expression of pAMPK and AMPK in ccRCC from 293 patients was analyzed by immunofluorescence, images were captured digitally and were analyzed using QuPath software. The effect of Nuc-7738 on the growth and confluence of nine renal cancer cell lines was assessed using SRB assay and Celigo scanner, under both 0.5% oxygen and normoxic conditions. Western blotting was used to assess changes in the ratio of pAMPK:AMPK caused by Nuc-7738 and results read using LiCor Odyssey. Effect of Nuc-7738 on AMPK activation in ex vivo ccRCC tissue slices was analyzed using immunofluorescence and QuPath software. Results: Whereas AMPK was widely expressed in ccRCC, pAMPK was focal and very heterogeneous. Cell lines by contrast strongly expressed pAMPK, but this was reduced when culture conditions were altered to be more physiologically appropriate through reduction of oxygen tension and lowering glucose levels. Nuc-7738 inhibited the growth of renal cancer cell lines under both hypoxic and normoxic conditions, and increased pAMPK levels were noted after 1 hour, 6 hours, 24 hours, and 48 hours treatment, with inhibition of mTOR activity predominantly observed after 48 hours. Nuc-7738 also increased pAMPK levels in ex vivo ccRCC tissue slices. Conclusion: Activation of AMPK was generally low in both primary ccRCC tissue and cell lines grown under physiologically appropriate conditions. Nuc-7738 caused activation of AMPK in ex vivo ccRCC tissue slices and in cell lines, and demonstrated efficacy against cell lines in both normoxic and hypoxic conditions. Renal cancer tissue typically has low expression of pAMPK, raising the prospect that AMPK modulation may offer a therapeutic option for ccRCC. These results suggest that inhibition of the mTOR pathway may be one of the anti-cancer mechanisms through which Nuc-7738 exerts its activity. Citation Format: Mary Kudsy, Mustafa Elshani, Sarah Puthur, In Hwa Um, Grant D. Stewart, Maeve Rahilly, Alex Chapman, Michelle Myers, David J. Harrison. Nuc-7738, a novel ProTide modification of 3’-deoxyadenosine, activates AMPK and kills renal cancer cells in vitro [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C122. doi:10.1158/1535-7163.TARG-19-C122
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abstract c059 inhibition of thymidylate synthase by the protide Nuc 3373 in vitro analysis and clinical validation
Molecular Cancer Therapeutics, 2019Co-Authors: Sarah P Blagden, Fiona G Mckissock, Janet Shirley Graham, Kristen Keon Ciombor, Francesca Aroldi, Lisa Rodgers, Michelle Myers, Jordan Berlin, T Jeffry R Evans, David J HarrisonAbstract:Background: Although 5-FU-based regimens such as FOLFOX and FOLFIRI remain the standard of care for treatment of patients with colorectal cancer (CRC), their clinical utility is limited by resistance mechanisms and the production of toxic by-products. Anti-cancer activity of 5-FU requires its conversion to the active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), which binds to and inhibits thymidylate synthase (TS), depleting dTMP, resulting in DNA damage. Due to breakdown by dihydropyrimidine dehydrogenase (DPD), a reliance on enzymatic activation and a short plasma half-life; 5-FU is a sub-optimal inhibitor of TS. Nuc-3373, a phosphoramidate transformation of FUDR-MP, was designed to bypass the key resistance mechanisms associated with the activation and breakdown of 5-FU. Herein, we characterize the impact of DPD on Nuc-3373 and the role of Nuc-3373 on TS biology in vitro. The NuTide:302 clinical study (NCT03428958) is investigating Nuc-3373 pharmacokinetics and dTMP levels. Methods: Sensitivity of nine CRC cell lines was assessed by sulforhodamine B assay and IC50values established, with two (one sensitive and one less sensitive) selected for further evaluation. Pharmacological inhibition by gimeracil was used to investigate DPD-induced catabolism in cells treated with 5-FU and Nuc-3373. Thymidine supplementation was used to indirectly assess the effect of Nuc-3373 on the de novopathway of dTMP synthesis. Immunocytochemistry and Western blot analysis were used to assess the localization and expression of TS. NuTide:302 is a three-part, Phase Ib study in patients with advanced CRC who have relapsed after ≥2 prior lines of 5-FU-containing therapies. Pharmacokinetic analyses via LC-MS from patient PBMCs are intended to determine whether leucovorin (LV) augments Nuc-3373 inhibition of TS and its depletion of the dTMP pool. Results: Unlike 5-FU-treated cells, inhibition of DPD by gimeracil had no significant effect on survival following Nuc-3373 treatment. Supplementation with exogenous thymidine rescued cells from Nuc-3373-induced death. No correlation was found between pre-treatment TS protein expression and the IC50for Nuc-3373. In selected cell lines treated with Nuc-3373, TS ternary complexes were detected for at least 72 hours. Treatment with Nuc-3373 was also associated with increased cytoplasmic TS expression. In the clinical setting, 22 patients have received Nuc-3373 in NuTide:302. The clinical impact of LV on TS inhibition and dTMP pool depletion in patient samples will be presented. Conclusions: Unlike 5-FU, Nuc-3373 was not catabolised by DPD, highlighting the ability of Nuc-3373 to bypass a key rate-limiting factor associated with 5-FU. Nuc-3373 targets the de novo pathway of dTMP synthesis and its cytotoxicity is not dependent on basal TS expression. Formation of long-lasting ternary complexes and increased cytoplasmic expression suggest that Nuc-3373 is a potent inhibitor of TS activity. Data describing whether LV potentiates the impact of Nuc-3373 on TS in patients will be presented. Citation Format: Sarah P Blagden, Fiona G McKissock, Janet S Graham, Kristen K Ciombor, Francesca Aroldi, Lisa J Rodgers, Michelle Myers, Jordan Berlin, T.R. Jeffry Evans, David J Harrison. Inhibition of thymidylate synthase by the ProTide Nuc-3373: in vitro analysis and clinical validation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C059. doi:10.1158/1535-7163.TARG-19-C059
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abstract c122 Nuc 7738 a novel protide modification of 3 deoxyadenosine activates ampk and kills renal cancer cells in vitro
Molecular Cancer Therapeutics, 2019Co-Authors: Mary Kudsy, Michelle Myers, In Hwa Um, Mustafa Elshani, Sarah Puthur, Grant D Stewart, Maeve A Rahilly, Alex Chapman, David J HarrisonAbstract:Background: Cordycepin or 3′-deoxyadenosine inhibits the growth of cancer cells by several possible mechanisms. The ProTide Nuc-7738, a phosphoramidate transformation of cordycepin, is designed to overcome cancer resistance mechanisms through direct release of 3’-deoxyadenosine monophosphate in cells. We hypothesized that it might activate AMP-activated protein kinase (AMPK), the key cellular energy sensor, and thus disrupt metabolic homeostasis in cancer cells. Clear cell renal cell carcinoma (ccRCC) is characterized by lipid accumulation due to dysregulation of the genes fundamental in metabolic pathways and so this disease might be a promising target for metabolic treatment. Phosphorylation of AMPK (pAMPK) is associated with downregulation of mTOR signalling, highlighting the potential of Nuc-7738 to regulate this important cancer pathway. Methods: The expression of pAMPK and AMPK in ccRCC from 293 patients was analyzed by immunofluorescence, images were captured digitally and were analyzed using QuPath software. The effect of Nuc-7738 on the growth and confluence of nine renal cancer cell lines was assessed using SRB assay and Celigo scanner, under both 0.5% oxygen and normoxic conditions. Western blotting was used to assess changes in the ratio of pAMPK:AMPK caused by Nuc-7738 and results read using LiCor Odyssey. Effect of Nuc-7738 on AMPK activation in ex vivo ccRCC tissue slices was analyzed using immunofluorescence and QuPath software. Results: Whereas AMPK was widely expressed in ccRCC, pAMPK was focal and very heterogeneous. Cell lines by contrast strongly expressed pAMPK, but this was reduced when culture conditions were altered to be more physiologically appropriate through reduction of oxygen tension and lowering glucose levels. Nuc-7738 inhibited the growth of renal cancer cell lines under both hypoxic and normoxic conditions, and increased pAMPK levels were noted after 1 hour, 6 hours, 24 hours, and 48 hours treatment, with inhibition of mTOR activity predominantly observed after 48 hours. Nuc-7738 also increased pAMPK levels in ex vivo ccRCC tissue slices. Conclusion: Activation of AMPK was generally low in both primary ccRCC tissue and cell lines grown under physiologically appropriate conditions. Nuc-7738 caused activation of AMPK in ex vivo ccRCC tissue slices and in cell lines, and demonstrated efficacy against cell lines in both normoxic and hypoxic conditions. Renal cancer tissue typically has low expression of pAMPK, raising the prospect that AMPK modulation may offer a therapeutic option for ccRCC. These results suggest that inhibition of the mTOR pathway may be one of the anti-cancer mechanisms through which Nuc-7738 exerts its activity. Citation Format: Mary Kudsy, Mustafa Elshani, Sarah Puthur, In Hwa Um, Grant D. Stewart, Maeve Rahilly, Alex Chapman, Michelle Myers, David J. Harrison. Nuc-7738, a novel ProTide modification of 3’-deoxyadenosine, activates AMPK and kills renal cancer cells in vitro [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C122. doi:10.1158/1535-7163.TARG-19-C122
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genome scale crispr cas9 screen determines factors modulating sensitivity to protide Nuc 1031
Scientific Reports, 2019Co-Authors: Awa Sarr, David J Harrison, Peter Mullen, In Hwa Um, Tsz Huen Chan, Paul A ReynoldsAbstract:Gemcitabine is a fluoropyrimidine analogue that is used as a mainstay of chemotherapy treatment for pancreatic and ovarian cancers, amongst others. Despite its widespread use, gemcitabine achieves responses in less than 10% of patients with metastatic pancreatic cancer and has a very limited impact on overall survival due to intrinsic and acquired resistance. Nuc-1031 (Acelarin), a phosphoramidate transformation of gemcitabine, was the first anti-cancer ProTide to enter the clinic. We find it displays important in vitro cytotoxicity differences to gemcitabine, and a genome-wide CRISPR/Cas9 genetic screening approach identified only the pyrimidine metabolism pathway as modifying cancer cell sensitivity to Nuc-1031. Low deoxycytidine kinase expression in tumour biopsies from patients treated with gemcitabine, assessed by immunostaining and image analysis, correlates with a poor prognosis, but there is no such correlation in tumour biopsies from a Phase I cohort treated with Nuc-1031.
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Genome-scale CRISPR/Cas9 screen determines factors modulating sensitivity to ProTide Nuc-1031
Scientific Reports, 2019Co-Authors: Awa Sarr, David J Harrison, Peter Mullen, In Hwa Um, Tsz Huen Chan, Paul A ReynoldsAbstract:Gemcitabine is a fluoropyrimidine analogue that is used as a mainstay of chemotherapy treatment for pancreatic and ovarian cancers, amongst others. Despite its widespread use, gemcitabine achieves responses in less than 10% of patients with metastatic pancreatic cancer and has a very limited impact on overall survival due to intrinsic and acquired resistance. Nuc-1031 (Acelarin), a phosphoramidate transformation of gemcitabine, was the first anti-cancer ProTide to enter the clinic. We find it displays important in vitro cytotoxicity differences to gemcitabine, and a genome-wide CRISPR/Cas9 genetic screening approach identified only the pyrimidine metabolism pathway as modifying cancer cell sensitivity to Nuc-1031. Low deoxycytidine kinase expression in tumour biopsies from patients treated with gemcitabine, assessed by immunostaining and image analysis, correlates with a poor prognosis, but there is no such correlation in tumour biopsies from a Phase I cohort treated with Nuc-1031.
Robert L Metzenberg - One of the best experts on this subject based on the ideXlab platform.
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Nuc 2 a component of the phosphate regulated signal transduction pathway in neurospora crassa is an ankyrin repeat protein
Molecular Genetics and Genomics, 1996Co-Authors: Yoav Peleg, Seogchan Kang, Rodolfo Aramayo, J G Hall, Robert L MetzenbergAbstract:In response to phosphorus limitation, the fungusNeurospora crassa synthesizes a number of enzymes that function to bring more phosphate into the cell. The Nuc-2 protein appears to sense the availability of phosphate and transmits the signal down-stream to the regulatory pathway. TheNuc-2+ gene has been cloned by its ability to restore growth of aNuc-2 mutant under restrictive conditions of high pH and low phosphate concentration. We mapped the cloned gene to the right arm of linkage group II, consistent with the chromosomal position of theNuc-2 mutation as determined by classical genetic mapping. TheNuc-2+ open reading frame is interrupted by five introns and codes for a protein of 1066 amino acid residues. Its predicted amino acid sequence has high similarity to that of its homolog inSaccharomyces cerevisiae, PHO81. Both proteins contain six ankyrin repeats, which have been implicated in the cyclin-dependent kinase inhibitory activity of PHO81. The phenotypes of aNuc-2 mutant generated by repeat-induced point mutation and of a strain harboring a UV-inducedNuc-2 allele are indistinguishable. Both are unable to grow under the restrictive conditions, a phenotype which is to some degree temperature dependent. TheNuc-2+ gene is transcriptionally regulated. A 15-fold increase in the level of theNuc-2+ transcript occurs in response to a decrease in exogenous phosphate concentration.
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translocation of neurospora crassa transcription factor Nuc 1 into the Nucleus is induced by phosphorus limitation
Fungal Genetics and Biology, 1996Co-Authors: Yoav Peleg, Randolph Addison, Rodolfo Aramayo, Robert L MetzenbergAbstract:Abstract Peleg, Y., Addison, R., Aramayo, R., and Metzenberg, R. L. 1996. Translocation of Neurospora crassa transcription factor Nuc-1 into the Nucleus is induced by phosphorus limitation. Fungal Genetics and Biology 20, 185–191. Nuc-1, a basic helix–loop–helix zipper protein, activates the expression of several genes involved in phosphorus acquisition in Neurospora crassa. In the present study we investigated whether posttranscriptional mechanisms control the activity of Nuc-1. The Nuc-1 level was higher (up to fivefold) in wild-type cells grown at low external phosphate concentration and in mutant strains expressing the phosphorus acquisition genes constitutively than in a wild-type strain grown at high external phosphate concentration. Using indirect immunofluorescence we demonstrated that Nuc-1 is localized at least predominantly in the cytosol when wild-type N. crassa is grown with an adequate supply of phosphate, whereas Nuc-1 is largely concentrated in the Nucleus upon limitation of external phosphate. In mutant strains expressing the phosphorus acquisition genes constitutively, Nuc-1 localization was also primarily in the Nucleus. Thus, subcellular compartmentation of regulatory proteins is an important mechanism in regulating gene expression in filamentous fungi.
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analysis of the dna binding and dimerization activities of neurospora crassa transcription factor Nuc 1
Molecular and Cellular Biology, 1994Co-Authors: Yoav Peleg, Robert L MetzenbergAbstract:Abstract Nuc-1, a positive regulatory protein of Neurospora crassa, controls the expression of several unlinked target genes involved in phosphorus acquisition. The carboxy-terminal end of the Nuc-1 protein has sequence similarity to the helix-loop-helix family of transcription factors. Bacterially expressed and in vitro-synthesized proteins, which consist of the carboxy-terminal portion of Nuc-1, bind specifically to upstream sequences of two of its target genes, pho2+ and pho-4+. These upstream sequences contain the core sequence, CACGTG, a target for many helix-loop-helix proteins. A large loop region (47 amino acids) separates the helix I and helix II domains. Mutations and deletion within the loop region did not interfere with the in vitro or in vivo functions of the protein. Immediately carboxy-proximal to the helix II domain, the Nuc-1 protein contains an atypical zipper domain which is essential for function. This domain consists of a heptad repeat of alanine and methionine rather than leucine residues. Analysis of mutant Nuc-1 proteins suggests that the helix II and the zipper domains are essential for the protein dimerization, whereas the basic and the helix I domains are involved in DNA binding. The helix I domain, even though likely to participate in dimer formation while Nuc-1 is bound to DNA, is not essential for in vitro dimerization.
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molecular analysis of Nuc 1 a gene controlling phosphorus acquisition in neurospora crassa
Molecular and Cellular Biology, 1990Co-Authors: Seogchan Kang, Robert L MetzenbergAbstract:Abstract In response to phosphorus starvation, Neurospora crassa makes several enzymes that are undetectable or barely detectable in phosphate-sufficient cultures. The Nuc-1+ gene, whose product regulates the synthesis of these enzymes, was cloned and sequenced. The Nuc-1+ gene encodes a protein of 824 amino acids with a predicted molecular weight of 87,429. The amino acid sequence shows homology with two yeast proteins whose functions are analogous to that of the Nuc-1 protein. Two Nuc-1+ transcripts of 3.2 and 3.0 kilobases were detected; they were present in similar amounts during growth at low or high phosphate concentrations. The Nuc-2+ gene encodes a product normally required for Nuc-1 function, and yet a Nuc-2 mutation can be complemented by overexpression of the Nuc-1+ gene. This implies physical interactions between Nuc-1 protein and the negative regulatory factor(s) PREG and/or PGOV. Analysis of Nuc-2 and Nuc-1; Nuc-2 strains transformed by the Nuc-1+ gene suggests that phosphate directly affects the level or activity of the negative regulatory factor(s) controlling phosphorus acquisition.
Yoshikazu Kurosawa - One of the best experts on this subject based on the ideXlab platform.
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organization of the human gene for Nucleobindin Nuc and its chromosomal assignment to 19q13 2 q13 4
Genomics, 1996Co-Authors: Keiji Miura, Momoki Hirai, Yoshiyuki Kanai, Yoshikazu KurosawaAbstract:Nucleobindin (Nuc) was first identified as a secreted protein of 55 kDa that promotes production of DNA-specific antibodies in lupus-prone MRL/lpr mice. Analysis of cDNA that encoded Nuc revealed that the protein is composed of a signal peptide, a DNA-binding site, two calcium-binding motifs (EF-hand motifs), and a leucine zipper. In the present study, we analysed the organization of the human gene for Nuc (Nuc). It consists of 13 exons that are distributed in a region of 32 kb. The functional motifs listed above are encoded in corresponding exons. Nuc was expressed in all organs examined. Comparison of Nucleotide sequences in the promotre regions between human and mouse NCU genes revealed several conserved sequences. Among them, two Sp1-binding sites and a CCAAT box are of particular interest. The promoter is of the TATA-less type, and transcription starts at multiple sites in both the human and the mouse genes. These features suggest that Nuc might normally play a role as a housekeeping gene. Nuc was located at human chromosome 19q13.2-q13.4. 25 refs., 4 figs., 1 tab.
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natural occurrence of Nuc in the sera of autoimmune prone mrl lpr mice
Biochemical and Biophysical Research Communications, 1993Co-Authors: Yoshiyuki Kanai, Keiji Miura, T Uehara, M Amagai, Osamu Takeda, Seiichi Tanuma, Yoshikazu KurosawaAbstract:Abstract We previously established a clone of cells termed KML1-7 which produces a soluble factor that boosts anti-DNA antibody production both in vitro and in vivo across the H-2 barrier. By using the purified protein, termed Nucleobindin (Nuc), we cloned cDNA and produced recombinant (r) Nuc in E.coli. Although the purified rNuc showed biological activities such as anti-DNA antibody boosting and DNA binding, there was no evidence that Nuc is really associated with autoimmune status in lupus-prone MRL/lpr mice. Here we report that identification of Nuc was successful from the sera of MRL/lpr mice, but not from those of the substrain MRL/n mice, which show no apparent autoimmune syndrome at the same age of MRL/lpr mice, by means of immunochemical as well as N-terminal amino-acid sequencing methods.
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Natural occurrence of Nuc in the sera of autoimmune-prone MRL/lpr mice.
Biochemical and Biophysical Research Communications, 1993Co-Authors: Yoshiyuki Kanai, Keiji Miura, T Uehara, M Amagai, Osamu Takeda, Seiichi Tanuma, Yoshikazu KurosawaAbstract:Abstract We previously established a clone of cells termed KML1-7 which produces a soluble factor that boosts anti-DNA antibody production both in vitro and in vivo across the H-2 barrier. By using the purified protein, termed Nucleobindin (Nuc), we cloned cDNA and produced recombinant (r) Nuc in E.coli. Although the purified rNuc showed biological activities such as anti-DNA antibody boosting and DNA binding, there was no evidence that Nuc is really associated with autoimmune status in lupus-prone MRL/lpr mice. Here we report that identification of Nuc was successful from the sera of MRL/lpr mice, but not from those of the substrain MRL/n mice, which show no apparent autoimmune syndrome at the same age of MRL/lpr mice, by means of immunochemical as well as N-terminal amino-acid sequencing methods.
Sarah P Blagden - One of the best experts on this subject based on the ideXlab platform.
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abstract c059 inhibition of thymidylate synthase by the protide Nuc 3373 in vitro analysis and clinical validation
Molecular Cancer Therapeutics, 2019Co-Authors: Sarah P Blagden, Fiona G Mckissock, Janet Shirley Graham, Kristen Keon Ciombor, Francesca Aroldi, Lisa Rodgers, Michelle Myers, Jordan Berlin, T Jeffry R Evans, David J HarrisonAbstract:Background: Although 5-FU-based regimens such as FOLFOX and FOLFIRI remain the standard of care for treatment of patients with colorectal cancer (CRC), their clinical utility is limited by resistance mechanisms and the production of toxic by-products. Anti-cancer activity of 5-FU requires its conversion to the active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), which binds to and inhibits thymidylate synthase (TS), depleting dTMP, resulting in DNA damage. Due to breakdown by dihydropyrimidine dehydrogenase (DPD), a reliance on enzymatic activation and a short plasma half-life; 5-FU is a sub-optimal inhibitor of TS. Nuc-3373, a phosphoramidate transformation of FUDR-MP, was designed to bypass the key resistance mechanisms associated with the activation and breakdown of 5-FU. Herein, we characterize the impact of DPD on Nuc-3373 and the role of Nuc-3373 on TS biology in vitro. The NuTide:302 clinical study (NCT03428958) is investigating Nuc-3373 pharmacokinetics and dTMP levels. Methods: Sensitivity of nine CRC cell lines was assessed by sulforhodamine B assay and IC50values established, with two (one sensitive and one less sensitive) selected for further evaluation. Pharmacological inhibition by gimeracil was used to investigate DPD-induced catabolism in cells treated with 5-FU and Nuc-3373. Thymidine supplementation was used to indirectly assess the effect of Nuc-3373 on the de novopathway of dTMP synthesis. Immunocytochemistry and Western blot analysis were used to assess the localization and expression of TS. NuTide:302 is a three-part, Phase Ib study in patients with advanced CRC who have relapsed after ≥2 prior lines of 5-FU-containing therapies. Pharmacokinetic analyses via LC-MS from patient PBMCs are intended to determine whether leucovorin (LV) augments Nuc-3373 inhibition of TS and its depletion of the dTMP pool. Results: Unlike 5-FU-treated cells, inhibition of DPD by gimeracil had no significant effect on survival following Nuc-3373 treatment. Supplementation with exogenous thymidine rescued cells from Nuc-3373-induced death. No correlation was found between pre-treatment TS protein expression and the IC50for Nuc-3373. In selected cell lines treated with Nuc-3373, TS ternary complexes were detected for at least 72 hours. Treatment with Nuc-3373 was also associated with increased cytoplasmic TS expression. In the clinical setting, 22 patients have received Nuc-3373 in NuTide:302. The clinical impact of LV on TS inhibition and dTMP pool depletion in patient samples will be presented. Conclusions: Unlike 5-FU, Nuc-3373 was not catabolised by DPD, highlighting the ability of Nuc-3373 to bypass a key rate-limiting factor associated with 5-FU. Nuc-3373 targets the de novo pathway of dTMP synthesis and its cytotoxicity is not dependent on basal TS expression. Formation of long-lasting ternary complexes and increased cytoplasmic expression suggest that Nuc-3373 is a potent inhibitor of TS activity. Data describing whether LV potentiates the impact of Nuc-3373 on TS in patients will be presented. Citation Format: Sarah P Blagden, Fiona G McKissock, Janet S Graham, Kristen K Ciombor, Francesca Aroldi, Lisa J Rodgers, Michelle Myers, Jordan Berlin, T.R. Jeffry Evans, David J Harrison. Inhibition of thymidylate synthase by the ProTide Nuc-3373: in vitro analysis and clinical validation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C059. doi:10.1158/1535-7163.TARG-19-C059
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nutide 302 a phase ib study to assess the safety pharmacokinetics and clinical activity of the protide Nuc 3373 when combined with standard agents used in colorectal cancer
Journal of Clinical Oncology, 2019Co-Authors: T Jeffry R Evans, Sarah P Blagden, Janet Shirley Graham, Kristen Keon Ciombor, Aimery De Gramont, Josep Tabernero, Jordan BerlinAbstract:TPS719Background: Although 5-fluorouracil-based chemotherapies (5-FU, capecitabine, and floxuridine) remain the cornerstone of combination therapies for colorectal cancer (CRC), their clinical utility is limited by key cancer resistance mechanisms associated with breakdown, transport, and activation. These agents require intracellular conversion to the active metabolite fluorodeoxyuridine-monophosphate (FUDR-MP) before they can exert their core anti-cancer activity through inhibition of the enzyme thymidylate synthase (TS). Nuc-3373 is a phosphoramidate transformation of FUDR-MP designed to bypass the key resistance mechanisms associated with 5-FU. Results from the first-in-human study of Nuc-3373 (NuTide:301) in patients with advanced solid tumours demonstrated a favourable PK/PD profile for Nuc-3373, with a longer plasma t1/2 (9.7 hours) than 5-FU (8-14 minutes) and much higher levels of the active anti-cancer metabolite, FUDR-MP (Ghazaly et alESMO, 2017). TS is efficiently inhibited and sequestered int...
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anti tumour activity of a first in class agent Nuc 1031 in patients with advanced cancer results of a phase i study
British Journal of Cancer, 2018Co-Authors: Sarah P Blagden, Ivana Rizzuto, Puvan Suppiah, Daniel Oshea, Markand Patel, Laura Spiers, Ajithkumar Sukumaran, Nishat BharwaniAbstract:Gemcitabine is used to treat a wide range of tumours, but its efficacy is limited by cancer cell resistance mechanisms. Nuc-1031, a phosphoramidate modification of gemcitabine, is the first anti-cancer ProTide to enter the clinic and is designed to overcome these key resistance mechanisms. Sixty-eight patients with advanced solid tumours who had relapsed after treatment with standard therapy were recruited to a dose escalation study to determine the recommended Phase II dose (RP2D) and assess the safety of Nuc-1031. Pharmacokinetics and anti-tumour activity was also assessed. Sixty-eight patients received treatment, 50% of whom had prior exposure to gemcitabine. Nuc-1031 was well tolerated with the most common Grade 3/4 adverse events of neutropaenia, lymphopaenia and fatigue occurring in 13 patients each (19%). In 49 response-evaluable patients, 5 (10%) achieved a partial response and 33 (67%) had stable disease, resulting in a 78% disease control rate. Cmax levels of the active intracellular metabolite, dFdCTP, were 217-times greater than those reported for equimolar doses of gemcitabine, with minimal toxic metabolite accumulation. The RP2D was determined as 825 mg/m2 on days 1, 8 and 15 of a 28-day cycle. Nuc-1031 was well tolerated and demonstrated clinically significant anti-tumour activity, even in patients with prior gemcitabine exposure and in cancers not traditionally perceived as gemcitabine-responsive.
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abstract 1855 Nuc 1031 overcomes resistance associated with gemcitabine in cancer patients
Cancer Research, 2018Co-Authors: Awa Sarr, Sarah P Blagden, Peter Mullen, In Hwa Um, Paul A Reynolds, David J HarrisonAbstract:Nuc-1031 (Nucana plc) is a phosphoramidate transformation of gemcitabine and the first anti-cancer compound based on ProTide technology (Slusarczyk et al., 2014). It overcomes the main resistance mechanisms associated with gemcitabine and is currently in Phase I, II and III trials for biliary, ovarian, and pancreatic cancers respectively. High expression of dCK is required for activation of gemcitabine, which competes directly with deoxycytidine (dC) as a substrate for dCK. We aimed to compare the mode of action of Nuc-1031 with gemcitabine. A panel of pancreatic and ovarian cancer cell lines were treated for 2h to 24h with Nuc-1031 or gemcitabine at the EC50 dose and were observed for 96h. Cell morphology and death were observed using time-lapse microscopy. Cell cycle analysis was performed using BrdU labeling and flow cytometry. Exogenous dC was added to the cells either when they were plated or at the time of the treatment, to observe the effect on the sensitivity of cells to Nuc-1031 and gemcitabine. Variation of expression of dCK was determined using RT-qPCR and western blotting. Cytospin, followed by immunocytochemistry, was performed to determine the intracellular localization of dCK. Expression of dCK was assessed in tissue samples from patients in the clinical studies using a modified Allred scoring system. After 48h, most cells treated with Nuc-1031 show a 24h period when they neither divide nor die, after which they then undergo apoptosis. By contrast, cells treated with gemcitabine start to die sooner, showing features of necrosis and apoptosis. A higher proportion of cells treated with Nuc-1031 exhibited a G2/M arrest compared to those treated with gemcitabine or control samples. Protein expression of dCK was increased in MiaPaCa2 cells soon after treatment with gemcitabine compared with Nuc-1031. The pool of endogenous Nucleotides was altered by addition of dC, inducing significant resistance to gemcitabine, both when dC was added at the time of the treatment and when cells were plated (EC50 respectively 2.5 and 9 times higher than in control cells). Nuc-1031 was associated with a moderate decrease in sensitivity when dC was added at the time of the treatment and when cells were plated (EC50 3 times higher than in control cells). Immunohistochemistry of 50 primary tumours from patients enrolled in a Phase 1 study for Nuc-1031 showed low-level dCK expression in almost all tumours studied. Our findings indicate that Nuc-1031 has a more targeted mode of action to induce cell death than gemcitabine. Also, although a small amount of dCK may be required for maximal sensitivity to Nuc-1031, dCK levels are not a limiting step for Nuc-1031 efficacy. Citation Format: Jennifer Bre, Awa Sarr, Peter Mullen, In Hwa Um, Sarah P. Blagden, Paul A. Reynolds, David J. Harrison. Nuc-1031 overcomes resistance associated with gemcitabine in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1855.
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abstract b46 Nuc 3373 a novel pyrimidine Nucleotide analogue that overcomes key cancer drug resistance limiting patient survival
Molecular Cancer Therapeutics, 2015Co-Authors: Essam Ghazaly, Magdalena Slusarczyk, Christopher Mcguigan, David J Harrison, Sarah P BlagdenAbstract:Background: Nuc-3373 is the first Nucleotide Analogue able to bypass the key drug resistance mechanisms associated with 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (FUDR) that severely hinder their clinical activity. The key cellular mechanisms causing drug resistance in cancer cells are the limited expression of activating kinases and Nucleoside transporters, and overexpression of catabolic enzymes. The anti-neoplastic activity of 5-FU is largely attributed to its active metabolite, 5-fluorodeoxyuridine monophosphate (FdUMP), which inhibits the enzyme thymidylate synthase. As it already bears the monophosphate moiety, Nuc-3373 is a pre-activated form of the active anti-cancer agent FdUMP. Here, we report potent in vitro and in vivobiological activity of Nuc-3373. Methods: The cytotoxicity of 5-FU and the ProTide Nuc-3373 was monitored with EC50in vitro viability assays using 4 colorectal (Colo-205-luc; HT-29; HCT-116 and SW620); 3 lung (H1975; H1703 and SK-MES-1); 2 ovarian (OVCAR3 and A2780); 1 acute lymphoblastic leukemia (CCRF-CEM) and 1 cervix (HeLa) human tumour cell lines. These assays were also performed in conditions mimicking cancer resistance, where the activating enzyme thymidine kinase (TK) and the Nucleoside transporter, hENT1, were mutated. Sensitivity of Nuc-3373 and 5-FU to dihydropyrimidine dehydrogenase (DPD) degradation was assessed by absorption spectroscopy and UPLC-MS/MS. Nuc-3373 anti-cancer activity was further evaluated in HT-29 nude mouse xenograft models. Results: Nuc-3373 was more cytotoxic than 5-FU, achieving 2 to 333 fold lower EC50 values in the majority of cancer cell lines tested. TK inhibition reduced the cytotoxicity of Nuc-3373 and FUDR by 4- and 136-fold respectively, suggesting Nuc-3373 is more independent of TK. Inhibition of Nucleoside transport decreased cytotoxic activity of Nuc-3373 only mildly compared to a 63-fold reduction in FUDR cytotoxicity. Nuc-3373 and 5-FU sensitivity to DPD degradation was assessed in cell lysates with or without the DPD inhibitor, gimeracil. Nuc-3373 concentrations remained unaffected in the cell lysate irrespective of gimeracil treatment. In contrast, when the experiment was conducted with 5-FU, gimeracil treatment significantly increased the 5-FU concentration (p = 0.039) suggesting that Nuc-3373, unlike 5-FU, is not a substrate for DPD metabolism. In colorectal cancer xenografts, Nuc-3373 demonstrated greater tumour growth inhibition (47%) than 5-FU (25%). A toxicology study in which Nuc-3373 was administered daily for 5 days/week for 4 consecutive weeks, at doses greater than 4 mg/kg/day in beagle dogs, compared favourably with that described following a single bolus intravenous administration of 5-FU. Conclusions: The novel ProTide, Nuc-3373, overcomes the key cancer resistance mechanisms associated with 5-FU. Nuc-3373 has efficacy in vitro and in vivo, and is resistant to DPD-mediated degradation. Results from formal toxicology assessments support the initiation of human clinical studies. A Phase I/II clinical study at Oxford University has been initiated to explore the safety, pharmacokinetic and clinical activity of Nuc-3373 in participants with advanced solid tumours. Citation Format: Essam A. Ghazaly, Magdalena Slusarczyk, Christopher McGuigan, David Harrison, Sarah P. Blagden. Nuc-3373: A novel pyrimidine Nucleotide analogue that overcomes key cancer drug resistance limiting patient survival. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B46.
Yoshiyuki Kanai - One of the best experts on this subject based on the ideXlab platform.
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organization of the human gene for Nucleobindin Nuc and its chromosomal assignment to 19q13 2 q13 4
Genomics, 1996Co-Authors: Keiji Miura, Momoki Hirai, Yoshiyuki Kanai, Yoshikazu KurosawaAbstract:Nucleobindin (Nuc) was first identified as a secreted protein of 55 kDa that promotes production of DNA-specific antibodies in lupus-prone MRL/lpr mice. Analysis of cDNA that encoded Nuc revealed that the protein is composed of a signal peptide, a DNA-binding site, two calcium-binding motifs (EF-hand motifs), and a leucine zipper. In the present study, we analysed the organization of the human gene for Nuc (Nuc). It consists of 13 exons that are distributed in a region of 32 kb. The functional motifs listed above are encoded in corresponding exons. Nuc was expressed in all organs examined. Comparison of Nucleotide sequences in the promotre regions between human and mouse NCU genes revealed several conserved sequences. Among them, two Sp1-binding sites and a CCAAT box are of particular interest. The promoter is of the TATA-less type, and transcription starts at multiple sites in both the human and the mouse genes. These features suggest that Nuc might normally play a role as a housekeeping gene. Nuc was located at human chromosome 19q13.2-q13.4. 25 refs., 4 figs., 1 tab.
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natural occurrence of Nuc in the sera of autoimmune prone mrl lpr mice
Biochemical and Biophysical Research Communications, 1993Co-Authors: Yoshiyuki Kanai, Keiji Miura, T Uehara, M Amagai, Osamu Takeda, Seiichi Tanuma, Yoshikazu KurosawaAbstract:Abstract We previously established a clone of cells termed KML1-7 which produces a soluble factor that boosts anti-DNA antibody production both in vitro and in vivo across the H-2 barrier. By using the purified protein, termed Nucleobindin (Nuc), we cloned cDNA and produced recombinant (r) Nuc in E.coli. Although the purified rNuc showed biological activities such as anti-DNA antibody boosting and DNA binding, there was no evidence that Nuc is really associated with autoimmune status in lupus-prone MRL/lpr mice. Here we report that identification of Nuc was successful from the sera of MRL/lpr mice, but not from those of the substrain MRL/n mice, which show no apparent autoimmune syndrome at the same age of MRL/lpr mice, by means of immunochemical as well as N-terminal amino-acid sequencing methods.
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Natural occurrence of Nuc in the sera of autoimmune-prone MRL/lpr mice.
Biochemical and Biophysical Research Communications, 1993Co-Authors: Yoshiyuki Kanai, Keiji Miura, T Uehara, M Amagai, Osamu Takeda, Seiichi Tanuma, Yoshikazu KurosawaAbstract:Abstract We previously established a clone of cells termed KML1-7 which produces a soluble factor that boosts anti-DNA antibody production both in vitro and in vivo across the H-2 barrier. By using the purified protein, termed Nucleobindin (Nuc), we cloned cDNA and produced recombinant (r) Nuc in E.coli. Although the purified rNuc showed biological activities such as anti-DNA antibody boosting and DNA binding, there was no evidence that Nuc is really associated with autoimmune status in lupus-prone MRL/lpr mice. Here we report that identification of Nuc was successful from the sera of MRL/lpr mice, but not from those of the substrain MRL/n mice, which show no apparent autoimmune syndrome at the same age of MRL/lpr mice, by means of immunochemical as well as N-terminal amino-acid sequencing methods.
