6 Methylpurine

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Abdalla E A Hassan - One of the best experts on this subject based on the ideXlab platform.

  • synthesis of 9 6 deoxy α l talofuranosyl 6 Methylpurine and 9 6 deoxy β d allofuranosyl 6 Methylpurine nucleosides
    Current protocols in human genetics, 2020
    Co-Authors: Abdalla E A Hassan, Reham A I Abouelkhair, Hend M Maaroof, John A Secrist
    Abstract:

    6-Methylpurine (MeP) is a cytotoxic adenine analog that does not exhibit selectivity when administered systemically and could be very useful in a gene therapy approach to cancer treatment involving Escherichia coli purine nucleoside phosphorylase (PNP). 9-(6-Deoxy-β-D-allofuranosyl)-6-Methylpurine [methyl(allo)-MePR, 18] and 9-(6-deoxy-α-L-talofuranosyl)-6-Methylpurine [methyl(talo)-MePR, 21] were synthesized as potential prodrugs for MeP in the E. coli PNP/prodrug cancer gene therapy approach. The detailed syntheses of [methyl(allo)-MePR] and [methyl(talo)-MePR] are described. The glycosyl donors, 1,2-di-O-acetyl-3,5-di-O-benzyl-α-D-allofuranose (12) and 1-O-acetyl-3-O-benzyl-2,5-di-O-benzoyl-α-L-talofuranose (16) were prepared from 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (4) in nine and eleven steps, respectively. Vorbruggen coupling of the latter glycosyl donors with 6-Methylpurine (3), followed by deprotection of the sugar hydroxyl groups, gave the title compounds in good overall yields. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of 6-Methylpurine Basic Protocol 2: Preparation of the D-allofuranose derivative (12) Basic Protocol 3: Preparation of 6-deoxy-α-L-talofuranoside Basic Protocol 4: Preparation of methyl(allo)-MePR (18) Basic Protocol 5: Preparation of methyl(talo)-MePR (21).

  • 6 Methylpurine derived sugar modified nucleosides synthesis and evaluation of their substrate activity with purine nucleoside phosphorylases
    Bioorganic Chemistry, 2016
    Co-Authors: William B Parker, Abdalla E A Hassan, Reham A I Abouelkhair, Paula W Allan, John A Secrist
    Abstract:

    6-Methylpurine (MeP) is cytotoxic adenine analog that does not exhibit selectivity when administered systemically, and could be very useful in a gene therapy approach to cancer treatment involving Escherichia coli PNP. The prototype MeP releasing prodrug, 9-(β-d-ribofuranosyl)-6-Methylpurine, MeP-dR has demonstrated good activity against tumors expressing E. coli PNP, but its antitumor activity is limited due to toxicity resulting from the generation of MeP from gut bacteria. Therefore, we have embarked on a medicinal chemistry program to identify non-toxic MeP prodrugs that could be used in conjunction with E. coli PNP. In this work, we report on the synthesis of 9-(6-deoxy-β-d-allofuranosyl)-6-Methylpurine (3) and 9-(6-deoxy-5-C-methyl-β-d-ribo-hexofuranosyl)-6-Methylpurine (4), and the evaluation of their substrate activity with several phosphorylases. The glycosyl donors; 1,2-di-O-acetyl-3,5-di-O-benzyl-α-d-allofuranose (10) and 1-O-acetyl-3-O-benzyl-2,5-di-O-benzoyl-6-deoxy-5-C-methyl-β-d-ribohexofuran-ose (15) were prepared from 1,2:5,6-di-O-isopropylidine-α-d-glucofuranose in 9 and 11 steps, respectively. Coupling of 10 and 15 with silylated 6-Methylpurine under Vorbruggen glycosylation conditions followed conventional deprotection of the hydroxyl groups furnished 5'-C-methylated-6-Methylpurine nucleosides 3 and 4, respectively. Unlike 9-(6-deoxy-α-l-talo-furanosyl)-6-Methylpurine, which showed good substrate activity with E. coli PNP mutant (M64V), the β-d-allo-furanosyl derivative 3 and the 5'-di-C-methyl derivative 4 were poor substrates for all tested glycosidic bond cleavage enzymes.

  • 6 Methylpurine derived sugar modified nucleosides synthesis and in vivo antitumor activity in d54 tumor expressing m64v escherichia coli purine nucleoside phosphorylase
    European Journal of Medicinal Chemistry, 2016
    Co-Authors: William B Parker, Abdalla E A Hassan, Reham A I Abouelkhair, Paula W Allan, John A Secrist
    Abstract:

    Impressive antitumor activity has been observed with fludarabine phosphate against tumors that express Escherichia coli purine nucleoside phosphorylase (PNP) due to the liberation of 2-fluoroadenine in the tumor tissue. 6-Methylpurine (MeP) is another cytotoxic adenine analog that does not exhibit selectivity when administered systemically, and could be very useful in a gene therapy approach to cancer treatment involving E. coli PNP. The prototype MeP releasing prodrug 9-(2-deoxy-β-d-ribofuranosyl)-6-Methylpurine (1) [MeP-dR] has demonstrated good activity against tumors expressing E. coli PNP, but its antitumor activity is limited due to toxicity resulting from the generation of MeP from gut bacteria. Therefore, we have embarked on a medicinal chemistry program to identify a combination of non-toxic MeP prodrugs and non-human adenosine glycosidic bond cleaving enzymes. The two best MeP-based substrates with M64V-E coli PNP, a mutant which was engineered to tolerate modification at the 5'-position of adenosine and its analogs, were 9-(6-deoxy-α-l-talofuranosyl)-6-Methylpurine (3) [methyl(talo)-MeP-R] and 9-(α-l-lyxofuranosyl)6-Methylpurine (4) [lyxo-MeP-R]. The detailed synthesis methyl(talo)-MeP-R and lyxo-MeP-R, and the evaluation of their substrate activity with 4 enzymes not normally associated with cancer patients is described. In addition, we have determined the intraperitoneal pharmacokinetic (ip-PK) properties of methyl(talo)-MeP-R and have determined its in vivo bystander activity in mice bearing D54 tumors that express M64V PNP. The observed good in vivo bystander activity of [methyl(talo)-MeP-R/M64V-E coli PNP combination suggests that these agents could be useful for the treatment of cancer.

  • designer gene therapy using an escherichia coli purine nucleoside phosphorylase prodrug system
    Chemistry & Biology, 2003
    Co-Authors: Eric M Bennett, William B Parker, Abdalla E A Hassan, John A Secrist, Paula W Allan, Ruchi Anand, Jeong S Hong, Dana N Levasseur, David T Mcpherson, Eric J Sorscher
    Abstract:

    Activation of prodrugs by Escherichia coli purine nucleoside phosphorylase (PNP) provides a method for selectively killing tumor cells expressing a transfected PNP gene. This gene therapy approach requires matching a prodrug and a known enzymatic activity present only in tumor cells. The specificity of the method relies on avoiding prodrug cleavage by enzymes already present in the host cells or the intestinal flora. Using crystallographic and computer modeling methods as guides, we have redesigned E. coli PNP to cleave new prodrug substrates more efficiently than does the wild-type enzyme. In particular, the M64V PNP mutant cleaves 9-(6-deoxy-alpha-L-talofuranosyl)-6-Methylpurine with a kcat/Km over 100 times greater than for native E. coli PNP. In a xenograft tumor experiment, this compound caused regression of tumors expressing the M64V PNP gene.

  • antitumor activity of 2 fluoro 2 deoxyadenosine against tumors that express escherichia coli purine nucleoside phosphorylase
    Cancer Gene Therapy, 2003
    Co-Authors: William B Parker, Eric J Sorscher, Abdalla E A Hassan, John A Secrist, Paula W Allan, William R Waud
    Abstract:

    The selective expression of Escherichia coli purine nucleoside phosphorylase (PNP) in solid tumors has been successfully used to activate two purine nucleoside analogs [9-(2-deoxy-β-D-ribofuranosyl)-6-Methylpurine (MeP-dR) and 9-β-D-arabinofuranosyl-2-fluoroadenine (F-araA)] resulting in lasting tumor regressions and cures. E. coli PNP also cleaves 2-fluoro-2′-deoxyadenosine (F-dAdo) to 2-F-adenine, which is the toxic purine analog liberated from F-araA that has high bystander activity and is active against nonproliferating tumor cells. As F-dAdo is 3000 times better than F-araA as a substrate for E. coli PNP, we have evaluated its antitumor activity against D54 gliomas that express E. coli PNP and have characterized its in vivo metabolism in order to better understand its mechanism of action with respect to the other two agents. Like MeP-dR and F-araA-5′-monophosphate (F-araAMP, a prodrug of F-araA), treatment of mice bearing D54 tumors that express E. coli PNP with F-dAdo resulted in excellent antitumor activity. Although F-dAdo was as active as MeP-dR and better than F-araAMP, it was not dramatically better than either compound because of its short plasma half-life and the limited activation of F-adenine to toxic metabolites. Regardless, these results indicated that F-dAdo was also an excellent prodrug for use with gene vectors that deliver E. coli PNP to tumor cells.

John A Secrist - One of the best experts on this subject based on the ideXlab platform.

  • synthesis of 9 6 deoxy α l talofuranosyl 6 Methylpurine and 9 6 deoxy β d allofuranosyl 6 Methylpurine nucleosides
    Current protocols in human genetics, 2020
    Co-Authors: Abdalla E A Hassan, Reham A I Abouelkhair, Hend M Maaroof, John A Secrist
    Abstract:

    6-Methylpurine (MeP) is a cytotoxic adenine analog that does not exhibit selectivity when administered systemically and could be very useful in a gene therapy approach to cancer treatment involving Escherichia coli purine nucleoside phosphorylase (PNP). 9-(6-Deoxy-β-D-allofuranosyl)-6-Methylpurine [methyl(allo)-MePR, 18] and 9-(6-deoxy-α-L-talofuranosyl)-6-Methylpurine [methyl(talo)-MePR, 21] were synthesized as potential prodrugs for MeP in the E. coli PNP/prodrug cancer gene therapy approach. The detailed syntheses of [methyl(allo)-MePR] and [methyl(talo)-MePR] are described. The glycosyl donors, 1,2-di-O-acetyl-3,5-di-O-benzyl-α-D-allofuranose (12) and 1-O-acetyl-3-O-benzyl-2,5-di-O-benzoyl-α-L-talofuranose (16) were prepared from 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (4) in nine and eleven steps, respectively. Vorbruggen coupling of the latter glycosyl donors with 6-Methylpurine (3), followed by deprotection of the sugar hydroxyl groups, gave the title compounds in good overall yields. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of 6-Methylpurine Basic Protocol 2: Preparation of the D-allofuranose derivative (12) Basic Protocol 3: Preparation of 6-deoxy-α-L-talofuranoside Basic Protocol 4: Preparation of methyl(allo)-MePR (18) Basic Protocol 5: Preparation of methyl(talo)-MePR (21).

  • 6 Methylpurine derived sugar modified nucleosides synthesis and evaluation of their substrate activity with purine nucleoside phosphorylases
    Bioorganic Chemistry, 2016
    Co-Authors: William B Parker, Abdalla E A Hassan, Reham A I Abouelkhair, Paula W Allan, John A Secrist
    Abstract:

    6-Methylpurine (MeP) is cytotoxic adenine analog that does not exhibit selectivity when administered systemically, and could be very useful in a gene therapy approach to cancer treatment involving Escherichia coli PNP. The prototype MeP releasing prodrug, 9-(β-d-ribofuranosyl)-6-Methylpurine, MeP-dR has demonstrated good activity against tumors expressing E. coli PNP, but its antitumor activity is limited due to toxicity resulting from the generation of MeP from gut bacteria. Therefore, we have embarked on a medicinal chemistry program to identify non-toxic MeP prodrugs that could be used in conjunction with E. coli PNP. In this work, we report on the synthesis of 9-(6-deoxy-β-d-allofuranosyl)-6-Methylpurine (3) and 9-(6-deoxy-5-C-methyl-β-d-ribo-hexofuranosyl)-6-Methylpurine (4), and the evaluation of their substrate activity with several phosphorylases. The glycosyl donors; 1,2-di-O-acetyl-3,5-di-O-benzyl-α-d-allofuranose (10) and 1-O-acetyl-3-O-benzyl-2,5-di-O-benzoyl-6-deoxy-5-C-methyl-β-d-ribohexofuran-ose (15) were prepared from 1,2:5,6-di-O-isopropylidine-α-d-glucofuranose in 9 and 11 steps, respectively. Coupling of 10 and 15 with silylated 6-Methylpurine under Vorbruggen glycosylation conditions followed conventional deprotection of the hydroxyl groups furnished 5'-C-methylated-6-Methylpurine nucleosides 3 and 4, respectively. Unlike 9-(6-deoxy-α-l-talo-furanosyl)-6-Methylpurine, which showed good substrate activity with E. coli PNP mutant (M64V), the β-d-allo-furanosyl derivative 3 and the 5'-di-C-methyl derivative 4 were poor substrates for all tested glycosidic bond cleavage enzymes.

  • 6 Methylpurine derived sugar modified nucleosides synthesis and in vivo antitumor activity in d54 tumor expressing m64v escherichia coli purine nucleoside phosphorylase
    European Journal of Medicinal Chemistry, 2016
    Co-Authors: William B Parker, Abdalla E A Hassan, Reham A I Abouelkhair, Paula W Allan, John A Secrist
    Abstract:

    Impressive antitumor activity has been observed with fludarabine phosphate against tumors that express Escherichia coli purine nucleoside phosphorylase (PNP) due to the liberation of 2-fluoroadenine in the tumor tissue. 6-Methylpurine (MeP) is another cytotoxic adenine analog that does not exhibit selectivity when administered systemically, and could be very useful in a gene therapy approach to cancer treatment involving E. coli PNP. The prototype MeP releasing prodrug 9-(2-deoxy-β-d-ribofuranosyl)-6-Methylpurine (1) [MeP-dR] has demonstrated good activity against tumors expressing E. coli PNP, but its antitumor activity is limited due to toxicity resulting from the generation of MeP from gut bacteria. Therefore, we have embarked on a medicinal chemistry program to identify a combination of non-toxic MeP prodrugs and non-human adenosine glycosidic bond cleaving enzymes. The two best MeP-based substrates with M64V-E coli PNP, a mutant which was engineered to tolerate modification at the 5'-position of adenosine and its analogs, were 9-(6-deoxy-α-l-talofuranosyl)-6-Methylpurine (3) [methyl(talo)-MeP-R] and 9-(α-l-lyxofuranosyl)6-Methylpurine (4) [lyxo-MeP-R]. The detailed synthesis methyl(talo)-MeP-R and lyxo-MeP-R, and the evaluation of their substrate activity with 4 enzymes not normally associated with cancer patients is described. In addition, we have determined the intraperitoneal pharmacokinetic (ip-PK) properties of methyl(talo)-MeP-R and have determined its in vivo bystander activity in mice bearing D54 tumors that express M64V PNP. The observed good in vivo bystander activity of [methyl(talo)-MeP-R/M64V-E coli PNP combination suggests that these agents could be useful for the treatment of cancer.

  • bis tbusate phosphotriester prodrugs of 8 azaguanosine and 6 Methylpurine riboside bis pom phosphotriester prodrugs of 2 deoxy 4 thioadenosine and its corresponding 9α anomer
    Nucleosides Nucleotides & Nucleic Acids, 2005
    Co-Authors: Jerry D Rose, William B Parker, John A Secrist
    Abstract:

    As an extension of previous work with bis(POM) nucleotide prodrugs, we report the synthesis and biological evaluation in tumor cell culture of the bis(pivaloyloxymethyl) phosphotriester prodrug of slightly cytotoxic 2'-deoxy-4'-thioadenosine and its alpha-anomer. We have experienced need for an alternative phosphate masking group, particularly with purine nucleosides. Accordingly, we report synthesis and biological evaluation of the bis(tBuSA TE) phosphotriester prodrugs of 8-azaguanosine and 6-Methylpurine riboside, nucleoside analogs with moderate to significant cytotoxicity. All four prodrugs were examined in tumor cell culture in parallel with the parent nucleosides. Synthetic routes and biological data are presented.

  • designer gene therapy using an escherichia coli purine nucleoside phosphorylase prodrug system
    Chemistry & Biology, 2003
    Co-Authors: Eric M Bennett, William B Parker, Abdalla E A Hassan, John A Secrist, Paula W Allan, Ruchi Anand, Jeong S Hong, Dana N Levasseur, David T Mcpherson, Eric J Sorscher
    Abstract:

    Activation of prodrugs by Escherichia coli purine nucleoside phosphorylase (PNP) provides a method for selectively killing tumor cells expressing a transfected PNP gene. This gene therapy approach requires matching a prodrug and a known enzymatic activity present only in tumor cells. The specificity of the method relies on avoiding prodrug cleavage by enzymes already present in the host cells or the intestinal flora. Using crystallographic and computer modeling methods as guides, we have redesigned E. coli PNP to cleave new prodrug substrates more efficiently than does the wild-type enzyme. In particular, the M64V PNP mutant cleaves 9-(6-deoxy-alpha-L-talofuranosyl)-6-Methylpurine with a kcat/Km over 100 times greater than for native E. coli PNP. In a xenograft tumor experiment, this compound caused regression of tumors expressing the M64V PNP gene.

William B Parker - One of the best experts on this subject based on the ideXlab platform.

  • 6 Methylpurine derived sugar modified nucleosides synthesis and evaluation of their substrate activity with purine nucleoside phosphorylases
    Bioorganic Chemistry, 2016
    Co-Authors: William B Parker, Abdalla E A Hassan, Reham A I Abouelkhair, Paula W Allan, John A Secrist
    Abstract:

    6-Methylpurine (MeP) is cytotoxic adenine analog that does not exhibit selectivity when administered systemically, and could be very useful in a gene therapy approach to cancer treatment involving Escherichia coli PNP. The prototype MeP releasing prodrug, 9-(β-d-ribofuranosyl)-6-Methylpurine, MeP-dR has demonstrated good activity against tumors expressing E. coli PNP, but its antitumor activity is limited due to toxicity resulting from the generation of MeP from gut bacteria. Therefore, we have embarked on a medicinal chemistry program to identify non-toxic MeP prodrugs that could be used in conjunction with E. coli PNP. In this work, we report on the synthesis of 9-(6-deoxy-β-d-allofuranosyl)-6-Methylpurine (3) and 9-(6-deoxy-5-C-methyl-β-d-ribo-hexofuranosyl)-6-Methylpurine (4), and the evaluation of their substrate activity with several phosphorylases. The glycosyl donors; 1,2-di-O-acetyl-3,5-di-O-benzyl-α-d-allofuranose (10) and 1-O-acetyl-3-O-benzyl-2,5-di-O-benzoyl-6-deoxy-5-C-methyl-β-d-ribohexofuran-ose (15) were prepared from 1,2:5,6-di-O-isopropylidine-α-d-glucofuranose in 9 and 11 steps, respectively. Coupling of 10 and 15 with silylated 6-Methylpurine under Vorbruggen glycosylation conditions followed conventional deprotection of the hydroxyl groups furnished 5'-C-methylated-6-Methylpurine nucleosides 3 and 4, respectively. Unlike 9-(6-deoxy-α-l-talo-furanosyl)-6-Methylpurine, which showed good substrate activity with E. coli PNP mutant (M64V), the β-d-allo-furanosyl derivative 3 and the 5'-di-C-methyl derivative 4 were poor substrates for all tested glycosidic bond cleavage enzymes.

  • 6 Methylpurine derived sugar modified nucleosides synthesis and in vivo antitumor activity in d54 tumor expressing m64v escherichia coli purine nucleoside phosphorylase
    European Journal of Medicinal Chemistry, 2016
    Co-Authors: William B Parker, Abdalla E A Hassan, Reham A I Abouelkhair, Paula W Allan, John A Secrist
    Abstract:

    Impressive antitumor activity has been observed with fludarabine phosphate against tumors that express Escherichia coli purine nucleoside phosphorylase (PNP) due to the liberation of 2-fluoroadenine in the tumor tissue. 6-Methylpurine (MeP) is another cytotoxic adenine analog that does not exhibit selectivity when administered systemically, and could be very useful in a gene therapy approach to cancer treatment involving E. coli PNP. The prototype MeP releasing prodrug 9-(2-deoxy-β-d-ribofuranosyl)-6-Methylpurine (1) [MeP-dR] has demonstrated good activity against tumors expressing E. coli PNP, but its antitumor activity is limited due to toxicity resulting from the generation of MeP from gut bacteria. Therefore, we have embarked on a medicinal chemistry program to identify a combination of non-toxic MeP prodrugs and non-human adenosine glycosidic bond cleaving enzymes. The two best MeP-based substrates with M64V-E coli PNP, a mutant which was engineered to tolerate modification at the 5'-position of adenosine and its analogs, were 9-(6-deoxy-α-l-talofuranosyl)-6-Methylpurine (3) [methyl(talo)-MeP-R] and 9-(α-l-lyxofuranosyl)6-Methylpurine (4) [lyxo-MeP-R]. The detailed synthesis methyl(talo)-MeP-R and lyxo-MeP-R, and the evaluation of their substrate activity with 4 enzymes not normally associated with cancer patients is described. In addition, we have determined the intraperitoneal pharmacokinetic (ip-PK) properties of methyl(talo)-MeP-R and have determined its in vivo bystander activity in mice bearing D54 tumors that express M64V PNP. The observed good in vivo bystander activity of [methyl(talo)-MeP-R/M64V-E coli PNP combination suggests that these agents could be useful for the treatment of cancer.

  • pnp anticancer gene therapy
    Current Topics in Medicinal Chemistry, 2005
    Co-Authors: Yang Zhang, William B Parker, Eric J Sorscher, Steven E Ealick
    Abstract:

    Escherichia coli purine nucleoside phosphorylase (PNP) catalyzes the cleavage of 9-(2-deoxy-β-Dribofuranosyl)- 6-Methylpurine (MeP-dR), while human PNP does not. MeP-dR is well tolerated while the cleavage product, 6-Methylpurine (MeP), is highly cytotoxic. This clinical profile suggests an anticancer gene therapy strategy in which solid tumors are transfected with the gene for E. coli PNP. Tumor cells expressing E. coli PNP will liberate MeP and be killed. Furthermore, MeP released from the cell via the purine transport system will enter nearby cells, resulting in bystander killing of tumor cells. To reduce toxicity resulting from activation of MeP-dR by intestinal tract flora, we redesigned the E. coli PNP active site to cleave prodrugs that are not cleaved by wild type E. coli PNP. It is possible that the variation of substrate specificity among enzymes that cleave nucleosides will have broader application in the gene therapy approach to prodrug activation. Here we review progress in the development of E. coli PNP anticancer gene therapy. We also review the structural basis for activity of nucleoside phosphorylases and suggest future directions for the development of activating enzymes for suicide gene therapy.

  • bis tbusate phosphotriester prodrugs of 8 azaguanosine and 6 Methylpurine riboside bis pom phosphotriester prodrugs of 2 deoxy 4 thioadenosine and its corresponding 9α anomer
    Nucleosides Nucleotides & Nucleic Acids, 2005
    Co-Authors: Jerry D Rose, William B Parker, John A Secrist
    Abstract:

    As an extension of previous work with bis(POM) nucleotide prodrugs, we report the synthesis and biological evaluation in tumor cell culture of the bis(pivaloyloxymethyl) phosphotriester prodrug of slightly cytotoxic 2'-deoxy-4'-thioadenosine and its alpha-anomer. We have experienced need for an alternative phosphate masking group, particularly with purine nucleosides. Accordingly, we report synthesis and biological evaluation of the bis(tBuSA TE) phosphotriester prodrugs of 8-azaguanosine and 6-Methylpurine riboside, nucleoside analogs with moderate to significant cytotoxicity. All four prodrugs were examined in tumor cell culture in parallel with the parent nucleosides. Synthetic routes and biological data are presented.

  • designer gene therapy using an escherichia coli purine nucleoside phosphorylase prodrug system
    Chemistry & Biology, 2003
    Co-Authors: Eric M Bennett, William B Parker, Abdalla E A Hassan, John A Secrist, Paula W Allan, Ruchi Anand, Jeong S Hong, Dana N Levasseur, David T Mcpherson, Eric J Sorscher
    Abstract:

    Activation of prodrugs by Escherichia coli purine nucleoside phosphorylase (PNP) provides a method for selectively killing tumor cells expressing a transfected PNP gene. This gene therapy approach requires matching a prodrug and a known enzymatic activity present only in tumor cells. The specificity of the method relies on avoiding prodrug cleavage by enzymes already present in the host cells or the intestinal flora. Using crystallographic and computer modeling methods as guides, we have redesigned E. coli PNP to cleave new prodrug substrates more efficiently than does the wild-type enzyme. In particular, the M64V PNP mutant cleaves 9-(6-deoxy-alpha-L-talofuranosyl)-6-Methylpurine with a kcat/Km over 100 times greater than for native E. coli PNP. In a xenograft tumor experiment, this compound caused regression of tumors expressing the M64V PNP gene.

Paula W Allan - One of the best experts on this subject based on the ideXlab platform.

  • 6 Methylpurine derived sugar modified nucleosides synthesis and evaluation of their substrate activity with purine nucleoside phosphorylases
    Bioorganic Chemistry, 2016
    Co-Authors: William B Parker, Abdalla E A Hassan, Reham A I Abouelkhair, Paula W Allan, John A Secrist
    Abstract:

    6-Methylpurine (MeP) is cytotoxic adenine analog that does not exhibit selectivity when administered systemically, and could be very useful in a gene therapy approach to cancer treatment involving Escherichia coli PNP. The prototype MeP releasing prodrug, 9-(β-d-ribofuranosyl)-6-Methylpurine, MeP-dR has demonstrated good activity against tumors expressing E. coli PNP, but its antitumor activity is limited due to toxicity resulting from the generation of MeP from gut bacteria. Therefore, we have embarked on a medicinal chemistry program to identify non-toxic MeP prodrugs that could be used in conjunction with E. coli PNP. In this work, we report on the synthesis of 9-(6-deoxy-β-d-allofuranosyl)-6-Methylpurine (3) and 9-(6-deoxy-5-C-methyl-β-d-ribo-hexofuranosyl)-6-Methylpurine (4), and the evaluation of their substrate activity with several phosphorylases. The glycosyl donors; 1,2-di-O-acetyl-3,5-di-O-benzyl-α-d-allofuranose (10) and 1-O-acetyl-3-O-benzyl-2,5-di-O-benzoyl-6-deoxy-5-C-methyl-β-d-ribohexofuran-ose (15) were prepared from 1,2:5,6-di-O-isopropylidine-α-d-glucofuranose in 9 and 11 steps, respectively. Coupling of 10 and 15 with silylated 6-Methylpurine under Vorbruggen glycosylation conditions followed conventional deprotection of the hydroxyl groups furnished 5'-C-methylated-6-Methylpurine nucleosides 3 and 4, respectively. Unlike 9-(6-deoxy-α-l-talo-furanosyl)-6-Methylpurine, which showed good substrate activity with E. coli PNP mutant (M64V), the β-d-allo-furanosyl derivative 3 and the 5'-di-C-methyl derivative 4 were poor substrates for all tested glycosidic bond cleavage enzymes.

  • 6 Methylpurine derived sugar modified nucleosides synthesis and in vivo antitumor activity in d54 tumor expressing m64v escherichia coli purine nucleoside phosphorylase
    European Journal of Medicinal Chemistry, 2016
    Co-Authors: William B Parker, Abdalla E A Hassan, Reham A I Abouelkhair, Paula W Allan, John A Secrist
    Abstract:

    Impressive antitumor activity has been observed with fludarabine phosphate against tumors that express Escherichia coli purine nucleoside phosphorylase (PNP) due to the liberation of 2-fluoroadenine in the tumor tissue. 6-Methylpurine (MeP) is another cytotoxic adenine analog that does not exhibit selectivity when administered systemically, and could be very useful in a gene therapy approach to cancer treatment involving E. coli PNP. The prototype MeP releasing prodrug 9-(2-deoxy-β-d-ribofuranosyl)-6-Methylpurine (1) [MeP-dR] has demonstrated good activity against tumors expressing E. coli PNP, but its antitumor activity is limited due to toxicity resulting from the generation of MeP from gut bacteria. Therefore, we have embarked on a medicinal chemistry program to identify a combination of non-toxic MeP prodrugs and non-human adenosine glycosidic bond cleaving enzymes. The two best MeP-based substrates with M64V-E coli PNP, a mutant which was engineered to tolerate modification at the 5'-position of adenosine and its analogs, were 9-(6-deoxy-α-l-talofuranosyl)-6-Methylpurine (3) [methyl(talo)-MeP-R] and 9-(α-l-lyxofuranosyl)6-Methylpurine (4) [lyxo-MeP-R]. The detailed synthesis methyl(talo)-MeP-R and lyxo-MeP-R, and the evaluation of their substrate activity with 4 enzymes not normally associated with cancer patients is described. In addition, we have determined the intraperitoneal pharmacokinetic (ip-PK) properties of methyl(talo)-MeP-R and have determined its in vivo bystander activity in mice bearing D54 tumors that express M64V PNP. The observed good in vivo bystander activity of [methyl(talo)-MeP-R/M64V-E coli PNP combination suggests that these agents could be useful for the treatment of cancer.

  • designer gene therapy using an escherichia coli purine nucleoside phosphorylase prodrug system
    Chemistry & Biology, 2003
    Co-Authors: Eric M Bennett, William B Parker, Abdalla E A Hassan, John A Secrist, Paula W Allan, Ruchi Anand, Jeong S Hong, Dana N Levasseur, David T Mcpherson, Eric J Sorscher
    Abstract:

    Activation of prodrugs by Escherichia coli purine nucleoside phosphorylase (PNP) provides a method for selectively killing tumor cells expressing a transfected PNP gene. This gene therapy approach requires matching a prodrug and a known enzymatic activity present only in tumor cells. The specificity of the method relies on avoiding prodrug cleavage by enzymes already present in the host cells or the intestinal flora. Using crystallographic and computer modeling methods as guides, we have redesigned E. coli PNP to cleave new prodrug substrates more efficiently than does the wild-type enzyme. In particular, the M64V PNP mutant cleaves 9-(6-deoxy-alpha-L-talofuranosyl)-6-Methylpurine with a kcat/Km over 100 times greater than for native E. coli PNP. In a xenograft tumor experiment, this compound caused regression of tumors expressing the M64V PNP gene.

  • a long acting suicide gene toxin 6 Methylpurine inhibits slow growing tumors after a single administration
    Journal of Pharmacology and Experimental Therapeutics, 2003
    Co-Authors: Vijayakrishna K Gadi, William B Parker, Paula W Allan, Sherrie D Alexander, William R Waud, Eric J Sorscher
    Abstract:

    We have demonstrated antitumor activity against refractory human glioma and pancreatic tumors with 6-Methylpurine (MeP) using either a suicide gene therapy strategy to selectively release 6-Methylpurine in tumor cells or direct intratumoral injection of 6-Methylpurine itself. A single i.p. injection in mice of the prodrug 9-beta-D-[2-deoxyribofuranosyl]-6-Methylpurine (MeP-dR; 134 mg/kg) caused sustained regression lasting over 70 days of D54 (human glioma) tumors transduced with the Escherichia coli purine nucleoside phosphorylase (PNP), and a single intratumoral injection of 6-Methylpurine (5-10 mg/kg) elicited prolonged delays of the growth of D54 tumors and CFPAC human pancreatic carcinoma. Because the D54 tumor doubling time is >15 days, the experiments indicate that prodrug activation by E. coli PNP engenders destruction of both dividing and nondividing tumor compartments in vivo and, therefore, address a fundamental barrier that has limited the development of suicide gene strategies in the past. A prolonged retention time of 6-Methylpurine metabolites in tumors was noted in vivo (T(1/2) >24 h compared with a serum half-life of <1 h). By high-pressure liquid chromatography, metabolites of [(3)H]MeP-dR were 5- to 6-fold higher in tumors expressing E. coli PNP. These experiments point to new endpoints for monitoring E. coli PNP suicide gene therapy, including intratumoral enzymatic activity, in situ (intratumoral) prodrug conversion, and tumor regressions after direct injection of a suicide gene toxin. The findings also help explain the strong in vivo bystander killing mechanism ascribed by several laboratories to E. coli PNP in the past.

  • antitumor activity of 2 fluoro 2 deoxyadenosine against tumors that express escherichia coli purine nucleoside phosphorylase
    Cancer Gene Therapy, 2003
    Co-Authors: William B Parker, Eric J Sorscher, Abdalla E A Hassan, John A Secrist, Paula W Allan, William R Waud
    Abstract:

    The selective expression of Escherichia coli purine nucleoside phosphorylase (PNP) in solid tumors has been successfully used to activate two purine nucleoside analogs [9-(2-deoxy-β-D-ribofuranosyl)-6-Methylpurine (MeP-dR) and 9-β-D-arabinofuranosyl-2-fluoroadenine (F-araA)] resulting in lasting tumor regressions and cures. E. coli PNP also cleaves 2-fluoro-2′-deoxyadenosine (F-dAdo) to 2-F-adenine, which is the toxic purine analog liberated from F-araA that has high bystander activity and is active against nonproliferating tumor cells. As F-dAdo is 3000 times better than F-araA as a substrate for E. coli PNP, we have evaluated its antitumor activity against D54 gliomas that express E. coli PNP and have characterized its in vivo metabolism in order to better understand its mechanism of action with respect to the other two agents. Like MeP-dR and F-araA-5′-monophosphate (F-araAMP, a prodrug of F-araA), treatment of mice bearing D54 tumors that express E. coli PNP with F-dAdo resulted in excellent antitumor activity. Although F-dAdo was as active as MeP-dR and better than F-araAMP, it was not dramatically better than either compound because of its short plasma half-life and the limited activation of F-adenine to toxic metabolites. Regardless, these results indicated that F-dAdo was also an excellent prodrug for use with gene vectors that deliver E. coli PNP to tumor cells.

Eric J Sorscher - One of the best experts on this subject based on the ideXlab platform.

  • pnp anticancer gene therapy
    Current Topics in Medicinal Chemistry, 2005
    Co-Authors: Yang Zhang, William B Parker, Eric J Sorscher, Steven E Ealick
    Abstract:

    Escherichia coli purine nucleoside phosphorylase (PNP) catalyzes the cleavage of 9-(2-deoxy-β-Dribofuranosyl)- 6-Methylpurine (MeP-dR), while human PNP does not. MeP-dR is well tolerated while the cleavage product, 6-Methylpurine (MeP), is highly cytotoxic. This clinical profile suggests an anticancer gene therapy strategy in which solid tumors are transfected with the gene for E. coli PNP. Tumor cells expressing E. coli PNP will liberate MeP and be killed. Furthermore, MeP released from the cell via the purine transport system will enter nearby cells, resulting in bystander killing of tumor cells. To reduce toxicity resulting from activation of MeP-dR by intestinal tract flora, we redesigned the E. coli PNP active site to cleave prodrugs that are not cleaved by wild type E. coli PNP. It is possible that the variation of substrate specificity among enzymes that cleave nucleosides will have broader application in the gene therapy approach to prodrug activation. Here we review progress in the development of E. coli PNP anticancer gene therapy. We also review the structural basis for activity of nucleoside phosphorylases and suggest future directions for the development of activating enzymes for suicide gene therapy.

  • designer gene therapy using an escherichia coli purine nucleoside phosphorylase prodrug system
    Chemistry & Biology, 2003
    Co-Authors: Eric M Bennett, William B Parker, Abdalla E A Hassan, John A Secrist, Paula W Allan, Ruchi Anand, Jeong S Hong, Dana N Levasseur, David T Mcpherson, Eric J Sorscher
    Abstract:

    Activation of prodrugs by Escherichia coli purine nucleoside phosphorylase (PNP) provides a method for selectively killing tumor cells expressing a transfected PNP gene. This gene therapy approach requires matching a prodrug and a known enzymatic activity present only in tumor cells. The specificity of the method relies on avoiding prodrug cleavage by enzymes already present in the host cells or the intestinal flora. Using crystallographic and computer modeling methods as guides, we have redesigned E. coli PNP to cleave new prodrug substrates more efficiently than does the wild-type enzyme. In particular, the M64V PNP mutant cleaves 9-(6-deoxy-alpha-L-talofuranosyl)-6-Methylpurine with a kcat/Km over 100 times greater than for native E. coli PNP. In a xenograft tumor experiment, this compound caused regression of tumors expressing the M64V PNP gene.

  • a long acting suicide gene toxin 6 Methylpurine inhibits slow growing tumors after a single administration
    Journal of Pharmacology and Experimental Therapeutics, 2003
    Co-Authors: Vijayakrishna K Gadi, William B Parker, Paula W Allan, Sherrie D Alexander, William R Waud, Eric J Sorscher
    Abstract:

    We have demonstrated antitumor activity against refractory human glioma and pancreatic tumors with 6-Methylpurine (MeP) using either a suicide gene therapy strategy to selectively release 6-Methylpurine in tumor cells or direct intratumoral injection of 6-Methylpurine itself. A single i.p. injection in mice of the prodrug 9-beta-D-[2-deoxyribofuranosyl]-6-Methylpurine (MeP-dR; 134 mg/kg) caused sustained regression lasting over 70 days of D54 (human glioma) tumors transduced with the Escherichia coli purine nucleoside phosphorylase (PNP), and a single intratumoral injection of 6-Methylpurine (5-10 mg/kg) elicited prolonged delays of the growth of D54 tumors and CFPAC human pancreatic carcinoma. Because the D54 tumor doubling time is >15 days, the experiments indicate that prodrug activation by E. coli PNP engenders destruction of both dividing and nondividing tumor compartments in vivo and, therefore, address a fundamental barrier that has limited the development of suicide gene strategies in the past. A prolonged retention time of 6-Methylpurine metabolites in tumors was noted in vivo (T(1/2) >24 h compared with a serum half-life of <1 h). By high-pressure liquid chromatography, metabolites of [(3)H]MeP-dR were 5- to 6-fold higher in tumors expressing E. coli PNP. These experiments point to new endpoints for monitoring E. coli PNP suicide gene therapy, including intratumoral enzymatic activity, in situ (intratumoral) prodrug conversion, and tumor regressions after direct injection of a suicide gene toxin. The findings also help explain the strong in vivo bystander killing mechanism ascribed by several laboratories to E. coli PNP in the past.

  • antitumor activity of 2 fluoro 2 deoxyadenosine against tumors that express escherichia coli purine nucleoside phosphorylase
    Cancer Gene Therapy, 2003
    Co-Authors: William B Parker, Eric J Sorscher, Abdalla E A Hassan, John A Secrist, Paula W Allan, William R Waud
    Abstract:

    The selective expression of Escherichia coli purine nucleoside phosphorylase (PNP) in solid tumors has been successfully used to activate two purine nucleoside analogs [9-(2-deoxy-β-D-ribofuranosyl)-6-Methylpurine (MeP-dR) and 9-β-D-arabinofuranosyl-2-fluoroadenine (F-araA)] resulting in lasting tumor regressions and cures. E. coli PNP also cleaves 2-fluoro-2′-deoxyadenosine (F-dAdo) to 2-F-adenine, which is the toxic purine analog liberated from F-araA that has high bystander activity and is active against nonproliferating tumor cells. As F-dAdo is 3000 times better than F-araA as a substrate for E. coli PNP, we have evaluated its antitumor activity against D54 gliomas that express E. coli PNP and have characterized its in vivo metabolism in order to better understand its mechanism of action with respect to the other two agents. Like MeP-dR and F-araA-5′-monophosphate (F-araAMP, a prodrug of F-araA), treatment of mice bearing D54 tumors that express E. coli PNP with F-dAdo resulted in excellent antitumor activity. Although F-dAdo was as active as MeP-dR and better than F-araAMP, it was not dramatically better than either compound because of its short plasma half-life and the limited activation of F-adenine to toxic metabolites. Regardless, these results indicated that F-dAdo was also an excellent prodrug for use with gene vectors that deliver E. coli PNP to tumor cells.

  • cell to cell contact is not required for bystander cell killing by escherichia coli purine nucleoside phosphorylase
    Journal of Biological Chemistry, 1998
    Co-Authors: Brian W Hughes, William B Parker, Paula W Allan, Scott A King, Eric J Sorscher
    Abstract:

    Abstract Expression of Escherichia coli purine nucleoside phosphorylase (PNP) activates prodrugs and kills entire populations of mammalian cells, even when as few as 1% of the cells express this gene. This phenomenon of bystander killing has been previously investigated for herpes simplex virus-thymidine kinase (HSV-TK) and has been shown to require cell to cell contact. Using silicon rings to separate E. coli PNP expressing cells from non-expressing cells sharing the same medium, we demonstrate that bystander cell killing by E. coli PNP does not require cell-cell contact. Initially, cells expressing E. coli PNP convert the non-toxic prodrug, 6-Methylpurine-2′-deoxyriboside (MeP-dR) to the highly toxic membrane permeable toxin, 6-Methylpurine (MeP). As the expressing cells die, E. coli PNP is released into the culture medium, retains activity, and continues precursor conversion extracellularly (as determined by reverse phase high performance liquid chromatography of both prodrug and toxin). Bystander killing can also be observed in the absence of extracellular E. coli PNP by removing the MeP-dR prior to death of the expressing cells. In this case, 100% of cultured cells die when as few as 3% of the cells of a population express E. coli PNP. Blocking nucleoside transport with nitrobenzylthioinosine reduces MeP-dR mediated cell killing but not MeP cell killing. These mechanisms differ fundamentally from those previously reported for the HSV-TK gene.

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