The unfolded protein response (UPR) is a pro-survival mechanism triggered by cellular stresses en... more The unfolded protein response (UPR) is a pro-survival mechanism triggered by cellular stresses encountered by tumor cells, such as hypoxia, acidosis, reactive oxygen species and even radiation and chemotherapy. In response to increased unfolded or mis-folded proteins in the endoplasmic reticulum (ER), the UPR initiates specific cell signaling pathways aiming to alleviate ER stress and restore ER proteostasis which include increasing the expression of chaperones to aid in protein folding, enhancing protein degradation and reducing global protein synthesis. However, if homeostasis cannot be restored, the UPR switches from survival to cell death. The UPR is a relatively novel therapeutic target for glioblastoma (GBM), and specifically GBM stem-like cells (GSCs) which maintain tumor heterogeneity, drive GBM tumor growth and therapy resistance. Here we tested a series of curcumin derivatives against three GSC lines and identified a trimethoxy bis-chalcone which promotes robust GSC death ...
Pediatric brain tumors remain a significant source of morbidity and mortality. Though development... more Pediatric brain tumors remain a significant source of morbidity and mortality. Though developments have been made in treating these malignancies, the blood–brain barrier, intra- and inter-tumoral heterogeneity, and therapeutic toxicity pose challenges to improving outcomes. Varying types of nanoparticles, including metallic, organic, and micellar molecules of varying structures and compositions, have been investigated as a potential therapy to circumvent some of these inherent challenges. Carbon dots (CDs) have recently gained popularity as a novel nanoparticle with theranostic properties. This carbon-based modality is highly modifiable, allowing for conjugation to drugs, as well as tumor-specific ligands in an effort to more effectively target cancerous cells and reduce peripheral toxicity. CDs are being studied pre-clinically. The ClinicalTrials.gov site was queried using the search terms: brain tumor and nanoparticle, liposome, micelle, dendrimer, quantum dot, or carbon dot. At t...
The current prognosis for glioblastoma is dismal. Treatment-resistant glioblastoma stem cells (GS... more The current prognosis for glioblastoma is dismal. Treatment-resistant glioblastoma stem cells (GSCs) and the failure of most drugs to reach therapeutic levels within the tumor remain formidable obstacles to successful treatment. Chalcones are aromatic ketones demonstrated to reduce malignant properties in cancers including glioblastoma. Nanomedicines can increase drug accumulation and tumor cell death. Carbon-dots are promising nanocarriers that can be easily functionalized with tumor-targeting ligands and anti-cancer drugs. Therefore, we synthesized a series of 4′-amino chalcones with the rationale that the amino group would serve as a “handle” to facilitate covalent attachment to carbon-dots and tested their cytotoxicity toward GSCs. We generated 31 chalcones (22 4′-amino and 9 4′ derivatives) including 5 novel chalcones, and found that 13 had an IC50 below 10 µM in all GSC lines. After confirming that the 4-amino group was not part of the active pharmacophore, chalcones were atta...
Glioblastoma (GBM) has a dismal prognosis and successful elimination of GBM stem cells (GSCs) is ... more Glioblastoma (GBM) has a dismal prognosis and successful elimination of GBM stem cells (GSCs) is a high-priority as these cells are responsible for tumor regrowth following therapy and ultimately patient relapse. Natural products and their derivatives continue to be a source for the development of effective anticancer drugs and have been shown to effectively target pathways necessary for cancer stem cell self-renewal and proliferation. We generated a series of curcumin inspired bis-chalcones and examined their effect in multiple patient-derived GSC lines. Of the 19 compounds synthesized, four analogs robustly induced GSC death in six separate GSC lines, with a half maximal inhibitory concentration (IC50) ranging from 2.7–5.8 μM and significantly reduced GSC neurosphere formation at sub-cytotoxic levels. Structural analysis indicated that the presence of a methoxy group at position 3 of the lateral phenylic appendages was important for activity. Pathway and drug connectivity analysis...
Resistance mechanisms employed by high-grade gliomas allow them to successfully evade current sta... more Resistance mechanisms employed by high-grade gliomas allow them to successfully evade current standard treatment of chemotherapy and radiation treatment. Withaferin A (WA), utilized in Ayurvedic medicine for centuries, is attracting attention for its antitumor capabilities. Here we review pertinent literature on WA as a high-grade glioma treatment, and discuss the cancerous mechanisms it affects. WA is relatively nontoxic and has shown potential in crossing the blood-brain barrier. WA prevents p53 alterations and inactivates overexpressed MDM2 through ARF and ROS production. Furthermore, WA upregulates Bax, inducing mitochondrial death cascades, inhibits mutated Akt, mTOR, and NF-jB pathways, and inhibits angiogenesis in tumors. Therapy with WA for high-grade gliomas is supported through the literature. Further investigation is warranted and encouraged to fully unearth its abilities against malignant gliomas.
Herein we report the facile conversion of 2 ,3 ,5-triO -acetylinosine to three different nucleosi... more Herein we report the facile conversion of 2 ,3 ,5-triO -acetylinosine to three different nucleoside analogs via reaction of hexamethylphosphorous triamide and an organic halide. Acetyl-protected 6-bromopurine riboside, 6-chloropurine riboside and N 6 ,N 6-dimethyladenosine can each be prepared in good yield from 2 ,3 ,5-tri-Oacetylinosine, HMPT and halide. The major product of the reaction is determined by the identity of the halide used and the reaction temperature.
Bioassay-directed fractionation of ethanolic extracts of the stem bark of Erythrina crista-galli ... more Bioassay-directed fractionation of ethanolic extracts of the stem bark of Erythrina crista-galli resulted in the isolation of a new pterocarpan, erycristin, and two previously known pterocarpans, sandwicensin and erythrabyssin-II. The structure of erycristin was determined by spectroscopic examination and by chemical transformation of sandwicensin. The absolute stereochemistry of erycristin was established by circular dichroism measurements. ErythrabyssindI diacetate was converted to erycristagallin diacetate, supporting the previous proposed structure of erycristagallin,
Advances in chemical synthesis and characterization of nucleic acids allows for atom-specific mod... more Advances in chemical synthesis and characterization of nucleic acids allows for atom-specific modification of complex RNAs, such as present in RNA editing substrates. By preparing substrates for ADARs by chemical synthesis, it is possible to subtly alter the structure of the edited nucleotide. Evaluating the effect these changes have on the rate of enzyme-catalyzed deamination reveals features of the editing reaction and guides the design of inhibitors. We describe the synthesis of select nucleoside analog phosphoramidites and their incorporation into RNAs that mimic known editing sites by solid phase synthesis, and
Encyclopedia of Reagents for Organic Synthesis, 2001
ABSTRACT (S) [64715-80-6] C10H15NO (MW 165.23) InChI = 1S/C10H15NO/c1-12-8-10(11)7-9-5-3-2-4-6-9/... more ABSTRACT (S) [64715-80-6] C10H15NO (MW 165.23) InChI = 1S/C10H15NO/c1-12-8-10(11)7-9-5-3-2-4-6-9/h2-6,10H,7-8,11H2,1H3/t10-/m1/s1 InChIKey = FAZIHZPDHJBQKN-SNVBAGLBSA-N (R) [59919-07-2] InChI = 1S/C10H15NO/c1-12-8-10(11)7-9-5-3-2-4-6-9/h2-6,10H,7-8,11H2,1H3/t10-/m0/s1 InChIKey = FAZIHZPDHJBQKN-JTQLQIEISA-N (chiral auxiliary for the enantioselective alkylation of ketones1 and aldehydes;2 can form chiral cuprate reagents3)Physical Data: (S) bp 55–59 °C/0.1 mmHg; mp HCl salt 151–152 °C; [α]d25 −14.4° (c 5.7, benzene); HCl salt [α]d25 +19.7° (c 2.5, EtOH).Preparative Method: these chiral methoxy amines are readily prepared from (S)- or (R)-phenylalanine via reduction followed by methylation.2Handling, Storage, and Precautions: conversion of the freshly distilled amine to its hydrochloride salt is a convenient way to store and handle the compound. The free amine reacts with atmospheric carbon dioxide to produce the respective carbonate. The free amine should be stored tightly sealed under argon or nitrogen immediately after distillation to avoid CO2 adsorption.
Herein, we report the use of triisopropyl phosphite (TIP) as an effective substitute for tripheny... more Herein, we report the use of triisopropyl phosphite (TIP) as an effective substitute for triphenylphosphine in the Mitsunobu reaction of nucleoside analogs. In addition, the use of triphenyl phosphite as an alternative reagent for the expensive hexamethylphosphorous triamide (HMPT) in the Véliz-Beal bromination protocol is reported.
The biosynthesis of coloradocin involves assembly of a polyketide chain similar to those of narge... more The biosynthesis of coloradocin involves assembly of a polyketide chain similar to those of nargenicin and nodusmicin and an apparent non-polyketide eleven carbon unit which incorporates acetate and succinate (but not propionate) by some undetermined pathway. Precursor incorporation was studied using single and double labeled C-13 precursors and label scrambling was suppressed by the simultaneous addition of other unlabeled substrates.
... FROM ROOT EXTRACTS OF ERYTHRINA VARIEGATA HANUMAIAH TELIKEPALLI, SITARAGHAV R GOLLAPUDI, ALI ... more ... FROM ROOT EXTRACTS OF ERYTHRINA VARIEGATA HANUMAIAH TELIKEPALLI, SITARAGHAV R GOLLAPUDI, ALI KESHAVARZ-SHOKRI, LILIAN VELAZQUEZ,* ROBERT A ... S, Dutta, SK and Bhattacharya, SK (1972) J. Pharm Sci 61, 1274 6 Mitscher, LA , Ward, JA , Drake ...
We report the synthesis of a 5′-DMT-2′-TBDMS-protected phosphoramidite of 6-trifluoromethylpurine... more We report the synthesis of a 5′-DMT-2′-TBDMS-protected phosphoramidite of 6-trifluoromethylpurine ribonucleoside (TFM P) and its use in the site-specific incorporation of 6-trifluoromethylpurine into RNA. Properties of TFM P-substituted RNA suggest it will be valuable in the study of RNA structure and the binding of RNA-modifying enzymes, particularly the RNA-editing adenosine deaminases.
The RNA-editing adenosine deaminases (ADARs) catalyze deamination of adenosine to inosine in doub... more The RNA-editing adenosine deaminases (ADARs) catalyze deamination of adenosine to inosine in double stranded structure found in various RNA substrates, including mRNAs. Here we describe the synthesis of a phosphoramidite of 2'-deoxy-2'-mercaptoadenosine and its incorporation into an ADAR substrate. Surprisingly, no deamination product was observed with this substrate indicating replacing the 2'-OH with a 2'-SH at the editing site is highly inhibitory. Modeling of nucleotide binding into the active site suggests the side chain of T375 of human ADAR2 to be in proximity of the 2'-substituent. Mutation of this residue to cysteine caused a greater that 100-fold reduction in deamination rate with the 2'-OH substrate.
ADARs are adenosine deaminases that act on RNA and are responsible for RNA-editing reactions that... more ADARs are adenosine deaminases that act on RNA and are responsible for RNA-editing reactions that occur in eukaryotic mRNAs, including the mRNAs of glutamate and serotonin receptors. ADARs capable of editing biologically relevant RNA substrates have been identified. In addition, the consequence of the RNA-editing reaction on the function of the gene product is known in several cases. However, our understanding of the chemical mechanism of the ADAR-catalyzed adenosine deamination in RNA is lagging. By studying analogues of a naturally occurring substrate for ADAR2, we infer features of the enzyme's active site and reaction mechanism. 8-Aza substitution at adenosine in various RNA substrates accelerates the rate of deamination at these sites by ADAR2 (2.8-17-fold). The magnitude of this "aza effect" depends on the RNA structural context of the reacting nucleotide. N 6-Methyladenosine in RNA is a slow substrate for ADAR2 (rate is 2% that of adenosine), with no product observed with N 6-ethyladenosine, suggesting a limited size of the leaving group pocket. 2,6-Diaminopurine ribonucleoside in RNA is not a substrate for ADAR, in contrast to adenosine deaminase (ADA), which catalyzes a similar reaction on nucleosides. This and other results indicate that ADAR2 uses a base recognition strategy different from that of ADA. Consistent with the large 8-aza effect observed for the ADAR2 reaction, we find that 8-azanebularine, as the free nucleoside, inhibits the ADAR2 reaction (IC 50) 15 (3 mM) with no inhibition observed with nebularine or coformycin.
Deamination at C6 of adenosine in RNA catalyzed by the ADAR enzymes generates inosine at the corr... more Deamination at C6 of adenosine in RNA catalyzed by the ADAR enzymes generates inosine at the corresponding position. Because inosine is decoded as guanosine during translation, this modification can lead to codon changes in messenger RNA. Hydration of 8-azanebularine across the C6-N1 double bond generates an excellent mimic of the transition state proposed for the hydrolytic deamination reaction catalyzed by ADARs. Here, we report the synthesis of a phosphoramidite of 8-azanebularine and its use in the preparation of RNAs mimicking the secondary structure found at a known editing site in the glutamate receptor B subunit pre-mRNA. The binding properties of analogue-containing RNAs indicate that a tight binding ligand for an ADAR can be generated by incorporation of 8azanebularine. The observed high-affinity binding is dependent on a functional active site, the presence of one, but not the other, of ADAR2's two double-stranded RNA-binding motifs (dsRBMs), and the correct placement of the nucleoside analogue into the sequence/structural context of a known editing site. These results advance our understanding of substrate recognition during ADARcatalyzed RNA editing and are important for structural studies of ADAR• RNA complexes.
ADARs are adenosine deaminases that act on RNA and are responsible for RNA-editing reactions that... more ADARs are adenosine deaminases that act on RNA and are responsible for RNA-editing reactions that occur in eukaryotic mRNAs, including the mRNAs of glutamate and serotonin receptors. 1 The deamination of adenosine in the mRNA results in inosine at that position. Since inosine is translated as guanosine, the ADAR reaction can lead to codon changes in the mRNA, resulting in the synthesis of variant protein structures (Scheme 1). In several cases, an ADAR capable of a particular editing reaction has been identified. 2-4 However, our understanding of the mechanism of the ADAR-catalyzed reaction is limited. Here we report the synthesis and analysis of substrate analogues designed to provide insight into the mechanistic relationship between the RNA-editing ADARs and the well-understood nucleoside-modifying enzyme adenosine deaminase (ADA). 5-8 These experiments illustrate significant mechanistic similarities and differences between the two classes of adenosine deaminase. ADAR2 efficiently deaminates specific adenosines in the glutamate receptor B subunit pre-mRNA, including one that converts an arginine codon to a glycine codon (the R/G site). 9 Adenosine deaminase (ADA) is a nucleoside-modifying enzyme that has been extensively characterized structurally and mechanistically. 5-8 ADA uses a zinc-activated water molecule to affect hydrolytic deamination of its nucleoside substrate. The oxygen in the product of the ADAR reaction has also been shown to be derived from water. 10 However, ADARs share little sequence homology with ADAs. 11 Furthermore, the potent ADA inhibitor, coformycin, does not inhibit ADARs, even at high concentra
The unfolded protein response (UPR) is a pro-survival mechanism triggered by cellular stresses en... more The unfolded protein response (UPR) is a pro-survival mechanism triggered by cellular stresses encountered by tumor cells, such as hypoxia, acidosis, reactive oxygen species and even radiation and chemotherapy. In response to increased unfolded or mis-folded proteins in the endoplasmic reticulum (ER), the UPR initiates specific cell signaling pathways aiming to alleviate ER stress and restore ER proteostasis which include increasing the expression of chaperones to aid in protein folding, enhancing protein degradation and reducing global protein synthesis. However, if homeostasis cannot be restored, the UPR switches from survival to cell death. The UPR is a relatively novel therapeutic target for glioblastoma (GBM), and specifically GBM stem-like cells (GSCs) which maintain tumor heterogeneity, drive GBM tumor growth and therapy resistance. Here we tested a series of curcumin derivatives against three GSC lines and identified a trimethoxy bis-chalcone which promotes robust GSC death ...
Pediatric brain tumors remain a significant source of morbidity and mortality. Though development... more Pediatric brain tumors remain a significant source of morbidity and mortality. Though developments have been made in treating these malignancies, the blood–brain barrier, intra- and inter-tumoral heterogeneity, and therapeutic toxicity pose challenges to improving outcomes. Varying types of nanoparticles, including metallic, organic, and micellar molecules of varying structures and compositions, have been investigated as a potential therapy to circumvent some of these inherent challenges. Carbon dots (CDs) have recently gained popularity as a novel nanoparticle with theranostic properties. This carbon-based modality is highly modifiable, allowing for conjugation to drugs, as well as tumor-specific ligands in an effort to more effectively target cancerous cells and reduce peripheral toxicity. CDs are being studied pre-clinically. The ClinicalTrials.gov site was queried using the search terms: brain tumor and nanoparticle, liposome, micelle, dendrimer, quantum dot, or carbon dot. At t...
The current prognosis for glioblastoma is dismal. Treatment-resistant glioblastoma stem cells (GS... more The current prognosis for glioblastoma is dismal. Treatment-resistant glioblastoma stem cells (GSCs) and the failure of most drugs to reach therapeutic levels within the tumor remain formidable obstacles to successful treatment. Chalcones are aromatic ketones demonstrated to reduce malignant properties in cancers including glioblastoma. Nanomedicines can increase drug accumulation and tumor cell death. Carbon-dots are promising nanocarriers that can be easily functionalized with tumor-targeting ligands and anti-cancer drugs. Therefore, we synthesized a series of 4′-amino chalcones with the rationale that the amino group would serve as a “handle” to facilitate covalent attachment to carbon-dots and tested their cytotoxicity toward GSCs. We generated 31 chalcones (22 4′-amino and 9 4′ derivatives) including 5 novel chalcones, and found that 13 had an IC50 below 10 µM in all GSC lines. After confirming that the 4-amino group was not part of the active pharmacophore, chalcones were atta...
Glioblastoma (GBM) has a dismal prognosis and successful elimination of GBM stem cells (GSCs) is ... more Glioblastoma (GBM) has a dismal prognosis and successful elimination of GBM stem cells (GSCs) is a high-priority as these cells are responsible for tumor regrowth following therapy and ultimately patient relapse. Natural products and their derivatives continue to be a source for the development of effective anticancer drugs and have been shown to effectively target pathways necessary for cancer stem cell self-renewal and proliferation. We generated a series of curcumin inspired bis-chalcones and examined their effect in multiple patient-derived GSC lines. Of the 19 compounds synthesized, four analogs robustly induced GSC death in six separate GSC lines, with a half maximal inhibitory concentration (IC50) ranging from 2.7–5.8 μM and significantly reduced GSC neurosphere formation at sub-cytotoxic levels. Structural analysis indicated that the presence of a methoxy group at position 3 of the lateral phenylic appendages was important for activity. Pathway and drug connectivity analysis...
Resistance mechanisms employed by high-grade gliomas allow them to successfully evade current sta... more Resistance mechanisms employed by high-grade gliomas allow them to successfully evade current standard treatment of chemotherapy and radiation treatment. Withaferin A (WA), utilized in Ayurvedic medicine for centuries, is attracting attention for its antitumor capabilities. Here we review pertinent literature on WA as a high-grade glioma treatment, and discuss the cancerous mechanisms it affects. WA is relatively nontoxic and has shown potential in crossing the blood-brain barrier. WA prevents p53 alterations and inactivates overexpressed MDM2 through ARF and ROS production. Furthermore, WA upregulates Bax, inducing mitochondrial death cascades, inhibits mutated Akt, mTOR, and NF-jB pathways, and inhibits angiogenesis in tumors. Therapy with WA for high-grade gliomas is supported through the literature. Further investigation is warranted and encouraged to fully unearth its abilities against malignant gliomas.
Herein we report the facile conversion of 2 ,3 ,5-triO -acetylinosine to three different nucleosi... more Herein we report the facile conversion of 2 ,3 ,5-triO -acetylinosine to three different nucleoside analogs via reaction of hexamethylphosphorous triamide and an organic halide. Acetyl-protected 6-bromopurine riboside, 6-chloropurine riboside and N 6 ,N 6-dimethyladenosine can each be prepared in good yield from 2 ,3 ,5-tri-Oacetylinosine, HMPT and halide. The major product of the reaction is determined by the identity of the halide used and the reaction temperature.
Bioassay-directed fractionation of ethanolic extracts of the stem bark of Erythrina crista-galli ... more Bioassay-directed fractionation of ethanolic extracts of the stem bark of Erythrina crista-galli resulted in the isolation of a new pterocarpan, erycristin, and two previously known pterocarpans, sandwicensin and erythrabyssin-II. The structure of erycristin was determined by spectroscopic examination and by chemical transformation of sandwicensin. The absolute stereochemistry of erycristin was established by circular dichroism measurements. ErythrabyssindI diacetate was converted to erycristagallin diacetate, supporting the previous proposed structure of erycristagallin,
Advances in chemical synthesis and characterization of nucleic acids allows for atom-specific mod... more Advances in chemical synthesis and characterization of nucleic acids allows for atom-specific modification of complex RNAs, such as present in RNA editing substrates. By preparing substrates for ADARs by chemical synthesis, it is possible to subtly alter the structure of the edited nucleotide. Evaluating the effect these changes have on the rate of enzyme-catalyzed deamination reveals features of the editing reaction and guides the design of inhibitors. We describe the synthesis of select nucleoside analog phosphoramidites and their incorporation into RNAs that mimic known editing sites by solid phase synthesis, and
Encyclopedia of Reagents for Organic Synthesis, 2001
ABSTRACT (S) [64715-80-6] C10H15NO (MW 165.23) InChI = 1S/C10H15NO/c1-12-8-10(11)7-9-5-3-2-4-6-9/... more ABSTRACT (S) [64715-80-6] C10H15NO (MW 165.23) InChI = 1S/C10H15NO/c1-12-8-10(11)7-9-5-3-2-4-6-9/h2-6,10H,7-8,11H2,1H3/t10-/m1/s1 InChIKey = FAZIHZPDHJBQKN-SNVBAGLBSA-N (R) [59919-07-2] InChI = 1S/C10H15NO/c1-12-8-10(11)7-9-5-3-2-4-6-9/h2-6,10H,7-8,11H2,1H3/t10-/m0/s1 InChIKey = FAZIHZPDHJBQKN-JTQLQIEISA-N (chiral auxiliary for the enantioselective alkylation of ketones1 and aldehydes;2 can form chiral cuprate reagents3)Physical Data: (S) bp 55–59 °C/0.1 mmHg; mp HCl salt 151–152 °C; [α]d25 −14.4° (c 5.7, benzene); HCl salt [α]d25 +19.7° (c 2.5, EtOH).Preparative Method: these chiral methoxy amines are readily prepared from (S)- or (R)-phenylalanine via reduction followed by methylation.2Handling, Storage, and Precautions: conversion of the freshly distilled amine to its hydrochloride salt is a convenient way to store and handle the compound. The free amine reacts with atmospheric carbon dioxide to produce the respective carbonate. The free amine should be stored tightly sealed under argon or nitrogen immediately after distillation to avoid CO2 adsorption.
Herein, we report the use of triisopropyl phosphite (TIP) as an effective substitute for tripheny... more Herein, we report the use of triisopropyl phosphite (TIP) as an effective substitute for triphenylphosphine in the Mitsunobu reaction of nucleoside analogs. In addition, the use of triphenyl phosphite as an alternative reagent for the expensive hexamethylphosphorous triamide (HMPT) in the Véliz-Beal bromination protocol is reported.
The biosynthesis of coloradocin involves assembly of a polyketide chain similar to those of narge... more The biosynthesis of coloradocin involves assembly of a polyketide chain similar to those of nargenicin and nodusmicin and an apparent non-polyketide eleven carbon unit which incorporates acetate and succinate (but not propionate) by some undetermined pathway. Precursor incorporation was studied using single and double labeled C-13 precursors and label scrambling was suppressed by the simultaneous addition of other unlabeled substrates.
... FROM ROOT EXTRACTS OF ERYTHRINA VARIEGATA HANUMAIAH TELIKEPALLI, SITARAGHAV R GOLLAPUDI, ALI ... more ... FROM ROOT EXTRACTS OF ERYTHRINA VARIEGATA HANUMAIAH TELIKEPALLI, SITARAGHAV R GOLLAPUDI, ALI KESHAVARZ-SHOKRI, LILIAN VELAZQUEZ,* ROBERT A ... S, Dutta, SK and Bhattacharya, SK (1972) J. Pharm Sci 61, 1274 6 Mitscher, LA , Ward, JA , Drake ...
We report the synthesis of a 5′-DMT-2′-TBDMS-protected phosphoramidite of 6-trifluoromethylpurine... more We report the synthesis of a 5′-DMT-2′-TBDMS-protected phosphoramidite of 6-trifluoromethylpurine ribonucleoside (TFM P) and its use in the site-specific incorporation of 6-trifluoromethylpurine into RNA. Properties of TFM P-substituted RNA suggest it will be valuable in the study of RNA structure and the binding of RNA-modifying enzymes, particularly the RNA-editing adenosine deaminases.
The RNA-editing adenosine deaminases (ADARs) catalyze deamination of adenosine to inosine in doub... more The RNA-editing adenosine deaminases (ADARs) catalyze deamination of adenosine to inosine in double stranded structure found in various RNA substrates, including mRNAs. Here we describe the synthesis of a phosphoramidite of 2'-deoxy-2'-mercaptoadenosine and its incorporation into an ADAR substrate. Surprisingly, no deamination product was observed with this substrate indicating replacing the 2'-OH with a 2'-SH at the editing site is highly inhibitory. Modeling of nucleotide binding into the active site suggests the side chain of T375 of human ADAR2 to be in proximity of the 2'-substituent. Mutation of this residue to cysteine caused a greater that 100-fold reduction in deamination rate with the 2'-OH substrate.
ADARs are adenosine deaminases that act on RNA and are responsible for RNA-editing reactions that... more ADARs are adenosine deaminases that act on RNA and are responsible for RNA-editing reactions that occur in eukaryotic mRNAs, including the mRNAs of glutamate and serotonin receptors. ADARs capable of editing biologically relevant RNA substrates have been identified. In addition, the consequence of the RNA-editing reaction on the function of the gene product is known in several cases. However, our understanding of the chemical mechanism of the ADAR-catalyzed adenosine deamination in RNA is lagging. By studying analogues of a naturally occurring substrate for ADAR2, we infer features of the enzyme's active site and reaction mechanism. 8-Aza substitution at adenosine in various RNA substrates accelerates the rate of deamination at these sites by ADAR2 (2.8-17-fold). The magnitude of this "aza effect" depends on the RNA structural context of the reacting nucleotide. N 6-Methyladenosine in RNA is a slow substrate for ADAR2 (rate is 2% that of adenosine), with no product observed with N 6-ethyladenosine, suggesting a limited size of the leaving group pocket. 2,6-Diaminopurine ribonucleoside in RNA is not a substrate for ADAR, in contrast to adenosine deaminase (ADA), which catalyzes a similar reaction on nucleosides. This and other results indicate that ADAR2 uses a base recognition strategy different from that of ADA. Consistent with the large 8-aza effect observed for the ADAR2 reaction, we find that 8-azanebularine, as the free nucleoside, inhibits the ADAR2 reaction (IC 50) 15 (3 mM) with no inhibition observed with nebularine or coformycin.
Deamination at C6 of adenosine in RNA catalyzed by the ADAR enzymes generates inosine at the corr... more Deamination at C6 of adenosine in RNA catalyzed by the ADAR enzymes generates inosine at the corresponding position. Because inosine is decoded as guanosine during translation, this modification can lead to codon changes in messenger RNA. Hydration of 8-azanebularine across the C6-N1 double bond generates an excellent mimic of the transition state proposed for the hydrolytic deamination reaction catalyzed by ADARs. Here, we report the synthesis of a phosphoramidite of 8-azanebularine and its use in the preparation of RNAs mimicking the secondary structure found at a known editing site in the glutamate receptor B subunit pre-mRNA. The binding properties of analogue-containing RNAs indicate that a tight binding ligand for an ADAR can be generated by incorporation of 8azanebularine. The observed high-affinity binding is dependent on a functional active site, the presence of one, but not the other, of ADAR2's two double-stranded RNA-binding motifs (dsRBMs), and the correct placement of the nucleoside analogue into the sequence/structural context of a known editing site. These results advance our understanding of substrate recognition during ADARcatalyzed RNA editing and are important for structural studies of ADAR• RNA complexes.
ADARs are adenosine deaminases that act on RNA and are responsible for RNA-editing reactions that... more ADARs are adenosine deaminases that act on RNA and are responsible for RNA-editing reactions that occur in eukaryotic mRNAs, including the mRNAs of glutamate and serotonin receptors. 1 The deamination of adenosine in the mRNA results in inosine at that position. Since inosine is translated as guanosine, the ADAR reaction can lead to codon changes in the mRNA, resulting in the synthesis of variant protein structures (Scheme 1). In several cases, an ADAR capable of a particular editing reaction has been identified. 2-4 However, our understanding of the mechanism of the ADAR-catalyzed reaction is limited. Here we report the synthesis and analysis of substrate analogues designed to provide insight into the mechanistic relationship between the RNA-editing ADARs and the well-understood nucleoside-modifying enzyme adenosine deaminase (ADA). 5-8 These experiments illustrate significant mechanistic similarities and differences between the two classes of adenosine deaminase. ADAR2 efficiently deaminates specific adenosines in the glutamate receptor B subunit pre-mRNA, including one that converts an arginine codon to a glycine codon (the R/G site). 9 Adenosine deaminase (ADA) is a nucleoside-modifying enzyme that has been extensively characterized structurally and mechanistically. 5-8 ADA uses a zinc-activated water molecule to affect hydrolytic deamination of its nucleoside substrate. The oxygen in the product of the ADAR reaction has also been shown to be derived from water. 10 However, ADARs share little sequence homology with ADAs. 11 Furthermore, the potent ADA inhibitor, coformycin, does not inhibit ADARs, even at high concentra
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Papers by Eduardo Veliz