I am originally an inorganic and organometallic chemist who has been working on Bioorganic Chemistry, Drug Discovery and Development, Nucleic Acids Chemistry and Biochemistry, and antiviral drug discovery for the past twenty years. I have spent half my career in industry and half in academia.
We wish to develop reagents for the sequencespecific cleavage of RNA by non-oxidative mechanisms.... more We wish to develop reagents for the sequencespecific cleavage of RNA by non-oxidative mechanisms. The molecules we have devised are synthetic ribozymes, and comprise a molecular recognition agent (such as single-stranded
Journal of The Chemical Society-dalton Transactions, 1985
ABSTRACT Co-condensation of molybdenum atoms with trimethylphosphine gives octahedral [ Mo(PMe3)6... more ABSTRACT Co-condensation of molybdenum atoms with trimethylphosphine gives octahedral [ Mo(PMe3)6](1) whose crystal structure has been determined. Treatment of (1) with dinitrogen, ethylene, carbon monoxide, iodine, or butadiene gives [Mo(N2)(PMe3)5], trans-[Mo(η-C2H4)2(PMe3)4]fac-[Mo(CO)3(PMe3)3], trans-[MoI2(PMe3)4], and cis-[Mo(η-C4H6)2(PMe3)2] respectively. Protonation of cis-[Mo(η-C4H6)2(PMe3)2] with tetrafluoroboric acid forms the compound cis-[[graphic omitted]CH2CHCHCH2)(η-C4H6)(PMe3)2]BF4. The presence of the Mo–H–C bond is shown by low-temperature n.m.r. spectra, and variable-temperature n.m.r. shows that the agostic hydrogen can scramble between the four terminal carbons of the two C4 ligands. The rate constants and activation parameters for the hydrogen-scrambling process have been determined and a mechanism is proposed. Reaction of cis-[Mo(η-C4H6)2(PMe3)2] with trifluoroacetic acid gives [Mo(η-MeC3H4)(η-C4H6)(PMe3)2(O2CCF3)]. Treatment of [Mo(PMe3)6] with cyclopentadiene forms [MoH(η-C5H5)(PMe3)3] which reacts with aqueous tetrafluoroboric acid giving [MoH2(η-C5H5)(PMe3)3]BF4 and [Mo(η-C5H5)(PMe3)2O]BF4. Reaction of [MoH(η-C5H5)(PMe3)3] with methyl iodide gives [MoH(η-C5H5)(PMe3)2I2].
Human papillomavirus (HPV) causes cervical cancer and other hyperproliferative diseases. There cu... more Human papillomavirus (HPV) causes cervical cancer and other hyperproliferative diseases. There currently are no approved antiviral drugs for HPV that directly decrease viral DNA load and that have low toxicity. We report the potent anti-HPV activity of two N-methylpyrrole-imidazole polyamides of the hairpin type, polyamide 1 (PA1) and polyamide 25 (PA25). Both polyamides have potent anti-HPV activity against 3 different genotypes when tested on cells maintaining HPV episomes. The compounds were tested against HPV16 (in W12 cells), HPV18 (in Ker4-18 cells), and HPV31 (tested in HPV31 maintaining cells). From a library of polyamides designed to recognize AT-rich DNA sequences such as those in or near E1 or E2 binding sites of the HPV16 origin of replication (ori), 4 polyamides were identified that possessed apparent IC 50 s ≤ 150 nM with no evidence of cytotoxicity and we report two highly-active compounds here. Treatment of epithelia engineered in organotypic cultures with these compounds also causes a dose-dependent loss of HPV episomal DNA that correlates with accumulation of compounds in the nucleus. Bromodeoxyuridine (BrdU) incorporation demonstrates that DNA synthesis in organotypic cultures is suppressed upon compound treatment, correlating with a loss of HPV16 and HPV18 episomes. PA1 and PA25 are currently in preclinical development as an antiviral compound for treatment of HPV-related disease, including cervical dysplasia. PA1 and related polyamides offer promise as antiviral agents and the ability to regulate HPV episomal levels in cells for the study of HPV biology. We also report that anti-HPV16 activity for Distamycin A, a natural product related to PA1, is accompanied by significant cellular toxicity.
A highly reproducible quantitative PCR (Q-PCR) assay was used to study the stability of human pap... more A highly reproducible quantitative PCR (Q-PCR) assay was used to study the stability of human papillomavirus (HPV) in undifferentiated keratinocytes that maintain viral episomes. The term "stability" refers to the ability of episomes to persist with little copy number variation in cells. In investigating the mechanism of action of PA25, a previously published compound that destabilizes HPV episomes, aphidicolin was also found to markedly decrease episome levels, but via a different pathway from that of PA25. Since aphidicolin is known to activate DNA damage response (DDR) pathways, effects of inhibitors and small interfering RNAs (siRNAs) acting within DDR pathways were investigated. Inhibitors of Chk1 and siRNA directed against ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia Rad3-related (ATR) pathways significantly reduced viral episomes, suggesting that these pathways play a role in maintaining HPV episome stability. Inhibitors of Chk2 and DNA-PK had no effect on episome levels. Pharmacological inhibition of ATM proteins had no effect on episome levels, but ATM knockdown by siRNA significantly reduced episome levels, suggesting that ATM proteins are playing an important role in HPV episome stability that does not require kinase activity. These results outline two pathways that trigger episome loss from cells and suggest the existence of a littleunderstood mechanism that mediates viral DNA elimination. Together, our results also indicate that HPV episomes have a stability profile that is remarkably similar to that of fragile sites; these similarities are outlined and discussed. This close correspondence may influence the preference of HPV for integration into fragile sites.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
There is a long history for the bioorganic and biomedical use of N-methyl-pyrrole-derived polyami... more There is a long history for the bioorganic and biomedical use of N-methyl-pyrrole-derived polyamides (PAs) that are higher homologs of natural products such as distamycin A and netropsin. This work has been pursued by many groups, with the Dervan and Sugiyama groups responsible for many breakthroughs. We have studied PAs since about 1999, partly in industry and partly in academia. Early in this program, we reported methods to control cellular uptake of polyamides in cancer cell lines and other cells likely to have multidrug resistance efflux pumps induced. We went on to discover antiviral polyamides active against HPV31, where SAR showed that a minimum binding size of about 10 bp of DNA was necessary for activity. Subsequently we discovered polyamides active against two additional high-risk HPVs, HPV16 and 18, a subset of which showed broad spectrum activity against HPV16, 18 and 31. Aspects of our results presented here are incompatible with reported DNA recognition rules. For example, molecules with the same cognate DNA recognition properties varied from active to inactive against HPVs. We have since pursued the mechanism of action of antiviral polyamides, and polyamides in general, with collaborators at NanoVir, the University of Missouri-St. Louis, and Georgia State University. We describe dramatic consequences of β-alanine positioning even in relatively small, 8-ring polyamides; these results contrast sharply with prior reports. This paper was originally presented by JKB as a Keynote Lecture in the
We have previously prepared ribozyme mimics and chemical nucleases from modified DNA containing p... more We have previously prepared ribozyme mimics and chemical nucleases from modified DNA containing pendant bipyridine and terpyridine groups. The ability of these modified DNA probes to support RNase H cleavage of complementary RNA is described. DNA/RNA duplexes ...
Abstracts of Papers American Chemical Society, Nov 8, 1997
ABSTRACT The objective of this project is to enhance sequence-specific hydrolysis of RNA by riboz... more ABSTRACT The objective of this project is to enhance sequence-specific hydrolysis of RNA by ribozyme mimics. These mimics may act as catalytic antisense reagents, recognizing harmful RNA sequences and cleaving those sequences by transesterification. We are optimizing ribozyme mimics by delivering the cleavage agent to the active site more efficiently. This will be accomplished by the use of electrostatics and groove-specific delivery. The total synthesis of novel phosphoramidites for automated DNA synthesis will be described. These new building blocks will probe the viability of using electrostatic attraction and specific-groove delivery to intensify cleavage by ribozyme mimics. Combining Watson-Crick base pairing with electrostatics and groove control yields three-way mol. recognition for a potential therapeutic agent. [on SciFinder(R)]
A series of polyamides based on pyrrole-imidazole polyamides was designed to interfere with viral... more A series of polyamides based on pyrrole-imidazole polyamides was designed to interfere with viral protein-viral DNA interactions at the origin of replication of the high risk human papillomavirus types 16, 18 and 31. The specific sites targeted were the E1 and E2 protein binding sites on the viral genomes. The initial library showed hits in cell culture screens. Activity was assessed by qPCR determination of viral DNA 48 h after treatment in cell culture. Dose response curves were determined in all cases, with at least triplicate points. Active compounds were further elaborated and modified in a series of subsequent libraries, with initial optimization achieving many sub-100 nM IC50s and apparent IC50's as low as 36 nM. Since the three viral genomes are not degenerate for polyamide binding in the target regions, we were surprised to find that a significant number of compounds showed broad-spectrum anti-HPV activity against all three viral subtypes investigated (16, 18 and 31). Although the MWs are high, excellent cell culture activity is seen, in agreement with related studies. Two compounds are in preclinical development and have been scaled up to >6 g. More recently, we have discovered a new class of polyamides functionalized at the N-terminus which are active to IC50s of ca. 10 nM. Most recently, important clues to the mechanism of action have been discovered: HPV-infected cells treated with active compounds show activation of one branch of the DNA Damage Response Pathway, while uninfected cells show no such response.
Journal of Biomolecular Structure & Dynamics, 2013
The accuracy of the Content should not be relied upon and should be independently verified with p... more The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden.
PA1 (dIm-PyPybPyPyPy-g-PyPybPyPyPyPyb-Ta) is a large (14-ring) hairpin polyamide that was designe... more PA1 (dIm-PyPybPyPyPy-g-PyPybPyPyPyPyb-Ta) is a large (14-ring) hairpin polyamide that was designed to recognize the DNA sequence 5'-W 2 GW 7-3', where W is either A or T. As is common among the smaller 6-8-ring hairpin polyamides (PAs), it binds its target recognition sequence with low nM affinity. However, in addition to its large size, it is distinct from these more extensively characterized PAs in its high tolerance for mismatches and antiviral properties. In ongoing attempts to understand the basis for these distinctions, we conducted thermodynamics studies of PA1-DNA interactions. The temperature dependence of binding affinity was measured using TAMRA-labeled hairpin DNAs containing a single target sequence. PA1 binding to either an ATAT/TATA or an AAAA/TTTT pattern is consistently entropically driven. This is in contrast to the A/T pattern-dependent driving forces for DNA binding by netropsin, distamycin, and smaller hairpin polyamides. Analysis of the salt dependence of PA1-DNA binding reveals that within experimental error, there is no dependence on ionic strength, indicating that the polyelectrolyte effect does not contribute to PA1-DNA binding energetics. This is similar to that observed for smaller PAs. PA1-DNA recognition sequence binding stoichiometries were determined at both nM (fluorescence) and mM (circular dichroism) concentrations. With all sequences and under both conditions, multiple PA1 molecules bind the small DNA hairpin that contains only a single recognition sequence. Implications for these observations are discussed.
... Chimica Acta 263 (1997) 49-52 J Transesterification of RNA by Cu (II) terpyridine Lisa A, Jen... more ... Chimica Acta 263 (1997) 49-52 J Transesterification of RNA by Cu (II) terpyridine Lisa A, Jenkins Autry, James K. Bashkin ... RNA; Transestenfication; Hydrolysis; Ribozymos mimics; Copper(II) complexes; Terp~dine complexes 1. Introduction The transesterification of RNA (Fig. ...
Journal of the American Society for Mass Spectrometry, 2006
The aim of this work is to establish a quantitative method to determine the ratio of [U-13 C] lab... more The aim of this work is to establish a quantitative method to determine the ratio of [U-13 C] labeled to unlabeled hexose monophosphates isolated from yeast extracts. This is accomplished by anion exchange chromatography and mobile phase desalting followed by electrospray (ESI) mass spectrometry. We test the method with the analysis of a sample of biological origin. Previously developed analytical techniques are not adequate to accomplish mass spectrometric analysis of these and other small monosaccharide systems because of interference from salt clusters. By lowering the ionic strength of the mobile phase and using a simplified injection system to the mass spectrometer, we were able to obtain data on the relative abundance of the hexose monophosphates.
ABSTRACT The transport and reactivity of oligonucleotides may be altered by attaching pendant pep... more ABSTRACT The transport and reactivity of oligonucleotides may be altered by attaching pendant peptides, and it is of interest to develop general synthetic methods for such bioconjugates. Two protecting group strategies are described for the synthesis of nucleotide peptides containing a lysine residue. The preparation of a lysine-nucleopeptide phosphoramidite reagent is described, along with its use in solid-phase DNA synthesis. Di- and trinucleotides were prepared with pendant and extensively characterized by NMR. These studies showed the peptide side chains to have survived DNA synthesis conditions; we then incorporated nucleopeptide residues into longer oligonucleotides. A similar approach is described for the preparation of oligonucleotide histidines. Previously reported histidine-nucleopeptides serve as precursors to phosphoramidites and to phosphodiester DNA building blocks. Both solution- and solid-phase techniques are presented for the preparation of histidine-containing oligonucleotides. The methodology developed here allows the incorporation of nucleopeptide residues at internal positions in a DNA sequence, using standard reagents. We present a complete description of the synthesis, purification, and characterization (via mass spectral and NMR methods) of the novel compounds.
H uman Papillomavirus (HPV) is a small, closed-circular dsDNA virus that infects mucosal and cuta... more H uman Papillomavirus (HPV) is a small, closed-circular dsDNA virus that infects mucosal and cutaneous epithelial tissues. HPV infections remain a major health issue, as viral persistence with one of the 15 isolated oncogenic HPV types has been identified as a key factor in the development of cervical cancer and HPV16 is implicated in oral cancers. Despite the fact that type-specific prophylactic vaccines, as well as nonspecific cyto-destructive and topical immune-stimulant treatments are available, the latter two with limitations from toxicity and other side effects, there are no specific antiviral treatments against HPV. We have employed a rational design of hairpin pyrrole-imidazole Polyamides (PAs) to target the AT-rich genomes of high-risk (oncogenic) HPV16, 18 and 31. PAs are synthetic agents that recognize the minor-groove of dsDNA in a reportedly sequence-dependent manner and impart structural alterations on DNA. We have synthesized a library of PAs that potently decrease the viral load in monolayer keratinocytes and organotypic rafts without cytotoxicity. We report that N-terminal substitution with a Tetramethylguanidinium (TMG) group of closely related anti-HPV polyamides can lead to improvement in the IC50 and/or IC90 antiviral parameters against HPV16, 18 and 31. Using DNase I footprinting and affinity cleavage methods, we have determined the binding affinities and sequence specificities of several lead compounds to the long control regions of HPV16 and HPV18. Although the dissociation constants are similar and do not account for the differences in antiviral activities, we observe differences in the binding distribution between non-TMG and TMG-substituted polyamides along the DNA. Biography Carlos H Castaneda previously worked as a Formulation Scientist in the design and development of immediate and extended release formulations at Mallinckrodt Pharmaceuticals. In 2012, he started his Graduate studies and is a PhD student at the
Nucleosome core particles (NCP) make up the fundamental repeating structures that package and reg... more Nucleosome core particles (NCP) make up the fundamental repeating structures that package and regulate access to eukaryotic chromatin. High resolution crystal structures deliver detailed snapshots of the NCP in its most stable conformations, which consists of 147 base-pairs of DNA tightly wrapped around an octamer of histone proteins. However, the intermediate states of the NCP, as the DNA unwinds from the histone core, are biologically interesting and can be challenging to characterize. To this end, we applied small angle x-ray scattering (SAXS)-a powerful technique for resolving global properties of macromolecules in solution. In order to resolve the ambiguity that arises from the different scattering properties between proteins and nucleic acids within complexes, we applied a contrast variation approach to effectively probe the DNA component of the NCP during salt-induced disassembly. We measured the equilibrium response of NCPs in increasing concentrations of NaCl and modelled the DNA conformations as it unwraps from the NCP. These results highlight the strength of SAXS with contrast variation as a platform for studying protein-nucleic acid complexes.
PA1 and PA25 are large hairpin polyamides that are effective in nearly eliminating HPV16 episomes... more PA1 and PA25 are large hairpin polyamides that are effective in nearly eliminating HPV16 episomes (DNA) in cell culture, and PA25 has broad spectrum activity against three cancer-causing forms of HPV (Edwards, T.
We wish to develop reagents for the sequencespecific cleavage of RNA by non-oxidative mechanisms.... more We wish to develop reagents for the sequencespecific cleavage of RNA by non-oxidative mechanisms. The molecules we have devised are synthetic ribozymes, and comprise a molecular recognition agent (such as single-stranded
Journal of The Chemical Society-dalton Transactions, 1985
ABSTRACT Co-condensation of molybdenum atoms with trimethylphosphine gives octahedral [ Mo(PMe3)6... more ABSTRACT Co-condensation of molybdenum atoms with trimethylphosphine gives octahedral [ Mo(PMe3)6](1) whose crystal structure has been determined. Treatment of (1) with dinitrogen, ethylene, carbon monoxide, iodine, or butadiene gives [Mo(N2)(PMe3)5], trans-[Mo(η-C2H4)2(PMe3)4]fac-[Mo(CO)3(PMe3)3], trans-[MoI2(PMe3)4], and cis-[Mo(η-C4H6)2(PMe3)2] respectively. Protonation of cis-[Mo(η-C4H6)2(PMe3)2] with tetrafluoroboric acid forms the compound cis-[[graphic omitted]CH2CHCHCH2)(η-C4H6)(PMe3)2]BF4. The presence of the Mo–H–C bond is shown by low-temperature n.m.r. spectra, and variable-temperature n.m.r. shows that the agostic hydrogen can scramble between the four terminal carbons of the two C4 ligands. The rate constants and activation parameters for the hydrogen-scrambling process have been determined and a mechanism is proposed. Reaction of cis-[Mo(η-C4H6)2(PMe3)2] with trifluoroacetic acid gives [Mo(η-MeC3H4)(η-C4H6)(PMe3)2(O2CCF3)]. Treatment of [Mo(PMe3)6] with cyclopentadiene forms [MoH(η-C5H5)(PMe3)3] which reacts with aqueous tetrafluoroboric acid giving [MoH2(η-C5H5)(PMe3)3]BF4 and [Mo(η-C5H5)(PMe3)2O]BF4. Reaction of [MoH(η-C5H5)(PMe3)3] with methyl iodide gives [MoH(η-C5H5)(PMe3)2I2].
Human papillomavirus (HPV) causes cervical cancer and other hyperproliferative diseases. There cu... more Human papillomavirus (HPV) causes cervical cancer and other hyperproliferative diseases. There currently are no approved antiviral drugs for HPV that directly decrease viral DNA load and that have low toxicity. We report the potent anti-HPV activity of two N-methylpyrrole-imidazole polyamides of the hairpin type, polyamide 1 (PA1) and polyamide 25 (PA25). Both polyamides have potent anti-HPV activity against 3 different genotypes when tested on cells maintaining HPV episomes. The compounds were tested against HPV16 (in W12 cells), HPV18 (in Ker4-18 cells), and HPV31 (tested in HPV31 maintaining cells). From a library of polyamides designed to recognize AT-rich DNA sequences such as those in or near E1 or E2 binding sites of the HPV16 origin of replication (ori), 4 polyamides were identified that possessed apparent IC 50 s ≤ 150 nM with no evidence of cytotoxicity and we report two highly-active compounds here. Treatment of epithelia engineered in organotypic cultures with these compounds also causes a dose-dependent loss of HPV episomal DNA that correlates with accumulation of compounds in the nucleus. Bromodeoxyuridine (BrdU) incorporation demonstrates that DNA synthesis in organotypic cultures is suppressed upon compound treatment, correlating with a loss of HPV16 and HPV18 episomes. PA1 and PA25 are currently in preclinical development as an antiviral compound for treatment of HPV-related disease, including cervical dysplasia. PA1 and related polyamides offer promise as antiviral agents and the ability to regulate HPV episomal levels in cells for the study of HPV biology. We also report that anti-HPV16 activity for Distamycin A, a natural product related to PA1, is accompanied by significant cellular toxicity.
A highly reproducible quantitative PCR (Q-PCR) assay was used to study the stability of human pap... more A highly reproducible quantitative PCR (Q-PCR) assay was used to study the stability of human papillomavirus (HPV) in undifferentiated keratinocytes that maintain viral episomes. The term "stability" refers to the ability of episomes to persist with little copy number variation in cells. In investigating the mechanism of action of PA25, a previously published compound that destabilizes HPV episomes, aphidicolin was also found to markedly decrease episome levels, but via a different pathway from that of PA25. Since aphidicolin is known to activate DNA damage response (DDR) pathways, effects of inhibitors and small interfering RNAs (siRNAs) acting within DDR pathways were investigated. Inhibitors of Chk1 and siRNA directed against ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia Rad3-related (ATR) pathways significantly reduced viral episomes, suggesting that these pathways play a role in maintaining HPV episome stability. Inhibitors of Chk2 and DNA-PK had no effect on episome levels. Pharmacological inhibition of ATM proteins had no effect on episome levels, but ATM knockdown by siRNA significantly reduced episome levels, suggesting that ATM proteins are playing an important role in HPV episome stability that does not require kinase activity. These results outline two pathways that trigger episome loss from cells and suggest the existence of a littleunderstood mechanism that mediates viral DNA elimination. Together, our results also indicate that HPV episomes have a stability profile that is remarkably similar to that of fragile sites; these similarities are outlined and discussed. This close correspondence may influence the preference of HPV for integration into fragile sites.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
There is a long history for the bioorganic and biomedical use of N-methyl-pyrrole-derived polyami... more There is a long history for the bioorganic and biomedical use of N-methyl-pyrrole-derived polyamides (PAs) that are higher homologs of natural products such as distamycin A and netropsin. This work has been pursued by many groups, with the Dervan and Sugiyama groups responsible for many breakthroughs. We have studied PAs since about 1999, partly in industry and partly in academia. Early in this program, we reported methods to control cellular uptake of polyamides in cancer cell lines and other cells likely to have multidrug resistance efflux pumps induced. We went on to discover antiviral polyamides active against HPV31, where SAR showed that a minimum binding size of about 10 bp of DNA was necessary for activity. Subsequently we discovered polyamides active against two additional high-risk HPVs, HPV16 and 18, a subset of which showed broad spectrum activity against HPV16, 18 and 31. Aspects of our results presented here are incompatible with reported DNA recognition rules. For example, molecules with the same cognate DNA recognition properties varied from active to inactive against HPVs. We have since pursued the mechanism of action of antiviral polyamides, and polyamides in general, with collaborators at NanoVir, the University of Missouri-St. Louis, and Georgia State University. We describe dramatic consequences of β-alanine positioning even in relatively small, 8-ring polyamides; these results contrast sharply with prior reports. This paper was originally presented by JKB as a Keynote Lecture in the
We have previously prepared ribozyme mimics and chemical nucleases from modified DNA containing p... more We have previously prepared ribozyme mimics and chemical nucleases from modified DNA containing pendant bipyridine and terpyridine groups. The ability of these modified DNA probes to support RNase H cleavage of complementary RNA is described. DNA/RNA duplexes ...
Abstracts of Papers American Chemical Society, Nov 8, 1997
ABSTRACT The objective of this project is to enhance sequence-specific hydrolysis of RNA by riboz... more ABSTRACT The objective of this project is to enhance sequence-specific hydrolysis of RNA by ribozyme mimics. These mimics may act as catalytic antisense reagents, recognizing harmful RNA sequences and cleaving those sequences by transesterification. We are optimizing ribozyme mimics by delivering the cleavage agent to the active site more efficiently. This will be accomplished by the use of electrostatics and groove-specific delivery. The total synthesis of novel phosphoramidites for automated DNA synthesis will be described. These new building blocks will probe the viability of using electrostatic attraction and specific-groove delivery to intensify cleavage by ribozyme mimics. Combining Watson-Crick base pairing with electrostatics and groove control yields three-way mol. recognition for a potential therapeutic agent. [on SciFinder(R)]
A series of polyamides based on pyrrole-imidazole polyamides was designed to interfere with viral... more A series of polyamides based on pyrrole-imidazole polyamides was designed to interfere with viral protein-viral DNA interactions at the origin of replication of the high risk human papillomavirus types 16, 18 and 31. The specific sites targeted were the E1 and E2 protein binding sites on the viral genomes. The initial library showed hits in cell culture screens. Activity was assessed by qPCR determination of viral DNA 48 h after treatment in cell culture. Dose response curves were determined in all cases, with at least triplicate points. Active compounds were further elaborated and modified in a series of subsequent libraries, with initial optimization achieving many sub-100 nM IC50s and apparent IC50's as low as 36 nM. Since the three viral genomes are not degenerate for polyamide binding in the target regions, we were surprised to find that a significant number of compounds showed broad-spectrum anti-HPV activity against all three viral subtypes investigated (16, 18 and 31). Although the MWs are high, excellent cell culture activity is seen, in agreement with related studies. Two compounds are in preclinical development and have been scaled up to >6 g. More recently, we have discovered a new class of polyamides functionalized at the N-terminus which are active to IC50s of ca. 10 nM. Most recently, important clues to the mechanism of action have been discovered: HPV-infected cells treated with active compounds show activation of one branch of the DNA Damage Response Pathway, while uninfected cells show no such response.
Journal of Biomolecular Structure & Dynamics, 2013
The accuracy of the Content should not be relied upon and should be independently verified with p... more The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden.
PA1 (dIm-PyPybPyPyPy-g-PyPybPyPyPyPyb-Ta) is a large (14-ring) hairpin polyamide that was designe... more PA1 (dIm-PyPybPyPyPy-g-PyPybPyPyPyPyb-Ta) is a large (14-ring) hairpin polyamide that was designed to recognize the DNA sequence 5'-W 2 GW 7-3', where W is either A or T. As is common among the smaller 6-8-ring hairpin polyamides (PAs), it binds its target recognition sequence with low nM affinity. However, in addition to its large size, it is distinct from these more extensively characterized PAs in its high tolerance for mismatches and antiviral properties. In ongoing attempts to understand the basis for these distinctions, we conducted thermodynamics studies of PA1-DNA interactions. The temperature dependence of binding affinity was measured using TAMRA-labeled hairpin DNAs containing a single target sequence. PA1 binding to either an ATAT/TATA or an AAAA/TTTT pattern is consistently entropically driven. This is in contrast to the A/T pattern-dependent driving forces for DNA binding by netropsin, distamycin, and smaller hairpin polyamides. Analysis of the salt dependence of PA1-DNA binding reveals that within experimental error, there is no dependence on ionic strength, indicating that the polyelectrolyte effect does not contribute to PA1-DNA binding energetics. This is similar to that observed for smaller PAs. PA1-DNA recognition sequence binding stoichiometries were determined at both nM (fluorescence) and mM (circular dichroism) concentrations. With all sequences and under both conditions, multiple PA1 molecules bind the small DNA hairpin that contains only a single recognition sequence. Implications for these observations are discussed.
... Chimica Acta 263 (1997) 49-52 J Transesterification of RNA by Cu (II) terpyridine Lisa A, Jen... more ... Chimica Acta 263 (1997) 49-52 J Transesterification of RNA by Cu (II) terpyridine Lisa A, Jenkins Autry, James K. Bashkin ... RNA; Transestenfication; Hydrolysis; Ribozymos mimics; Copper(II) complexes; Terp~dine complexes 1. Introduction The transesterification of RNA (Fig. ...
Journal of the American Society for Mass Spectrometry, 2006
The aim of this work is to establish a quantitative method to determine the ratio of [U-13 C] lab... more The aim of this work is to establish a quantitative method to determine the ratio of [U-13 C] labeled to unlabeled hexose monophosphates isolated from yeast extracts. This is accomplished by anion exchange chromatography and mobile phase desalting followed by electrospray (ESI) mass spectrometry. We test the method with the analysis of a sample of biological origin. Previously developed analytical techniques are not adequate to accomplish mass spectrometric analysis of these and other small monosaccharide systems because of interference from salt clusters. By lowering the ionic strength of the mobile phase and using a simplified injection system to the mass spectrometer, we were able to obtain data on the relative abundance of the hexose monophosphates.
ABSTRACT The transport and reactivity of oligonucleotides may be altered by attaching pendant pep... more ABSTRACT The transport and reactivity of oligonucleotides may be altered by attaching pendant peptides, and it is of interest to develop general synthetic methods for such bioconjugates. Two protecting group strategies are described for the synthesis of nucleotide peptides containing a lysine residue. The preparation of a lysine-nucleopeptide phosphoramidite reagent is described, along with its use in solid-phase DNA synthesis. Di- and trinucleotides were prepared with pendant and extensively characterized by NMR. These studies showed the peptide side chains to have survived DNA synthesis conditions; we then incorporated nucleopeptide residues into longer oligonucleotides. A similar approach is described for the preparation of oligonucleotide histidines. Previously reported histidine-nucleopeptides serve as precursors to phosphoramidites and to phosphodiester DNA building blocks. Both solution- and solid-phase techniques are presented for the preparation of histidine-containing oligonucleotides. The methodology developed here allows the incorporation of nucleopeptide residues at internal positions in a DNA sequence, using standard reagents. We present a complete description of the synthesis, purification, and characterization (via mass spectral and NMR methods) of the novel compounds.
H uman Papillomavirus (HPV) is a small, closed-circular dsDNA virus that infects mucosal and cuta... more H uman Papillomavirus (HPV) is a small, closed-circular dsDNA virus that infects mucosal and cutaneous epithelial tissues. HPV infections remain a major health issue, as viral persistence with one of the 15 isolated oncogenic HPV types has been identified as a key factor in the development of cervical cancer and HPV16 is implicated in oral cancers. Despite the fact that type-specific prophylactic vaccines, as well as nonspecific cyto-destructive and topical immune-stimulant treatments are available, the latter two with limitations from toxicity and other side effects, there are no specific antiviral treatments against HPV. We have employed a rational design of hairpin pyrrole-imidazole Polyamides (PAs) to target the AT-rich genomes of high-risk (oncogenic) HPV16, 18 and 31. PAs are synthetic agents that recognize the minor-groove of dsDNA in a reportedly sequence-dependent manner and impart structural alterations on DNA. We have synthesized a library of PAs that potently decrease the viral load in monolayer keratinocytes and organotypic rafts without cytotoxicity. We report that N-terminal substitution with a Tetramethylguanidinium (TMG) group of closely related anti-HPV polyamides can lead to improvement in the IC50 and/or IC90 antiviral parameters against HPV16, 18 and 31. Using DNase I footprinting and affinity cleavage methods, we have determined the binding affinities and sequence specificities of several lead compounds to the long control regions of HPV16 and HPV18. Although the dissociation constants are similar and do not account for the differences in antiviral activities, we observe differences in the binding distribution between non-TMG and TMG-substituted polyamides along the DNA. Biography Carlos H Castaneda previously worked as a Formulation Scientist in the design and development of immediate and extended release formulations at Mallinckrodt Pharmaceuticals. In 2012, he started his Graduate studies and is a PhD student at the
Nucleosome core particles (NCP) make up the fundamental repeating structures that package and reg... more Nucleosome core particles (NCP) make up the fundamental repeating structures that package and regulate access to eukaryotic chromatin. High resolution crystal structures deliver detailed snapshots of the NCP in its most stable conformations, which consists of 147 base-pairs of DNA tightly wrapped around an octamer of histone proteins. However, the intermediate states of the NCP, as the DNA unwinds from the histone core, are biologically interesting and can be challenging to characterize. To this end, we applied small angle x-ray scattering (SAXS)-a powerful technique for resolving global properties of macromolecules in solution. In order to resolve the ambiguity that arises from the different scattering properties between proteins and nucleic acids within complexes, we applied a contrast variation approach to effectively probe the DNA component of the NCP during salt-induced disassembly. We measured the equilibrium response of NCPs in increasing concentrations of NaCl and modelled the DNA conformations as it unwraps from the NCP. These results highlight the strength of SAXS with contrast variation as a platform for studying protein-nucleic acid complexes.
PA1 and PA25 are large hairpin polyamides that are effective in nearly eliminating HPV16 episomes... more PA1 and PA25 are large hairpin polyamides that are effective in nearly eliminating HPV16 episomes (DNA) in cell culture, and PA25 has broad spectrum activity against three cancer-causing forms of HPV (Edwards, T.
Polyamides (PAs) are powerful DNA ligands that can bind the minor groove of DNA with high affinit... more Polyamides (PAs) are powerful DNA ligands that can bind the minor groove of DNA with high affinity and specificity. While the characterization of PA-DNA behavior has focused principally on hairpin PAs 6–8 rings in size, there is increasing evidence that their behavior does not necessarily reflect the complexities that are emerging from studies of larger hairpin PAs, particularly concerning sequence mismatch tolerance and observed but unaddressed high PA-target site binding stoichiometries. To explore these complexities in more detail, kinetics studies of binding a large anti-HPV hairpin polyamide to an isolated DNA recognition site are described. Using a fluorescence assay, two distinct binding phases are observed for the first time in hairpin PA literature. PA14 concentration dependence analysis indicates that the faster binding event is diffusion-controlled; the apparent, second event is significantly slower (350–1500 fold). Both association phases are sampled in 1:1 complexes, consistent with cooperative binding of two PA molecules even under this condition. Fitting of the slow phase to a biexponential model yields two λon,app that differ by 4-5-fold, which is consistent with the high mismatch tolerance and binding site stoichiometry previously observed. A/T patterns in the recognition sequence do not affect these decay constants significantly. Dissociation decay constants are among the slowest reported for hairpin PAs (10−3 s−1), independent of A/T pattern, and may point to the efficacy of PA14 as an antiviral.
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Papers by James Bashkin