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Vern L. Schramm - One of the best experts on this subject based on the ideXlab platform.
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genetic resistance to purine Nucleoside Phosphorylase inhibition in plasmodium falciparum
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Rodrigo G Ducati, Hilda A Namanjamagliano, Rajesh K Harijan, Eduardo J Fajardo, Andras Fiser, Johanna P Daily, Vern L. SchrammAbstract:Plasmodium falciparum causes the most lethal form of human malaria and is a global health concern. The parasite responds to antimalarial therapies by developing drug resistance. The continuous development of new antimalarials with novel mechanisms of action is a priority for drug combination therapies. The use of transition-state analog inhibitors to block essential steps in purine salvage has been proposed as a new antimalarial approach. Mutations that reduce transition-state analog binding are also expected to reduce the essential catalytic function of the target. We have previously reported that inhibition of host and P. falciparum purine Nucleoside Phosphorylase (PfPNP) by DADMe-Immucillin-G (DADMe-ImmG) causes purine starvation and parasite death in vitro and in primate infection models. P. falciparum cultured under incremental DADMe-ImmG drug pressure initially exhibited increased PfPNP gene copy number and protein expression. At increased drug pressure, additional PfPNP gene copies appeared with point mutations at catalytic site residues involved in drug binding. Mutant PfPNPs from resistant clones demonstrated reduced affinity for DADMe-ImmG, but also reduced catalytic efficiency. The catalytic defects were partially overcome by gene amplification in the region expressing PfPNP. Crystal structures of native and mutated PfPNPs demonstrate altered catalytic site contacts to DADMe-ImmG. Both point mutations and gene amplification are required to overcome purine starvation induced by DADMe-ImmG. Resistance developed slowly, over 136 generations (2136 clonal selection). Transition-state analog inhibitors against PfPNP are slow to induce resistance and may have promise in malaria therapy.
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catalytic site design for inverse heavy enzyme isotope effects in human purine Nucleoside Phosphorylase
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Rajesh K Harijan, Steven D Schwartz, Dimitri Antoniou, Ioanna Zoi, Vern L. SchrammAbstract:Heavy-enzyme isotope effects ( 15 N-, 13 C-, and 2 H-labeled protein) explore mass-dependent vibrational modes linked to catalysis. Transition path-sampling (TPS) calculations have predicted femtosecond dynamic coupling at the catalytic site of human purine Nucleoside Phosphorylase (PNP). Coupling is observed in heavy PNPs, where slowed barrier crossing caused a normal heavy-enzyme isotope effect ( k chem light / k chem heavy > 1.0). We used TPS to design mutant F159Y PNP, predicted to improve barrier crossing for heavy F159Y PNP, an attempt to generate a rare inverse heavy-enzyme isotope effect ( k chem light / k chem heavy
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immucillin h a purine Nucleoside Phosphorylase transition state analog causes non lethal attenuation of growth in staphylococcus aureus
Bioinformation, 2013Co-Authors: Christopher F Stratton, Vern L. SchrammAbstract:Purine Nucleoside Phosphorylase (PNP; EC: 2.4.2.1) is a key enzyme involved in the purine salvage pathway. A recent bioinformatic study by Yadav, P. K. et al. (Bioinformation 2012, 8(14), 664–672) reports PNP as an essential enzyme and potential drug target in community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). We conducted an analysis using the methodology outlined by the authors, but were unable to identify PNP as an essential gene product in CA-MRSA. In addition, the treatment of Staphylococcus aureus cultures with immucillin-H, a powerful inhibitor of PNP, resulted in the non-lethal attenuation of growth, suggesting that PNP activity is not essential for cell viability.
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mass modulation of protein dynamics associated with barrier crossing in purine Nucleoside Phosphorylase
Journal of Physical Chemistry Letters, 2012Co-Authors: Dimitri Antoniou, Vern L. Schramm, Steven D SchwartzAbstract:The role of protein dynamics on different time scales in enzyme catalysis remains an area of active debate. The connection between enzyme dynamics on the femtosecond time scale and transition state formation has been demonstrated in human purine Nucleoside Phosphorylase (PNP) through the study of a mass-altered enzyme. Isotopic substitution in human PNP (heavy PNP) decreased the rate of on-enzyme chemistry but did not alter either the transition state structure or steady-state kinetic parameters. Here we investigate the underlying atomic motions associated with altered barrier crossing probability for heavy PNP. Transition path sampling was employed to illuminate the molecular differences between barrier crossing in light and heavy enzymes. The mass effect is apparent in promoting vibrations that polarize the N-ribosidic bond, and that promote the stability of the purine leaving group. These motions facilitate barrier crossing.
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conformational dynamics in human purine Nucleoside Phosphorylase with reactants and transition state analogues
Journal of Physical Chemistry B, 2010Co-Authors: Jennifer S Hirschi, Karunesh Arora, Charles L Brooks, Vern L. SchrammAbstract:Dynamic motions of human purine Nucleoside Phosphorylase (hPNP) in complex with transition-state analogues and reactants were studied using 10 ns explicit solvent molecular dynamics simulations. hPNP is a homotrimer that catalyzes the phosphorolysis of purine 6-oxyNucleosides. The ternary complex of hPNP includes the binding of a ligand and phosphate to the active site. Molecular dynamics simulations were performed on the ternary complex of six ligands including the picomolar transition-state analogues, Immucillin-H (Kd = 56 pM), DADMe-Immucillin-H (Kd = 8.5 pM), DATMe-Immucillin-H (Kd = 8.6 pM), SerMe-Immucillin-H (Kd = 5.2 pM), the substrate inosine, and a complex containing only phosphate. Protein−inhibitor complexes of the late transition-state inhibitors, DADMe-Imm-H and DATMe-Imm-H, are inflexible. Despite the structural similarity of SerMe-Imm-H and DATMe-Imm-H, the protein complex of SerMe-Imm-H is flexible, and the inhibitor is highly mobile within the active sites. All inhibitors exhibit an incr...
Peter C Tyler - One of the best experts on this subject based on the ideXlab platform.
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conformational states of human purine Nucleoside Phosphorylase at rest at work and with transition state analogues
Biochemistry, 2010Co-Authors: Achelle A Edwards, Peter C Tyler, Gary B Evans, Jeremiah D Tipton, Michael Brenowitz, Mark R Emmett, Alan G Marshall, Vern L. SchrammAbstract:Human purine Nucleoside Phosphorylase (PNP) is a homotrimer binding tightly to the transition state analogues Immucillin-H (ImmH; Kd = 56 pM) and DATMe-ImmH-Immucillin-H (DATMe-ImmH; Kd = 8.6 pM). ImmH binds with a larger entropic penalty than DATMe-ImmH, a chemically more flexible inhibitor. The testable hypothesis is that PNP conformational states are more relaxed (dynamic) with DATMe-ImmH, despite tighter binding than with ImmH. PNP conformations are probed by peptide amide deuterium exchange (HDX) using liquid chromatography high-resolution Fourier transform ion cyclotron resonance mass spectrometry and by sedimentation rates. Catalytically equilibrating Michaelis complexes (PNP·PO4·inosine ↔ PNP·Hx·R-1-P) and inhibited complexes (PNP·PO4·DATMe-ImmH and PNP·PO4·ImmH) show protection from HDX at 9, 13, and 15 sites per subunit relative to resting PNP (PNP·PO4) in extended incubations. The PNP·PO4·ImmH complex is more compact (by sedimentation rate) than the other complexes. HDX kinetic analysis of liga...
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altered enthalpy entropy compensation in picomolar transition state analogues of human purine Nucleoside Phosphorylase
Biochemistry, 2009Co-Authors: Achelle A Edwards, Peter C Tyler, Gary B Evans, Keith Clinch, Jennifer M Mason, Vern L. SchrammAbstract:Human purine Nucleoside Phosphorylase (PNP) belongs to the trimeric class of PNPs and is essential for catabolism of deoxyguanosine. Genetic deficiency of PNP in humans causes a specific T-cell immune deficiency, and transition state analogue inhibitors of PNP are in development for treatment of T-cell cancers and autoimmune disorders. Four generations of Immucillins have been developed, each of which contains inhibitors binding with picomolar affinity to human PNP. Full inhibition of PNP occurs upon binding to the first of three subunits, and binding to subsequent sites occurs with negative cooperativity. In contrast, substrate analogue and product bind without cooperativity. Titrations of human PNP using isothermal calorimetry indicate that binding of a structurally rigid first-generation Immucillin (Kd = 56 pM) is driven by large negative enthalpy values (ΔH = −21.2 kcal/mol) with a substantial entropic (−TΔS) penalty. The tightest-binding inhibitors (Kd = 5−9 pM) have increased conformational flexibil...
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transition state interactions revealed in purine Nucleoside Phosphorylase by binding isotope effects
Journal of the American Chemical Society, 2008Co-Authors: Andrew S Murkin, Peter C Tyler, Vern L. SchrammAbstract:The binding of [5‘-3H]inosine to human purine Nucleoside Phosphorylase results in an equilibrium binding isotope effect (BIE) of 1.5%, and transition state formation causes an intrinsic KIE of 4.7%. These values reflect atomic vibrational distortions in the 5‘-C−H bonds upon formation of the Michaelis complex and transition state. The degree of atomic distortion for catalysis is compared to that for binding of transition state analogues. Similar radiolabeled forms of the transition-state analogues ImmH and DADMe-ImmH gave large 5‘-3H BIEs of 12.6% and 29.2%, respectively. Greater bond distortions occur upon complex formation with transition-state analogues, supporting weaker distortional forces at the transition state than in the formation of complexes with transition-state analogues.
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l enantiomers of transition state analogue inhibitors bound to human purine Nucleoside Phosphorylase
Journal of the American Chemical Society, 2008Co-Authors: Agnes Rinaldomatthis, Peter C Tyler, Steven C Almo, Richard H Furneaux, Gary B Evans, Andrew S Murkin, Udupi A Ramagopal, Keith Clinch, Simon P H Mee, Vern L. SchrammAbstract:Human purine Nucleoside Phosphorylase (PNP) was crystallized with transition-state analogue inhibitors Immucillin-H and DADMe-Immucillin-H synthesized with ribosyl mimics of l-stereochemistry. The inhibitors demonstrate that major driving forces for tight binding of these analogues are the leaving group interaction and the cationic mimicry of the transition state, even though large geometric changes occur with d-Immucillins and l-Immucillins bound to human PNP.
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inhibition and structure of trichomonas vaginalis purine Nucleoside Phosphorylase with picomolar transition state analogues
Biochemistry, 2007Co-Authors: Agnes Rinaldomatthis, Peter C Tyler, Richard H Furneaux, Gary B Evans, Corin Wing, Mahmoud Ghanem, Hua Deng, Peng Wu, Arti Gupta, Steven C AlmoAbstract:Trichomonas vaginalis is a parasitic protozoan purine auxotroph possessing a unique purine salvage pathway consisting of a bacterial type purine Nucleoside Phosphorylase (PNP) and a purine Nucleoside kinase. Thus, T. vaginalis PNP (TvPNP) functions in the reverse direction relative to the PNPs in other organisms. Immucillin-A (ImmA) and DADMe-Immucillin-A (DADMe-ImmA) are transition state mimics of adenosine with geometric and electrostatic features that resemble early and late transition states of adenosine at the transition state stabilized by TvPNP. ImmA demonstrates slow-onset tight-binding inhibition with TvPNP, to give an equilibrium dissociation constant of 87 pM, an inhibitor release half-time of 17.2 min, and a Km/Kd ratio of 70,100. DADMe-ImmA resembles a late ribooxacarbenium ion transition state for TvPNP to give a dissociation constant of 30 pM, an inhibitor release half-time of 64 min, and a Km/Kd ratio of 203,300. The tight binding of DADMe-ImmA supports a late SN1 transition state. Despite...
Steven E Ealick - One of the best experts on this subject based on the ideXlab platform.
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structural basis for substrate specificity of escherichia coli purine Nucleoside Phosphorylase
Journal of Biological Chemistry, 2003Co-Authors: Eric M Bennett, Paula W. Allan, William B. Parker, Steven E EalickAbstract:Purine Nucleoside Phosphorylase catalyzes reversible phosphorolysis of purine Nucleosides and 2′-deoxypurine Nucleosides to the free base and ribose (or 2′-deoxyribose) 1-phosphate. Whereas the human enzyme is specific for 6-oxopurine riboNucleosides, the Escherichia coli enzyme accepts additional substrates including 6-oxopurine riboNucleosides, 6-aminopurine riboNucleosides, and to a lesser extent purine arabinosides. These differences have been exploited in a potential suicide gene therapy treatment for solid tumors. In an effort to optimize this suicide gene therapy approach, we have determined the three-dimensional structure of the E. coli enzyme in complex with 10 Nucleoside analogs and correlated the structures with kinetic measurements and computer modeling. These studies explain the preference of the enzyme for ribose sugars, show increased flexibility for active site residues Asp204 and Arg24, and suggest that interactions involving the 1- and 6-positions of the purine and the 4′- and 5′-positions of the ribose provide the best opportunities to increase prodrug specificity and enzyme efficiency.
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the crystal structure of escherichia coli purine Nucleoside Phosphorylase a comparison with the human enzyme reveals a conserved topology
Structure, 1997Co-Authors: Chen Mao, Thomas A. Krenitsky, William J Cook, Min Zhou, George W Koszalka, Steven E EalickAbstract:Abstract Background: Purine Nucleoside Phosphorylase (PNP) from Escherichia coli is a hexameric enzyme that catalyzes the reversible phosphorolysis of 6-amino and 6-oxopurine (2′-deoxy)riboNucleosides to the free base and (2′-deoxy)ribose-1-phosphate. In contrast, human and bovine PNPs are trimeric and accept only 6-oxopurine Nucleosides as substrates. The difference in the specificities of these two enzymes has been utilized in gene therapy treatments in which certain prodrugs are cleaved by E. coli PNP but not the human enzyme. The trimeric and hexameric PNPs show no similarity in amino acid sequence, even though they catalyze the same basic chemical reaction. Structural comparison of the active sites of mammalian and E. coli PNPs would provide an improved basis for the design of potential prodrugs that are specific for E. coli PNP. Results: The crystal structure of E. coli PNP at 2.0 A resolution shows that the overall subunit topology and active-site location within the subunit are similar to those of the subunits from human PNP and E. coli uridine Phosphorylase. Nevertheless, even though the overall geometry of the E. coli PNP active site is similar to human PNP, the active-site residues and subunit interactions are strikingly different. In E. coli PNP, the purine- and ribose-binding sites are generally hydrophobic, although a histidine residue from an adjacent subunit probably forms a hydrogen bond with a hydroxyl group of the sugar. The phosphate-binding site probably consists of two mainchain nitrogen atoms and three arginine residues. In addition, the active site in hexameric PNP is much more accessible than in trimeric PNP. Conclusions: The structures of human and E. coli PNP define two possible classes of Nucleoside Phosphorylase, and help to explain the differences in specificity and efficiency between trimeric and hexameric PNPs. This structural data may be useful in designing prodrugs that can be activated by E. coli PNP but not the human enzyme.
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Purine Nucleoside Phosphorylase. 2. Catalytic mechanism.
Biochemistry, 1997Co-Authors: Mark D. Erion, Johanna D. Stoeckler, Wayne C. Guida, Richard L. Walter, Steven E EalickAbstract:X-ray crystallography, molecular modeling, and site-directed mutagenesis were used to delineate the catalytic mechanism of purine Nucleoside Phosphorylase (PNP). PNP catalyzes the reversible phosphorolysis of purine Nucleosides to the corresponding purine base and ribose 1-phosphate using a substrate-assisted catalytic mechanism. The proposed transition state (TS) features an oxocarbenium ion that is stabilized by the cosubstrate phosphate dianion which itself functions as part of a catalytic triad (Glu89-His86-PO4). Participation of phosphate in the TS accounts for the poor hydrolytic activity of PNP and is likely to be the mechanistic feature that differentiates Phosphorylases from glycosidases. The proposed PNP TS also entails a hydrogen bond between N7 and a highly conserved Asn. Hydrogen bond donation to N7 in the TS stabilizes the negative charge that accumulates on the purine ring during glycosidic bond cleavage. Kinetic studies using N7-modified analogs provided additional support for the hydrogen...
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purine Nucleoside Phosphorylase 1 structure function studies
Biochemistry, 1997Co-Authors: Mark D. Erion, Kenji Takabayashi, Harry B Smith, Janine Kessi, Sylvia Wagner, Sybille Honger, Spencer L Shames, Steven E EalickAbstract:To probe the catalytic mechanism of human purine Nucleoside Phosphorylase (PNP), 13 active-site mutants were constructed and characterized by steady-state kinetics. In addition, microtiter plate assays were developed for both the phosphorolytic and synthetic reactions and used to determine the kinetic parameters of each mutant. Mutations in the purine binding site exhibited the largest effects on enzymatic activity with the Asn243Ala mutant resulting in a 1000-fold decrease in the kcat for inosine phosphorolysis. This result in combination with the crystallographic location of the Asn243 side chain suggested a potential transition state (TS) structure involving hydrogen bond donation by the carboxamido group of Asn243 to N7 of the purine base. Analogous to the oxyanion hole of serine proteases, this hydrogen bond was predicted to aid catalysis by preferentially stabilizing the TS as a consequence of the increase in negative charge on N7 that occurs during glycosidic bond cleavage and the associated increa...
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structure based design of inhibitors of purine Nucleoside Phosphorylase 3 9 arylmethyl derivatives of 9 deazaguanine substituted on the methylene group
Journal of Medicinal Chemistry, 1993Co-Authors: Shri Niwas, Wayne C. Guida, Jerry D Rose, Subramaniam Ananthan, Mark D Allen, Secrist Ja Rd, Yarlagadda S Babu, Charles E Bugg, Steven E EalickAbstract:: X-ray crystallography and computer-assisted molecular modeling (CAMM) studies aided in the design of a potent series of mammalian purine Nucleoside Phosphorylase (PNP) inhibitors. Enhanced potency was achieved by designing substituted 9-(arylmethyl)-9-deazaguanine analogs that interact favorably with all three of the binding subsites of the PNP active site, namely the purine binding site, the hydrophobic pocket, and the phosphate binding site. The most potent PNP inhibitor prepared during our investigation, (S)-9-[1-(3-chlorophenyl)-2-carboxyethyl]-9-deazaguanine (18b), was shown to have an IC50 of 6 nM, whereas the corresponding (R)-isomer was 30-fold less potent.
Gary B Evans - One of the best experts on this subject based on the ideXlab platform.
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conformational states of human purine Nucleoside Phosphorylase at rest at work and with transition state analogues
Biochemistry, 2010Co-Authors: Achelle A Edwards, Peter C Tyler, Gary B Evans, Jeremiah D Tipton, Michael Brenowitz, Mark R Emmett, Alan G Marshall, Vern L. SchrammAbstract:Human purine Nucleoside Phosphorylase (PNP) is a homotrimer binding tightly to the transition state analogues Immucillin-H (ImmH; Kd = 56 pM) and DATMe-ImmH-Immucillin-H (DATMe-ImmH; Kd = 8.6 pM). ImmH binds with a larger entropic penalty than DATMe-ImmH, a chemically more flexible inhibitor. The testable hypothesis is that PNP conformational states are more relaxed (dynamic) with DATMe-ImmH, despite tighter binding than with ImmH. PNP conformations are probed by peptide amide deuterium exchange (HDX) using liquid chromatography high-resolution Fourier transform ion cyclotron resonance mass spectrometry and by sedimentation rates. Catalytically equilibrating Michaelis complexes (PNP·PO4·inosine ↔ PNP·Hx·R-1-P) and inhibited complexes (PNP·PO4·DATMe-ImmH and PNP·PO4·ImmH) show protection from HDX at 9, 13, and 15 sites per subunit relative to resting PNP (PNP·PO4) in extended incubations. The PNP·PO4·ImmH complex is more compact (by sedimentation rate) than the other complexes. HDX kinetic analysis of liga...
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altered enthalpy entropy compensation in picomolar transition state analogues of human purine Nucleoside Phosphorylase
Biochemistry, 2009Co-Authors: Achelle A Edwards, Peter C Tyler, Gary B Evans, Keith Clinch, Jennifer M Mason, Vern L. SchrammAbstract:Human purine Nucleoside Phosphorylase (PNP) belongs to the trimeric class of PNPs and is essential for catabolism of deoxyguanosine. Genetic deficiency of PNP in humans causes a specific T-cell immune deficiency, and transition state analogue inhibitors of PNP are in development for treatment of T-cell cancers and autoimmune disorders. Four generations of Immucillins have been developed, each of which contains inhibitors binding with picomolar affinity to human PNP. Full inhibition of PNP occurs upon binding to the first of three subunits, and binding to subsequent sites occurs with negative cooperativity. In contrast, substrate analogue and product bind without cooperativity. Titrations of human PNP using isothermal calorimetry indicate that binding of a structurally rigid first-generation Immucillin (Kd = 56 pM) is driven by large negative enthalpy values (ΔH = −21.2 kcal/mol) with a substantial entropic (−TΔS) penalty. The tightest-binding inhibitors (Kd = 5−9 pM) have increased conformational flexibil...
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l enantiomers of transition state analogue inhibitors bound to human purine Nucleoside Phosphorylase
Journal of the American Chemical Society, 2008Co-Authors: Agnes Rinaldomatthis, Peter C Tyler, Steven C Almo, Richard H Furneaux, Gary B Evans, Andrew S Murkin, Udupi A Ramagopal, Keith Clinch, Simon P H Mee, Vern L. SchrammAbstract:Human purine Nucleoside Phosphorylase (PNP) was crystallized with transition-state analogue inhibitors Immucillin-H and DADMe-Immucillin-H synthesized with ribosyl mimics of l-stereochemistry. The inhibitors demonstrate that major driving forces for tight binding of these analogues are the leaving group interaction and the cationic mimicry of the transition state, even though large geometric changes occur with d-Immucillins and l-Immucillins bound to human PNP.
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inhibition and structure of trichomonas vaginalis purine Nucleoside Phosphorylase with picomolar transition state analogues
Biochemistry, 2007Co-Authors: Agnes Rinaldomatthis, Peter C Tyler, Richard H Furneaux, Gary B Evans, Corin Wing, Mahmoud Ghanem, Hua Deng, Peng Wu, Arti Gupta, Steven C AlmoAbstract:Trichomonas vaginalis is a parasitic protozoan purine auxotroph possessing a unique purine salvage pathway consisting of a bacterial type purine Nucleoside Phosphorylase (PNP) and a purine Nucleoside kinase. Thus, T. vaginalis PNP (TvPNP) functions in the reverse direction relative to the PNPs in other organisms. Immucillin-A (ImmA) and DADMe-Immucillin-A (DADMe-ImmA) are transition state mimics of adenosine with geometric and electrostatic features that resemble early and late transition states of adenosine at the transition state stabilized by TvPNP. ImmA demonstrates slow-onset tight-binding inhibition with TvPNP, to give an equilibrium dissociation constant of 87 pM, an inhibitor release half-time of 17.2 min, and a Km/Kd ratio of 70,100. DADMe-ImmA resembles a late ribooxacarbenium ion transition state for TvPNP to give a dissociation constant of 30 pM, an inhibitor release half-time of 64 min, and a Km/Kd ratio of 203,300. The tight binding of DADMe-ImmA supports a late SN1 transition state. Despite...
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synthesis of a transition state analogue inhibitor of purine Nucleoside Phosphorylase via the mannich reaction
Organic Letters, 2003Co-Authors: Gary B Evans, Peter C Tyler, Richard H Furneaux, Vern L. SchrammAbstract:The expeditious convergent synthesis of the potent human purine Nucleoside Phosphorylase inhibitor DADMe-Immucillin-G (3) was achieved via the Mannich reaction. The Mannich chemistry of a series of deazapurines and amine hydrochlorides was also investigated.
Eric J Sorscher - One of the best experts on this subject based on the ideXlab platform.
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in vivo antitumor activity of intratumoral fludarabine phosphate in refractory tumors expressing e coli purine Nucleoside Phosphorylase
Cancer Chemotherapy and Pharmacology, 2012Co-Authors: Eric J Sorscher, William R. Waud, Paula W. Allan, Jeong S Hong, William B. ParkerAbstract:Purpose Systemically administered fludarabine phosphate (F-araAMP) slows growth of human tumor xenografts that express Escherichia coli purine Nucleoside Phosphorylase (PNP). However, this treatment has been limited by the amount of F-araAMP that can be administered in vivo. The current study was designed to (1) determine whether efficacy of this overall strategy could be improved by intratumoral administration of F-araAMP, (2) test enhancement of the approach with external beam radiation, and (3) optimize recombinant adenovirus as a means to augment PNP delivery and bystander killing in vivo.
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designer gene therapy using an escherichia coli purine Nucleoside Phosphorylase prodrug system
Chemistry & Biology, 2003Co-Authors: Eric M Bennett, William B. Parker, Paula W. Allan, John A. Secrist, Abdalla E A Hassan, Jeong S Hong, Ruchi Anand, Dana N Levasseur, David T Mcpherson, Eric J SorscherAbstract: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.
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antitumor activity of 2 fluoro 2 deoxyadenosine against tumors that express escherichia coli purine Nucleoside Phosphorylase
Cancer Gene Therapy, 2003Co-Authors: William B. Parker, Paula W. Allan, John A. Secrist, Abdalla E A Hassan, Eric J Sorscher, William R. WaudAbstract:Antitumor activity of 2-fluoro-2′-deoxyadenosine against tumors that express Escherichia coli purine Nucleoside Phosphorylase
