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Naegleria fowleri is a pathogenic free-living amoeba that is commonly found in warm freshwater and can cause a rapidly fulminant disease known as primary amoebic meningoencephalitis (PAM). New drugs are urgently needed to treat PAM, as the fatality rate is >97%. Until recently, few advances have been made in the discovery of new drugs for N. fowleri, and one drawback is the lack of validated tools and methods to enhance drug discovery and diagnostics research. In this study, we aimed to validate alternative methods to assess cell proliferation that are commonly used for other cell types and develop a novel drug screening assay to evaluate drug efficacy on N. fowleri replication. EdU (5-ethynyl-2′-deoxyuridine) is a pyrimidine analog of thymidine that can be used as a quantitative endpoint for cell proliferation. EdU incorporation is detected via a copper catalyzed click reaction with an Alexa Fluor-linked azide. EdU incorporation in replicating N. fowleri was validated using fluorescence microscopy, and quantitative methods for assessing EdU incorporation were developed by using an imaging flow cytometer. Currently used PAM therapeutics inhibited N. fowleri replication and EdU incorporation in vitro. EdA (7-deaza-2′-deoxy-7-ethynyladenosine), an adenine analog, also was incorporated by N. fowleri but was more cytotoxic than EdU. In summary, EdU incorporation could be used as a complimentary method for drug discovery for these neglected pathogens.

Naegleria fowleri is a pathogenic free-living amoeba that is commonly found in warm, freshwater and can cause a rapidly fulminant disease known as primary amoebic meningoencephalitis (PAM). New drugs are urgently needed to treat PAM, as the fatality rate is >97%. Until recently, few advances have been made in the discovery of new drugs for N. fowleri and one drawback is the lack of validated tools and methods to enhance drug discovery and diagnostics research. In this study we aimed to validate alternative methods to assess cell proliferation that are commonly used for other cell types and develop a novel drug screening assay to evaluate drug efficacy on N. fowleri replication. EdU (5-ethynyl-2′-deoxyuridine) is a pyrimidine analog of thymidine that can be used as a quantitative endpoint for cell proliferation. EdU incorporation is detected via a copper catalyzed click reaction with an Alexa Fluor linked azide. EdU incorporation in replicating N. fowleri was validated using fluorescence microscopy and quantitative methods for assessing EdU incorporation were developed by using an imaging flow cytometer. Currently used PAM therapeutics inhibited N. fowleri replication and EdU incorporation in vitro. EdA (5′ethynyl-2′-deoxyadenosine), an adenine analog, also was incorporated by N. fowleri, but was more cytotoxic than EdU. In summary, EdU incorporation could be used as a complimentary method for drug discovery for these neglected pathogens.

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

We used base-appended base modification to develop a new adenine analog, which incorporates an adenine derivative at position 7 of adenine. Using the systematic evolution of ligands by exponential enrichment method with a modified DNA library including this analog, we obtained Aad1, an aptamer that binds strongly to human β-defensin 2, a biomarker of physical stress found in saliva. The dissociation constant of Aad1 with respect to human β-defensin 2 was found to be low (6.8 nM), and was found to bind specifically to human β-defensin 2 in saliva spiked with the protein, as confirmed using pull-down with magnetic beads. To our knowledge, there are no prior reports of nucleic-acid aptamers that bind specifically to human β-defensin 2. However, our results indicated that such adenine analog-containing DNA libraries are extremely effective in the acquisition of high-affinity aptamers.

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

Toll-like receptor (TLR) 7/8 agonists have been applied in combination with chemo-, radio- or immunotherapy for lymphoma, and used as topical drugs for the treatment of viral skin lesions and skin tumors. In the present study, the role of an adenine analog, 9-(4-carboxyphenyl)-8-hydroxy-2-(2-methoxyethoxy)-adenine [termed Gao Dong (GD)], a novel TLR7 agonist, in the activation of cytokine-induced killer/natural killer (CIK/NK) cells was determined. The results of the present study indicated that GD was able to activate CIK/NK cells. The proportion of GD-induced CD3+CD56+ CIK and CD3−CD56+ NK cells was ~4% higher respectively compared with the control. Notably, combination therapy with CIK/NK cells stimulated by GD, markedly suppressed the proliferation of the chronic myelogenous leukemia K562 cell line. Following GD treatment, the cytotoxicity improved by ~25 and 21% when the effector/target ratio was 20:1 and 10:1, respectively. The results of the present study suggested a novel protocol for CIK/NK cell proliferation and revealed that GD may serve as a potent innate and adaptive immunomodulator in immunocyte culture.

Human adenoviruses (AdV) cause generally mild infections of the respiratory and GI tracts as well as some other tissues. However, AdV can cause serious infection in severely immunosuppressed individuals, especially pediatric patients undergoing allogeneic hematopoietic stem cell transplantation, where mortality rates are up to 80% with disseminated disease. Despite the seriousness of AdV disease, there are no drugs approved specifically to treat AdV infections. We report here that USC-087, an N-alkyl tyrosinamide phosphonate ester prodrug of HPMPA, the adenine analog of cidofovir, is highly effective against multiple AdV types in cell culture. USC-087 is also effective against AdV-C6 in our immunosuppressed permissive Syrian hamster model. In this model, hamsters are immunosuppressed by treatment with high dose cyclophosphamide. Injection of AdV-C6 (or AdV-C5) intravenously leads to a disseminated infection that resembles the disease seen in humans, including death. We have tested the efficacy of orally-administered USC-087 against the median lethal dose of intravenously administered AdV-C6. USC-087 completely prevented or significantly decreased mortality when administered up to 4 days post challenge. USC-087 also prevented or significantly decreased liver damage caused by AdV-C6 infection, and suppressed virus replication even when administered 4 days post challenge. These results imply that USC-087 is a promising candidate for drug development against HAdV infections.

Racemic syntheses of novel 5′-deoxy-3′,5′,5′-trifluoro-apiose nucleoside phosphonic acid analogs were carried out from 1,3-dihydroxyacetone, to be used as antiviral agents. Nucleophilic displacement of the triflate intermediate with diethyl (lithiodifluoromethyl)phosphonate was performed to yield the corresponding (α,α-difluoroalkyl) phosphonate. Condensation successfully proceeded from a glycosyl donor under Vorbruggen conditions to yield the nucleoside phosphonate analogs. Ammonolysis and hydrolysis of the phosphonates yielded the corresponding nucleoside phosphonic acid analogs. Furthermore, the lipophilic prodrug of the adenine derivative was synthesized to increase cellular uptake. The synthesized nucleoside analogs were screened for their antiviral activity against human immunodeficiency virus (HIV)-1. The bis(SATE) prodrug of the adenine analog exhibited significant in vitro activity against HIV-1.

The RNA Polymerase II C-terminal domain (CTD) kinase CDK12 has been implicated as a tumor suppressor and regulator of DNA damage response genes. Although much has been learned about CDK12 and its activity, due to the lack of a specific inhibitor and the complications posed by long term RNAi depletion, much is still unknown about the particulars of CDK12 function. Therefore gaining a better understanding of CDK12’s roles at the molecular level will be challenging without the development of additional tools. In order to address these issues we have used the CRISPR/Cas gene engineering system to create a mammalian cell line in which the only functional copy of CDK12 is selectively inhibitable by a cell-permeable adenine analog (analog-sensitive CDK12). Inhibition of CDK12 results in a perturbation of the phosphorylation patterns on the CTD and an arrest in cellular proliferation. This cell line should serve as a powerful tool for future studies.*

Efficient synthetic route to novel 4'-trifluoromethylated 5'-deoxycarbocyclic nucleoside phosphonic acids was described from α-trifluoromethyl-α,β-unsaturated ester. Coupling of purine nucleosidic bases with cyclopentanol using a Mitsunobu reaction gave the nucleoside intermediates which were further phosphonated and hydrolyzed to reach desired nucleoside analogs. Synthesized nucleoside analogs were tested for anti-HIV activity as well as cytotoxicity. Adenine analog 22 shows significant anti-HIV activity (EC50 = 8.3 μM) up to 100 μM.

Novel 5′-deoxy-5′,5′-difluoro-threose purine phosphonic acid analogs were designed and synthesized from 2-propanone-1,3-diacetate. Direct displacement of the triflate intermediate 12 with diethyl (lithiodifluoromethyl)phosphonate provided the corresponding (α,α-difluoroalkyl) phosphonate 13. Condensation successfully proceeded from a glycosyl donor 14 under Vorbruggen conditions to provide the nucleoside phosphonate analogs 15α, 15β, 18α, and 18β, respectively. Ammonolysis and hydrolysis of the phosphonates 15β and 18β yielded the nucleoside phosphonic acid analogs 16, 17, 19b, and 20. Also, synthesis of prodrugs of adenine derivative was performed to increase cellular uptake. The synthesized nucleoside analogs were subjected to antiviral screening against human immunodeficiency virus (HIV)-1. Bis(SATE) prodrug 22 of the adenine analog exhibited significant in vitro activity against HIV-1.

8-Vinyladenosine (8VA) is an adenine analog that effectively base pairs with thymine in duplex DNA. With a red-shifted absorption band centered around 290 nm excited 8VA emits Stokes-shifted fluorescence centered at 370 nm. The fluorescence quantum yield of 8VA* is sensitive to base stacking, making it an effective reporter of DNA structure and dynamics. We have performed cyclic voltammetry (CV) studies on 8VA in aprotic organic solvents to explore the mechanism of fluorescence quenching. Excited state oxidation and reduction potentials were calculated from the experimentally determined ground state one electron redox potentials. The Rehm–Weller equation was used to obtain the free energies of electron transfer for native nucleobase monophosphate oxidation and reduction paired with 8VA*. The magnitude and sign of the free energies have been used to assess whether electron transfer is thermodynamically plausible. The results suggest that electron transfer to or from 8VA* may not be the dominant mechanism of quenching for AMP, CMP, and perhaps GMP. In contrast, TMP may be an effective electron acceptor. Taken together, the results suggest that 8VA* quenching by nucleobase monophosphates in aqueous solution proceeds through a different mechanism than ET.

We tested pairwise combinations of classical base analog mutagens in Escherichia coli to study possible mutagen synergies. We examined the cytidine analogs zebularine (ZEB) and 5-azacytidine (5AZ), the adenine analog 2-aminopurine (2AP), and the uridine/thymidine analog 5-bromodeoxyuridine (5BrdU). We detected a striking synergy with the 2AP plus ZEB combination, resulting in hypermutability, a 35-fold increase in mutation frequency (to 53,000 × 10 −8 ) in the rpoB gene over that with either mutagen alone. A weak synergy was also detected with 2AP plus 5AZ and with 5BrdU plus ZEB. The pairing of 2AP and 5BrdU resulted in suppression, lowering the mutation frequency of 5BrdU alone by 6.5-fold. Sequencing the mutations from the 2AP plus ZEB combination showed the predominance of two new hot spots for A·T→G·C transitions that are not well represented in either single mutagen spectrum, and one of which is not found even in the spectrum of a mismatch repair-deficient strain. The strong synergy between 2AP and ZEB could be explained by changes in the dinucleoside triphosphate (dNTP) pools. IMPORTANCE Although mutagens have been widely studied, the mutagenic effects of combinations of mutagens have not been fully researched. Here, we show that certain pairwise combinations of base analog mutagens display synergy or suppression. In particular, the combination of 2-aminopurine and zebularine, analogs of adenine and cytidine, respectively, shows a 35-fold increased mutation frequency compared with that of either mutagen alone. Understanding the mechanism of synergy can lead to increased understanding of mutagenic processes. As combinations of base analogs are used in certain chemotherapy regimens, including those involving ZEB and 5AZ, these results indicate that testing the mutagenicity of all drug combinations is prudent.

3-Methyladenine and 7-methyladenine are biomarkers of DNA damage from exposure to methylating agents. For example, the concentration of 3-methyladenine increases significantly in the urine of cigarette smokers. Surface-enhanced Raman spectroscopy (SERS) has shown much potential for detection of biomolecules, including DNA. Much work has been dedicated to the canonical nucleobases, with comparatively fewer investigations of modified DNA and modified DNA nucleobases. Herein, Raman spectroscopy and SERS are used to examine the adsorption orientations of 3-methyladenine and 7-methyladenine on Ag nanoparticles. Density functional theory (DFT) calculations at the B3LYP level are used to support the conclusions via simulated spectra of the nucleobases and of Ag+/nucleobase complexes. The results herein show that 7-methyladenine adsorbs upright via its N3 and N9 atoms side, similarly to adenine. 3-Methyladenine adsorbs in a very tilted or flat orientation on the Ag nanoparticles. These findings will be useful for future SERS or other nanoparticle-based bioanalytical assays for detection of these methyladenines or other modified nucleobases.

Porcine reproductive and respiratory syndrome virus (PRRSV), a common viral pathogen, causes huge annual economic losses to the swine industry worldwide. After triggering by specific ligands, the Toll-like receptor 7 (TLR7), a type of pattern-recognition receptor (PRR), induces antiviral cytokines production. Previously, we synthesized an adenine analog, designated SZU101, a TLR7-specific ligand. In this study, we assessed the inhibitory effect of SZU101 on PRRSV infection in vitro. SZU101 significantly suppressed PRRSV infection in primary porcine alveolar macrophages (PAMs) in a dose-dependent manner. Moreover, SZU101-induced inhibition involved NF-κB pathway activation in PAMs to initiate expression of TLR7-mediated cytokines and induce expression of downstream signaling IFN-stimulated genes (ISGs). Chloroquine, a TLR7 inhibitor, and BAY 11-7082, an NF-κB inhibitor, reversed both the SZU101-induced antiviral effect and induction of cytokine genes and ISGs expression. Therefore, SZU101 antiviral effects depend at least in part on TLR7-NF-κB signaling pathway. Additionally, administration of SZU101 enhanced the humoral and cell-mediated immune responses against PRRSV antigens in mice. Given these results, SZU101 holds promise as an antiviral agent and a vaccine adjuvant to prevent PRRSV infection in pigs.

Genetic instability is determined not only by exogenous factors but also by the properties of DNA itself (1, 2). Such inherent mutagenicity was established through association of fragile X syndrome, spinal and bulbar muscular atrophy, and myotonic dystrophy with abnormally long CNG (N = A, T, G, or C) repeats, and subsequently ~ 30 inherited neurological diseases have been linked with expansion of tri-and tetranucleotide repeats beyond critical thresholds (3–6). Linkage between these inherited diseases and sequence length is substantiated by its correlation with phenotypic severity and progression. Furthermore, these mutations are dynamically transmitted, as repeat tracts progressively lengthen through succeeding generations. Beyond the primary information gleaned from DNA sequence and length, secondary structure is the deeper key to this class of genetic diseases, as self-folded conformations are favored by repeated sequences (7). Stem-loop hairpins are favored by CNG repeats and are implicated in repeat expansion via replication, repair, and recombination (5). To illustrate, hairpins that form on single-stranded Okazaki fragments can disrupt coordinated synthesis on the template strands, thereby extending the nascent strand on the leading template through repeated pausing and restarting of DNA polymerase (8). These self-folded moieties can also preferentially recognize and sequester proteins involved in DNA repair, thereby disrupting normal pathways that maintain the DNA integrity (9, 10). Thus, establishing the structures of these alternative forms of DNA is necessary to understand their potential broad-scale biological function. Within double-stranded DNA, repeated sequences self-associate to produce two distinct structures (11). Slipped forms occur when strands have identical lengths of complementary repeats, and the structures are distinguished from canonical duplex DNA by enhanced sensitivity to nucleases and anomalously fast electrophoretic mobility (12). Their diverse range of structures is stable to challenging environmental changes, thereby suggesting their biological viability. Slipped intermediates form when opposing strands have different lengths, and the resulting three-way junctions are implicated in DNA expansion during replication (13). Within these structures, repeat conformation depends on base sequence, with CTG repeats favoring self-associated hairpins while CAG repeats favor open, solvent-exposed loops, and this difference may originate in the lower thermodynamic stability of CAG vs CTG repeats (7). Our studies show that tract length also dictates conformation. Toward this goal, the adenine isomer 2-aminopurine is used to develop structural and energetic models of repeated CAG sequences, which are prevalent in many inherited neurological diseases (14). Fluorescent nucleobase analogs are powerful tools for assessing DNA structure and function, and 2-aminopurine is widely used for these purposes (15, 16). Single substitutions with this fluorescent adenine analog do not alter global conformations of CAG based structures, as demonstrated by electrophoresis, spectroscopic, and energetic studies. Structural motifs are identified from their characteristic fluorescence intensities and thermal stability by utilizing the strong effect of base stacking on the fluorescence quantum yield of 2-aminopurine (17, 18). The studies described in this paper are motivated by earlier studies of (CAG)8 (19, 20). The isolated oligonucleotide behaves as the expected stem-loop hairpin, as supported from intensities in the stem region that are comparable to a duplex DNA analog, and intensities in the central loop are comparable to or exceed those of a single-stranded DNA analog. However, when this same sequence is integrated into duplex DNA, the hairpin structure is lost to yield a repeat loop that is highly and uniformly solvent-exposed. Motivated by how DNA context determines conformation in repeated DNA, we sought to understand how tract length influences secondary structure in slipped intermediate forms of DNA. Utilizing dual structural and energetic perspectives offered by 2-aminopurine substitutions, the secondary structure adopted by (CAG)15 in a three-way junction was determined. Our major finding is that this longer 15 repeat sequence retains the inherent hairpin structure of the isolated oligonucleotide when it is incorporated into the three-way junction. This behavior sharply contrasts with the open loop adopted by the analogous (CAG)8 structure, and such length dependence suggests that repeat length dictates secondary structure and potential biological functions of long repeat tracts.

The fluorescent adenine analogue 2-aminopurine (2AP) has been used extensively to monitor conformational changes and macromolecular binding events involving nucleic acids because its fluorescence properties are highly sensitive to changes in chemical environment. Furthermore, site-specific incorporation of 2AP permits local DNA and RNA conformational events to be discriminated from the global structural changes monitored by UV/Vis spectroscopy and circular dichroism. However, while the steady-state fluorescence properties of 2AP have been well defined in diverse settings, interpretation of 2AP fluorescence lifetime parameters has been hampered by the heterogeneous nature of multi-exponential 2AP intensity decays observed across populations of microenvironments. To resolve this problem, we have tested the utility of multi-exponential versus continuous Lorentzian lifetime distribution models to describe fluorescence intensity decays from 2AP in diverse chemical backgrounds and within the context of RNA. Heterogeneity was introduced into 2AP intensity decays by mixing solvents of differing polarities, or by adding quenchers under high viscosity to evaluate the transient effect. Heterogeneity of 2AP fluorescence within the context of a synthetic RNA hairpin was introduced by structural remodeling using a magnesium salt. In each case, except folded RNA which required a bimodal distribution, 2AP lifetime properties were well described by single Lorentzian distribution functions, abrogating the need to introduce additional discrete lifetime subpopulations. Rather, heterogeneity in fluorescence decay processes was accommodated by the breadth of each distribution. This approach also permitted solvent relaxation effects on 2AP emission to be assessed by comparing lifetime distributions at multiple wavelengths. Together, these studies provide a new perspective for the interpretation of 2AP fluorescence lifetime properties, which will further the utility of this fluorophore in analyses of the complex and heterogeneous structural microenvironments associated with nucleic acids.

We report the crystal structure of the DNA adenine-N6 methyltransferase, M.TaqI, complexed with DNA, showing the fluorescent adenine analog, 2-aminopurine, flipped out of the DNA helix and occupying virtually the same position in the active site as the natural target adenine. Time-resolved fluorescence spectroscopy of the crystalline complex faithfully reports this state: base flipping is accompanied by the loss of the very short (similar to 50 ps) lifetime component associated with fully base-stacked 2-aminopurine in DNA, and 2-aminopurine is subject to considerable quenching by pi-stacking interactions with Tyr108 in the catalytic motif IV (NPPY). This proves 2-aminopurine to be an excellent probe for studying base flipping by M.TaqI and suggests similar quenching in the active sites of DNA and RNA adenine-N6 as well as DNA cytosine-N4 methyltransferases sharing the conserved motif IV. In solution, the same distinctive fluorescence response confirms complete destacking from DNA and is also observed when the proposed key residue for base flipping by M.TaqI, the target base partner thymine, is substituted by an abasic site analog. The corresponding cocrystal structure shows 2-aminopurine in the active site of M.TaqI, demonstrating that the partner thymine is not essential for base flipping. However, in this structure, a shift of the 3' neighbor of the target base into the vacancy left after base flipping is observed, apparently replicating a stabilizing role of the missing partner thymine. Time-resolved fluorescence and acrylamide quenching measurements of M.TaqI complexes in solution provide evidence for an alternative binding site for the extra-helical target base within M.TaqI and suggest that the partner thymine assists in delivering the target base into the active site.

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

2-Aminopurine (2-AP) is an adenine analog shown to cause cells to bypass chemical- and radiation-induced cell cycle arrest through as-yet unidentified mechanisms. 2-AP has also been shown to act as a kinase inhibitor. Tumor suppressor p53 plays an important role in the control of cell cycle and apoptosis in response to genotoxic stress. We were interested in examining the effect of 2-AP on p53 phosphorylation and its possible consequences on checkpoint control in cells subjected to various forms of DNA damage. Here, we show that 2-AP suppresses p53 phosphorylation in response to gamma radiation, adriamycin, or ultraviolet treatment. This is partly explained by the ability of the kinase inhibitor to inhibit ATM or ATR activities in vitro and impair ATM- or ATR-dependent p53 phosphorylation in vivo. However, 2-AP is also capable of inhibiting p53 phosphorylation in cells deficient in ATM, DNA-PK, or ATR suggesting the existence of multiple pathways by which this kinase inhibitor modulates p53 activation. Biologically, the 2-AP-mediated inhibition of p53 stabilization enables wild-type p53-containing cells to bypass adriamycin-induced G(2)/M arrest. In the long term, however, 2-AP facilitates cells to resist DNA damage-induced cell death independently of p53.

The adenine analog erythro-9-(2-hydroxy-3-nonyl)adenine, EHNA, a tight reversible inhibitor (KI = 1.6 x 10(-9) M) of adenosine deaminase (EC 3.5.4.4) (ADase), was modified into the fluorescent etheno derivative epsilon-EHNA. The latter is a competitive inhibitor of adenosine deaminase [KI = (2.80 +/- 0.01)10(-6) M], having the fluorescent properties of epsilon-adenines. Affinity to the active site, monitored by both steady-state and dynamic fluorescence polarization, was confirmed by competition experiments with 2'-deoxycoformycin, the substrate adenosine and EHNA. The epsilon-adenine moiety of epsilon-EHNA librates at the shallow active site of ADase. The low absorptivity of epsilon-EHNA required the measurement of fluorescence excitation spectra. Computer subtraction of fluorescence excitation spectrum of ADase from that of its equimolar complex with epsilon-EHNA revealed the corrected excitation spectrum of epsilon-EHNA at the active site of the enzyme. This spectrum mimics that of epsilon-EHNA at pH 5.5 in buffer solution, implying its protonation at the active site of the enzyme. These results are in agreement with the presence of acidic amino acids that are essential to the catalytic mechanism.

Conventional reactions of the new multidentate ligand 7-amine-1,2,4-triazolo[1,5-a]pyrimidine (7atp, 1) with copper(II) salts lead to four novel multidimensional coordination complexes [Cu(7atp)(mal)(H(2)O)(2)]·H(2)O (2), [Cu(2)(μ-7atp)(4)(H(2)O)(2)](ClO(4))(4)·3H(2)O (3), {[Cu(7atp)(2)(μ-ox)]·3H(2)O}(n) (4) and {[Cu(7atp)(2)(μ-suc)]·2H(2)O}(n) (5), where ox(2-), mal(2-) and suc(2-) mean oxalate, malonate and succinate, respectively. In these compounds, the 7atp ligand coordinates monodentately through its atom N3, except for compound 3, which displays N3-N4 coordination mode, giving rise to all to structures with diverse topologies and dimensionality. Compound 2 is a mononuclear entity, 3 consists of dinuclear species, 4 is a zig-zag chain with oxalate as a bridging ligand and 5 is a succinate-bridged mono-dimensional system. All polynuclear metal complexes show antiferromagnetic interactions of with J values ranging from -0.12 to -49.5 cm(-1). The ligand donor capabilities have been estimated by topological analyses of the electron density (QTAIM) and electron localization function (ELF), obtained by DFT calculations. The compounds are the first structurally characterized copper(II) complexes containing the 7atp ligand.

Adenosine analogs in which the sugar unit was replaced by a 1,4-dioxane sugar equivalent, were prepared by coupling the 1,4-dioxane sugar analog as its anomeric acetates, with N6-benzoyl protected adenine. The 1,4-dioxane system was obtained in an enantioselective synthesis from (R,R)-dimethyl tartrate. Using standard phosphorimidite methodology, the adenine analog was further reacted with a 1,4-dioxane uridine analog to give the corresponding, protected dinucleotide, set-up for further condensation into larger oligonucleotides. This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The structure of the title ionic compound, (C5H6N5)2[Cu(C2O4)2]·2H2O, consists of a centrosymmetric copper(II) oxalate dianion, two monoprotonated molecules of the adenine analog 7-amino-1,2,4-triazolo[1,5-a]pyrimidine (7atp) and two water molecules of crystallization. The CuII ion, located on an inversion center, exhibits a sligthly distorted square-planar coordination geometry, in which two oxalate anions bind in a bidentate fashion. The triazolopyrimidine ligand is protonated at the N atom in position 4, instead of its most basic N atom in position 3. This fact may be explained by the network stability, which is provided through the formation of a two-dimensional wave-like network parallel to (50\overline1) by N-H...O, O-H...N and O-H...O hydrogen bonds. These nets are further connected via C-H...O interactions., Financial support from the Junta de Andalucı´a (FQM-3705 and FQM-4228) and the Spanish Ministry of Education (FPU fellowship to ABC) is gratefully acknowledged.

2-Aminopurine (2AP) is a fluorescent adenine analog that probes mainly base stacking in nucleic acids. We labeled the loop or the stem of the RNA hairpin gacUACGguc with 2AP to study folding thermodynamics and kinetics at both loci. Thermal melts and fast laser temperature jumps detected by 2AP fluorescence monitored the stability and folding/unfolding kinetics. The observed thermodynamic and kinetic traces of the stem and loop mutants, though strikingly different at a first glance, can be fitted to the same free-energy landscape. The differences between the two probe locations arise because base stacking decreases upon unfolding in the stem, whereas it increases in the loop. We conclude that 2AP is a conservative adenine substitution for mapping out the contributions of different RNA structural elements to the overall folding process. Molecular dynamics (MD) totaling 0.6 μsec were performed to look at the conformations populated by the RNA at different temperatures. The combined experimental data, and MD simulations lead us to propose a minimal four-state free-energy landscape for the RNA hairpin. Analysis of this landscape shows that a sequential folding model is a good approximation for the full folding dynamics. The frayed state formed initially from the native state is a heterogeneous ensemble of structures whose stem is frayed either from the end or from the loop.

Summary Fluorescent reporter groups have served for many years as sensitive probes of macromolecular structure. Such probes can be especially useful in comparative studies such as detection of conformational changes and discrimination among structural models. Spectroscopic methods such as fluorescence are attractive because they are rapid, require small amounts of material, are nondestructive, can be carried out with commonly available equipment, and are relatively inexpensive. In addition, there is a rich library of theoretical and practical materials available to aid in data interpretation. The intrinsic fluorescence of most nucleic acids is too low to be useful in structural studies. Thus, it is necessary to incorporate a suitable reporter group to utilize fluorescence methods involving polynucleotide structure. A highly fluorescent adenine analog, 2aminopurine, has long served in this capacity. The present article describes our use of 2aminopurine as a probe of loop structures in quadruplex DNA. In particular, we show how knowledge of the relative intensity of 2-aminopurine emission as well as its sensitivity to exogenous quenching molecules such as acrylamide can aid in comparing crystal and solution structures of an oligonucleotide model of the human telomere and in discrimination among models containing tandem repeats of the telomeric quadruplex.

Vacuolar-type H(+)-ATPase was solubilized from tonoplasts of mung bean (Vigna radiata L.) and purified on a Mono Q anion-exchange column by fast protein liquid chromatography. The purified enzyme was inactivated by the reactive adenine analog, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). This inactivation was reversed by addition of dithiothreitol (DTT). Inactivation by NBD-Cl was prevented by Mg-ADP, a competitive inhibitor of ATPase. [(14)C]NBD-Cl predominantly modified the 68-kilodalton subunit and the degree of (14)C incorporation was decreased in the presence of Mg-ADP or upon subsequent addition of DTT. The loss of activity followed pseudo first-order kinetics with respect to NBD-Cl concentration, and double log plots of pseudo first-order rate constants versus reagent concentration yielded a straight line with a slope of 0.957. The NBD-modified/inactivated enzyme showed an absorbance maximum at 418 nanometers and a fluorescence emission peak at 515 nanometers. The absorption and fluorescence emission spectra of the NBD-modified enzyme were essentially the same as those of the model compound, N-acetyl-S-NBD cysteine. Absorbance by the modified enzyme at 418 nanometers disappeared upon addition of DTT, which coincided with the restoration of ATPase activity and the decrease in bound [(14)C]NBD-Cl. These findings show that NBD-Cl modifies an essential cysteine residue(s) at or near the catalytic site in the 68-kilodalton subunit of tonoplast H(+)-ATPase and that the modification closely correlates with the loss of ATPase activity.

Background The gene for methylthioadenosine phosphorylase (MTAP) lies on 9p21, close to the gene CDKN2A that encodes the tumor suppressor proteins p16 and p14ARF. MTAP and CDKN2A are homozygously co-deleted, with a frequency of 35 to 70%, in lung and pancreatic cancer, glioblastoma, osteosarcoma, soft-tissue sarcoma, mesothelioma, and T-cell acute lymphoblastic leukemia. In normal cells, but not in tumor cells lacking MTAP, MTAP cleaves the natural substrate, 5′-deoxy-5′-methylthioadenosine (MTA), to adenine and 5-methylthioribose-1-phosphate (MTR-1-P), which are then converted to adenine nucleotides and methionine. This distinct difference between normal MTAP-positive cells and tumor MTAP-negative cells led to several proposals for therapy. We offer a novel strategy in which both MTA and a toxic adenine analog, such as 2,6-diaminopurine (DAP), 6-methylpurine (MeP), or 2-fluoroadenine (F-Ade), are administered. In MTAP-positive cells, abundant adenine, generated from supplied MTA, competitively blocks the conversion of an analog, by adenine phosphoribosyltransferase (APRT), to its active nucleotide form. In MTAP-negative tumor cells, the supplied MTA cannot generate adenine; hence conversion of the analog is not blocked. Principal Findings We show that this combination treatment – adenine analog plus MTA – kills MTAP-negative A549 lung tumor cells, while MTAP-positive human fibroblasts (HF) are protected. In co-cultures of the breast tumor cell line, MCF-7, and HF cells, MCF-7 is inhibited or killed, while HF cells proliferate robustly. 5-fluorouracil (5-FU) and 6-thioguanine (6-TG) may also be used with our strategy. Though neither analog is activated by APRT, in MTAP-positive cells, adenine produced from supplied MTA blocks conversion of 5-FU and 6-TG to their toxic nucleotide forms by competing for 5-phosphoribosyl-1-pyrophosphate (PRPP). The combination of MTA with 5-FU or 6-TG, in the treatment of MTAP-negative tumors, may produce a significantly improved therapeutic index. Conclusion We describe a selective strategy to kill tumor cells lacking MTAP.

The new fluorinated adenine analog, fludarabine, has been tested for efficacy in many tumor types over the past ten years. Two other similar nucleoside analogs are currently available for commercial use. Cytarabine is used principally as an antileukemic agent, and vidarabine as an antiviral. Unlike vidarabine, fludarabine is resistant to deactivation by adenosine deaminase. Data from Phase I and II trials suggest that fludarabine is potentially effective in a number of leukemias, including acute lymphocytic leukemia, acute nonlymphocytic leukemia, and chronic lymphocytic leukemia (CLL). Unfortunately, the doses required to achieve adequate response in the acute leukemias (>75mg/m2) were above the maximum tolerated dose, resulting in intolerable granulocytopenia, thrombocytopenia, and a life-threatening neurotoxic syndrome. In CLL, however, the dose required to achieve a satisfactory response is well within tolerated limits. Long-term survival statistics are not yet available, but historical perspective strongly correlates response to other agents with increased survival times. Toxicities seen at dose regimens of 15–40 mg/m2/d for five consecutive days include somnolence, metabolic acidosis, confusion, fatigue, nausea, vomiting, increase in serum creatinine and aminotransferase concentrations, and pulmonary and hepatic abnormalities. Mild to severe hematologic toxicity has been observed at all dose levels.

Synthesis of the pyrazolo[3,4-d]pyrimidin-3-one congeners of guanosine, adenosine and inosine is described. Glycosylation of 3-methoxy-6-methylthio-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (13) with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose (16) in the presence of boron trifluoride etherate gave 3-methoxy-6-methylthio-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidin-4(5H)-one (17) which, after successive treatments with 3-chloroperoxybenzoic acid and methanolic ammonia, afforded 6-amino-3-methoxy-1-β-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-4(5H)one (18). The guanosine analog, 6-amino-1-β-D-ribofuranosylpyrazolo[3,4-d]pyrimidine-3,4(2H,5H)-dione (21), was made by sodium iodide-chlorotrimethylsilane treatment of 6-amino-3-methoxy-1-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidin-4(5H)one (19), followed by sugar deprotection. Treatment of the adenine analog, 4-amino-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one (11), according to the high temperature glycosylation procedure yielded a mixture of N-1 and N-2 ribosyl-attached isomers. Deprotection of the individual isomers afforded 4-amino-3-hydroxy-1-βribofuranosylpyrazolo-[3,4-d]pyrimidine (26) and 4-amino-2-β-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-3(7H)-one (27). The structures of 26 and 27 were established by single crystal X-ray diffraction analysis. The inosine analog, 1-β-D-ribofuranosylpyrazolo[3,4-d]pyrimidine-3,4(2H,5H)-dione (28), was synthesized enzymatically by direct ribosylation of 1H-pyrazolo[3,4-d]pyrimidine-3,4(2H,5H)-dione (8) with ribose-1-phosphate in the presence of purine nucleoside phosphorylase, and also by deamination of 26 with adenosine deaminase.

The nucleocapsid protein (NC) plays an important role in HIV-1, mainly through interactions with the genomic RNA and its DNA copies. Though the structures of several complexes of NC with oligonucleotides (ODNs) are known, detailed information on the ODN dynamics in the complexes is missing. To address this, we investigated the steady state and time-resolved fluorescence properties of 2-aminopurine (2Ap), a fluorescent adenine analog introduced at positions 2 and 5 of AACGCC and AATGCC sequences. In the absence of NC, 2Ap fluorescence was strongly quenched in the flexible ODNs, mainly through picosecond to nanosecond dynamic quenching by its neighboring bases. NC strongly restricted the ODN flexibility and 2Ap local mobility, impeding the collisions of 2Ap with its neighbors and thus, reducing its dynamic quenching. Phe(16)-->Ala and Trp(37)-->Leu mutations largely decreased the ability of NC to affect the local dynamics of 2Ap at positions 2 and 5, respectively, while a fingerless NC was totally ineffective. The restriction of 2Ap local mobility was thus associated with the NC hydrophobic platform at the top of the folded fingers. Since this platform supports the NC chaperone properties, the restriction of the local mobility of the bases is likely a mechanistic component of these properties.

DNA Photolyase is a flavoprotein that uses light to repair cyclobutylpyrimidine dimers in DNA. From considerations of the crystal structure of the protein, it has been hypothesized that the dimer lesion is flipped out of the DNA double helix into the substrate binding pocket. We have used a fluorescent adenine analog, 2-aminopurine (2-Ap), as a probe of local double helical structure upon binding of the substrate to the protein. Our results show that the local structure around the thymidine lesion changes dramatically upon binding to Photolyase. This is consistent with base flipping of the lesion into the protein binding cavity with concomitant destacking of the opposing complementary 2-Ap nucleotide.

We have used B6C3F1 mice heterozygous at Aprt (adenine phosphoribosyltransferase) as a model to study in vivo loss of heterozygosity (LOH) in normal splenic T-lymphocytes. APRT-deficient T-cells were selected in medium containing 50 μg/ml 2,6-diaminopurine (DAP), an adenine analog that is toxic only to cells with APRT enzyme activity. DAP-resistant (DAPr) T-cell variants were recovered at an average frequency of 3 × 10−5 from 21 B6C3F1 Aprt+/− mice. Allele-specific PCR of Aprt showed that about 70% of 122 DAPr colonies were caused by loss of the nontargeted Aprt allele (Aprt+). Analysis of microsatellite markers along the length of chromosome 8 suggested that mitotic recombination, or chromosome loss, with or without duplication of the remaining chromosome are the predominant mechanisms resulting in loss of Aprt+. DNA sequencing of Aprt RT-PCR products from the DAPr variants that retained Aprt+ indicated that point mutation as well as other mechanisms could cause this second class of variants. The high spontaneous frequency of in vivo Aprt LOH in mouse T-cells, mediated by LOH mechanisms that are also known to produce human cancers, suggests that the Aprt heterozygous mouse is a valid model for studying the diversity of mechanisms for in vivo somatic mutagenesis. Environ. Mol. Mutagen. 35:150–157, 2000 © 2000 Wiley-Liss, Inc.

Mice heterozygous at Aprt (adenine phosphoribosyltransferase) were used as a model to study in vivo loss of heterozygosity (LOH) in normal fibroblasts. Somatic cell variants that exhibited functional loss of the wild-type Aprt in vivo were recovered as APRT-deficient cell colonies after culturing in selection medium containing 2,6-diaminopurine (DAP), an adenine analog that is toxic only to cells with APRT enzyme activity. DAP-resistant (DAP r ) fibroblast variants were recovered at a median frequency of 12 × 10 −5 from individual ears from progeny of crosses between mouse strains 129/Sv and C3H/HeJ. The frequency of DAP r variants varied greatly among individual ears, suggesting that they preexisted in vivo and arose at various times during development . Polymorphic molecular markers and a cytological marker on the centromere of chromosome 8 made it possible to discriminate between each of six possible mechanistic pathways of LOH. The majority (about 80%) of the DAP r variants were a consequence of mitotic recombination. The prevalence of mitotic recombination in regions proximal to Aprt did not correlate with meiotic map distances. In particular, there was a higher than expected frequency of crossovers within the interval 59 cM to 67 cM. The high spontaneous frequency of Aprt LOH, mediated primarily by mitotic recombination, is fully consistent with our previous results with human peripheral T cells from individuals known to be heterozygous at APRT . Thus, this Aprt heterozygote mouse is a valid model for studying somatic mutagenesis and mitotic recombination in vivo .

Modification of the (Ca2+ + Mg2+)-ATPase protein of rabbit skeletal sarcoplasmic reticulum (SR) with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, NBD-Cl, at 4 degrees C for 5 min caused a 63% loss of the Ca(2+)-dependent ATPase activity when 1 mol of the adenine analog was incorporated per 10(5) g of protein. At 25 degrees C, above the lipid phase transition, the extent of labeling was 3-fold higher although the Ca(2+)-ATPase activity was inhibited to the same extent. MgATP protected the ATPase activity at 4 degrees C and 25 degrees C but there was little change in the extent of labeling at 4 degrees C suggesting that changes in the fluidity of the lipid moiety made different sites on the ATPase protein accessible to the reagent. At 4 degrees C, addition of sodium deoxycholate enhanced the inactivation (6% ATPase activity remained) but the labeling of the SR-ATPase protein did not increase significantly. Incubation with MgATP prior to solubilization with deoxycholate resulted in the protection of the Ca(2+)-ATPase activity and only a small decrease in the labeling occurred. At 25 degrees C, a similar pattern was found with deoxycholate but the loss of ATPase activity was less dramatic and the extent of labeling by NBD-Cl was greater than that at 4 degrees C. MgATP induced changes in the conformation of the ATPase protein protecting essential cysteine residues while shifting the reaction of NBD-Cl with the ATPase protein to non-essential sites in the absence or presence of deoxycholate. An analysis of tryptic digests of the NBD-ATPase protein showed that MgATP shifted the labeling from the A2 subfragment to the A1 subfragment in the absence of deoxycholate and from the A1 subfragment to the A2 subfragment in the presence of deoxycholate. The reagent, NBD-Cl, can distinguish between different temperature dependent conformational states of the ATPase protein.

A continuous fluorescence-based assay is described for measuring helicase-mediated unwinding of duplex DNA. The assay utilizes an oligonucleotide substrate containing the fluorescent adenine analog, 2-aminopurine, at regular intervals. 2-Aminopurine forms a Watson-Crick-type base pair with thymine and does not distort normal B-form DNA. Fluorescence of the 2-aminopurines within this oligonucleotide is quenched 2-fold upon its hybridization to a complementary strand. Unwinding of this substrate by the T4 dda helicase restores the fluorescence of the 2-aminopurines and is easily followed using stopped-flow or steady-state fluorescence spectroscopy. The flourescence-based assay provides rate data comparable to that obtained from conventional discontinuous assays using labeled substrates and additionally furnishes a means for following a single turnover. This assay should prove useful for defining the mechanism by which helicases unwind duplex DNA.

A single dose of 4-aminopyrazolo(3,4-d) pyrimidine (4APP), an adenine analog, was orally administered at various concentrations of groups of mice and after 24 h, the plasma-alpha-amylase activity and cholesterol levels had significantly decreased in a dose-dependent manner. There was no change, however, in pancreatic superoxide dismutase activity or the lipid peroxide level. Histologically, zymogen degranulation was found after a high oral dose of 4APP (175 mg/kg). The same decrease in alpha-amylase activity occurred in mice that had received ip administration of 4APP, and in mice that were pretreated with dimethyl sulfoxide. Based on these results, it appears that 4APP decreases plasma alpha-amylase activity dose-dependently, possibly by pancreatic injury, and that free radicals play no part in the observed changes.

Colony formation by variant Chinese hamster cells highly resistant to adenine analogs and deficient in adenine phosphoribosyltransferase (APRT) activity was measured after co-cultivation with APRT + , CHO-K1 cells in medium containing one of three different adenine analogs. Depending upon the density of APRT + cells and the specific adenine analog, large differences in the recovery of APRT − colonies were observed. The particular adenine analog and APRT + cell density were more significant factors in the recovery of APRT − colonies than the concentration of the analog or the level of APRT activity. The number of wild-type cells (CHO-K1) required to inhibit formation of APRT − colonies by 50% (mean lethal density; MLD 50 ) with 65 μg/ml 8-aza-adenine (AzA) as the selective drug was 8.0 × 10 5 cells/100 mm dish (1.5 × 10 4 /cm 2 ). With 100 μg/ml 2,6-diaminopurine (DAP) the MLD 50 for CHO-K1 was 4.0 × 10 5 cells/100 mm dish (7.3 × 10 3 /cm 2 ). The MLD 50 for CHO-K1 when the DAP concentration was decreased to 50 μg/ml was only slightly higher, 5 × 10 5 cells/100 mm dish (9.1 × 10 3 /cm 2 ). The most toxic effect was observed with 2-fluoroadenine (FA). The MLD 50 for CHO-K1 in 2 μg/ml FA was 4.5 × 10 4 cells/100 mm dish (8.2 × 10 2 /cm 2 ), a cell density which permits minimal direct contact between APRT + and APRT − cells. The toxic effects of FA on individually resistant, APRT − cells were found to be mediated by metabolites released into the medium by dying APRT + cells. This metabolite toxicity to APRT − cells was also demonstrated in mixtures with cells having only 8% of wild-type APRT activity. The MLD 50 for these APRT + (8%) cells in 2 μg/ml FA was 7.5 × 10 4 cells/100 dish (1.4 × 10 3 /cm 2 ), a small difference from the MLD 50 for cells with wild-type levels of APRT activity. The differences in the recovery of APRT − colonies from mixtures with APRT + cells in these three adenine analogs are critical to the design of procedures for the selection of APRT − cells from populations of APRT + cells and emphasize the importance of establishing the parameters of metabolic cooperation, not only in terms of cell density but also with regard to the particular selective agent, in any experiment designed to determine precise mutation rates or to test putative mutagens upon mammalian cells in culture.

The reduction of plasma testosterone (T) in male rats treated with 4-aminopyrazolo(3,4-d)pyrimidine (4-APP) has been recently shown to be associated with a fall in plasma LH, and appears to be secondary to this impairment of gonadotropin secretion rather than to the decrease in lipoprotein cholesterol caused by the adenine analog. The effects of 4-APP on pituitary function and the mechanism of the fall in gonadotropin secretion were analyzed in adult male rats treated ip with 5, 10, 25, and 50 mg 4-APP/kg BW for 3 days. Twenty four hours later, serum cholesterol levels were reduced by 68 +/- 5% (mean +/- SE) in animals treated with 5 mg 4-APP/kg BW and by up to 84 +/- 1% after higher doses. Basal levels of serum T were not affected by 5 mg/kg APP but were significantly reduced by higher doses (P less than 0.05). Serum T responses to human CG (100 IU) stimulation in APP-treated rats were reduced in absolute magnitude, but were enhanced in terms of percentage increase above baseline. The three highest doses of 4-APP reduced serum levels of LH (51-79%) and FSH (21-39%). However, serum gonadotropin responses to GnRH stimulation were augmented by 32 +/- 9% for LH and 27 +/- 12% for FSH after 4-APP treatment. Serum levels of TSH were significantly (P less than 0.05) reduced by 25 and 50 mg/kg 4-APP and serum PRL was lowered by only the higher dose. Serum GH was lowered and ACTH increased by 5 mg/kg 4-APP. The pituitary contents of LH, FSH, and TSH were unchanged after 4-APP treatment, but PRL and GH contents were slightly but significantly increased (P less than 0.05). Pituitary receptors for GnRH (88 +/- 14 fmol/mg protein) were reduced by in vivo treatment with 25 and 50 mg/kg 4-APP to 52 +/- 5 and 57 +/- 7 fmol/mg, but not by 10 mg/kg 4-APP. No change in pituitary GnRH receptor content was observed after 48-h exposure to 10(-7) or to 10(-5) M 4-APP in vitro. The medial hypothalamic GnRH content was unchanged by 4-APP treatment. The dissociated effects of 4-APP on serum cholesterol and T levels, and the maintenance of T responses to human CG, provide further evidence that testicular androgen biosynthesis is not solely dependent on circulating cholesterol. The greater sensitivity of pituitary gonadotropins to 4-APP inhibition is evidence for a selective action of this drug on pituitary hormone release in vivo. Although the hypothalamic content of GnRH was unaltered by 4-APP treatment, the decrease in pituitary GnRH receptors observed in vivo, together with the fall in circulating LH and FSH and retention of gonadotropin responses to GnRH stimulation, suggest that the primary effect of 4-APP is to inhibit GnRH synthesis and/or release from the hypothalamus.

The reaction of yeast tRNAPhe with methyl chlorotetrolate, ClCH2-C≡C-COOCH3, was studied. This reagent converts adenine and cytosine rings into derivatives in which an additional heterocycle bearing the alkylating chloromethyl group is fused to the original base; these derivatives can exist in two isomeric forms. Modified nucleosides of this type can be easily identified by reverse-phase HPLC. It was found that under native conditions, the modification of tRNA involves the anticodon loop and the 3′-end. The isomers of adenine derivatives formed in the anticodon loop were different from those formed in the 3′-end. It is suggested that the isomeric structure of the derivatives is related to the fine conformational differences between these two regions of tRNAPhe. Methyl chlorotetrolate could thus be used as a conformational probe of single-stranded nucleic acids. Preliminary assays showed that modified tRNAPhe binds irreversibly to yeast phenylalanyl-tRNA synthetase.

An adenine analog 8-[m-(m-fluorosulfonylbenzamido)benzylthio]adenine (FSB-adenine) reacts covalently with sheep heart phosphofructokinase. Under conditions optimal for allosteric kinetics the modified enzyme is less sensitive to inhibition by ATP and insensitive to activation by AMP, cyclic AMP, and ADP. The concentration of fructose-6-P necessary for half-maximal activity is markedly decreased, while the cooperativity to the same substrate is not changed under the same conditions. The modified enzyme is more stable at pH 6.5 when compared with the native enzyme. Changes in the allosteric kinetics of the enzyme are proportional to the extent of modification reaching maximal effect when 3.2 mol of the reagent were bound/mol of tetrameric enzyme. Affinity labeling of the enzyme by the adenine derivative does not affect significantly the catalytic site. This is evidenced by the demonstration that under assay conditions optimal for Michaelian kinetics neither the Km for ATP nor for fructose-6-P is significantly changed following chemical modification. Maximal activity of the modified enzyme was 60% of the native enzyme. ADP gives the best protection, while AMP gives less protection against modification by the reagent. ATP slows the rate of the reaction and causes a slight decrease in maximum binding of the reagent to the enzyme. Modification of the enzyme caused a marked reduction of AMP and ADP binding. The evidence indicates that the modified site is a nucleotide mono- and diphosphate activation site.

tRNAPhe in which the adenine and cytosine rings in the aminoacyl arm and in the anticodon loop were converted to alkylating derivatives by mild treatment with methyl chlorotetrolate was used to study the tRNAPhe-yeast phenylalanyl-tRNAPhe synthetase interaction. At neutral pH, modified tRNA inhibited the enzyme competitively. At pH 9 this binding is accompanied by irreversible inactivation of the enzyme due to alkylation of the α subunit of the synthetase. Such a derivatization of tRNA could probably be used to investigate the interaction of other tRNAs with their cognate synthetases.

With L-15 as the base medium, drug-resistant variants were isolated from two amphibian tissue culture strains: the Xenopus laevis A8 diploid cell line and the ICR 2A cell line of Rana pipiens. Four different classes of variants were obtained: (1) A8 cells resistant to chloramphenicol, an inhibitor of mitochondrial protein synthesis; (2) A8 cells resistant to ouabain, an inhibitor of the Na+/K+-activated ATPase of the plasma membrane; (3) ICR 2A cells resistant to low (20 μg/ml) and high (300 μg/ml) levels of bromodeoxyuridine (BUdR), a thymidine analog which interferes with the pyrimidine salvage pathway; and (4) ICR 2A cells resistant to 2,6-diaminopurine (DAP), an adenine analog which interferes with the purine salvage pathway. Unlike the other variants, isolation of BUdR resistant cells is a 2-step process. Resistance to low levels of BUdR is phenotypically expressed by a reduction in thymidine transport activities while resistance to high levels of this compound is evidenced by greatly reduced levels of thymidine kinase activity. DAP-resistant cells, which are characterized by reduced levels of adenine phosphoribosyl transferase (APRT) activity, do not die in AAT (adenine, aminopterin, thymidine) selection medium. This suggests that these cells utilize adenine efficiently. With MEM as the base medium, an asparagine independent clone was isolated from the ICR 2A cell line. When compared with the wild type, this variant exhibited a slightly reduced growth rate in the presence or absence of asparagine.

Among three unrelated patients with recurrent 2,8-dihydroxyadenine urolithiasis, two completely lacked adenine phosphoribosyltransferase (APRT) in both erythrocytes and proliferative T cells. The third patient possessed significant enzyme activities in both hemolysates and T-cell extracts at levels comparable to heterozygotes for complete APRT deficiency. Despite significant APRT activites in cell extracts, cultured T cells from the third patient were at least 100-fold more resistant than normal T cells to an adenine analog, 6-methylpurine, whose cytotoxicity is dependent on APRT. These data indicate that APRT activity in T cells from the third patient is positive in cell extracts, but apparently not operating in viable cells. Although the cells from the patients with complete APRT deficiency were as resistant to 6-methylpurine as the cells from the third patient, the cells from the heterozygotes for complete APRT deficiency were almost as sensitive as normal T cells. Therefore, adenine metabolism in the third patient but not in the heterozygotes seems to be as severely impaired as in the patients with complete APRT deficiency, which is quite consistent with the clinical manifestations in these individuals.

DNA polymerase I* is a form of the DNA polymerase I isolated from Escherichia coli which are expressing recA/lexA (SOS) functions. Induction of recA or polA1 cells by nalidixic acid does not result in the appearance of pol I*, but lexA or recA mutants that are constitutive for SOS functions constitutively express pol I* and mutants which lack functional recA protein produce pol I* when they carry a lexA mutation which renders the lexA repressor inoperative. Pol I* has been induced by nalidixic acid in dinA, dinD, dinF, and umuC mutants. Polymerase I* has a lower affinity for single-stranded DNA-agarose than polymerase I and it sediments through sucrose gradients in a dispersed manner between 6.6-10.5 S, whereas polymerase I sediments at 5 S. Whereas pol I* migrates significantly faster than pol I in nondenaturing polyacrylamide gels, the active polypeptide of both forms migrates at the same rate in denaturing polyacrylamide gels. Compared with polymerase I, polymerase I* has an enhanced capacity to incorporate the adenine analog, 2-amino-purine, into activated salmon sperm DNA and a relatively low fidelity in replicating synthetic polydeoxyribonucleotides. Both the 3'----5' (proofreading) and 5'----3' (nick-translational) exonuclease activities of pol I* and pol I are indistinguishable. Estimates of processivity give a value of approximately 6 for both forms of the enzyme.

The biological activities of several previously synthesized [J. A. Montgomery et al. , J. med. Chem. 17 , 1197 (1974)] adenine-substituted analogs of 5′-deoxy-5′-methylthio- or -deoxy-5′-ethyl-thioadenosine, including the 2-fluoroadenine, 2-chloroadenine, 2,6-diaminopurine, 8-azaadenine, and 4-aminopyrazolo[3,4- d ]pyrimidine-containing derivatives, have been reexamined. It is demonstrated that many of these analogs are cleaved to their respective free base analogs by 5t - deoxy-5′-methyl-thioadenosine phosphorylase (MTAPase), an enzyme associated with polyamine biosynthesis, and that this reaction is necessary for the cytotoxic action of these MTA analogs to be fully expressed. Evidence to support this includes: (1) the growth of two MTAPase-containing human colon carcinoma cell lines (the HCT-15 and DLD-1 lines) was inhibited by these analogs, whereas an MTAPase-deflcient cell line, the CCRF-CEM human T-cell leukemia, was relatively insensitive to their cytotoxic action; (2) extracts of the MTAPase-containing colon carcinoma cell lines were able to cleave these analogs to their respective free base analogs; in contrast, extracts of MTAPase-deficient CCRF-CEM cells were unable to cleave these analogs; (3) intact colon carcinoma cells converted these MTA analogs to their corresponding 5′-phosphorylated analog nucleotides, whereas CCRF-CEM cells did not, at least to detectable levels; and (4) the MTA analog, 5′-deoxy-5′-ethylthio-4-aminopyrazolo[3,4- d ]pyrimidine ribonucleoside, which is not a substrate of MTAPase, did not form analog nucleotides and was essentially noncytotoxic to all cell lines tested, whereas the corresponding adenine analog, 4-aminopyrazolo[3,4- d ]pyrimidine, readily formed analog nucleotides and was highly cytotoxic to all the lines. It is postulated that the corresponding adenine analog 5'-phosphorylated nucleotides are the primary active metabolites of these MTA analogs, having been formed by the cleavage of these nucleosides to free adenine analogs by MTAPase, followed by the conversion of these base analogs to analog nucleotides by adenine phosphoribosyltransferase and the enzymes of adenine nucleotide phosphorylation. This pathway represents a novel drug-activation system for the synthesis of analog nucleotides and has the potential to be exploited chemotherapeutically.

The purified tonoplast H+-ATPase from oat roots (Avena sativa L. var. Lang) consists of at least three different polypeptides with masses 72, 60, and 16 kDa. We have used covalent modifiers (inhibitors) and polyclonal antibodies to identify the catalytic subunit of the H+-pumping ATPase. The inactivation of ATPase activity by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (Nbd-Cl, an adenine analog) was protected by MgATP or MgADP, and showed kinetic properties consistent with active site-directed inhibition. Under similar conditions, [14C]Nbd-Cl preferentially labeled the 72-kDa polypeptide of the purified ATPase. This binding was reduced by MgATP or 2' (3')-)O-(2,4,6-trinitrophenyl) ATP. Nbd-Cl probably modified cysteinyl--SH or tyrosyl--OH groups, as dithiothreitol reversed both ATPase inactivation and [14C]Nbd-Cl binding to the 72-kDa subunit. The finding that N-ethylmaleimide inhibition of ATPase activity was protectable by nucleotides is consistent with the idea of sulfhydryl groups in the ATP-binding site. Polyclonal antibody made to the 72-kDa polypeptide specifically reacted (Western blot) with a 72-kDa polypeptide from both tonoplast-enriched membranes and the purified tonoplast ATPase, but it did not cross-react with the mitochondrial or Escherichia coli F1-ATPase. The antibody inhibited tonoplast ATPase and H+-pumping activities. We conclude from these results that the 72-kDa polypeptide of the tonoplast H+-ATPase contains an ATP- (or nucleotide-) binding site that may constitute the catalytic domain.