Nucleobase-modified nucleosides and nucleotides: Applications in biochemistry, synthetic biology, and drug discovery.

. DNA is often referred to as the "molecule of life" since it contains the genetic blueprint for all forms of life on this planet. The core building blocks composing DNA are deoxynucleotides. While the deoxyribose sugar and phosphate group are ubiquitous, it is the composition and spatial arrangement of the four natural nucleobases, adenine (A), cytosine (C), guanine (G), and thymine (T), that provide diversity in the coding information present in DNA. The ability of DNA to function as the genetic blueprint has historically been attributed to the formation of proper hydrogen bonding interactions made between complementary nucleobases. However, recent chemical and biochemical studies using nucleobase-modified nucleotides that contain "non-hydrogen bonding" functional groups have challenged many of the dogmatic views for the necessity of hydrogen-bonding interactions for DNA stability and function. Based on years of exciting research, this area has expanded tremendously and is thus too expansive to provide a comprehensive review on the topic. As such, this review article provides an opinion highlighting how nucleobase-modified nucleotides are being applied in diverse biomedical fields, focusing on three exciting areas of research. The first section addresses how these analogs are used as mechanistic probes for DNA polymerase activity and fidelity during replication. This section outlines the synthetic logic and medicinal chemistry approaches used to replace hydrogen-bonding functional groups to examine the contributions of shape/size, nucleobase hydrophobicity, and pi-electron interactions. The second section extends these mechanistic studies to provide insight into how nucleobase-modified nucleosides are used in synthetic biology. One example is through expansion of the genetic code in which changing the composition of DNA makes it possible to site-specifically incorporate unnatural amino acids bearing unique functional groups into enzymes and receptors. The final section describes results of pre-clinical studies using nucleobase-modified nucleosides as potential therapeutic agents against diseases such as cancer.

Reimagining the Power of Nucleic Acids as Therapeutic and Diagnostic Agents.

The central dogma of molecular biology proposes that in a typical cell, the flow of genetic information proceeds from DNA to RNA to polypeptide [...].

An artificial nucleoside that simultaneously detects and combats drug resistance to doxorubicin.

OBJECTIVES: Doxorubicin is a DNA-damaging agent used to treat hematological cancers. Unfortunately, drug resistance can occur by defective DNA repair activity coupled with the ability of DNA polymerases to misreplicate unrepaired DNA lesions. This study demonstrates that the efficacy of doxorubicin can be improved by using an artificial nucleoside to efficiently and selectively inhibit this activity. METHODS: In vitro studies using acute lymphoblastic leukemia cell lines define the mechanism of cell death caused by combining an artificial nucleoside with doxorubicin. RESULTS: Flow cytometry experiments demonstrate that combining an artificial nucleoside with doxorubicin potentiates the cell killing effects of the drug by increasing apoptosis. The potentiation effect correlates with expression of TdT, a specialized DNA polymerase overexpressed in acute lymphoblastic leukemia. Cell cycle experiments demonstrate that this combination blocks cells at S-phase prior to inducing apoptosis. Finally, the unique chemical composition of the nucleoside analog was used to visualize the replication of damaged DNA in TdT-positive cells. This represents a potential diagnostic tool to easily identify doxorubicin-resistant cancer cells. CONCLUSION: Studies demonstrate that a novel artificial nucleoside improves the therapeutic efficacy of doxorubicin, thereby reducing the risk of potential side effects caused by the DNA-damaging agent.

Outcomes of Anterior Cruciate Ligament Reconstruction Using Biologic Augmentation in Patients 21 Years of Age and Younger.

PURPOSE: To report on the outcomes of a subset of patients ≤21 years of age after anterior cruciate ligament (ACL) reconstruction coupled with biologic augmentation using platelet-rich plasma (PRP) and a porous collagen carrier. METHODS: A cohort of patients was retrospectively reviewed after ACL reconstruction with hamstring autograft tendon. All reconstructive surgeries combined biologic augmentation in which the ACL graft was coupled with PRP contained within porous collagen membrane. Patients were included if they maintained a minimum follow-up period of 24 months. Outcomes were assessed through patient-reported questionnaires and physical examination in the clinical setting. Patient-reported outcomes including International Knee Documentation Committee (IKDC), Lysholm, Tegner, and Single Assessment Numeric Evaluation (SANE) scores were collected. ACL stability was evaluated using Lachman and KT-1000 testing. Patients were also evaluated for return to play at the same level of competition, family history of ACL injury, and time to complete rehabilitation. RESULTS: A total of 194 patients were initially eligible; 143 (74%) patients with 151 knees were ultimately evaluated. The average patient age was 16 years; 79 patients were female and 64 were male. Follow-up duration averaged 52 months. IKDC and Lysholm scores averaged 91 and 91; the average SANE score was 94. The KT-1000 side-to-side difference averaged 1.2 mm. The average time to complete physical therapy was 22 weeks, and 132 patients (92%) returned to their preinjury level of competition. There were 23 cases of contralateral ACL injury (15%) and 7 cases of ACL reinjury necessitating revision surgery (5%). CONCLUSIONS: Biologic augmentation with hamstring autograft in ACL reconstruction shows a decreased rate of second ACL injury, specifically with regard to ACL revision surgery. The patients in this study also show higher return to preinjury level of competition at a faster rate than other studies have shown. LEVEL OF EVIDENCE: Level IV, Therapeutic Case Series.

A non-natural nucleotide uses a specific pocket to selectively inhibit telomerase activity.

Telomerase, a unique reverse transcriptase that specifically extends the ends of linear chromosomes, is up-regulated in the vast majority of cancer cells. Here, we show that an indole nucleotide analog, 5-methylcarboxyl-indolyl-2'-deoxyriboside 5'-triphosphate (5-MeCITP), functions as an inhibitor of telomerase activity. The crystal structure of 5-MeCITP bound to the Tribolium castaneum telomerase reverse transcriptase reveals an atypical interaction, in which the nucleobase is flipped in the active site. In this orientation, the methoxy group of 5-MeCITP extends out of the canonical active site to interact with a telomerase-specific hydrophobic pocket formed by motifs 1 and 2 in the fingers domain and T-motif in the RNA-binding domain of the telomerase reverse transcriptase. In vitro data show that 5-MeCITP inhibits telomerase with a similar potency as the clinically administered nucleoside analog reverse transcriptase inhibitor azidothymidine (AZT). In addition, cell-based studies show that treatment with the cell-permeable nucleoside counterpart of 5-MeCITP leads to telomere shortening in telomerase-positive cancer cells, while resulting in significantly lower cytotoxic effects in telomerase-negative cell lines when compared with AZT treatment.

Artificial Nucleosides as Diagnostic Probes to Measure Translesion DNA Synthesis.

The misreplication of damaged DNA, a biological process termed translesion DNA synthesis (TLS), produces a large number of adverse effects on human health. This chapter describes the application of an artificial nucleoside/nucleotide system that functions as a biochemical probe to quantify TLS activity under in vitro and in vivo conditions. For in vitro studies, the artificial nucleotide, 3-ethynyl-5-nitroindolyl-2'-deoxyriboside triphosphate (3-Eth-5-NITP), is used as it is efficiently inserted opposite an abasic site, a highly pro-mutagenic DNA lesion produced by several types of DNA-damaging agents. The placement of the ethynyl moiety allows the incorporated nucleoside triphosphate to be selectively tagged with azide-containing fluorophores via "click" chemistry. This reaction provides a facile way to quantify the extent of nucleotide incorporation opposite this and other noninstructional DNA lesions. The corresponding nucleoside, 3-Eth-5-NIdR, can be used to monitor TLS activity in hematological and adherent cancer cells treated with compounds that produce noninstructional DNA lesions. As described above, visualizing the replication of these lesions is achieved using copper-catalyzed "click" chemistry to tag the ethynyl moiety present on the nucleotide with fluorogenic probes. This technique represents a new diagnostic approach to quantify TLS activity inside cells. In addition, the application of this "clickable" nucleoside provides a chemical probe to identify cells that become drug resistant by the facile replication of noninstructional DNA lesions produced by DNA-damaging agents.

Administration of a Nucleoside Analog Promotes Cancer Cell Death in a Telomerase-Dependent Manner.

Telomerase, the end-replication enzyme, is reactivated in malignant cancers to drive cellular immortality. While this distinction makes telomerase an attractive target for anti-cancer therapies, most approaches for inhibiting its activity have been clinically ineffective. As opposed to inhibiting telomerase, we use its activity to selectively promote cytotoxicity in cancer cells. We show that several nucleotide analogs, including 5-fluoro-2'-deoxyuridine (5-FdU) triphosphate, are effectively incorporated by telomerase into a telomere DNA product. Administration of 5-FdU results in an increased number of telomere-induced foci, impedes binding of telomere proteins, activates the ATR-related DNA-damage response, and promotes cell death in a telomerase-dependent manner. Collectively, our data indicate that telomerase activity can be exploited as a putative anti-cancer strategy.

Inhibition of Translesion DNA Synthesis as a Novel Therapeutic Strategy to Treat Brain Cancer.

Temozolomide is a DNA-alkylating agent used to treat brain tumors, but resistance to this drug is common. In this study, we provide evidence that efficacious responses to this drug can be heightened significantly by coadministration of an artificial nucleoside (5-nitroindolyl-2'-deoxyriboside, 5-NIdR) that efficiently and selectively inhibits the replication of DNA lesions generated by temozolomide. Conversion of this compound to the corresponding nucleoside triphosphate, 5-nitroindolyl-2'-deoxyriboside triphosphate, in vivo creates a potent inhibitor of several human DNA polymerases that can replicate damaged DNA. Accordingly, 5-NIdR synergized with temozolomide to increase apoptosis of tumor cells. In a murine xenograft model of glioblastoma, whereas temozolomide only delayed tumor growth, its coadministration with 5-NIdR caused complete tumor regression. Exploratory toxicology investigations showed that high doses of 5-NIdR did not produce the side effects commonly seen with conventional nucleoside analogs. Collectively, our results offer a preclinical pharmacologic proof of concept for the coordinate inhibition of translesion DNA synthesis as a strategy to improve chemotherapeutic responses in aggressive brain tumors.Significance: Combinatorial treatment of glioblastoma with temozolomide and a novel artificial nucleoside that inhibits replication of damaged DNA can safely enhance therapeutic responses. Cancer Res; 78(4); 1083-96. ©2017 AACR.

Inhibiting DNA Polymerases as a Therapeutic Intervention against Cancer.

Inhibiting DNA synthesis is an important therapeutic strategy that is widely used to treat a number of hyperproliferative diseases including viral infections, autoimmune disorders, and cancer. This chapter describes two major categories of therapeutic agents used to inhibit DNA synthesis. The first category includes purine and pyrmidine nucleoside analogs that directly inhibit DNA polymerase activity. The second category includes DNA damaging agents including cisplatin and chlorambucil that modify the composition and structure of the nucleic acid substrate to indirectly inhibit DNA synthesis. Special emphasis is placed on describing the molecular mechanisms of these inhibitory effects against chromosomal and mitochondrial DNA polymerases. Discussions are also provided on the mechanisms associated with resistance to these therapeutic agents. A primary focus is toward understanding the roles of specialized DNA polymerases that by-pass DNA lesions produced by DNA damaging agents. Finally, a section is provided that describes emerging areas in developing new therapeutic strategies targeting specialized DNA polymerases.

A Comparative Analysis of Translesion DNA Synthesis Catalyzed by a High-Fidelity DNA Polymerase.

Translesion DNA synthesis (TLS) is the ability of DNA polymerases to incorporate nucleotides opposite and beyond damaged DNA. TLS activity is an important risk factor for the initiation and progression of genetic diseases such as cancer. In this study, we evaluate the ability of a high-fidelity DNA polymerase to perform TLS with 8-oxo-guanine (8-oxo-G), a highly pro-mutagenic DNA lesion formed by reactive oxygen species. Results of kinetic studies monitoring the incorporation of modified nucleotide analogs demonstrate that the binding affinity of the incoming dNTP is controlled by the overall hydrophobicity of the nucleobase. However, the rate constant for the polymerization step is regulated by hydrogen-bonding interactions made between the incoming nucleotide with 8-oxo-G. Results generated here for replicating the miscoding 8-oxo-G are compared to those published for the replication of the non-instructional abasic site. During the replication of both lesions, binding of the nucleotide substrate is controlled by energetics associated with nucleobase desolvation, whereas the rate constant for the polymerization step is influenced by the physical nature of the DNA lesion, that is, miscoding versus non-instructional. Collectively, these studies highlight the importance of nucleobase desolvation as a key physical feature that enhances the misreplication of structurally diverse DNA lesions.

Inhibiting translesion DNA synthesis as an approach to combat drug resistance to DNA damaging agents.

Anti-cancer agents exert therapeutic effects by damaging DNA. Unfortunately, DNA polymerases can effectively replicate the formed DNA lesions to cause drug resistance and create more aggressive cancers. To understand this process at the cellular level, we developed an artificial nucleoside that visualizes the replication of damaged DNA to identify cells that acquire drug resistance through this mechanism. Visualization is achieved using "click" chemistry to covalently attach azide-containing fluorophores to the ethynyl group present on the nucleoside analog after its incorporation opposite damaged DNA. Flow cytometry and microscopy techniques demonstrate that the extent of nucleotide incorporation into genomic DNA is enhanced by treatment with DNA damaging agents. In addition, this nucleoside analog inhibits translesion DNA synthesis and synergizes the therapeutic activity of certain anti-cancer agents such as temozolomide. The combined diagnostic and therapeutic activities of this synthetic nucleoside analog represent a new paradigm in personalized medicine.

Inhibition of yeast ribonucleotide reductase by Sml1 depends on the allosteric state of the enzyme.

Sml1 is an intrinsically disordered protein inhibitor of Saccharomyces cerevisiae ribonucleotide reductase (ScRR1), but its inhibition mechanism is poorly understood. RR reduces ribonucleoside diphosphates to their deoxy forms, and balances the nucleotide pool. Multiple turnover kinetics show that Sml1 inhibition of dGTP/ADP- and ATP/CDP-bound ScRR follows a mixed inhibition mechanism. However, Sml1 cooperatively binds to the ES complex in the dGTP/ADP form, whereas with ATP/CDP, Sml1 binds weakly and noncooperatively. Gel filtration and mutagenesis studies indicate that Sml1 does not alter the oligomerization equilibrium and the CXXC motif is not involved in the inhibition. The data suggest that Sml1 is an allosteric inhibitor.

The use of modified and non-natural nucleotides provide unique insights into pro-mutagenic replication catalyzed by polymerase eta.

This report evaluates the pro-mutagenic behavior of 8-oxo-guanine (8-oxo-G) by quantifying the ability of high-fidelity and specialized DNA polymerases to incorporate natural and modified nucleotides opposite this lesion. Although high-fidelity DNA polymerases such as pol δ and the bacteriophage T4 DNA polymerase replicating 8-oxo-G in an error-prone manner, they display remarkably low efficiencies for TLS compared to normal DNA synthesis. In contrast, pol η shows a combination of high efficiency and low fidelity when replicating 8-oxo-G. These combined properties are consistent with a pro-mutagenic role for pol η when replicating this DNA lesion. Studies using modified nucleotide analogs show that pol η relies heavily on hydrogen-bonding interactions during translesion DNA synthesis. However, nucleobase modifications such as alkylation to the N2 position of guanine significantly increase error-prone synthesis catalyzed by pol η when replicating 8-oxo-G. Molecular modeling studies demonstrate the existence of a hydrophobic pocket in pol η that participates in the increased utilization of certain hydrophobic nucleotides. A model is proposed for enhanced pro-mutagenic replication catalyzed by pol η that couples efficient incorporation of damaged nucleotides opposite oxidized DNA lesions created by reactive oxygen species. The biological implications of this model toward increasing mutagenic events in lung cancer are discussed.

Physiological enzymology: The next frontier in understanding protein structure and function at the cellular level.

Historically, the study of proteins has relied heavily on characterizing the activity of a single purified protein isolated from other cellular components. This classic approach allowed scientists to unambiguously define the intrinsic kinetic and chemical properties of that protein. The ultimate hope was to extrapolate this information toward understanding how the enzyme or receptor behaves within its native cellular context. These types of detailed in vitro analyses were necessary to reduce the innate complexities of measuring the singular activity and biochemical properties of a specific enzyme without interference from other enzymes and potential competing substrates. However, recent developments in fields encompassing cell biology, molecular imaging, and chemical biology now provide the unique chemical tools and instrumentation to study protein structure, function, and regulation in their native cellular environment. These advancements provide the foundation for a new field, coined physiological enzymology, which quantifies the function and regulation of enzymes and proteins at the cellular level. In this Special Edition, we explore the area of Physiological Enzymology and Protein Function through a series of review articles that focus on the tools and techniques used to measure the cellular activity of proteins inside living cells. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions.

Visualizing nucleic acid metabolism using non-natural nucleosides and nucleotide analogs.

Nucleosides and their corresponding mono-, di-, and triphosphates play important roles in maintaining cellular homeostasis. In addition, perturbations in this homeostasis can result in dysfunctional cellular processes that cause pathological conditions such as cancer and autoimmune diseases. This review article discusses contemporary research areas applying nucleoside analogs to probe the mechanistic details underlying the complexities of nucleoside metabolism at the molecular and cellular levels. The first area describes classic and contemporary approaches used to quantify the activity of nucleoside transporters, an important class of membrane proteins that mediate the influx and efflux of nucleosides and nucleobases. A focal point of this section is describing how biophotonic nucleosides are replacing conventional assays employing radiolabeled substrates to study the mechanism of these proteins. The second section describes approaches to understand the utilization of nucleoside triphosphates by cellular DNA polymerases during DNA synthesis. Emphasis here is placed on describing how novel nucleoside analogs such as 5-ethynyl-2'-deoxyuridine are being used to quantify DNA synthesis during normal replication as well as during the replication of damaged DNA. In both sections, seminal research articles relevant to these areas are described to highlight how these novel probes are improving our understanding of these biological processes. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions.

A Genetic Selection for dinB Mutants Reveals an Interaction between DNA Polymerase IV and the Replicative Polymerase That Is Required for Translesion Synthesis.

Translesion DNA synthesis (TLS) by specialized DNA polymerases (Pols) is a conserved mechanism for tolerating replication blocking DNA lesions. The actions of TLS Pols are managed in part by ring-shaped sliding clamp proteins. In addition to catalyzing TLS, altered expression of TLS Pols impedes cellular growth. The goal of this study was to define the relationship between the physiological function of Escherichia coli Pol IV in TLS and its ability to impede growth when overproduced. To this end, 13 novel Pol IV mutants were identified that failed to impede growth. Subsequent analysis of these mutants suggest that overproduced levels of Pol IV inhibit E. coli growth by gaining inappropriate access to the replication fork via a Pol III-Pol IV switch that is mechanistically similar to that used under physiological conditions to coordinate Pol IV-catalyzed TLS with Pol III-catalyzed replication. Detailed analysis of one mutant, Pol IV-T120P, and two previously described Pol IV mutants impaired for interaction with either the rim (Pol IVR) or the cleft (Pol IVC) of the ß sliding clamp revealed novel insights into the mechanism of the Pol III-Pol IV switch. Specifically, Pol IV-T120P retained complete catalytic activity in vitro but, like Pol IVR and Pol IVC, failed to support Pol IV TLS function in vivo. Notably, the T120P mutation abrogated a biochemical interaction of Pol IV with Pol III that was required for Pol III-Pol IV switching. Taken together, these results support a model in which Pol III-Pol IV switching involves interaction of Pol IV with Pol III, as well as the ß clamp rim and cleft. Moreover, they provide strong support for the view that Pol III-Pol IV switching represents a vitally important mechanism for regulating TLS in vivo by managing access of Pol IV to the DNA.

A metal-containing nucleoside that possesses both therapeutic and diagnostic activity against cancer.

Nucleoside transport is an essential process that helps maintain the hyperproliferative state of most cancer cells. As such, it represents an important target for developing diagnostic and therapeutic agents that can effectively detect and treat cancer, respectively. This report describes the development of a metal-containing nucleoside designated Ir(III)-PPY nucleoside that displays both therapeutic and diagnostic properties against the human epidermal carcinoma cell line KB3-1. The cytotoxic effects of Ir(III)-PPY nucleoside are both time- and dose-dependent. Flow cytometry analyses validate that the nucleoside analog causes apoptosis by blocking cell cycle progression at G2/M. Fluorescent microscopy studies show rapid accumulation in the cytoplasm within 4 h. However, more significant accumulation is observed in the nucleus and mitochondria after 24 h. This localization is consistent with the ability of the metal-containing nucleoside to influence cell cycle progression at G2/M. Mitochondrial depletion is also observed after longer incubations (Δt ∼48 h), and this effect may produce additional cytotoxic effects. siRNA knockdown experiments demonstrate that the nucleoside transporter, hENT1, plays a key role in the cellular entry of Ir(III)-PPY nucleoside. Collectively, these data provide evidence for the development of a metal-containing nucleoside that functions as a combined therapeutic and diagnostic agent against cancer.

Current and emerging strategies to increase the efficacy of ionizing radiation in the treatment of cancer.

INTRODUCTION: Ionizing radiation (IR) is an important therapeutic modality used in approximately 50% of all cancer patients and is particularly effective against solid tumors that cannot be removed by surgery or that are refractory to standard anticancer agents. IR is often combined with other chemotherapeutic agents with the goal of sensitizing cancer cells to the cytotoxic effects of IR to produce a synergistic cell-killing effect. AREAS COVERED: This review article describes current and emerging therapeutic agents that are designed to increase the therapeutic efficacy of IR. This includes a discussion of how IR causes cell death by damaging nucleic acid. The involvement of various DNA repair pathways, cell-cycle-dependent kinases and apoptotic pathways is also described. This mechanistic information provides the framework to understand how combining therapeutic modalities with IR produces synergistic effects as well as to explain how emerging therapeutic strategies are being designed to inhibit or activate these pathways. Biochemical mechanisms and clinical applications of these chemical entities are discussed. Finally, brief descriptions are provided for several emerging chemical entities that show promise as potential adjunctive agents to sensitize cells to the effects of IR. EXPERT OPINION: Using DNA damaging agents or kinase inhibitors to potentiate the cytotoxic effects of IR has significantly improved patient outcomes. However, several advancements in instrumentation as well as new molecular targets are changing the landscape of applying IR as a therapeutic modality.