DNA Polymerases as Therapeutic Targets

DNA Polymerases as Therapeutic Targets

The alphaherpesvirus varicella-zoster virus (VZV) causes chickenpox and shingles. Concomitant with this momentous therapeutic advance, the mitochondrial toxicities of ART were recognized as an important clinical entity. Normal cellular enzymes do not phosphorylate acyclovir to any significant degree. High throughput screening of large compound collections for inhibitors of specific viral enzymes or inhibition of viral growth in cell culture have identified a number of new HCMV inhibitors at several pharmaceutical companies. The initial phase of the process depends on the enzyme thymidine kinase viral-coded. They are also endowed with fluorescent properties when exposed to light with short UV wavelength. Provided there are no safety issues to stop their progress, this new class of compound will be a major advance in the herpesvirus antiviral field.

The viral DNA polymerase binds strongly to the acyclo-GMP-terminated template, and in thereby inactivated. 2 Aluminum Lake, FD&C Red No. The pkas of acyclovir are 2.27 and 9.25. Product Type Name HUMAN PRESCRIPTION DRUG Indicates the type of product, such as Human Prescription Drug or Human OTC Drug. This data element corresponds to the “Document Type” of the SPL submission for the listing. The complete list of codes and translations can be found at www.fda.gov/edrls under Structured Product Labeling Resources. The complete list of codes and translations can be found at www.fda.gov/edrls under Structured Product Labeling Resources.

Proprietary Name Valacyclovir Hydrochloride Also known as the trade name. Non Proprietary Name Valacyclovir Hydrochloride Sometimes called the generic name, this is usually the active ingredient(s) of the product. Meanwhile, nucleoside analogues are not natural substrates and show low affinity for nucleoside transporters. The complete list of codes and translations can be found www.fda.gov/edrls under Structured Product Labeling Resources. Route Name ORAL The translation of the Route Code submitted by the firm, indicating route of administration. The complete list of codes and translations can be found at www.fda.gov/edrls under Structured Product Labeling Resources. 1).

The CC50, EC50, and selectivity index values for these drugs are shown in Table 1 (means ± standard errors of three or more experiments). This result was intriguing since the orthopoxviruses encode thymidine kinase (TK) homologs that are closely related to the human cytosolic TK (TK1) (15). After the resolution of the primary infection, latent virus resides in sensory neurons. In regard to the phosphorylation of ACV and GCV by the gammaherpesvirus TK, different groups have reported conflicting findings. With the aim of better understanding the modes of action of these two novel antivirals, drug-resistant viruses were selected in vitro, and enzymatic TK assays were performed. This specificity excludes also the Simian varicella virus, which, despite being closely related to VZV, is not inhibited by BCNAs [18]. Perhaps the most direct approach, however, is the use of nucleoside analogs such as AZT (6) or fludaribine (7) that target the enzymatic activity of the DNA polymerase, the workhorse of the DNA replication apparatus (Step 4).

The phenotypes of three strains with the following TK substitutions were described previously: first, the amino acid exchange Q303stop, observed in two patients with AIDS developing persistent zoster under ACV treatment (17, 18); second, the T256M substitution found after in vitro adaption to ACV (19); and third, the W225R substitution in one strain cultivated from a patient specimen (5). Despite differences between the three classes of human herpesviruses, the mechanisms by which they replicate their DNA during productive (‘lytic’) infection are largely conserved. It directly inhibits HSV DNA polymerase and HIV reverse transcriptase by reversibly blocking the pyrophosphate binding site (pyrophosphate is a necessary co-factor in the functioning of these enzymes). DNA replication initially produces head-to-tail linear concatemers, and branched concatemers accumulate later in the replication cycle through recombination and/or reinitiation mechanisms. aciclovir, BVDU) is available. The first point for generating high catalytic efficiency and fidelity occurs through the binding of dNTP to the polymerase:DNA complex (Step 2). The binding affinity (Kd value) for a nucleotide opposite its correct pairing partner is generally ∼10 μM while Kd values for incorrect nucleotides are usually 10-fold higher.

Reaching a complete CPE, infected cell cultures were harvested by three freezing and thawing cycles and subsequent sonification for 30 s. This conformational change step reflects an “induced-fit” mechanism that also imposes discrimination against nucleotide misinsertion as misaligned intermediates perturb the geometry of the polymerase’s active site to prevent the chemical step (8). T-tropic or X4 HIV strains use the CXCR4, and M-tropic or R5 HIV strains use the CCR5 to enter the cells. In 2 trials, acyclovir tablets were administered at 20 mg/kg 4 times daily (up to 3,200 mg per day) for 5 days. Because certain molecules like ganciclovir have much higher affinity for HSV1TK than for VZVTK, we also decided to mutate residues His97 and Tyr21 to the corresponding HSV1TK residues, respectively, Tyr132 and His58 (Fig. Confluent HEL 299 cells in 12-well tissue culture plates were inoculated with the virus at 30 to ∼40 plaque-forming units/well. Piette, University of Liège, Liège, Belgium) as a template.

Except for 3′-Ara-C, all oligonucleotides were purchased from either Midland (Midland, TX, USA) or Integrated DNA Technologies (Coralville, IA, USA). Kinetic mechanism for RNA- and DNA-dependent polymerases. Individual steps along the pathway for DNA polymerization are numbered and identified. AraC is an important drug in the treatment of acute myeloic leukemia (AML). Following phosphoryl transfer, there is a second conformational change (step 5) that is required for pyrophosphate release (step 6). After pyrophosphate (PPi) is released, the polymerase can either remain bound to the nucleic acid substrate to continue primer elongation (step 8) or dissociate from the elongated primer (step 7) to initiate DNA synthesis on another usable primer template. The ability to incorporate multiple nucleotides without dissociating from DNA defines the processivity of the polymerase.

Alterations in various kinetic steps can occur to influence the behavior of the polymerase and alter the effectiveness of certain chemotherapeutic agents. One example is the ability of a polymerase to reverse the polymerization step (, Step 4) via a process known as pyrophosphorolysis to remove inappropriately incorporated nucleotides (11). Other kinetic steps include the dNTP binding step (, Step 2) and the conformational change preceding chemistry (, Step 3). This mechanistic framework highlights the importance of kinetic steps that can be targeted to inhibit replication and prevent proliferation. One potential stage for therapeutic intervention is the use of competitive inhibitors to block dNTP binding to the polymerase’s active site (, Step 2). Unfortunately, developing a reversible inhibitor that binds tightly and selectively to a DNA polymerase is an extraordinarily difficult task. Much of this challenge arises from the promiscuous and relatively weak binding affinity (Kd ∼ 10 μM) of natural nucleotides to the polymerase.

DNA Polymerases as Therapeutic Targets
Furthermore, “correct” polymerization occurs with an incredibly high commitment to catalysis (Keq ∼106) since forward kinetic steps such as phosphoryl transfer (kpol ∼100 sec−1) are generally much faster than the corresponding reverse step of pyrophosphorolysis (kpyro ∼0.0001 sec−1) (12). These two features coupled with additional pharmacokinetic problems such as drug absorption (13) and metabolism (14) of nucleoside triphosphates have hindered this conventional approach. An alternative strategy is to take advantage of the polymerase’s high commitment to catalysis and trick the enzyme into using a potential suicide inhibitor. This Trojan Horse strategy outlined in represents the current paradigm in the design and implementation of many nucleoside analogs targeting prokaryotic and eukaryotic DNA polymerases. The polymerase is provided with a modified nucleotide such as AZT-TP that is incorporated into DNA as efficiently as its natural counterpart, dTTP. The conversion of the colorimetric substrate was determined by measuring the absorbance at 570 nm, and EC50s were calculated by standard methods (29). These findings provide a strong rationale for using proteasome/HDAC inhibitor combination for the therapy of PEL.

A kinase-inactive form of the Halo-EBV PK plasmid (in which lysine residue 102 was changed to an isoleucine residue) was also constructed by using site-directed mutagenesis. Our discussion begins by analyzing the development and application of several viral DNA polymerase inhibitors as this represents a major effort in modern pharmaceutical science. 5′-O-L-valyl ester of L-BHDU (2) was synthesized by coupling Boc-L-valine with L-BHDU (1) in the presence of 4-dimethylaminopyridine (DMAP) and 1,3-diisopropylcarbodiimide (DIC) to provide the Boc-protected valyl esters of L-BHDU. AZT-TP is an effective chain-terminator due to the simple replacement of the hydroxyl group with an azide (-N3) which prevents subsequent primer elongation. Viruses are responsible for a wide variety of human diseases ranging from simple ailments including the common cold, chickenpox, and cold sores to more serious health threats such as Aquired Immune Deficiency Syndrome (AIDS), Severe Acute Respiratory Syndrome (SARS), and cervical cancer (15). In addition, an alarming number of other diseases such as progressive multifocal leukoencephalopathy (PML) and multiple sclerosis may also be linked with viral infections (16). A universal feature for the virulence and propagation of any virus is the requirement for high levels of DNA synthesis.

For example, reverse transcription of the viral single-stranded (+) RNA genome of the human immunodeficiency virus (HIV) into double-stranded DNA is catalyzed by the retroviral polymerase, reverse transcriptase (RT). This RNA- and DNA-dependent polymerase is a prime target for anti-viral drug development since it plays an essential role in the HIV life cycle. To date, two distinct types of RT inhibitors with unique pharmacodynamic behavior have been developed for clinical use. There was no statistically significant difference in the incidence of tumors between treated and control animals, nor did acyclovir shorten the latency of tumors. Testicular atrophy and aspermatogenesis were observed in rats and dogs at higher dose levels. All of these nucleoside analogs share a common feature that distinguishes them from their natural counterparts – the absence of the 3′-OH group that is required for subsequent primer elongation. As previously illustrated in , AZT-TP is an effective chain-terminator due to the simple replacement of the hydroxyl group with an azide (-N3).

Since the coding potential of the nucleobase remains identical to that of the natural nucleoside, the binding affinities for chain-terminators such as AZT as well as d4T (Stavudine) and ddC (Zalcitabine) are nearly identical to their natural counterparts (17-19). However, the maximal rate constant for the incorporation of AZT-TP is significantly slower than dTTP incorporation (compare kpol values of 0.7 vs ∼20 sec−1 for AZT-TP and dTTP, respectively) (17). Thus, alterations to the ribose moiety appear to influence the phosphoryl transfer step and/or the ability of the polymerase to mediate the conformational change preceding this kinetic step. Regardless, the kpol/Kd values for these modified nucleotides are comparable to their natural counterparts and the inhibition of viral replication via chain-termination corroborates clinical data showing viral load reductions upon treatment with these analogs. Acyclovir () is a unique guanine analog that is widely used in the treatment of herpes simplex and herpes zoster (shingles) infections (reviewed in 20). Acyclovir differs significantly from the other aforementioned anti-viral agents as the 2′-deoxyribose sugar is replaced by an open-chain structure which accounts for its chain-termination capabilities. However, this mofication confers two additional pharmacodynamic properties that make it a highly effective anti-viral agent.

The first is an unusually high selectively for conversion into acyclo-guanosine monophosphate (acylo-GMP) by the virally-encoded thymidine kinase as opposed to the host kinase. The 3,000-fold higher activity of the viral enzyme leads to higher concentrations of acylo-GMP in infected cells as opposed to healthy, unfected cells. Secondly, the triphosphate form of acyclovir (acyclo-GTP) is incorporated 100-fold more efficiently by the viral polymerase compared to the host enzyme. Latently infected Akata cells were induced to undergo a productive infection by the addition of 50 μg/ml goat anti-human immunoglobulin G antibody, and a sample of 4 × 104 cells was added to each well. Since the discovery of iododeoxyuridine (IDU) in 1959, followed by trifluorothymidine (TFT), nucleoside analogues have been the mainstay of herpesvirus antiviral therapy.17 IDU, for topical treatment of herpes keratitis (HK), was one of the first effective antiviral compounds to be licensed. In contrast, 5-ethyl-2′-deoxyuridine (EDU) showed weak antiviral activity against both MHV-68 and EBV (EC50 of 43 μM and 74 μM, respectively). Inhibition of TK-catalyzed dThd phosphorylation.Since herpesvirus TK mutants showed a modest decrease in sensitivity to KAY-2-41 and KAH-39-149, we further investigated the role of the viral TK in the mode of action of these nucleoside analogs.

(21). Despite their widespread use, the therapeutic utility of most nucleoside analogs is often restricted by major complications including the development of resistance and the induction of adverse side effects. The primer-probe sequences are provided in Table S1 in the supplemental material. Viral DNA synthesis initiates at one of the three viral origins of replication with UL9 and ICP8 acting in conjunction to distort the AT rich origin spacer region. A second mechanism occurs via the generation of selective point mutations in RT that hinders incorporation of the chain-terminator but that have no effect on the utilization of natural nucleotides. Vero cell monolayers were treated with compound (50 or 5 μM) or a DMSO vehicle control and simultaneously infected at a multiplicity of infection (MOI) of 0.1. This simple mutation decreases the binding affinity of the chain-terminator but has no effect on the binding of the natural substrate, dTTP.

The generation of point mutations also accounts for the third most common mechanism of resistance – enhanced enzymatic removal of the chain-terminator from the viral genome via pyrophosphorolysis, the reversal of polymerization (11). Adverse side effects arise from the inherently low selectivity of these nucleoside analogs as they can be effectively utilized by either viral or host DNA polymerases to inhibit DNA synthesis. Intensity of chemiluminescence signals was quantified using the LumiImager system (Roche). In contrast, most eukaryotic polymerases have robust exonuclease activities that can rapidly excise the analog and allow for replication of the host’s genome. The specificity in their antiviral action is determined by a specific virus-encoded thymidine kinase (TK), which ensures and confines the specific phosphorylation of these nucleoside analogs to the virus-infected cells. In the former case, chronic administration of certain nucleoside analogs can cause side effects resembling heritable mitochondrial diseases (24). Therefore, the residues Glu59 and Arg84 were mutated to their equivalents in HSV1TK, respectively, serine and valine, aimed at destabilizing the VZVTK loop conformation.

The RT of the resultant peak and its UV spectrum was consistent with l-BVOddU in the HPLC profile. A second level of selectivity may be represented by the VZV TK-associated dTMP kinase. We compared processing of 3′-ACV, 3′-Ara-C, 3′-AZT and 3′-Y in the two different contexts, and in each case, TDP1 prefers the single-stranded to the double-stranded substrates by 3–80-folds (Figure 2), consistent with previous reports for 3′-Y (30,31). It is clear that a diminution in exonuclease proofreading by human polymerases can cause an increased risk in toxicity. However, a different complication arises if the viral polymerase efficiently excises the chain-terminator from the viral genome via pyrophosphorolysis (11). As described earlier, pyrophosphorolysis plays an important role in drug resistance as the efficient removal of these chain terminators allows RT to re-initiate viral DNA synthesis. During long-term treatment with AZT, a set of mutations including M41L, D67N, K70R, T215F/Y, and K219Q develop within the active site of HIV RT that confer resistance (27).

The structures of these mutants complexed with chain-terminated DNA (reviewed in 28) has been instrumental in understanding the mechanism of this resistance. In this model, RT can position the 3’-OH of the primer into two distinct locations denoted as the priming site (P-site) or the nucleotide-binding site (N-site). During normal replication, RT binds the 3’-OH in the P site while dNTP binding occurs in the N-site. After phosphoryl transfer, the newly extended primer is transferred from the N-site to the P-site and the catalytic cycle continues. However, the dynamics of this cycle are altered if a chain-terminator is incorporated as the absence of the 3′-OH on a chain-terminated primer prevents elongation. Thus, the binding of the next correct dNTP in the N-site typically causes the formation of a dead-end ternary complex to prevent DNA synthesis. However, the aforementioned mutations allows RT to partition the chain-terminated primer from the P-site back into the N-site.

This movement allows for PPi binding and subsequent pyrophosphorolysis to excise the chain-terminator so that DNA synthesis can resume. Although PPi is the natural substrate for this excision reaction, ATP can also act as the acceptor substrate (29). ATP appears to interact directly within the active site through stacking interactions between the purine moiety and the aromatic ring of Y215 (30). Since pyrophosphorolysis plays an important role in drug resistance, significant effort has been placed in developing molecules to inhibit this process. One example is foscarnet, a structural mimic of PPi, that selectively inhibits the binding of pyrophosphate to various viral DNA polymerases (31). Foscarnet is widely used as an anti-viral agent against cytomegalovirus retinitis and HSV type 1 and 2 infections. However, its use in HIV therapy is limited as chronic administration causes electrolyte imbalance and nephrotoxicity due to alterations in calcium, magnesium, potassium, and phosphate levels.

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