An Analysis of Enzyme Kinetics Data for Mitochondrial DNA Strand Termination by Nucleoside Reverse Transcription Inhibitors

Nucleoside analogs used in antiretroviral treatment have been associated with mitochondrial toxicity. The polymerase-γ hypothesis states that this toxicity stems from the analogs' inhibition of the mitochondrial DNA polymerase (polymerase-γ) leading to mitochondrial DNA (mtDNA) depletion. We have constructed a computational model of the interaction of polymerase-γ with activated nucleoside and nucleotide analog drugs, based on experimentally measured reaction rates and base excision rates, together with the mtDNA genome size, the human mtDNA sequence, and mitochondrial dNTP concentrations. The model predicts an approximately 1000-fold difference in the activated drug concentration required for a 50% probability of mtDNA strand termination between the activated di-deoxy analogs d4T, ddC, and ddI (activated to ddA) and the activated forms of the analogs 3TC, TDF, AZT, FTC, and ABC. These predictions are supported by experimental and clinical data showing significantly greater mtDNA depletion in cell culture and patient samples caused by the di-deoxy analog drugs. For zidovudine (AZT) we calculated a very low mtDNA replication termination probability, in contrast to its reported mitochondrial toxicity in vitro and clinically. Therefore AZT mitochondrial toxicity is likely due to a mechanism that does not involve strand termination of mtDNA replication

Structure-Activity-Resistance Relationships of Novel Nucleoside and Nucleotide HIV-1 Reverse Transcriptase Inhibitors

Nucleos(t)ide reverse transcriptase inhibitors (N(t)RTI) are essential components of combination antiretroviral therapy for treatment of human immunodeficiency virus type-1 (HIV-1) infection. N(t)RTI are analogs of natural 2’-deoxyribonucleos(t)ides that lack a 3’-hydroxyl. Once metabolized by host kinases to the active form, their incorporation into viral DNA by HIV-1 reverse transcriptase (RT) results in chain termination of DNA synthesis. N(t)RTI efficacy is undermined primarily by rapid selection of resistant/cross-resistant HIV-1 variants. Consequently, the development of novel N(t)RTI with activity against a broad range of N(t)RTI-resistant HIV-1 is of critical importance. Rational design of novel N(t)RTI with knowledge of analog structure-activity-resistance relationships with the RT target enzyme is the most promising approach. We hypothesized that uncovering knowledge of how N(t)RTI base, sugar, and phosphate structures influence activity and resistance phenotypes would aid in the rational design of new N(t)RTI with improved activity and resistance profiles. Therefore, a combination of biochemical, antiviral, molecular modeling, and cellular pharmacology analyses provided a detailed characterization of structure-activity-resistance relationships for inhibition of wild-type and NRTI-resistant HIV-1 by novel N(t)RTI. First, we studied two novel nucleoside phosphonate NtRTI, (R)-6-[2-phosphonylmethoxy]propoxy]-2,4-diaminopyrimidine (PMEO-DAPym) and (5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl)phosphonate (GS-9148). We showed the diphosphate (-DP) form, PMEO-DAPym-DP acts as a purine mimetic that is recognized by RT as an adenosine analog and unambiguously incorporated across from thymine (DNA) or uracil (RNA). Studies indicated that PMEO-DAPym-DP and GS-9148-DP were superior to tenofovir-DP against both discrimination and excision RT resistance mechanisms. Next, we examined structure-activity-resistance relationships of 6-modified, 3’-azido-2’,3’-dideoxyguanosine (3’-azido-ddG) NRTI analogs. In RT-mediated DNA synthesis assays the triphosphate (-TP) form of each analog behaved as an adenosine mimetic for incorporation by HIV-1 RT. Importantly, the structure-activity relationships for incorporation and ATP-mediated excision were different, suggesting that new analogs can be designed that are efficiently incorporated but poorly excised by RT. RS-788, a 5’-monophosphate prodrug of 3’-azido-2’,3’-dideoxy-2,6-diaminopurine (3’-azido-2,6-DA-P), displayed potent activity against multi-NRTI-resistant HIV-1 and unique cellular metabolism. RS-788 was metabolized ~1:1 to both 3’-azido-2,6-DA-P and 3’-azido-ddG, thus delivering two distinct metabolites, each of which are potent RT chain-terminators that are incorporated opposite different bases, thymine and cytosine, respectively. Combinations of 3’-azido-2,6-DA-P+3’-azido-ddG synergistically inhibited multi-NRTI-resistant RT DNA synthesis

HIV-1 Polymerase Inhibition by Nucleoside Analogs: Cellular- and Kinetic Parameters of Efficacy, Susceptibility and Resistance Selection

Nucleoside analogs (NAs) are used to treat numerous viral infections and cancer. They compete with endogenous nucleotides (dNTP/NTP) for incorporation into nascent DNA/RNA and inhibit replication by preventing subsequent primer extension. To date, an integrated mathematical model that could allow the analysis of their mechanism of action, of the various resistance mechanisms, and their effect on viral fitness is still lacking. We present the first mechanistic mathematical model of polymerase inhibition by NAs that takes into account the reversibility of polymerase inhibition. Analytical solutions for the model point out the cellular- and kinetic aspects of inhibition. Our model correctly predicts for HIV-1 that resistance against nucleoside analog reverse transcriptase inhibitors (NRTIs) can be conferred by decreasing their incorporation rate, increasing their excision rate, or decreasing their affinity for the polymerase enzyme. For all analyzed NRTIs and their combinations, model-predicted macroscopic parameters (efficacy, fitness and toxicity) were consistent with observations. NRTI efficacy was found to greatly vary between distinct target cells. Surprisingly, target cells with low dNTP/NTP levels may not confer hyper-susceptibility to inhibition, whereas cells with high dNTP/NTP contents are likely to confer natural resistance. Our model also allows quantification of the selective advantage of mutations by integrating their effects on viral fitness and drug susceptibility. For zidovudine triphosphate (AZT-TP), we predict that this selective advantage, as well as the minimal concentration required to select thymidine-associated mutations (TAMs) are highly cell-dependent. The developed model allows studying various resistance mechanisms, inherent fitness effects, selection forces and epistasis based on microscopic kinetic data. It can readily be embedded in extended models of the complete HIV-1 reverse transcription process, or analogous processes in other viruses and help to guide drug development and improve our understanding of the mechanisms of resistance development during treatment

Kinetics and specificity of human mitochondrial DNA polymerase gamma and HIV-1 reverse transcriptase

textThe human mitochondrial DNA (mtDNA) genome must be faithfully maintained by the mitochondrial DNA replication machinery. Deficiencies in mtDNA maintenance result in the accumulation of mutations and deletions, which have been associated with a number of neuromuscular degenerative disorders including, mtDNA depletion syndrome, Alpers syndrome, progressive external opthalmoplegia (PEO), and sensory ataxic neuropathy, dysarthria, and opthalmoparesis (SANDO). The mtDNA replication machinery is comprised of a nuclearly-encoded DNA polymerase gamma (Pol γ), single-stranded DNA binding protein (mtSSB), and a hexameric mtDNA helicase. In this work, we employed quantitative pre-steady state kinetic techniques to establish the mechanisms responsible for the replication of the human mitochondrial DNA by Pol γ and explored the effects of point mutations that are observed in heritable diseases. With our biochemical characterization of mutants of Pol γ, we have shown unique characteristics that would lead to profound physiological consequences over time. Additionally, we have made significant progress towards reconstitution of the mitochondrial DNA replisome by monitoring DNA polymerization that is dependent on helicase unwinding of double stranded DNA. Overall, this work provides a better understanding of the mechanism of mtDNA replication and has important implications toward understanding the role of mitochondrial DNA replication in mitochondrial disease, ageing and cancer. In addition to the work on the mtDNA replisome, we have applied pre-steady state kinetic techniques to better understand the mechanism of RNA-dependent DNA polymerization by HIV reverse transcriptase (HIV-RT). This enzyme is responsible for the replication of the viral genome in HIV and is a common target for anti-HIV drugs. We have characterized the role of enzyme conformational changes in the kinetics of incorporation of correct nucleotide and the Nucleotide Reverse Transcriptase Inhibitor (NRTI) AZT by wild-type enzyme, as well as a mutant with clinical resistance to AZT. This work provides a better understanding of the complete mechanism of RNA-dependent DNA polymerization, the changes in the mechanism in the presence of inhibitor and the development of resistance to this nucleoside analog; and thereby this work contributes to the long-term goal of designing more effective drugs that can possibly deter resistance and be used successfully for treatment of HIV.Cellular and Molecular Biolog

Enzyme Kinetics of the Mitochondrial Deoxyribonucleoside Salvage Pathway Are Not Sufficient to Support Rapid mtDNA Replication

Using a computational model, we simulated mitochondrial deoxynucleotide metabolism and mitochondrial DNA replication. Our results indicate that the output from the mitochondrial salvage enzymes alone is inadequate to support a mitochondrial DNA replication duration of as long as 10 hours. We find that an external source of deoxyribonucleoside diphosphates or triphosphates (dNTPs), in addition to those supplied by mitochondrial salvage, is essential for the replication of mitochondrial DNA to complete in the experimentally observed duration of approximately 1 to 2 hours. For meeting a relatively fast replication target of 2 hours, almost two-thirds of the dNTP requirements had to be externally supplied as either deoxyribonucleoside di- or triphosphates, at about equal rates for all four dNTPs. Added monophosphates did not suffice. However, for a replication target of 10 hours, mitochondrial salvage was able to provide for most, but not all, of the total substrate requirements. Still, additional dGTPs and dATPs had to be supplied. Our analysis of the enzyme kinetics also revealed that the majority of enzymes of this pathway prefer substrates that are not precursors (canonical deoxyribonucleosides and deoxyribonucleotides) for mitochondrial DNA replication, such as phosphorylated ribonucleotides, instead of the corresponding deoxyribonucleotides. The kinetic constants for reactions between mitochondrial salvage enzymes and deoxyribonucleotide substrates are physiologically unreasonable for achieving efficient catalysis with the expected in situ concentrations of deoxyribonucleotides

Towards an understanding of the side effects of anti-HIV drugs using Caenorhabditis elegans

Since the discovery of HIV-1 as a cause for AIDS, many antiretroviral drugs - such as the nucleoside reverse transcriptase inhibitors (NRTIs) and the protease inhibitors (PIs) - have been developed to target viral replication. The therapeutic use of a combination of drugs, more commonly known as Highly Active Anti-Retroviral Therapy (HAART), has significantly improved the quality and length of patient lives. Overshadowing this success, however, is the problem that HIV-1 infected patients are afflicted with drug induced adverse events, some of which can be life threatening. Most adverse events seem to be related to tissues with high-energy demand and have predominantly been found to be caused by mitochondrial toxicity. In this thesis the nematode Caenorhabditis elegans is used as a model system to study the adverse side effects of HIV-1 antiretroviral medicines administered alone or in combination. Using an array of established and novel molecular techniques, drugs that have similar chemical structure and modes of action are shown to each have distinct toxicity profiles. Evidence is shown in support of an earlier proposal that there are modes to NRTI toxicity beyond the polymerase-γ theory and a novel hypothesis that NRTIs cause premature and accelerated aging is assessed. Interestingly, the observed mitochondrial dysfunction and toxicity phenotypes of both NRTIs and PIs could be attenuated by antioxidants. Taken together, this project has endeavoured to shed some light on the mechanisms behind HIV drug toxicity and ultimately benefit the development of new, effective, and less toxic compounds

Mechanisms of telomerase inhibition by oxidized and therapeutic dNTPs

Telomeres cap chromosome ends and are essential for genome stability and human health, but they shorten in most human somatic cells with cell division due to the end replication problem. Telomerase a specialized reverse transcriptase that lengthens telomeres by adding GGTTAG repeats to chromosome ends and is upregulated in most human cancers to enable limitless proliferation. Here, we uncover two distinct mechanisms by which naturally occurring oxidized and therapeutic dNTP DNA precursors inhibit telomerase-mediated telomere elongation. We conducted a series of direct telomerase extension assays in the presence of modified dNTPs on various telomeric substrates. We provide direct evidence that telomerase can add the metabolized form of NRTIs, dideoxyadenosine 5’ triphosphate (ddITP) and 3’-azido-3’deoxythymidine triphosphate (AZT-TP), to the telomeric end, causing chain termination. In contrast, telomerase continues elongation after inserting oxidized 2-OH-dATP or the therapeutic 6-thioguanine metabolite, 6-thio-dGTP, but insertion disrupts translocation and inhibits further repeat addition. Kinetics reveal that telomerase poorly selects against 6-thio-dGTP, inserting with similar catalytic efficiency as dGTP. Furthermore, telomerase processivity factor POT1-TPP1 fails to restore processive elongation in the presence of inhibitory dNTPs. These findings reveal mechanisms for targeting telomerase with modified dNTPs in cancer therapy

Mechanistic insights into the roles and activities of polymerases in host and viral replication

Polymerases are vital enzymes in the continuation of life, responsible for the replication of genetic material and the conversion of genetic information to necessary products. A large subset of these polymerases is dedicated to the high-fidelity replication and repair of DNA in the cell cycle of organisms. In addition, viruses utilize polymerases in order to produce DNA or RNA used to synthesize products for virion assembly. With such an important role, polymerases have been a focus in many therapeutic studies of cancer and antiviral treatments. This dissertation focuses on three different polymerases, PrimPol, human immunodeficiency virus (HIV) reverse transcriptase (RT), and DNA polymerase α (Polα). The goal of this work was to understand their overall mechanisms and roles not only in the context of replication and repair, but also in antiviral therapies. HIV treatment, typically referred to as highly active antiretroviral therapy (HAART), consists of drugs that target various enzymes important for viral life cycle. A major fraction of these compounds, which target RT, can be classified into nucleoside (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). One prevailing issue with NRTIs is that administration of these drugs may cause off-target toxicity within patients, affecting adherence to treatment regimens. This off-target toxicity can be attributed to the incorporation of NRTIs by host polymerases, such as the mitochondrial polymerase γ (Polγ). To this end, I investigated the possibility of PrimPol, a recently characterized polymerase, in mediating the mitochondrial toxicity effects seen in HIV+ patients taking tenofovir (TFV)-containing treatments. Using gel-based kinetic assays, I validated that the active metabolite form of tenofovir is a substrate for PrimPol. Cellular-based assays using overexpression and knockdown PrimPol renal cells suggests that PrimPol likely plays a protective role against tenofovir-induced toxicity through its repriming activity, despite the in vitro incorporation evidence. Given this potential role of PrimPol in TFV toxicity, I biochemically assessed a PrimPol active site mutant in an HIV+ patient taking TFV. The mutant appears to have drastically reduced polymerase activity and complete loss of priming activity, which may predispose this patient to TFV toxicity. With NNRTIs, there are continuous development efforts to improve pharmacokinetic properties and combat drug resistance. To this end, a series of 2-naphthyl phenyl ether compounds were developed to target the Y181C mutation of RT. Interestingly, early structures of RT with these class of compounds showed two different binding modes that affected potency against the mutant. By solving structures of 2-naphthyl phenyl ether derivatives with WT and Y181C RT, we determined that the compounds that interact with W229 retain potency against the mutant. These studies will be important to consider in the development process of next generation NNRTIs. Polα, in complex with Primase, is similar to PrimPol by possessing the ability to carry out de novo synthesis of nucleic acid primers. The primary role of the Polα-Pri complex in the primosome is to produce Okazaki fragments during DNA replication in a coordinated manner. Where primase initiates the primer with ribonucleotides, Polα continues the initial primer with deoxyribonucleotides. Interestingly, recent evidence shows that after replication mutations are left over from Polα, which is low-fidelity and lacks a proofreading mechanism. To gain insight on Polα’s activity during replication, we solved the structure of Polα with two replication-like substrates (Polα:dNTP:RNA/DNA or DNA/DNA) and kinetically characterized its activity with these substrates. We observed that with the RNA/DNA structure, a kink in n-4 sugar on the RNA primer correlated to a decrease in activity of the enzyme. Our kinetic characterization also revealed that with the DNA/DNA strand, Polα had increased incorporation efficiency but lower processivity. Our studies provide evidence of how different nucleotide substrates may regulate polymerase activity during replication. Taken together, the studies of three different polymerases presented here provide a mechanistic and functional understanding of these polymerases in diseases and potential treatments. Ultimately, these findings will contribute to the development of therapies in diseases where polymerases play a vital role