Christal D. Sohl

Mechanism of Interaction of Human Mitochondrial DNA Polymerase γ with the Novel Nucleoside Reverse Transcriptase Inhibitor 4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine Indicates a Low Potential for Host Toxicity

ABSTRACT The potent antiretroviral 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is a promising experimental agent for treating HIV infection. Pre-steady-state kinetics were used to characterize the interaction of EFdA-triphosphate (EFdA-TP) with human mitochondrial DNA polymerase γ (Pol γ) to assess the potential for toxicity. Pol γ incorporated EFdA-TP 4,300-fold less efficiently than dATP, with an excision rate similar to ddATP. This strongly indicates EFdA is a poor Pol γ substrate, suggesting minimal Pol γ-mediated toxicity, although this should be examined under clinical settings.

Fluorescence Resonance Energy Transfer Studies of DNA Polymerase β: THE CRITICAL ROLE OF FINGERS DOMAIN MOVEMENTS AND A NOVEL NON-COVALENT STEP DURING NUCLEOTIDE SELECTION*

Background: DNA Pol β participates in base excision repair by choosing correct dNTP to fill single-nucleotide gaps in DNA. Results: Pol β experiences a non-covalent step with correct dNTP selection. Conclusion: Correct and incorrect dNTP incorporation by Pol β are different. Significance: FRET-based system of Pol β elucidates a mechanism of substrate choice necessary for understanding the molecular basis of human disease. During DNA repair, DNA polymerase β (Pol β) is a highly dynamic enzyme that is able to select the correct nucleotide opposite a templating base from a pool of four different deoxynucleoside triphosphates (dNTPs). To gain insight into nucleotide selection, we use a fluorescence resonance energy transfer (FRET)-based system to monitor movement of the Pol β fingers domain during catalysis in the presence of either correct or incorrect dNTPs. By labeling the fingers domain with ((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS) and the DNA substrate with Dabcyl, we are able to observe rapid fingers closing in the presence of correct dNTPs as the IAEDANS comes into contact with a Dabcyl-labeled, one-base gapped DNA. Our findings show that not only do the fingers close after binding to the correct dNTP, but that there is a second conformational change associated with a non-covalent step not previously reported for Pol β. Further analyses suggest that this conformational change corresponds to the binding of the catalytic metal into the polymerase active site. FRET studies with incorrect dNTP result in no changes in fluorescence, indicating that the fingers do not close in the presence of incorrect dNTP. Together, our results show that nucleotide selection initially occurs in an open fingers conformation and that the catalytic pathways of correct and incorrect dNTPs differ from each other. Overall, this study provides new insight into the mechanism of substrate choice by a polymerase that plays a critical role in maintaining genome stability.

Balancing antiviral potency and host toxicity: Identifying a nucleotide inhibitor with an optimal kinetic phenotype for HIV-1 reverse transcriptase

Two novel thymidine analogs, 3′-fluoro-3′-deoxythymidine (FLT) and 2′,3′-didehydro-3′-deoxy-4′-ethynylthymidine (Ed4T), have been investigated as nucleoside reverse transcriptase inhibitors (NRTIs) for treatment of HIV infection. Ed4T seems very promising in phase II clinical trials, whereas toxicity halted FLT development during this phase. To understand these different molecular mechanisms of toxicity, pre–steady-state kinetic studies were used to examine the interactions of FLT and Ed4T with wild-type (WT) human mitochondrial DNA polymerase γ (pol γ), which is often associated with NRTI toxicity, as well as the viral target protein, WT HIV-1 reverse transcriptase (RT). We report that Ed4T-triphosphate (TP) is the first analog to be preferred over native nucleotides by RT but to experience negligible incorporation by WT pol γ, with an ideal balance between high antiretroviral efficacy and minimal host toxicity. WT pol γ could discriminate Ed4T-TP from dTTP 12,000-fold better than RT, with only an 8.3-fold difference in discrimination being seen for FLT-TP. A structurally related NRTI, 2′,3′-didehydro-2′,3′-dideoxythymidine, is the only other analog favored by RT over native nucleotides, but it exhibits only a 13-fold difference (compared with 12,000-fold for Ed4T) in discrimination between the two enzymes. We propose that the 4′-ethynyl group of Ed4T serves as an enzyme selectivity moiety, critical for discernment between RT and WT pol γ. We also show that the pol γ mutation R964C, which predisposes patients to mitochondrial toxicity when receiving 2′,3′-didehydro-2′,3′-dideoxythymidine to treat HIV, produced some loss of discrimination for FLT-TP and Ed4T-TP. These molecular mechanisms of analog incorporation, which are critical for understanding pol γ-related toxicity, shed light on the unique toxicity profiles observed during clinical trials.

Pools and Pols: Mechanism of a mutator phenotype

The maintenance of the human genome is dependent upon several cellular processes including DNA replication. Ordinarily, DNA replication is an exceptionally faithful process, with approximately one error occurring for every 109–1010 nucleotides (1, 2). High-fidelity replicative DNA polymerases with exonucleolytic proofreading activity, along with DNA mismatch repair machinery, are responsible for accurate DNA synthesis. DNA polymerase δ (pol δ) and polymerase e (pol e) are two essential replicative lagging and leading strand polymerases, respectively, that ensure efficient and high-fidelity genome replication (3–5). Replicative polymerase variants have recently been identified in human tumors that harbor enormous numbers of mutations. In recent studies reported in PNAS, Mertz et al. and Williams et al. provide evidence that this hypermutator phenotype results from expansion of deoxyribonucleoside triphosphate (dNTP) pools (6, 7).

Probing the structural and molecular basis of nucleotide selectivity by human mitochondrial DNA polymerase γ

Significance Nucleoside analog reverse transcriptase inhibitors (NRTIs) are the cornerstones of treatment for fighting HIV infection. Unfortunately, they also cause drug toxicity by inhibiting human mitochondrial DNA polymerase (Pol γ). Identification of structural differences between the intended target (RT) and adverse reaction target (Pol γ) will provide critical information for designing more potent drugs with lower toxicity. Here, we reveal structural and mechanistic differences between Pol γ and RT by studying NRTIs that have comparable efficacy on RT but significantly different affinities for Pol γ. We identified critical discriminator residues in Pol γ that are fully responsible for its differential response to emtricitabine. More importantly, the topological equivalent residue in RT is essential for activity, thus identifying this region as a hot-spot for inhibitor design. Nucleoside analog reverse transcriptase inhibitors (NRTIs) are the essential components of highly active antiretroviral (HAART) therapy targeting HIV reverse transcriptase (RT). NRTI triphosphates (NRTI-TP), the biologically active forms, act as chain terminators of viral DNA synthesis. Unfortunately, NRTIs also inhibit human mitochondrial DNA polymerase (Pol γ), causing unwanted mitochondrial toxicity. Understanding the structural and mechanistic differences between Pol γ and RT in response to NRTIs will provide invaluable insight to aid in designing more effective drugs with lower toxicity. The NRTIs emtricitabine [(-)-2,3′-dideoxy-5-fluoro-3′-thiacytidine, (-)-FTC] and lamivudine, [(-)-2,3′-dideoxy-3′-thiacytidine, (-)-3TC] are both potent RT inhibitors, but Pol γ discriminates against (-)-FTC-TP by two orders of magnitude better than (-)-3TC-TP. Furthermore, although (-)-FTC-TP is only slightly more potent against HIV RT than its enantiomer (+)-FTC-TP, it is discriminated by human Pol γ four orders of magnitude more efficiently than (+)-FTC-TP. As a result, (-)-FTC is a much less toxic NRTI. Here, we present the structural and kinetic basis for this striking difference by identifying the discriminator residues of drug selectivity in both viral and human enzymes responsible for substrate selection and inhibitor specificity. For the first time, to our knowledge, this work illuminates the mechanism of (-)-FTC-TP differential selectivity and provides a structural scaffold for development of novel NRTIs with lower toxicity.

Mutations in human DNA polymerase γ confer unique mechanisms of catalytic deficiency that mirror the disease severity in mitochondrial disorder patients

Human mitochondrial DNA polymerase γ (pol γ) is solely responsible for the replication and repair of the mitochondrial genome. Unsurprisingly, alterations in pol γ activity have been associated with mitochondrial diseases such as Alpers syndrome and progressive external ophthalmoplegia. Thus far, predicting the severity of mitochondrial disease based the magnitude of deficiency in pol γ activity has been difficult. In order to understand the relationship between disease severity in patients and enzymatic defects in vitro, we characterized the molecular mechanisms of four pol γ mutations, A957P, A957S, R1096C and R1096H, which have been found in patients suffering from aggressive Alpers syndrome to mild progressive external ophthalmoplegia. The A957P mutant showed the most striking deficiencies in the incorporation efficiency of a correct deoxyribonucleotide triphosphate (dNTP) relative to wild-type pol γ, with less, but still significant incorporation efficiency defects seen in R1096H and R1096C, and only a small decrease in incorporation efficiency observed for A957S. Importantly, this trend matches the disease severity observed in patients very well (approximated as A957P ≫ R1096C ≥ R1096H ≫ A957S, from most severe disease to least severe). Further, the A957P mutation conferred a two orders of magnitude loss of fidelity relative to wild-type pol γ, indicating that a buildup of mitochondrial genomic mutations may contribute to the death in infancy seen with these patients. We conclude that characterizing the unique molecular mechanisms of pol γ deficiency for physiologically important mutant enzymes is important for understanding mitochondrial disease and for predicting disease severity.

Erratum: Probing the structural and molecular basis of nucleotide selectivity by human mitochondrial DNA polymerase γ (Proceedings of the National Academy of Sciences of the United States of America (2015) 112 (8596-8601) DOI: 10.1073/pnas.1421733112)

BIOCHEMISTRY Correction for “Probing the structural and molecular basis of nucleotide selectivity by human mitochondrial DNA polymerase γ,” by Christal D. Sohl, Michal R. Szymanski, Andrea C. Mislak, Christie K. Shumate, Sheida Amiralaei, Raymond F. Schinazi, Karen S. Anderson, and Y. Whitney Yin, which appeared in issue 28, July 14, 2015, of Proc Natl Acad Sci USA (112:8596–8601; first published June 29, 2015; 10.1073/pnas.1421733112). The authors note that Fig. 5 appeared incorrectly. The corrected figure and its legend appear below.

Abstract 2436: Understanding the molecular mechanism of targeted kinase inhibitor resistance mediated by the FGFR1 gatekeeper mutation

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Human fibroblast growth factor receptors (FGFRs) can serve as drivers of oncogenesis and are a target of several inhibitors in clinical trials. Unfortunately, resistance severely hampers the long-term success of tyrosine kinase inhibitors (TKIs), with mutations at the gatekeeper residue often serving as an early means of resistance to cancer therapy, resulting in tumor progression. Here we show the first crystal structures of the FGFR1 gatekeeper mutation, V561M FGFR1, and use kinetic and structural methods to explore the mechanisms of the catalytic activation we observe for this mutation. We show that while the FGFR1 gatekeeper mutation confers resistance to two inhibitors currently in clinical trials, the extent of change in efficacy differ, indicating two unique resistance profiles. We show that some affinity for one inhibitor is maintained by the V561M mutation due to a flexible linker that allows multiple binding conformations. This is the first example showing the same inhibitor binding in unique ways to the WT and gatekeeper mutant forms of FGFR, highlighting some regions of drug design flexibility both within the binding pocket and in the linker of the inhibitor. Identifying this flexibility in the context of the structural features of an active conformation of V561M FGFR1, the form most kinase inhibitors target, will provide critical insights for designing future inhibitors effective against the FGFR gatekeeper mutations. Citation Format: Christal D. Sohl, Molly Ryan, BeiBei Luo, Kathleen Frey, Karen S. Anderson. Understanding the molecular mechanism of targeted kinase inhibitor resistance mediated by the FGFR1 gatekeeper mutation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2436. doi:10.1158/1538-7445.AM2015-2436