Browsing by Author "Chang, Caleb"

Now showing 1 - 6 of 6
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    Early Drug Discovery and Development of Novel Cancer Therapeutics Targeting DNA Polymerase Eta (POLH)
    (Frontiers Media S.A., 2021) Wilson, David M.; Duncton, Matthew A.J.; Chang, Caleb; Lee Luo, Christie; Georgiadis, Taxiarchis M.; Pellicena, Patricia; Deacon, Ashley M.; Gao, Yang; Das, Debanu
    Polymerase eta (or Pol η or POLH) is a specialized DNA polymerase that is able to bypass certain blocking lesions, such as those generated by ultraviolet radiation (UVR) or cisplatin, and is deployed to replication foci for translesion synthesis as part of the DNA damage response (DDR). Inherited defects in the gene encoding POLH (a.k.a., XPV) are associated with the rare, sun-sensitive, cancer-prone disorder, xeroderma pigmentosum, owing to the enzyme’s ability to accurately bypass UVR-induced thymine dimers. In standard-of-care cancer therapies involving platinum-based clinical agents, e.g., cisplatin or oxaliplatin, POLH can bypass platinum-DNA adducts, negating benefits of the treatment and enabling drug resistance. POLH inhibition can sensitize cells to platinum-based chemotherapies, and the polymerase has also been implicated in resistance to nucleoside analogs, such as gemcitabine. POLH overexpression has been linked to the development of chemoresistance in several cancers, including lung, ovarian, and bladder. Co-inhibition of POLH and the ATR serine/threonine kinase, another DDR protein, causes synthetic lethality in a range of cancers, reinforcing that POLH is an emerging target for the development of novel oncology therapeutics. Using a fragment-based drug discovery approach in combination with an optimized crystallization screen, we have solved the first X-ray crystal structures of small novel drug-like compounds, i.e., fragments, bound to POLH, as starting points for the design of POLH inhibitors. The intrinsic molecular resolution afforded by the method can be quickly exploited in fragment growth and elaboration as well as analog scoping and scaffold hopping using medicinal and computational chemistry to advance hits to lead. An initial small round of medicinal chemistry has resulted in inhibitors with a range of functional activity in an in vitro biochemical assay, leading to the rapid identification of an inhibitor to advance to subsequent rounds of chemistry to generate a lead compound. Importantly, our chemical matter is different from the traditional nucleoside analog-based approaches for targeting DNA polymerases.
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    In crystallo observation of three metal ion promoted DNA polymerase misincorporation
    (Springer Nature, 2022) Chang, Caleb; Lee Luo, Christie; Gao, Yang
    Error-free replication of DNA is essential for life. Despite the proofreading capability of several polymerases, intrinsic polymerase fidelity is in general much higher than what base-pairing energies can provide. Although researchers have investigated this long-standing question with kinetics, structural determination, and computational simulations, the structural factors that dictate polymerase fidelity are not fully resolved. Time-resolved crystallography has elucidated correct nucleotide incorporation and established a three-metal-ion-dependent catalytic mechanism for polymerases. Using X-ray time-resolved crystallography, we visualize the complete DNA misincorporation process catalyzed by DNA polymerase η. The resulting molecular snapshots suggest primer 3´-OH alignment mediated by A-site metal ion binding is the key step in substrate discrimination. Moreover, we observe that C-site metal ion binding preceded the nucleotidyl transfer reaction and demonstrate that the C-site metal ion is strictly required for misincorporation. Our results highlight the essential but separate roles of the three metal ions in DNA synthesis.
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    Observing one-divalent-metal-ion-dependent and histidine-promoted His-Me family I-PpoI nuclease catalysis in crystallo
    (eLife Sciences Publications Ltd, 2024) Chang, Caleb; Zhou, Grace; Gao, Yang
    Metal-ion-dependent nucleases play crucial roles in cellular defense and biotechnological applications. Time-resolved crystallography has resolved catalytic details of metal-ion-dependent DNA hydrolysis and synthesis, uncovering the essential roles of multiple metal ions during catalysis. The histidine-metal (His-Me) superfamily nucleases are renowned for binding one divalent metal ion and requiring a conserved histidine to promote catalysis. Many His-Me family nucleases, including homing endonucleases and Cas9 nuclease, have been adapted for biotechnological and biomedical applications. However, it remains unclear how the single metal ion in His-Me nucleases, together with the histidine, promotes water deprotonation, nucleophilic attack, and phosphodiester bond breakage. By observing DNA hydrolysis in crystallo with His-Me I-PpoI nuclease as a model system, we proved that only one divalent metal ion is required during its catalysis. Moreover, we uncovered several possible deprotonation pathways for the nucleophilic water. Interestingly, binding of the single metal ion and water deprotonation are concerted during catalysis. Our results reveal catalytic details of His-Me nucleases, which is distinct from multi-metal-ion-dependent DNA polymerases and nucleases.
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    Primer terminal ribonucleotide alters the active site dynamics of DNA polymerase η and reduces DNA synthesis fidelity
    (Elsevier, 2023) Chang, Caleb; Lee Luo, Christie; Eleraky, Sarah; Lin, Aaron; Zhou, Grace; Gao, Yang
    DNA polymerases catalyze DNA synthesis with high efficiency, which is essential for all life. Extensive kinetic and structural efforts have been executed in exploring mechanisms of DNA polymerases, surrounding their kinetic pathway, catalytic mechanisms, and factors that dictate polymerase fidelity. Recent time-resolved crystallography studies on DNA polymerase η (Pol η) and β have revealed essential transient events during the DNA synthesis reaction, such as mechanisms of primer deprotonation, separated roles of the three metal ions, and conformational changes that disfavor incorporation of the incorrect substrate. DNA-embedded ribonucleotides (rNs) are the most common lesion on DNA and a major threat to genome integrity. While kinetics of rN incorporation has been explored and structural studies have revealed that DNA polymerases have a steric gate that destabilizes ribonucleotide triphosphate binding, the mechanism of extension upon rN addition remains poorly characterized. Using steady-state kinetics, static and time-resolved X-ray crystallography with Pol η as a model system, we showed that the extra hydroxyl group on the primer terminus does alter the dynamics of the polymerase active site as well as the catalysis and fidelity of DNA synthesis. During rN extension, Pol η error incorporation efficiency increases significantly across different sequence contexts. Finally, our systematic structural studies suggest that the rN at the primer end improves primer alignment and reduces barriers in C2′-endo to C3′-endo sugar conformational change. Overall, our work provides further mechanistic insights into the effects of rN incorporation on DNA synthesis.
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    Sugar ring alignment and dynamics underline cytarabine and gemcitabine inhibition on Pol η catalyzed DNA synthesis
    (Elsevier, 2024) Chang, Caleb; Zhou, Grace; Lee Luo, Christie; Eleraky, Sarah; Moradi, Madeline; Gao, Yang
    Nucleoside analogue drugs are pervasively used as antiviral and chemotherapy agents. Cytarabine and gemcitabine are anti-cancer nucleoside analogue drugs that contain C2′ modifications on the sugar ring. Despite carrying all the required functional groups for DNA synthesis, these two compounds inhibit DNA extension once incorporated into DNA. It remains unclear how the C2′ modifications on cytarabine and gemcitabine affect the polymerase active site during substrate binding and DNA extension. Using steady-state kinetics, static and time-resolved X-ray crystallography with DNA polymerase η (Pol η) as a model system, we showed that the sugar ring C2′ chemical groups on cytarabine and gemcitabine snugly fit within the Pol η active site without occluding the steric gate. During DNA extension, Pol η can extend past gemcitabine but with much lower efficiency past cytarabine. The Pol η crystal structures show that the -OH modification in the β direction on cytarabine locks the sugar ring in an unfavorable C2′-endo geometry for product formation. On the other hand, the addition of fluorine atoms on gemcitabine alters the proper conformational transition of the sugar ring for DNA synthesis. Our study illustrates mechanistic insights into chemotherapeutic drug inhibition and resistance and guides future optimization of nucleoside analogue drugs.
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    Understanding the Mechanisms of DNA Polymerases and Nucleases with Time-Resolved X-ray Crystallography
    (2024-04-18) Chang, Caleb; Gao, Yang
    Divalent metal ions, especially Mg2+, play pivotal roles in an enzyme’s ability to manipulate the highly stable structure of DNA. DNA and RNA polymerases, as well as numerous nucleases are clear examples of such enzymes, and are integral to critical cellular functions. Thus, these proteins represent important drug targets for various diseases and biotechnological tools for genome editing. Understanding the molecular mechanism of Mg2+-promoted DNA synthesis and cleavage is crucial for engineering and efficiently targeting of these enzymes. Time-resolved X-ray crystallography enables the visualization of catalytic processes and aids in dissecting catalytic molecular mechanisms. This technique tracks active conformations and intermediate states during catalysis by initiating chemical reactions in protein crystals synchronously via light activation or substrate diffusion. In my thesis work, I applied diffusion-based time-resolved crystallography to investigate two representative enzymes: DNA polymerase η, which is a Y-family DNA polymerase that participates during DNA replication to bypass bulky DNA lesions; and I-PpoI endonuclease, which is one-metal dependent His-Me nuclease that has an active site homologous to the HNH active site of the Cas9 nuclease. In elucidating the catalytic mechanism of DNA polymerases, wild-type and mutant variants of DNA polymerase η were generated and analyzed using kinetic assays. Over 100 crystal structures of DNA polymerase η complexed with a wide variety of deoxyribonucleotides, ribonucleotides, and nucleoside analogue drugs were determined with resolutions ranging from 1.4 to 2.8 Å, providing high-resolution visualization of canonical DNA synthesis and polymerase targeting. The structural alignments revealed the essential role of the third divalent metal ion and the dynamics of the primer end, including the sugar ring, correlated to substrate discrimination and efficient chemistry. Similarly, I tracked the reaction process of I-PpoI with kinetic assays and time-resolved crystallography. More than 40 crystal structures of I-PpoI at various pH and metal ion concentrations were determined. The intermediate structures revealed the involvement of one and only one divalent metal ion in DNA hydrolysis. DNA cleavage assays unveiled several possible deprotonation pathways for the nucleophilic water molecule. Importantly, metal ion binding and water deprotonation were found to be highly correlated during catalysis. These results offer mechanistic insights that can be instrumental in enhancing the bioengineering and targeting of polymerases and nucleases.