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Item A New Drug Discovery Platform: Application to DNA Polymerase Eta and Apurinic/Apyrimidinic Endonuclease 1(MDPI, 2023-11-23) Das, Debanu; Duncton, Matthew A. J.; Georgiadis, Taxiarchis M.; Pellicena, Patricia; Clark, Jennifer; Sobol, Robert W.; Georgiadis, Millie M.; King-Underwood, John; Jobes, David V.; Chang, Caleb; Gao, Yang; Deacon, Ashley M.; Wilson, David M., III; Biochemistry and Molecular Biology, School of MedicineThe ability to quickly discover reliable hits from screening and rapidly convert them into lead compounds, which can be verified in functional assays, is central to drug discovery. The expedited validation of novel targets and the identification of modulators to advance to preclinical studies can significantly increase drug development success. Our SaXPyTM (“SAR by X-ray Poses Quickly”) platform, which is applicable to any X-ray crystallography-enabled drug target, couples the established methods of protein X-ray crystallography and fragment-based drug discovery (FBDD) with advanced computational and medicinal chemistry to deliver small molecule modulators or targeted protein degradation ligands in a short timeframe. Our approach, especially for elusive or “undruggable” targets, allows for (i) hit generation; (ii) the mapping of protein–ligand interactions; (iii) the assessment of target ligandability; (iv) the discovery of novel and potential allosteric binding sites; and (v) hit-to-lead execution. These advances inform chemical tractability and downstream biology and generate novel intellectual property. We describe here the application of SaXPy in the discovery and development of DNA damage response inhibitors against DNA polymerase eta (Pol η or POLH) and apurinic/apyrimidinic endonuclease 1 (APE1 or APEX1). Notably, our SaXPy platform allowed us to solve the first crystal structures of these proteins bound to small molecules and to discover novel binding sites for each target.Item Building better polymerases: Engineering the replication of expanded genetic alphabets(Elsevier, 2020-12-11) Ouaray, Zahra; Benner, Steven A.; Georgiadis, Millie M.; Richards, Nigel G. J.; Biochemistry and Molecular Biology, School of MedicineDNA polymerases are today used throughout scientific research, biotechnology, and medicine, in part for their ability to interact with unnatural forms of DNA created by synthetic biologists. Here especially, natural DNA polymerases often do not have the "performance specifications" needed for transformative technologies. This creates a need for science-guided rational (or semi-rational) engineering to identify variants that replicate unnatural base pairs (UBPs), unnatural backbones, tags, or other evolutionarily novel features of unnatural DNA. In this review, we provide a brief overview of the chemistry and properties of replicative DNA polymerases and their evolved variants, focusing on the Klenow fragment of Taq DNA polymerase (Klentaq). We describe comparative structural, enzymatic, and molecular dynamics studies of WT and Klentaq variants, complexed with natural or noncanonical substrates. Combining these methods provides insight into how specific amino acid substitutions distant from the active site in a Klentaq DNA polymerase variant (ZP Klentaq) contribute to its ability to replicate UBPs with improved efficiency compared with Klentaq. This approach can therefore serve to guide any future rational engineering of replicative DNA polymerases.Item Development of selective inhibitors for human aldehyde dehydrogenase 3A1 (ALDH3A1) for the enhancement of cyclophosphamide cytotoxicity(Wiley, 2014-03) Parajuli, Bibek; Georgiadis, Taxiarchis M.; Fishel, Melissa L.; Hurley, Thomas D.; Biochemistry & Molecular Biology, School of MedicineAldehyde dehydrogenase 3A1 (ALDH3A1) plays an important role in many cellular oxidative processes, including cancer chemoresistance, by metabolizing activated forms of oxazaphosphorine drugs such as cyclophosphamide (CP) and its analogues, such as mafosfamide (MF), ifosfamide (IFM), and 4-hydroperoxycyclophosphamide (4-HPCP). Compounds that can selectively target ALDH3A1 could permit delineation of its roles in these processes and could restore chemosensitivity in cancer cells that express this isoenzyme. Here we report the detailed kinetic and structural characterization of an ALDH3A1-selective inhibitor, CB29, previously identified in a high-throughput screen. Kinetic and crystallographic studies demonstrate that CB29 binds within the aldehyde substrate-binding site of ALDH3A1. Cellular proliferation of ALDH3A1-expressing lung adenocarcinoma (A549) and glioblastoma (SF767) cell lines, as well as ALDH3A1 non-expressing lung fibroblast (CCD-13Lu) cells, is unaffected by treatment with CB29 and its analogues alone. However, sensitivity toward the anti-proliferative effects of mafosfamide is enhanced by treatment with CB29 and its analogue in the tumor cells. In contrast, the sensitivity of CCD-13Lu cells toward mafosfamide was unaffected by the addition of these same compounds. CB29 is chemically distinct from the previously reported small-molecule inhibitors of ALDH isoenzymes and does not inhibit ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, or ALDH2 isoenzymes at concentrations up to 250 μM. Thus, CB29 is a novel small molecule inhibitor of ALDH3A1, which might be useful as a chemical tool to delineate the role of ALDH3A1 in numerous metabolic pathways, including sensitizing ALDH3A1-positive cancer cells to oxazaphosphorines.Item Discovery and characterization of small molecule inhibitors of the aldehyde dehydrogenase 1/2 family(2016-12) Buchman, Cameron D.; Hurley, Thomas D.; Elmendorf, Jeffrey S.; Hoang, Quyen; Wek, Ronald C.The human aldehyde dehydrogenase (ALDH) superfamily consists of 19 isoenzymes that are critical for normal physiology as well as the removal of toxic aldehydes. Members of the ALDH1/2 family have vital roles in cell signaling during early development, ethanol metabolism, and the removal of aldehydes derived from oxidative stress. We sought to develop selective compounds toward ALDH2 to help determine its individual contribution to biological function, as many of the ALDH1/2 family possess overlapping substrate preferences. A high-throughput screen of over 100,000 compounds uncovered a class of aromatic lactones which inhibit the ALDH1/2 enzyme family. The lactones were then characterized using a combination of enzyme kinetics, X-ray crystallography, and cell culture experiments. We found that many of the lactones are over ten times more potent toward ALDH2 than daidzin, a previously described ALDH2 inhibitor. Our ability to produce many more ALDH isoenzymes allowed us to determine that daidzin is not as selective as previously believed, inhibiting ALDH2, ALDH1B1, and ALDH1A2 with equal potency. This inhibition pattern was seen with several of the aromatic lactones as well. Structural studies show that many of the lactones bind between key aromatic residues in the ALDH1/2 enzyme substrate-binding sites. One lactone in particular mimics the position of an aldehyde substrate and alters the position of the catalytic cysteine to interfere with the productive binding of NAD+ for enzyme catalysis. Further characterization of related compounds led to the realization that the mechanism of inhibition, potency, and selectivity differs amongst the lactones based off the substituents on the aromatic scaffold and its precise binding location. Two of these compounds were found to be selective for one of the ALDH1/2 family members, BUC22, selective for ALDH1A1, and BUC27, selective for ALDH2. BUC22 demonstrates ten-fold selectivity for ALDH1A1 over ALDH1A2 and does not inhibit the remaining ALDH1/2 enzymes. Additionally, treatment with BUC22 led to decreased growth of triple-negative breast cancer cells in culture. BUC27 inhibits ALDH2 with the same potency as daidzin. Both BUC22 and BUC27 could be further developed to use as chemical tools to better understand the functional roles of ALDH1A1 and ALDH2 in biological systems.Item Discovery, Characterization, and Development of Small Molecule Inhibitors of Glycogen Synthase(2020-06) Tang, Buyun; Hurley, Thomas D.; Roach, Peter J.; Georgiadis, Millie M.; Johnson, Steven M.; Elmendorf, Jeffrey S.The over-accumulation of glycogen appears as a hallmark in various glycogen storage diseases (GSDs), including Pompe, Cori, Andersen, and Lafora disease. Glycogen synthase (GS) is the rate-limiting enzyme for glycogen synthesis. Recent evidence suggests that suppression of glycogen accumulation represents a potential therapeutic approach for treating these diseases. Herein, we describe the discovery, characterization, and development of small molecule inhibitors of GS through a multicomponent study including biochemical, biophysical, and cellular assays. Adopting an affinity-based fluorescence polarization assay, we identified a substituted imidazole molecule (H23), as a first-in-class inhibitor of yeast glycogen synthase 2 (yGsy2) from the 50,000 ChemBridge DIVERSet library. Structural data derived from X-ray crystallography at 2.85 Å, and enzyme kinetic data, revealed that H23 bound within the uridine diphosphate glucose binding pocket of yGsy2. Medicinal chemistry efforts examining over 500 H23 analogs produced structure-activity relationship (SAR) profiles that led to the identification of potent pyrazole and isoflavone compounds with low micromolar potency against human glycogen synthase 1 (hGYS1). Notably, several of the isoflavones demonstrated cellular efficacy toward suppressing glycogen accumulation. In an alternative effort to screen inhibitors directly against human GS, an activity-based assay was designed using a two-step colorimetric approach. This assay led to the identification of compounds with submicromolar potency to hGYS1 from a chemical library comprised of 10,000 compounds. One of the hit molecules, hexachlorophene, was crystallized bound to the active site of yGsy2. The structure was determined to 3.15 Å. Additional kinetic, mutagenic, and SAR studies validated the binding of hexachlorophene in the catalytic pocket and its non-competitive mode of inhibition. In summary, these two novel assays provided feasible biochemical platforms for large-scale screening of small molecule modulators of GS. The newly-developed, potent analogs possess diverse promising scaffolds for drug development efforts targeting GS activity in GSDs associated with excess glycogen accumulation.Item High-resolution crystal structure of human asparagine synthetase enables analysis of inhibitor binding and selectivity(Springer Nature, 2019-09-17) Zhu, Wen; Radadiya, Ashish; Bisson, Claudine; Wenzel, Sabine; Nordin, Brian E.; Martínez-Márquez, Francisco; Imasaki, Tsuyoshi; Sedelnikova, Svetlana E.; Coricello, Adriana; Baumann, Patrick; Berry, Alexandria H.; Nomanbhoy, Tyzoon K.; Kozarich, John W.; Jin, Yi; Rice, David W.; Takagi, Yuichiro; Richards, Nigel G. J.; Biochemistry and Molecular Biology, School of MedicineExpression of human asparagine synthetase (ASNS) promotes metastatic progression and tumor cell invasiveness in colorectal and breast cancer, presumably by altering cellular levels of L-asparagine. Human ASNS is therefore emerging as a bona fide drug target for cancer therapy. Here we show that a slow-onset, tight binding inhibitor, which exhibits nanomolar affinity for human ASNS in vitro, exhibits excellent selectivity at 10 μM concentration in HCT-116 cell lysates with almost no off-target binding. The high-resolution (1.85 Å) crystal structure of human ASNS has enabled us to identify a cluster of negatively charged side chains in the synthetase domain that plays a key role in inhibitor binding. Comparing this structure with those of evolutionarily related AMP-forming enzymes provides insights into intermolecular interactions that give rise to the observed binding selectivity. Our findings demonstrate the feasibility of developing second generation human ASNS inhibitors as lead compounds for the discovery of drugs against metastasis.Item Identification and characterization of small-molecule inhibitors of aldehyde dehydrogenase 1A1(2015-01) Morgan, Cynthia A.; Hurley, Thomas D., 1961-; Georgiadis, Millie M.; Harrington, Maureen A.; Sullivan, William J.The human genome encodes 19 members of the aldehyde dehydrogenase (ALDH) superfamily, critical enzymes involved in the metabolism of aldehyde substrates. A major function of the ALDH1A subfamily is the oxidation of retinaldehyde to retinoic acid, a key regulator of numerous cell growth and differentiation pathways. ALDH1A1 has been identified as a biomarker for both normal stem cells and cancer stem cells. Small molecule probes are needed to better understand the role of this enzyme in both normal and disease states. However, there are no commercially available, small molecules that selectively inhibit ALDH1A1. Our goal is to identify and characterize small molecule inhibitors of ALDH1A1 as chemical tools and as potential therapeutics. To better understand the basis for selective inhibition of ALDH1A1, we characterized N,N-diethylaminobenzaldehyde (DEAB), which is a commonly used inhibitor of ALDH1A1 and purported to be selective. DEAB serves as the negative control for the Aldefluor assay widely utilized to identify stem cells. Rather than being a selective inhibitor for ALDH1A1, we found that DEAB is a slow substrate for multiple ALDH isoenzymes, and depending on the rate of turnover, DEAB behaves as either a traditional substrate or as an inhibitor. Due to its very slow turnover, DEAB is a potent inhibitor of ALDH1A1 with respect to propionaldehyde oxidation, but it is not a good candidate for the development of selective ALDH1A1 inhibitors because of its promiscuity. Next, to discover novel selective inhibitors, we used an in vitro, high-throughput screen of 64,000 compounds to identify 256 hits that either activate or inhibit ALDH1A1 activity. We have characterized two structural classes of compounds, CM026 and CM037, using enzyme kinetics and X-ray crystallographic structural data. Both classes contained potent and selective inhibitors for ALDH1A1. Structural studies of ALDH1A1 with CM026 showed that CM026 binds at the active site, and its selectivity is achieved by a single residue substitution. Importantly, CM037 selectively inhibits proliferation of ALDH+ ovarian cancer cells. The discovery of these two selective classes of ALDH1A1 inhibitors may be useful in delineating the role of ALDH1A1 in biological processes and may seed the development of new chemotherapeutic agents.Item Identification, kinetic and structural characterization of small molecule inhibitors of aldehyde dehydrogenase 3a1 (Aldh3a1) as an adjuvant therapy for reversing cancer chemo-resistance(2013-10) Parajuli, Bibek; Hurley, Thomas D., 1961-; Zhang, Zhong-Yin; Georgiadis, Millie M.; Zhang, Jian-TingALDH isoenzymes are known to impact the sensitivity of certain neoplastic cells toward cyclophosphamides and its analogs. Despite its bone marrow toxicity, cyclophos-phamide is still used to treat various recalcitrant forms of cancer. When activated, cyclo-phosphamide forms aldophosphamide that can spontaneously form the toxic phospho-ramide mustard, an alkylating agent unless detoxified by ALDH isozymes to the carbox-yphosphamide metabolite. Prior work has demonstrated that the ALDH1A1 and ALDH3A1 isoenzymes can convert aldophosphamide to carboxyphosphamide. This has also been verified by over expression and siRNA knockdown studies. Selective small molecule inhibitors for these ALDH isoenzymes are not currently available. We hypothe-sized that novel and selective small molecule inhibitors of ALDH3A1 would enhance cancer cells’ sensitivity toward cyclophosphamide. If successful, this approach can widen the therapeutic treatment window for cyclophosphamides; permitting lower effective dos-ing regimens with reduced toxicity. An esterase based absorbance assay was optimized in a high throughput setting and 101, 000 compounds were screened and two new selective inhibitors for ALDH3A1, which have IC50 values of 0.2 µM (CB7) and 16 µM (CB29) were discovered. These two compounds compete for aldehyde binding, which was vali-dated both by kinetic and crystallographic studies. Structure activity relationship dataset has helped us determine the basis of potency and selectivity of these compounds towards ALDH3A1 activity. Our data is further supported by mafosfamide (an analog of cyclo-phosphamide) chemosensitivity data, performed on lung adenocarcinoma (A549) and gli-oblastoma (SF767) cell lines. Overall, I have identified two compounds, which inhibit ALDH3A1’s dehydrogenase activity selectively and increases sensitization of ALDH3A1 positive cells to aldophosphamide and its analogs. This may have the potential in improving chemotherapeutic efficacy of cyclophosphamide as well as to help us understand better the role of ALDH3A1 in cells. Future work will focus on testing these compounds on other cancer cell lines that involve ALDH3A1 expression as a mode of chemoresistance.Item Induced magnetoelectric coupling at a ferroelectric-ferromagnetic interface(2013-11-08) Carvell, Jeffrey David; Ruihua, Cheng; Joglekar, Yogesh; Decca, Ricardo; Petrache, Horia; Hu, JiangpingPreparation and characterization of multiferroic materials in which ferroelectricity and ferromagnetism coexist would be a milestone for functionalized materials and devices. First, electric properties of polyvinylidene (PVDF) films fabricated using the Langmuir-Schaefer method have been studied. Films of different thickness were deposited on silicon substrates and analyzed using several techniques. X-ray diffraction (XRD) data showed that PVDF films crystallize at an annealing temperature above 130 °C. Polarization versus electric field (PE) ferroelectric measurements were done for samples prepared with electrodes. PE measurements show that the coercivity of the films increases as the maximum applied electric field increases. The coercivity dependence on the frequency of the applied electric field can be fitted as . The results also show that the coercivity decreases with increasing the thickness of PVDF film due to the pinning effect. Next, we have demonstrated that those PVDF properties can be controlled by applying an external magnetic field. Samples were created in a layered heterostructure, starting with a Fe thin film, PVDF above that, and followed by another thin film of Fe. Extended X-ray absorption fine structure (EXAFS) spectroscopy was used to study the interface between PVDF polymer films and ferromagnetic iron thin films. Conventional EXAFS was applied to identify the structure of a Fe film sandwiched between two PVDF layers. An electric signal was then applied to the polymer to study the effects polarizing the polymer has on the Fe atoms at the interface. This shows that the Fe atoms diffuse into the PVDF layer at the interface between the two layers. Polarizing the film causes further diffusion of Fe atoms into the polymer. We also found that as the applied magnetic field is changed, the switching of electric polarization for the PVDF displayed a dependence on the external magnetic field. We also noticed that both the coercivity and polarization for the PVDF polymer display hysteretic features as the applied magnetic field is changed. We also found that the thickness of both the iron layers and the PVDF layer has an effect on the magnetoelectric coupling in our samples. The same strain applied to a thicker PVDF layer becomes tougher to flip the polarization compared to a thinner PVDF layer. As the iron film thickness increases, the strain also increases, and the polarization of the PVDF polymer is more easily flipped. We also found that the magnetoelectric sensitivity increases as both the PVDF and iron layers increase in thickness. We have shown that it is possible to control the ferroelectric properties of a PVDF film by tuning the magnetic field in a heterostructure. Our experiments show a coupling between the electric polarization and applied magnetic field in multiferroic heterostructures much larger than any previously reported values. Previous reports have used inorganic materials for the ferroelectric layer. Organic polymers have an electric dipole originating at the molecular level due to atoms with different electronegativity that are free to rotate. To flip the polarization, the chains must rotate and the position of the atoms must change. This increases the force felt locally by those chains. Using this polymer, we are able to increase the magnetoelectric coupling.Item Mechanistic and Structural Insight to an Evolved Benzoylformate Decarboxylase with Enhanced Pyruvate Decarboxylase Activity(MDPI, 2016-12) Andrews, Forest H.; Wechsler, Cindy; Rogers, Megan P.; Meyer, Danilo; Tittmann, Kai; McLeish, Michael J.; Department of Chemistry and Chemical Biology, School of ScienceBenzoylformate decarboxylase (BFDC) and pyruvate decarboxylase (PDC) are thiamin diphosphate-dependent enzymes that share some structural and mechanistic similarities. Both enzymes catalyze the nonoxidative decarboxylation of 2-keto acids, yet differ considerably in their substrate specificity. In particular, the BFDC from P. putida exhibits very limited activity with pyruvate, whereas the PDCs from S. cerevisiae or from Z. mobilis show virtually no activity with benzoylformate (phenylglyoxylate). Previously, saturation mutagenesis was used to generate the BFDC T377L/A460Y variant, which exhibited a greater than 10,000-fold increase in pyruvate/benzoylformate substrate utilization ratio compared to that of wtBFDC. Much of this change could be attributed to an improvement in the Km value for pyruvate and, concomitantly, a decrease in the kcat value for benzoylformate. However, the steady-state data did not provide any details about changes in individual catalytic steps. To gain insight into the changes in conversion rates of pyruvate and benzoylformate to acetaldehyde and benzaldehyde, respectively, by the BFDC T377L/A460Y variant, reaction intermediates of both substrates were analyzed by NMR and microscopic rate constants for the elementary catalytic steps were calculated. Herein we also report the high resolution X-ray structure of the BFDC T377L/A460Y variant, which provides context for the observed changes in substrate specificity.