Browsing by Subject "Enzymology"

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    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.
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    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-Ting
    ALDH 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.