Indiana University-Purdue University Indianapolis (IUPUI) / 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. / 2021-07-01

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<p>The current rise in multi-drug resistant Gram-negative bacterial infections is of particularconcern. Gram-negative pathogens are difficult to treat due to their intrinsic resistome.The outer membrane (OM) of Gram-negative bacteria serves as a permeability barrier tomany antibiotics, due in large part to the lipopolysaccharide (LPS) component that isunique to these organisms, and in addition to, the OM is lined with a number of multidrugresistant efflux pumps. As the clinical effectiveness of first line therapies declines inthe face of this resistance, novel strategies to discover new antibiotics are required. Theidentification of new antibiotic targets is one method currently being applied to meet thischallenge. This work examines the permeability barrier of Escherichia coli as a possibletarget for antibiotic adjuvants. A structure-function analysis of GmhA and GmhB, whichcatalyze the first and third conserved steps in LPS ADP-heptose biosynthesis, wasperformed. The active site residues of each of these enzymes were identified viacrystallographic, mutagenic, and kinetic analyses. Potential mechanisms have beenproposed, offering insight into the function of these potential adjuvant targets. In addition,a whole screen of E. coli was performed to identify compounds that potentiatenovobiocin, an antibiotic with limited activity against Gram-negative pathogens due toOM permeability. Four small molecules were found that were able to synergize withnovobiocin. One of these, A22, is known to alter bacterial cell shape, suggesting a newpathway for antibiotic adjuvants to combat Gram-negative infection. Together, thesestudies highlight the varied targets available for novel therapeutic strategies.</p> / Doctor of Philosophy (PhD)

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No / A central theme within the concept of enzyme-directed bioreductive drug development is the potential to predict tumour response based on the profiling of enzymes involved in the bioreductive activation process. Mitomycin C (MMC) is the prototypical bioreductive drug that is reduced to active intermediates by several reductases including NAD(P)H:quinone oxidoreductase (NQO1) and NADPH cytochrome P450 reductase (P450R). The purpose of our study was to determine whether NQO1 and P450R protein expression in a panel of low-grade, human superficial bladder tumours correlates with clinical response to MMC. A retrospective clinical study was conducted in which the response to MMC of 92 bladder cancer patients was compared to the immunohistochemical expression of NQO1 and P450R protein in archived paraffin-embedded bladder tumour specimens. A broad spectrum of NQO1 protein levels exists in bladder tumours between individual patients, ranging from intense to no immunohistochemical staining. In contrast, levels of P450R were similar with most tumours having moderate to high levels. All patients were chemotherapy naïve prior to receiving MMC and clinical response was defined as the time to first recurrence. A poor correlation exists between clinical response and NQO1, P450R or the expression patterns of various combinations of the 2 proteins. The results of our study demonstrate that the clinical response of superficial bladder cancers to MMC cannot be predicted on the basis of NQO1 and/or P450R protein expression and suggest that other factors (other reductases or post DNA damage events) have a significant bearing on tumour response.

As L-asparaginases bacterianas (L-asparaginase amidohidrolase, E.C.3.S.1.1) são enzimas de alto valor terapêutico devido ao seu uso no tratamento de leucemia linfocítica aguda. A L-asparaginase da Escherichia coli por ser uma enzima periplásmica com alta afinidade, é particularmente efetiva em certas terapias de câncer infantil. Muitos agentes terapêuticos recentes são proteínas e peptídeos que surgiram do design molecular de drogas e tecnologia de DNA recombinante. Numerosos estudos têm demonstrado que aditivos preservam a estrutura e a atividade biológica de cada molécula destas proteínas. Entretanto, o mecanismo de proteção, pelo qual estes aditivos funcionam, não tem sido totalmente elucidado. O objetivo do presente trabalho é investigar detalhadamente o efeito da liofilização sobre a estrutura e a atividade enzimática da L-asparaginase, tanto em células íntegras de Escherichia coli, como na enzima purificada. Até recentemente a maneira para se avaliar o comportamento de um aditivo e o comportamento da água na estabilização de uma proteína durante a liofilização consistia na medida dos parâmetros de atividade após a reidratação, porém atualmente modernas técnicas de Ressonância Magnética Nuclear (RMN) de baixa resolução são utilizadas para se entender o comportamento da água nas interações com proteínas. E a Espectroscopia de Infravermelho por Transformada de Fourier apresenta grande potencial no estudo de estabilização de proteínas durante a liofilização. Utilizou-se o cálculo de porcentagem de retenção de atividade para expressar os valores de atividade enzimática estudados. Estes cálculos foram realizados para os sistemas congelados e para os sistemas congelados e liofilizados. Os sistemas foram tratados em velocidades de congelamento diferentes (20°C/min, SOC/min e 2°C/min), sendo em seguida liofilizados por 24 horas. São apresentados resultados sobre o efeito das diferentes velocidades de congelamento e o efeito da liofilização nos diferentes sistemas aditivo-enzima e aditivo-célula. Utilizaram-se os aditivos: sacarose, maltose, lactose, inositol, manitol e trealose testados em diferentes concentrações (30, 90 e 150mM). Para identificar quais as condições e os aditivos que apresentaram uma crioproteção satisfatória. Nos sistemas maltose-enzima e trealose-enzima observou-se um aumento da crioproteção com o aumento da concentração de aditivo. Para os sistemas maltose-enzima, congelados lentamente, os resultados de retenção de atividade foram: 8,67%, 14,02% e 30,80% respectivamente para 30, 90 e 150mM. O sistema enzima-maltose (150mM) congelado rapidamente e liofilizado apresentou a maior retenção de atividade (111,11 %) e também o maior valor de T2 (81&#181;s) nos resultados referentes a RMN. Nos sistemas trealose-enzima nas concentrações de 90 e 150mM apresentaram retenção de atividade 89,93% e 79,74%, respectivamente. / Bacterial L-asparaginase (L-asparaginase amidohydrolase, E.C. 3.5.1.1) are enzymes of high therapeutic value due to their use in the treatment of lymphocytic acute leukemia. Escherichia coli L-asparaginase is a periplasmic enzyme of high affinity, particularly effective in some kinds of childhood cancer therapies. Several studies have showed that there are specific stabilizing additives preserve the structure and the biological activity of protein molecules (lyoprotectant). However, the protection mechanism for these excipients has not been totally elucidated yet. The aim of this work was investigate the effect of freeze-drying on the enzymatic activity of L-asparaginase using both the purified enzyme as well as intact cells of Escherichia coli. Until recently the way to evaluate the behavior of an addictive one and the behavior of the water in the stabilization of a protein during the freeze-drying consisted of the measure of the activity parameters after the reidratação, even so now modem techniques of the Nuclear Magnetic Resonance of low resolution have been used to understand the behavior of the water in the interactions with proteins. It is Infrared Spectroscopy for Fourier Transformed it presents a great potential in the study of stabilization of proteins during the freeze-drying. The calculation of percentage of activity retention was used to express the studied values of enzymatic activity. These calculations were accomplished for the frozen systems and for the frozen systems and freeze-dryied. The systems were treated in three speeds of different freezing (20°C/min, 5°C/min and 2°C/min), being the freeze-drying for 24 hours. Results are presented on the effect of the freeze-drying in the different systems addictive-enzyme and addictive-cell. The addictive ones were used: sucrose, maltose, lactose, inositol, manitol and trehalose tested in different concentrations (30, 90 and 150mM), to identify which the conditions and the addictive ones that presented a satisfactory cryoprotection. For the systems enzyme-maltose, frozen slowly, the results of activity retention were: 8,67%, 14,02% e 30,80% to 30,90 e 150mM,respectively . The system enzyme-maltose (150mM) frozen quickly and freeze-dryied presented the largest activity retention (111,11 %) and also the largest value of T2 (81 &#181;s) in the referring results NMR. The systems enzyme-trealose in the concentrations of 90 and 150mM they presented retention of activity 89,93% and 79,74%, respectively.

As L-asparaginases bacterianas (L-asparaginase amidohidrolase, E.C.3.S.1.1) são enzimas de alto valor terapêutico devido ao seu uso no tratamento de leucemia linfocítica aguda. A L-asparaginase da Escherichia coli por ser uma enzima periplásmica com alta afinidade, é particularmente efetiva em certas terapias de câncer infantil. Muitos agentes terapêuticos recentes são proteínas e peptídeos que surgiram do design molecular de drogas e tecnologia de DNA recombinante. Numerosos estudos têm demonstrado que aditivos preservam a estrutura e a atividade biológica de cada molécula destas proteínas. Entretanto, o mecanismo de proteção, pelo qual estes aditivos funcionam, não tem sido totalmente elucidado. O objetivo do presente trabalho é investigar detalhadamente o efeito da liofilização sobre a estrutura e a atividade enzimática da L-asparaginase, tanto em células íntegras de Escherichia coli, como na enzima purificada. Até recentemente a maneira para se avaliar o comportamento de um aditivo e o comportamento da água na estabilização de uma proteína durante a liofilização consistia na medida dos parâmetros de atividade após a reidratação, porém atualmente modernas técnicas de Ressonância Magnética Nuclear (RMN) de baixa resolução são utilizadas para se entender o comportamento da água nas interações com proteínas. E a Espectroscopia de Infravermelho por Transformada de Fourier apresenta grande potencial no estudo de estabilização de proteínas durante a liofilização. Utilizou-se o cálculo de porcentagem de retenção de atividade para expressar os valores de atividade enzimática estudados. Estes cálculos foram realizados para os sistemas congelados e para os sistemas congelados e liofilizados. Os sistemas foram tratados em velocidades de congelamento diferentes (20°C/min, SOC/min e 2°C/min), sendo em seguida liofilizados por 24 horas. São apresentados resultados sobre o efeito das diferentes velocidades de congelamento e o efeito da liofilização nos diferentes sistemas aditivo-enzima e aditivo-célula. Utilizaram-se os aditivos: sacarose, maltose, lactose, inositol, manitol e trealose testados em diferentes concentrações (30, 90 e 150mM). Para identificar quais as condições e os aditivos que apresentaram uma crioproteção satisfatória. Nos sistemas maltose-enzima e trealose-enzima observou-se um aumento da crioproteção com o aumento da concentração de aditivo. Para os sistemas maltose-enzima, congelados lentamente, os resultados de retenção de atividade foram: 8,67%, 14,02% e 30,80% respectivamente para 30, 90 e 150mM. O sistema enzima-maltose (150mM) congelado rapidamente e liofilizado apresentou a maior retenção de atividade (111,11 %) e também o maior valor de T2 (81&#181;s) nos resultados referentes a RMN. Nos sistemas trealose-enzima nas concentrações de 90 e 150mM apresentaram retenção de atividade 89,93% e 79,74%, respectivamente. / Bacterial L-asparaginase (L-asparaginase amidohydrolase, E.C. 3.5.1.1) are enzymes of high therapeutic value due to their use in the treatment of lymphocytic acute leukemia. Escherichia coli L-asparaginase is a periplasmic enzyme of high affinity, particularly effective in some kinds of childhood cancer therapies. Several studies have showed that there are specific stabilizing additives preserve the structure and the biological activity of protein molecules (lyoprotectant). However, the protection mechanism for these excipients has not been totally elucidated yet. The aim of this work was investigate the effect of freeze-drying on the enzymatic activity of L-asparaginase using both the purified enzyme as well as intact cells of Escherichia coli. Until recently the way to evaluate the behavior of an addictive one and the behavior of the water in the stabilization of a protein during the freeze-drying consisted of the measure of the activity parameters after the reidratação, even so now modem techniques of the Nuclear Magnetic Resonance of low resolution have been used to understand the behavior of the water in the interactions with proteins. It is Infrared Spectroscopy for Fourier Transformed it presents a great potential in the study of stabilization of proteins during the freeze-drying. The calculation of percentage of activity retention was used to express the studied values of enzymatic activity. These calculations were accomplished for the frozen systems and for the frozen systems and freeze-dryied. The systems were treated in three speeds of different freezing (20°C/min, 5°C/min and 2°C/min), being the freeze-drying for 24 hours. Results are presented on the effect of the freeze-drying in the different systems addictive-enzyme and addictive-cell. The addictive ones were used: sucrose, maltose, lactose, inositol, manitol and trehalose tested in different concentrations (30, 90 and 150mM), to identify which the conditions and the addictive ones that presented a satisfactory cryoprotection. For the systems enzyme-maltose, frozen slowly, the results of activity retention were: 8,67%, 14,02% e 30,80% to 30,90 e 150mM,respectively . The system enzyme-maltose (150mM) frozen quickly and freeze-dryied presented the largest activity retention (111,11 %) and also the largest value of T2 (81 &#181;s) in the referring results NMR. The systems enzyme-trealose in the concentrations of 90 and 150mM they presented retention of activity 89,93% and 79,74%, respectively.

The nuclear lamina protein, lamin A is produced by proteolytic cleavage of a 74 kDa precursor protein, prelamin A. The conversion of this precursor to mature lamin A is mediated by a specific endoprotease, prelamin A endoprotease. Subnuclear fractionation indicates that the prelamin A endoprotease is localized at the nuclear membrane. The enzyme appears to be an integral membrane protein, as it can only be removed from the nuclear envelope with detergent. It is effectively solubilized by the detergent n-octyl-beta-D-glucopyranoside and can be partially-purified (approximately 1200-fold) by size exclusion and cation exchange (Mono S) chromatography. Prelamin A endoprotease from HeLa cells was eluted from Mono S with 0.3 M sodium chloride as a single peak of activity. SDS-PAGE analysis of this prelamin A endoprotease preparation shows that it contains one major polypeptide at 65 kDa and smaller amounts of a second 68 kDa polypeptide. Inhibition of the enzyme activity in this preparation by specific serine protease inhibitors is consistent with the enzyme being a serine protease.