Descripción del PLC SLC 500 de Allen Bradley

Effect of Inhibitors on Related Enzymes

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β-Glucuronidase is in the family 2 of glycosyl hydrolases, all of which use a conserved mechanism of enzymatic cleavage (24-27). Additional enzymes outside of this family, such as β-galactosidase, β-mannosidase, and β-glucosidase, also utilize an acid/base and a nucleophile to hydrolyze a variety of sugar-linked metabolites and compounds (28-31). Each of these enzymes contains the generic α/β-barrel and has an overall similar structure to E. coli β-glucuronidase (see Table 3.2). With these similarities, it is crucial to determine if our novel inhibitors affect the enzymatic activity of these other sugar hydrolyzing enzymes. Many of these enzymes can be purchased from commercial retailers, as well as the substrates that can be used to detect activity via fluorescence- or absorbance-based detection.

Additional High-Throughput Screening

The original high-throughput screening of two libraries at NCCU-BRITE institute yielded many hits that have been further characterized in-house through activity assays and crystallization studies. However, additional screening of other compound libraries could yield a number of other hits. The newly developed Center for Integrative Chemical Biology and Drug Discovery at UNC contains a number of widely used chemical compound libraries that could be used to further screen for additional β-glucuronidase inhibitors. Newly discovered compounds should be characterized in a similar way as inhibitors 1-4 of these previous studies.

FIGURE LEGENDS

Figure 4.1 SDS-PAGE gels of purified S. agalactiae β-glucuronidase, C. perfringens β- glucuronidase, and B. fragilis β-glucuronidase. Each protein was first purified by IMAC, followed by S200 gel filtration. Both the S. agalactiae and C. perfringens enzyme achieved >95% purity. The B. fragilis β-glucuronidase was determined to be >80% pure, however, other bands found on the gel were believed to be degraded protein.

Figure 4.2 (A) Initial crystal hit of S. agalactiae β-glucuronidase. This condition was composed to 20% (w/v) PEG3350 and 0.2 M potassium thiocyanate as well as 10-15 mg/mL protein. (B) Initial crystal hit of C. perfringens β-glucuronidase. These crystals were grown in the presence of 30% (w/v) PEG400 and 0.1 M MES pH 6.5 and 10-15 mg/mL protein.

Figure 4.3 Optimized crystals of the S. agalactiae β-glucuronidase. A grid optimization screen was prepared by varying the precipitant (PEG3350) and salt (potassium thiocyanate) concentration, as well as testing a range of protein to crystallant drop ratios to achieve well formed crystals.

Figure 4.4 (A) Overall structures of the two solved versions of S. agalactiae β-

glucuronidase. One structure was determined in the space group I222 (left), with a 2.6 Å data limit, and one monomer in the ASU. The other crystal structure was solved in the space group P21212, at 2.3 Å, with 2 monomers in the ASU. (B) Monomer overlay of the E. coli structure and the S. agalactiae structure. The secondary structure of these two structures proved relatively similar with an overall r.m.s.d of 1.58 Å over all atoms. Deviations were mostly localized in the N-terminal region (β-sandwich domain) of each protein.

Figure 4.5 (A) Structural overlay of the two S. agalactiae structures, focusing on the proposed loop region of each structure, showing both conformations. The I222 loop region swings further out into the solvent region, and actually crosses paths with a symmetry mate in the crystal packing. The P21212 loop is positioned much closer to the active site opening. These two conformations suggest that this loop is mobile, and upon ligand binding, most likely adopts the position seen in the P21212 structure. (B) Overlay of the two S. agalactiae and inhibitor 2-bound E. coli β-glucuronidase structures, focusing on the bacterial loop region. Although the loops in the S. agalactiae proved to be disordered in the structures, they appear to follow the same path as the E. coli loop, most notably that of the P21212 structure.

Figure 4.6 (A) DCF-AG conversion to DCF catalyzed by bacterial β-glucuronidases; using inhibitor 1 prevents this reaction. Ideally, inhibitor 1 will prevent the

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hydrolysis of the glucuronidated-DCF to form DCF, which would prevent formation of ulcers. (B) HPLC results of the in vitro assay using DCF-AG as a substrate, as well as inhibitor 1 to disrupt enzyme activity. The bar graph represents a dose-response of DCF formation, resulting from DCF-AG hydrolysis by β-glucuronidase, with increasing inhibitor 1 concentrations. DCF formation is severely disrupted with increasing amounts of the inhibitor, with a calculated IC50 of ~164 + 11 nM.

Figure 4.1 SDS-PAGE gels of S200 gel purified S. agalactiae β-glucuronidase, C. perfringens β-glucuronidase, and B. fragilis β-glucuronidase.

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Figure 4.2 (A) Initial crystal hit of S. agalactiae β-glucuronidase. (B) Initial crystal hit of C. perfringens β-glucuronidase.

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Figure 4.3 Optimized crystals of the S. agalactiae β-glucuronidase.

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Figure 4.4 (A) Overall structures of the two solved versions of S. agalactiae β- glucuronidase. (B) Monomer overlay of the E. coli structure and the S. agalactiae structure.

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Figure 4.5 (A) Structural overlay of the two S. agalactiae structures, focusing on the proposed loop region of each structure, showing both conformations. (B) Overlay of the two S. agalactiae and the inhibitor 2-bound E. coli β- glucuronidase structures, focusing on the bacterial loop region.

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Figure 4.6 (A) DCF-AG conversion to DCF catalyzed by bacterial β-glucuronidases; using inhibitor 1 prevents this reaction. (B) HPLC results of the in vitro assay using DCF-AG as a substrate (DCF formation detected as μV*min), as well as inhibitor 1 to disrupt enzyme activity.

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REFERENCES

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2. F. Guarner, J. R. Malagelada, Gut flora in health and disease. Lancet 361, 512 (2003). 3. G. L. Simon, S. L. Gorbach, The human intestinal microflora Dig Dis Sci 31, 147S

(Sep, 1986).

4. C. L. Sears, A dynamic partnership: celebrating our gut flora Anaerobe 11, 247 (Oct, 2005).

5. M. Allison, A. Howatson, C. Torrance, F. Lee, R. Russell, Gastrointestinal damage associated with the use of nonsteroidal antiinflammtory drugs New Engl. J. Med. 327, 749 (1992).

6. I. Bjarnason, J. Hayllar, A. J. McPherson, A. S. Russell, Side effects of nonsteroidal anti-inflammatory drugs on the small and large intestine in humans Gastroenterology 104, 1832 (1993).

7. M. M. Wolfe, D. R. Lichtenstein, G. Singh, Gastrointestinal toxicity of nonsteroidal antiinflammatory drugs New Engl. J. Med. 340, 1888 (1999).

8. N. M. Davies, J. Y. Saleh, N. M. Skjodt, Detection and prevention of NSAID-induced enteropathy J. Pharmacy Pharm. Sci. 3, 137 (2000).

9. H. Koga, T. Matsumoto, M. Iida, Epidemiology, diagnosis and treatments of non- steroidal anti-inflammatory drug-induced enteropathy: A review of the literature Gastroenterol. Endosc. 50, 189 (2008).

10. C. Scarpignato, R. H. Hunt, Nonsteroidal antiinflammatory drug-related injury to the gastrointestinal tract: Clinical picture, pathogenesis, and prevention Gastroenterol. Clin. N. Am. 39, 433 (2010).

11. P. J. Fortun, C. J. Hawkey, Nonsteroidal antiinflammatory drugs and the small intestine Curr. Opin. Gastroenterol. 23, 134 (2007).

12. L. Maiden, Capsule endoscopic diagnosis of nonsteroidal antiinflammatory drug- induced enteropathy J. Gastroenterol. 44, 64 (2009).

13. R. Sidhu, L. K. Brunt, S. R. Morley, D. S. Sanders, M. E. McAlindon, Undisclosed use of non-steroidal anti-inflammatory drugs may underlie small bowel injury observed by capsule endoscopy Clin. Gastroenterol. Hepatol. 8, 992 (2010).

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14. S. Seitz, U. A. Boelsterli, Diclofenac acyl glucuronide, a major biliary metabolite, is directly involved in small intestinal injury in rats Gastroenterology 115, 1476 (1998). 15. M. Treinen-Moslen, M. F. Kanz, Intestinal tract injury by drugs: Importance of

metabolite delivery by yellow bile road Pharmacol. Ther. 112, 649 (2006).

16. U. A. Boelsterli, V. Ramirez-Alcantara, NSAID acyl glucuronides and enteropathy Curr. Drug Metab. 12, 245 (2011).

17. R. G. Dickinson, A. R. King, Rearrangement of diflunisal acyl glucuronide into its β- glucuronidase-resistant isomers facilitates transport through the small intestine to the colon of the rat Life Sci. 70, 25 (2001).

18. V. Pavillard, V. Charasson, A. Laroche-Clary, I. Soubeyran, J. Robert, Cellular parameters predictive of the clinical response of colorectal cancers to irinotecan. A preliminary study Anticancer Res 24, 579 (Mar-Apr, 2004).

19. Z. Otwinowski, W. Minor, Charles W. Carter, Jr., in Methods Enzymol. (Academic Press, 1997), vol. Volume 276, pp. 307-326.

20. A. J. McCoy et al., Phaser crystallographic software J Appl Crystallogr 40, 658 (Aug 1, 2007).

21. B. D. Wallace et al., Alleviating cancer drug toxicity by inhibiting a bacterial enzyme Science 330, 831 (2010).

22. S. Seitz, A. Kretz-Rommel, R. P. J. Oude Elferink, U. A. Boelsterli, Selective protein adduct formation of diclofenac glucuronide is critically dependent on the rat

canalicular conjugate export pump (Mrp2) Chem. Res. Toxicol. 11, 513 (1998). 23. J. L. Hyatt et al., Planarity and constraint of the carbonyl groups in 1,2-diones are

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CHAPTER 5.

Background on Xenobiotic Nuclear Receptors and Formation of the PXR.1/RXRα

Complex

INTRODUCTION

A vast array of enzymes are responsible for the biotransformation and metabolism of xenobiotics compounds in the human body. The metabolism of xenobiotics occurs by three different methods, classified as Phase I, Phase II, or Phase III (1-5). Phase I metabolism includes oxidation, reduction, hydrolysis, and hydration (6). Phase II involves conjugation of the xenobiotics by hydrophilic compounds to increase water solubility to facilitate excretion and removal from the body (6). Phase III uses membrane-bound drug pumps that serve to inactivate and clear chemical compounds (5).

In the liver, the heme-containing family of enzymes known as cytochrome P450’s are mono-oxygenases involved in endobiotic and xenobiotics clearance. Furthermore, they prepare xenobiotic compounds to be shuttled to the Phase II mechanism of metabolism (6). This system is utilized for the metabolism of not only xenobiotics compounds (drugs and other foreign compounds), but also of endogenous chemicals such as steroids, bile acids, hormones, and fatty acids (5, 7). There are 57 known P450s expressed in the human genome,

and of these, 15 are found to be involved in xenobiotic metabolism, including carcinogens, food additives, pollutants, pesticides, and other chemicals (7). However, it has been noted that in the absence of such xeno- or endobiotic compounds, the basal expression of the P450 family is quite low. Furthermore, in the presence of these substrates, their expression is greatly elevated (8, 9).

These drug metabolizing enzymes are critically involved in clinically significant drug-drug interactions, and the mechanism of these interactions are derived from the drug- induced increase in expression of the P450 enzymes. As such, other drugs that are metabolized by the now induced protein will have altered characteristics, possibly resulting in adverse or negative effects. This upregulation of the P450 class of enzymes is mediated by a group of proteins known as orphan nuclear receptors. These orphan nuclear receptors are related to nuclear hormone receptors, but are distinguished in that there are no previously identified physiological ligands (5).

Over the last several years, it has come to light that two closely related nuclear receptors, the pregnane x receptor (PXR) and the constitutive androstane receptor (CAR), are xenobiotic sensors that regulate the induction of the drug clearance pathways involving the P450 family of enzymes. CHAPTER 5 and 6 will focus on the orphan nuclear receptor PXR.