Research Advances: Less Expensive and More Convenient Gaucher's

Chemical Education Today

Reports from Other Journals

Research Advances by Angela G. King

Less Expensive and More Convenient Gaucher’s Disease Treatment Prospects for eventual development of a less costly and more convenient treatment for Gaucher’s (go-SHAYZ) disease have brightened with new research findings involving chemical chaperones. Gaucher’s disease is rare, but ranks as the most common lysosomal storage disorder and genetic disorder affecting Jewish people of Eastern European ancestry. Individuals with Gaucher’s disease, which can be fatal, produce the enzyme glucocerebrosidase (GC) with a mutation that results in misfolding. GC is a liposomal hydrolase that breaks down glucosylceramide to ceramide and glucose. If GC is not functioning properly, glucosylceramide accumulates in lyposomes of monocyte-macrophage cells, resulting in medical problems. Patients experience a range of health problems, including anemia, bone fractures, and sometimes lung and brain disorders. Scientists, led by Jeffrey W. Kelly of The Scripps Research Institute (TSRI), have confirmed experiments they reported initially in 2002 that chemical chaperones can partially correct the genetic defect responsible for most cases of Gaucher’s disease. Using patient-derived cell lines, researchers have extended those earlier studies to provide new insights into the defect and how chaperones correct it. Like aspirin, penicillin, and most other existing drugs, chemical chaperones are small molecules—natural and synthetic substances with a low molecular weight. The chaperones help the target proteins alter the environment of the pH-neutral endoplasmic reticulum (ER) where GC is folded prior to being trafficked to the acidic lysosome. In the case of Gaucher’s disease, the molecular chaperones overcome folding barriers caused by mutations in GC. Once the chaperone helps the mutant GC fold and get to the lysosome, the mutant protein is both stable and functional. “Gaucher’s disease patients can now be treated with enzyme replacement therapy”, Kelly explained. “The hope is that this current strategy could be replaced with a small molecule chemical chaperone therapy wherein the cost would be reduced by at least one hundred-fold.” Although enzyme replacement therapy is highly effective in treating Gaucher’s disease, it must be given intravenously rather than by mouth, and treatment often costs $100,000–$750,000 annually for each patient. An oral treatment based on the new research could cut those costs to $1000–$7500 annually. Kelly said researchers are now trying to identify an optimal small molecule that could be suitable for use as a chemical chaperone in clinical trials.

More Information 1. Sawkar, Anu R.; Schmitz, Martina; Zimmer, Klaus-Peter; Reczek, David; Edmunds, Tim; Balch, William E.; Kelly, Jeffrey W. Chemical Chaperones and Permissive Temperatures

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Alter the Cellular Localization of Gaucher’s Disease Associated Glucocerebrosidase Variants. Chemical Biology 2006, 1, 235–251. 2. This Journal has previously published two articles on protein folding. See Bowen, Robert; Hartung, Richard; Gindt, Yvonne M. A Simple Protein Purification and Folding Experiment for General Chemistry Laboratory. J. Chem. Educ. 2000, 77, 1456–1457 and Jones, Colleen M. An Introduction to Research in Protein Folding for Undergraduates. J. Chem. Educ. 1997, 74, 1306–1310. 3. Descriptions of ongoing research under the guidance of Jeffrey Kelly can be found online at http://www.scripps.edu/skaggs/ kelly/research.php?a=l&cid=3 (accessed Oct 2006).

HO HO

OH N

HO HO HO

OH N C6H13

HO HO HO

OH N

HO NH O HO HO

OH N

HO O

Figure 1. Glucocerebrosidase inhibitors/GC chaperones used in this study (top to bottom): N-butyl-deoxynojirimycin (Miglustat); Nnonyl-deoxynojirimycin; N -hexanoic acid adamantly amide deoxynojirimycin; N-pentyl adamantly ether deoxynojirimycin. Structures provided by A. King.

Vol. 83 No. 12 December 2006

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Reports from Other Journals Structural Loop Regions: Key to Multidrug Resistance Transporters?

microbial genomes. These transporters are distinctive in their ability to recognize and expel a highly diverse range of amphipathic compounds. Amphipathic molecules contain both hydrophobic and hydrophilic groups—molecules that

repel or are attracted to water, respectively. The X-ray structure of the EmrD transporter—determined with data collected at the Stanford Synchrotron Radiation Laboratory and the Advanced

Scientists at The Scripps Research Institute have determined the X-ray structure of EmrD, a multidrug transporter protein from Escherichia coli (E. coli), common bacteria known to cause several food-borne illnesses. Proteins like EmrD that expel drugs from cells contribute significantly to the continued rise in multidrug-resistant bacteria, and the re-emergence of drug-resistant strains of diseases such as tuberculosis that were once thought to have been eradicated. This new study could potentially help researchers find new ways to avoid the problem of multidrug resistance and enhance the potency of existing drug compounds. “The development of antibiotics to treat infectious disease is being seriously undermined by the emergence of drugresistant bacteria”, says Geoffrey A. Chang, who led the study. “Multidrug resistance develops in part through the expulsion of drugs by integral membrane transporters like EmrD. Determining the structure of this transporter will add significantly to our general understanding of the mechanism of drug transport through the cell membrane and provide the structural basis for how these proteins go about selecting specific drugs to expel.” Multidrug-resistant bacterial infections raise the cost of medical treatment and are far more expensive to treat than normal infections. Treating drug-resistant tuberculosis, for example, requires so-called second-line drugs if standard treatment fails. According to the Centers for Disease Control (CDC), secondline drugs can cost as much as “$33,000 per patient in industrialized countries compared to $84 for first-line drugs”. In addition, the CDC noted, second-line drugs need to be taken for longer periods of time—from 18 to 36 months—and may require substantial patient monitoring, making these medical treatments difficult, if not impossible, to “be available in many of the resource-poor nations where drug-resistant tuberculosis is now emerging.” EmrD belongs to the Major Facilitator Superfamily, a group of transporters that are among the most prevalent in

Figure 2. Stereoimages of crystallography and structure of EmrD. (A) A portion of the experimental electron density map is shown for H3, H6, and L6-7. The map is contoured to 1␴. (B) Side view of EmrD. The N and C termini are indicated. (C) View of EmrD looking toward the cytoplasm showing the molecular two-fold axis relating the N- and C-terminal halves. Transmembrane helices are indicated. From Yin, Yong; He, Xiao; Szewczyk, Paul; Nguyen, That; Chang, Geoffrey. Structure of the Multidrug Transporter EmrD from Escherichia coli. Science 2006, 312, 741–744. Reprinted with permission from AAAS.

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Figure 3. A potential mechanism for hydrophobic substrate transport by EmrD. (A) The drug can enter the internal cavity of the transporter either through the inner membrane leaflet (path 1) or through the cytoplasm (path 2). Substrate recognition and binding may be facilitated through the selectivity filter and the internal cavity containing hydrophobic residues. (B) The drug is transported

through a rocker-switch alternating-access model coupled with H⫹ antiport. (C) The drug is transported across the lipid bilayer. From Yin, Yong; He, Xiao; Szewczyk, Paul; Nguyen, That; Chang, Geoffrey. Structure of the Multidrug Transporter EmrD from Escherichia coli. Science 2006, 312, 741–744. Reprinted with permission from AAAS.

Light Source at the University of California, Berkeley— revealed an interior composed primarily of hydrophobic residues. This finding is consistent with its role of transporting hydrophobic or lipophilic molecules—and similar to the interior of another multidrug transporter, EmrE, which Chang and his colleagues uncovered in a study that was published last year in the journal Science. This internal cavity is the “most notable difference” between EmrD and most non-Major Facilitator Superfamily multidrug transporters that, the new study noted, typically transport “a relatively narrow range of structurally related” compounds. The hydrophobic residues in the EmrD internal cavity are likely to contribute to the general mechanism transporting various compounds through the cell membrane, and may play “an important role in dictating a level of drug specificity” through a number of molecular interactions. The study also suggests that EmrD intercepts and binds cyanide m-chlorophenyl hydrazone, a known efflux pump inhibitor, before it reaches the cell cytoplasm. This binding is likely facilitated by hydrophobic interactions within the internal cavity of EmrD. The researchers speculate that cyanide m-chlorophenyl hydrazone is either expelled from the bacterial cell or into the periplasmic space—the space between the outer membrane and the plasma membrane in gram-negative bacteria like E. coli. “While EmrD and EmrE are completely different proteins from different molecular families”, Chang said, “both are multidrug transporters that help bacteria develop multidrug resistance. Together with MsbA, another MDR structure that our laboratory is studying, this new X-ray structure adds another important view of some general structural features across multi-drug resistant transporter families.”

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1. Yin, Yong; He, Xiao; Szewczyk, Paul; Nguyen, That; Chang, Geoffrey. Structure of the Multidrug Transporter EmrD from Escherichia coli. Science 2006, 312, 741–744. 2. This Journal has previously described undergraduate laboratories focused on X-ray crystallography. See Garrett, Elizabeth; Wehr, Audrey; Hedge, Rebecca; Roberts, David L.; Roberts, Jacqueline R. A Novel and Innovative Biochemistry Laboratory: Crystal Growth of Hen Egg White Lysozyme. J. Chem. Educ. 2002, 79, 366–368; and García-Ruiz, Juan Manuel; Moreno, Abel; Otálora, F.; Rondón, D.; Viedma, C.; Zauscher, F. Teaching Protein Crystallization by the Gel Acupuncture Method. J. Chem. Educ. 1998, 75, 442–446. 3. More information on Geoffrey Chang’s research projects can be found at http://www.scripps.edu/research/faculty.php?rec_ id=7431 (accessed Oct 2006).

New Method Identifies Proteins in Old Artwork Artists’ paint contains two main types of components: pigment (mainly inorganic powders that impart what we perceive as color) and binders (substances that hold the pigment particles to each other as well as to the surface the paint was applied to). Eggs, milk, and glue have all been historically employed as binders, both individually and in mixtures. Art historians have long sought to identify binder proteins to gain insight into both the creation process and restoration approaches. Various techniques have been used in attempts to identify proteins present in paint binders, including the Naphthol Blue Black-based method, amino acid thin-layer chromatography, UV–vis spectroscopy, Raman spectroscopy, and gas chromatography. Using these methods provided some


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Reports from Other Journals success, although each method has limitations. Now researchers in France, led by Cécile Cren-Olivé, have developed a new methodology utilizing nanochromatography and tandem mass spectrometry to identify proteins in paint samples from works of art. The team first constructed painting models using the formulation of old paintings: lead white as pigment, linseed oil, and egg protein. They next identified the conditions needed to extract protein from paint binding without protein hydrolysis using hen egg albumin and then whole egg protein in their painting models. The best extraction conditions were afforded using 1% trifluoroacetic acid and samples ground in single-use commercial microsample DNA extraction kits consisting of synthetic resin, mortar, and pestle. Following grinding, three 15-minute ultrasonic baths, and centrifugation, the supernatant was ready for analysis to identify and quantify proteins present in the binder. Linear mode MALDI-TOF mass spectrometry confirmed the efficiency of the developed extraction procedure. The researchers then turned to proteomics analysis for identifying extracted protein by developing a protocol for enzymatic hydrolysis, peptide purification, tandem mass spectrometry analysis, and comparison with protein databanks working with samples in the 10 ␮g range. A sample from a fresh egg painting model was extracted by the proven procedure and subjected to enzymatic digestion with trypsin. The resulting peptides were identified by MALDI-TOF analysis by matching peptide mass fingerprints with those from known peptides and proteins. Researchers confirmed the results by separating the peptides produced by digestion using nanochromatography and on-line analysis by a Q-q-TOF mass spectrometer. The combined new methodologies of extraction and protein identification were applied to samples from Renaissance paintings: Benedetto Bonfigli’s triptych—The Virgin and

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Child, St. John the Baptist, St. Sebastian (15th century)— and Niccolo di Pietro Gerini’s painting, The Virgin and Child (14th century). The new extraction and identification protocols were able to identify for the first time, with no uncertainty, the presence of whole egg (white and yolk) proteins in the paintings’ binder. These advances in analytical chemistry and their applications to art history and conservation could be considered by those engaged in the field as a work of art in their own right.

More Information 1. Tokarski, Caroline; Martin, Elisabeth; Rolando, Christian; Cren-Olivé, Cécile. Identification of Proteins in Renaissance Paintings by Proteomics. Anal. Chem. 2006, 78, 1494–1502. 2. C&EN has covered the collaboration between scientists and curators needed for art conservation. See http://pubs.acs.org/cen/ coverstory/7931/7931art.html (accessed Oct 2006). 3. Wellesley College has a collection of resources on chemistry and art available online at http://www.wellesley.edu/Chemistry/ Chem&Art/chemandart.html (accessed Oct 2006). 4. Instructions for making your own paints have been published as JCE Classroom Activity #38 in this Journal. See Gettys, Nancy S. Pigments of Your Imagination: Making Artist’s Paints J. Chem. Educ. 2001, 78, 1320A–B. 5. This Journal has published several articles on chemistry and art, including a very recent one describing a program utilizing art-related content to reduce chemophobia in 11–18 year olds. See Kafetzopoulos, C.; Spyrellis, N.; LymperopoulouKaraliota, A. The Chemistry of Art and the Art of Chemistry. J. Chem. Educ. 2006, 83, 1484–1488.

Angela G. King is Senior Lecturer in Chemistry at Wake Forest University, P.O. Box 7486, Winston-Salem, NC 27109; [email protected].


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Research Advances: Less Expensive and More Convenient Gaucher's

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