Research Advances - American Chemical Society

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Research Advances by Angela G. King

Potential New Drug for Cocaine Addiction and Overdose

Figure 1. Chemists have developed a substance that could help fight addictions and overdoses of cocaine (shown above). Image courtesy of U.S. Drug Enforcement Administration.

782

(A) (-)Cocaine G116 1. 97

G117

1.79

1.92

2.12

1.06

1.13

1.46

H438

S198 A199

1.67

E325

(B) (-)Cocaine G116 1.97

G117 1.69

1.93

2.09

(C)

1.13 1.43

S198

S199

Relative Energy (kcal/mol)

Chemists report development of what they term the most powerful tool ever discovered for eliminating cocaine from the body, an advance that could lead to the world’s first effective medicine for fighting overdoses and addictions of the illicit drug (Figure 1). In the recent study, Chang-Guo Zhan and colleagues point out that no effective medication currently exists to treat cocaine abuse. One of the most promising approaches focuses on substances that mimic butyrylcholinesterase (BChE), a natural blood enzyme that accelerates the conversion of cocaine into biologically inactive metabolites. Cocaine is a chiral compound, with (−)-cocaine responsible for affecting the central nervous system; its enantiomer, (+)-cocaine, is biologically inactive. However, natural BChE has a low catalytic efficiency against street cocaine, allowing (−)-cocaine a plasma half-life of 45–90 min even for a tiny dose of (−)-cocaine. Since cocaine’s effects on the central nervous system peak within minutes of administration, native BChE cannot clear the drug before positive reinforcement begins, making cocaine the most reinforcing drug known. Because of its low kcat, native BChE is ineffective for medical use, the researchers note. The researchers employed a novel systematic computational design approach involving transition-state simulations and activation-energy calculations to identify the mutant-type BChE that would increase the enzyme’s reaction rate with cocaine. Previous work had identified the rate-determining step of (−)-cocaine metabolism in humans as the formation of a prereactive BChE-(−)-cocaine complex known as ES. Researchers generated mutant BChE altered at residues 328 and 332 that significantly increased the rate of this step by decreasing steric hindrance. In fact, the activation energy decreased to the point that the first step of the chemical process became the rate determining step, instead of the formation of ES. Zhan and his team set their goal of further increasing the catalytic efficiency of cocaine hydrolysis by increasing the rate

1.06 1.60

H438 E325

A328W/Y332A A199S/F227A/S287G/A328W/Y332G

20 18 16 14 12 10 8 6 4 2 0 -2

TS1

TS1

ΔE‡=16.21

kcal/mol

INT1 INT1

ΔE‡=10.43 kcal/mol ES ES 5

4.5

4

3.5

3

2.5

2

1.5

1

0.5

0

Reaction Coordinate (Å)

Figure 2. Results from the QM/MM reaction coordinate calculations. (A) Optimized geometry of the transition state (TS1) for the first reaction step of (−)-cocaine hydrolysis catalyzed by A328W/Y332G BChE. (B) Optimized geometry of the transition state (TS1) for the first reaction step of (−)-cocaine hydrolysis catalyzed by A199S/F227A/S287G/A328W/Y332G BChE. (C) Calculated potential energy surface along the reaction coordinate (R1 − R2 + R3) of the enzymatic hydrolysis of (−)-cocaine. R1 is the length of the C…O transition bond between the hydroxyl oxygen of S198 and carbonyl carbon of (−)-cocaine benzoyl ester. R2 refers to the length of the O…H transition bond in the S198 hydroxyl group. R3 represents the length of the N…H transition bond between the hydroxyl hydrogen of S198 and the nitrogen of H438 side chain. Reprinted with permission from J. Am. Chem. Soc. 2008, 130, 12148–12155. Copyright 2008 American Chemical Society.

Journal of Chemical Education • Vol. 86 No. 7 July 2009 • www.JCE.DivCHED.org • © Division of Chemical Education


of the new rate-determining step, without sacrificing the efficiency in ES formation afforded by the mutant A328W/ Y332G enzyme. They used molecular dynamics (MD) simulations for virtual screening of BChE mutants (Figure 2). The screening involved estimating the total interaction energy (TIE) between the whole oxyanion hole of the enzyme and all (−)-cocaine atoms in the structure being analyzed. More sophisticated hybrid quantum mechanics/molecular mechanics calculations were used to determine the effects of the interactions on the ratedetermining reaction step’s energy barrier. In vitro and in vivo experimental tests followed the virtual mutant screening. The researchers’ efforts resulted in the most potent, stable mutant BChE structure ever produced. In lab studies, that mutant form of BChE metabolized cocaine 2,000 times faster than the body’s natural version of BChE, the scientists say, noting that reducing the concentration of cocaine in the blood is a key to fighting overdose in humans. The substance also prevented convulsions and death when injected into mice that had been given overdoses of cocaine, they note. But in addition to fighting cocaine addiction, this work may pave the way for future computationally based rational enzyme design that could have huge implications in drug discovery. For More Information 1. Zheng, Fang; Yang, Wenchao; Ko, Mei-Chuan; Liu, Junjun; Cho, Hoon; Gao, Daquan; Tong, Min; Tai, Hsin-Hsiung; Woods, James H.; Zhan, Chang-Guo. Most Efficient Cocaine Hydrolase Designed by Virtual Screening of Transition States. J. Am. Chem. Soc. 2008, 130, 12148–12155. 2. More information on Zhan’s research can be found at http:// pharmacy.mc.uky.edu/faculty/Chang-GuoZhan.php (accessed Apr 2009). 3. This Journal has published teaching activities regarding cocaine for both analytical and organic chemistry classes. See J. Chem. Educ. 2002, 79, 1254 and 1986, 63, 594, respectively. 4. Educators may also be interested in science-rich material available on the National Institute on Drug Abuse (NIDA) Web site. For an example involving the science of cocaine addiction, see http://www. nida.nih.gov/pubs/Teaching/Teaching.html (accessed Apr 2009). 5. The Pharmacology Education Partnership (PEP) Project has free online resources that have been formally shown to increase student learning in chemistry and biology through the integration of pharmacology. Free online materials, including a unit using cocaine and crack to reinforce acid–base concepts, are available at http://www. thepepproject.net (accessed Apr 2009).

Marijuana Components Show Promise in Battling Superbugs Scientists in Italy and the United Kingdom have determined that substances in marijuana show promise for fighting deadly drug-resistant bacterial infections, including so-called “superbugs”, without causing the drug’s mood-altering effects. Besides serving as infection-fighting drugs, the plant substances (Figure 3) also could provide a more environmentally friendly alternative to synthetic antibacterial substances now widely used in personal care items, including soaps and cosmetics.

R

OH

HO R = Terpenyl

Figure 3. Compounds from marijuana could lead to effective treatment for infection. Reprinted with permission from J. Nat. Prod. 2008, 71, 1427–1430. Copyright 2008 American Chemical Society.

In the new study, Giovanni Appendino and colleagues point out that new antibacterials are urgently needed to treat infections caused by multi-drug resistant clinically relevant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), but only one new class of antibacterial has been identified in the last 30 years. The authors also say plants in general are an untapped resource of new antibacterials, especially considering that cross-resistance to plant and microbial antibacterial agents is uncommon. While scientists have known for years that marijuana contains antibacterial substances, little research has been done on those ingredients, including studies on their ability to fight antibiotic resistant infections. To close that gap, researchers tested the five most common cannabinoids on different strains of methicillin-resistant MRSA, a “superbug” increasingly resistant to antibiotics. The assayed cannabinoids included cannabidiol (1b, CBD), cannabichromene (2, CBC), cannabigerol (3b, CBG), Δ9tetrahydrocannabinol (4b, THC), and cannabinol (5, CBN) (Figure 4). These five compounds could be obtained in >98% purity by isolation from a C. sativa strain that produced only a single major cannabinoid, (THC, CBD, CBG), total synthesis (CBC), or semisynthesis (CBN) from a related natural product. All five substances showed potent antibacterial activity against these drug-resistant strains, as did some synthetic non-natural cannabinoids. The scientists also showed that these substances appear to kill bacteria by mechanisms different from those of conventional antibiotics, making them more likely to avoid bacterial resistance. At least two of the tested cannabinoids (CBD and CBG) have no known mood-altering effects, suggesting that they could be developed into marijuana-based drugs without causing a “high”. The research team also assessed the antibacterial activity of pre-cannabinoids such as 1a, the carboxylic acids that lead to cannabinoids through thermal degradation, as well as componds produced through the acetylation or methylation (1c–e and 3c–e) of these carboxylic acids. These altered cannabinoids, and also abnormal synthetic cannabinoids such as 6, demonstrated antibacterial activity comparable to the natural products, reveal-

© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 86 No. 7 July 2009 • Journal of Chemical Education

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Reports from Other Journals more information see J. Chem. Educ. 2007, 84, 378. 3. This Journal has previously published an article detailing the use of EMIT to quantitatively detect marijuana in urine, serum, or plasma. See J. Chem. Educ. 1989, 66, 346. 4. NIDA offers information and literature on marijuana abuse in our society. See http://www.nida.nih.gov/DrugPages/Marijuana.html (accessed Apr 2009).

ing tolerance for structural modification within the family of antibacterial cannabinoid compounds. The researchers efforts may lead to use of a semi-pure mixture of cannabinoids as an inexpensive and biodegradable alternative to synthetic antibacterial agents that are not only less effective, but also questioned with regard to their environmental profile. For More Information

Molecular Conformation: Smelly Science Shakespeare wrote “a rose by any other name would smell as sweet.” But would it if the molecules that generate its fragrance were to change their shape (Figure 5)? That’s what Kevin Ryan, Assistant Professor of Chemistry at The City College of New York (CCNY) and collaborators

1. Appendino, Giovanni; Gibbons, Simon; Giana, Anna; Pagani, Alberto; Grassi, Gianpaolo; Stavri, Michael; Smith, Eileen; Rahman, M. Mukhlesur. Antibacterial Cannabinoids from Cannabis sativa: A Structure–Activity Study. J. Nat. Prod. 2008, 71, 1427–1430. 2. Research Advances has previously reported new research in using a marijuana component as treatment for Alzheimer’s disease. For

OR2

H

OH R3

H O R1

O

R1

R 2

R3

1a 1b 1c 1d 1e 1f 1g

H H Ac Me Me H H

H H Ac H Me H H

COOH H H H H COOMe COOCH2CH2Ph

2

OR2

Figure 4. Assayed natural and synthetic cannabinoids. Reprinted with permission from J. Nat. Prod. 2008, 71, 1427–1430. Copyright 2008 American Chemical Society.

R3

OH R

O R1

O

R1

R 2

R3

3a 3b 3c 3d 3e 3f 3g

H H Ac Me Me H H

H H Ac H Me H H

COOH H H H H COOMe COOCH2CH2Ph

4a COOH 4b H

OH

R

H H

O 5

784

HO

OH

6



set out to investigate. Their findings shed new insight into how our sense of smell works and have potential applications in the design of flavors and fragrances. When odor-producing molecules, known as odorants, pass through the nose, they trigger intracellular changes in a subset of the approximately 400 different varieties of olfactory sensory neurons (OSN) housed in the nose’s internal membrane tissue, Ryan explained. The unique reaction pattern produced, known as the olfactory code, is sent as a signal to the brain, which leads to perception of distinct odors. Ryan and his team, including biologists Stuart Firestein and Zita Peterlin of Columbia University, wanted to learn how these receptor cells respond when odorants change their shape. They studied the odorant octanal, an eight-carbon aldehyde that occurs in many flowers and citrus fruits. Octanal is a structurally flexible molecule that can sample many conformers by rotating its bonds. The researchers designed and synthesized eight-carbon aldehydes that resembled octanal, but had their conformations locked by adding one additional bond (Figure 6). These molecules were tested on genetically engineered OSNs known to respond to octanal. The aldehyde molecules that could stretch to their greatest length triggered strong activity in the OSNs. Those mol-

Figure 5. Conformationally restricted odorants above rat olfactory sensory neurons. Image courtesy of Kevin Ryan and David Bauer.

Figure 6. Conformationally restricted octanal analogs. (A) Lengths refer to the distance measured from the carbonyl to the most distant carbon. (B) Synthetic routes to compounds 1–6. Reproduced with permission. Reprinted from Peterlin, Zita; Li, Yadi; Sun, Guangxing; Shah, Rohan; Firestein, Stuart; Ryan, Kevin. The Importance of Odorant Conformation to the Binding and Activation of a Representative Olfactory Receptor. Chemistry & Biology 2008, 15, 1317–1327. Copyright 2008 with permission from Elsevier.

© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 86 No. 7 July 2009 • Journal of Chemical Education

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Reports from Other Journals ecules whose carbon chains were constrained into a U shape blocked the receptor and left the cell unable to sense octanal (Figure 7). “Conformationally constrained odorants were more selective in the number of OSNs they activated”, Ryan noted. “The results indicate that these odorant molecules might be able to alter fragrance mixture odors in two ways: by muting the activity of flexible odorants present in a mixture and by activating a smaller subset of OSNs than chemically related flexible odorants. This would produce a different olfactory code signature.” Olfactory receptors belong to the G-protein coupled receptor (GPCR) class of proteins, a family of molecules found in cell membranes throughout the body. Ryan pointed out that half of all commercial pharmaceuticals work by interaction with proteins within this family. Thus, the findings could also have applications to GPCR drug design.

2. More information on Ryan’s research is found at http://www. sci.ccny.cuny.edu/~kr107/index2/index.html (accessed Apr 2009). 3. This Journal has previously published undergraduate labs involving bioassays of molecules and conformational restrictions. See J. Chem. Educ. 2006, 83, 934 and 2004, 81, 121 for examples. 4. http://faculty.washington.edu/chudler/chems.html will take you to Neuroscience for Kids, featuring an excellent educational site on olfactory science (accessed Apr 2009). 5. http://www.leffingwell.com/chirality/chirality.htm is a great site for looking at the effect of chirality on odorant character and http:// www.thegoodscentscompany.com/rawmatex.html will help teachers and students looking at the structures of odor compounds (both sites accessed Apr 2009).

For More Information

Full text (PDF) with links to cited URLs and JCE articles

1. Peterlin, Zita; Li, Yadi; Sun, Guangxing; Shah, Rohan; Fire stein, Stuart; Ryan, Kevin. The Importance of Odorant Conformation to the Binding and Activation of a Representative Olfactory Receptor. Chemistry & Biology 2008, 15, 1317–1327.

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

Supporting JCE Online Material

http://www.jce.divched.org/Journal/Issues/2009/Jul/abs782.html Abstract and keywords

Figure 7. Summary of OR-17 binding activation by octanal conformation mimics. (A) Structures, maximum lengths, and inhibition/activation constants. Regions of the structures responsible for binding and activation are indicated (left) as is the 6.5–6.9 Å length requirement for activation (right). Except for C4, which had neither type of activity, dashes in the IC50 row indicate that the compound was not tested for antagonism because it is strongly activating. Dashes in the EC50 row indicate the compound had no activity within its solubility range. (B) Schematic depiction of octanal’s conformation on OR-17’s activation. Reprinted from Peterlin, Zita; Li, Yadi; Sun, Guangxing; Shah, Rohan; Firestein, Stuart; Ryan, Kevin. The Importance of Odorant Conformation to the Binding and Activation of a Representative Olfactory Receptor. Chemistry & Biology 2008, 15, 1317–1327. Copyright 2008 with permission from Elsevier.

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