CARMEN DRAHL/C&EN
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HIGH THROUGHPUT
NCGC research scientist Wendy Lea demonstrates a screen for potential anesthetics.
ANESTHESIA’S AWAKENING Chemistry informs the quest to UNDERSTAND ANESTHETICS and make them better CARMEN DRAHL, C&EN WASHINGTON
Medicinal chemists are used to using model species to test their compounds. Weiser. It had been several minutes since Some go with mice, others rats, still othhe’d administered propofol, a potent ers zebrafish. But for people general anesthetic, and one of like Eckenhoff and Weiser, a his charges just refused to go OH Penn pharmacology graduate under. Most didn’t budge in student, the search for drugs is, response to a quick tap. But one and requires, a different animal. quickly darted away. “He’s slowing down,” Weiser Such hunts typically start with Propofol told his supervisor, University of a biological target known to Pennsylvania Perelman School be implicated in a disease. But of Medicine anesthesiologist Roderic G. in anesthesia, those kinds of target-based Eckenhoff. “But he’s still got a startle reflex.” approaches are in their infancy, which is So Weiser made a few adjustments, part of the reason for the tests on tadpoles. filling a widemouthed glass pipette with In anesthesia, target discovery and drug a solution of propofol in pond water and adding some to his charges’ plastic dish. “There’s always one resistant tadpole,” he said, gently tapping the petri dish on his laboratory benchtop. IT WAS BUSINESS as usual for Brian
discovery are two sides of the same coin. Each year, more than 40 million patients in North America alone undergo a procedure that requires anesthesia, according to the American Society of Anesthesiologists. The earliest general anesthetics, ether and chloroform, have given way to newer agents. Anesthesia is the safest it’s ever been. But that doesn’t mean anesthetics are risk-free drugs. “One of the problems with anesthetics is that they have a number of side effects,” Eckenhoff says. “That’s why you see a specialty of anesthesiology, for managing the side effects, for monitoring for them, for keeping patients safe.”
“It’s almost a defining characteristic of life that we can be anesthetized.”
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Highly trained people administer anesthetics because they affect breathing, blood pressure, and heart function. A growing body of evidence suggests anesthetics could also have cognitive effects long after they have left the body. In particular, children who have been under general anesthesia multiple times by the age of four appear to carry a higher risk of developing learning
disabilities. In animal tests several teams have shown that commonly used anesthetics boost aggregation of amyloid-β, the peptide thought to be the culprit behind Alzheimer’s disease. “Safety is an interesting issue with anesthetics,” says R. Adron Harris, a pharmacology expert at the University of Texas, Austin. One way to assess drug safety is by
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calculating what’s called the therapeutic index, he says. “That’s the ratio of the dose that would kill you to the dose that gives you the desired effect.” For most drugs that number is 10 or greater, with some antibiotics having ratios of more than 100. But anesthetics clock in lower, with several below 5. “By that measure, they’re quite dangerous,” Harris says. “But in fact mortality due to anesthesia is pretty low. That speaks to the skills of anesthesiologists,” he says. He thinks a new anesthetic with a better therapeutic index would be especially welcome in regions where the cost of having many highly skilled anesthesia professionals is prohibitive. “Anesthetics are imperfect,” says Edmond I. Eger, a professor of anesthesia at the University of California, San Francisco Medical Center. “But it’s possible they can be improved.” BACK AT PENN, the stubborn tadpole fi-
nally went under. Satisfied, Weiser got ready to reverse the propofol’s effect. With the pipette, he removed as much of the propofol solution as possible and disposed of it, being careful not to disturb the immobile amphibians. Then, with a whir of the pipette’s electric motor, he added fresh pond water to the dish and once again began to wait. Tadpoles have been a mainstay of anesthesia research for more than a century. They are relatively cheap as far as lab animals go, and their response to anesthetics is an excellent mimic of humans’. In fact, tadpoles helped bolster one of the earliest theories about how anesthesia worked. As late as the 1980s, most practitioners thought anesthetics interacted nonspecifically with the lipids in nerve cell membranes. This theory grew out of the work of researchers Charles Ernest Overton and Hans Horst Meyer, who at the turn of the 20th century independently observed that anesthetics that were more soluble in oil had greater anesthetic power in tadpoles. This nonspecific mechanism inspired a decidedly empirical approach to the search for new anesthetics, says Neil L. Harrison, a pharmacologist at Columbia University. “Rather than anesthetics being rationally designed, they were discovered semi-serendipitously,” he says. “People in drug companies used to get some mice and inject them with whatever happened to be sitting on the shelf.” But in 1984, Imperial College London biophysicists Nicholas P. Franks and William R. Lieb learned something that
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COVE R STORY
“Anesthetics are imperfect, but it’s possible they can be improved.”
opened new avenues for anesthesia drug discovery. The pair found that at clinically relevant concentrations, anesthetics inhibit the glowing action of the firefly luciferase protein, suggesting anesthetics exert their effects by interacting with proteins (Nature, DOI: 10.1038/310599a0). As with many results that run counter to convention, the work didn’t catch on at first. “It was the
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beginning of quite a struggle,” Franks says. “It really took many years thereafter before the work was thoroughly accepted.” It wasn’t until the 1990s that the tide
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began to turn, and today the majority of researchers and physicians think proteins are key to anesthetic action, Eger says. “Nick Franks really caused a sea change in the thinking about how anesthetics work,” he adds. After Franks’s and Lieb’s work, more researchers started looking for the protein targets of anesthetics. It’s an important quest en route to better anesthetics, explains Richard Olsen, a pharmacologist at the University of California, Los Angeles. “Students of anesthesia know that you can define five or six different aspects of the anesthetized state,” including immobility, amnesia, analgesia, unconsciousness, and sedation, Olsen says. Each of these effects could be due to the anesthetic interacting with a different protein. If that were the case, it might be possible to tune anesthetics for different applications simply by tweaking affinities for target receptors, Olsen adds. Olsen consults for multiple companies developing sleep aids.
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Olsen suggests because they still don’t fully understand how anesthetics work. For the injectable anesthetics propofol and etomidate, the picture is reasonably clear. They largely act at one target, the γ-aminobutyric acid type A (GABAA) receptor. GABAA is a chloride ion channel with more than a dozen subtypes in different parts of the nervous system. When the receptor responds to the neurotransmitter GABA, it admits chloride ions and prevents the neuron from generating an electrical signal. Anesthetics enhance the action of GABA, in essence quieting brain function. Knowing that the injectable anesthetic alphaxalone acts on GABAA is helpful for anesthesiologist Alex S. Evers and chemist Douglas F. Covey of Washington University in St. Louis. They are working toward next-generation versions of alphaxalone, a steroid anesthetic pulled from the market in the 1980s because of allergic reactions to its lipid formulation. Steroid anesthetics have several advantages over other classes—for example, they don’t lower blood pressure or suppress breathing, Evers says. The St. Louis team has had a long-standing collaboration to explore the chemical features of steroids that lend anesthetic
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COU RT ESY O F DA N IEL EM ERSO N
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activity while developing water-soluble steroids that will be easier to formulate. “Recently we think we’ve learned enough to make a better steroid anesthetic,” Covey says. Covey and Evers both advise Sage Pharmaceuticals, a start-up with an interest in anesthetic steroids. The pair’s most advanced alphaxalone analog, a sterol with a nitrile group on its D ring, has a structure consistent with cri-
a consulting relationship with Baxter Healthcare, a maker of inhaled anesthetics. Eckenhoff and his collaborators at Penn are just one of a list of teams trying to make sense of anesthetics’ mechanisms. Penn chemist William P. Dailey’s group provides light-activated versions of inhaled or injectable anesthetics, such as m-azipropofol, to Eckenhoff, who can then use ultraviolet light to covalently link the anesthetic to any proteins it binds, whether in vitro or in a live tadpole (J. NC H O Med. Chem., DOI: 10.1021/ O O jm1004072). Every target a of the protein apoferritin, which is cheap H H light-activated anesthetic deand commercially available, could mimic tects is subjected to follow-up anesthetic binding to at least one receptor, H H H H tests to confirm that they are GABAA (J. Biol. Chem., DOI: 10.1074/jbc. HO HO relevant to the mechanism of M109.017814). But to measure compound H H anesthesia, Eckenhoff says. binding to apoferritin, his team was using Alphaxalone nitrile analog Alphaxalone Fishing for targets is a time-consuming isothermal titration one thing, but using that calorimetry technique that was unlikely to teria that Covey and Evers have learned are knowledge gained to search find use in high-throughput important for inducing anesthesia (J. Med. for new anesthetics, whether screens. OH Chem., DOI: 10.1021/jm2002487). It also injected or inhaled, is harder Enter Ivan J. Dmochowski, a has comparable activity to alphaxalone in than it seems. Little threePenn chemist with expertise in N mice. “We’ve filed a provisional patent apdimensional structural data on both ferritin and fluorescence. N plication on the compound,” Covey says. anesthesia-receptive binding Together, he and Eckenhoff CF3 pockets exist. One exception identified the first fluorescent m-Azipropofol MUCH OF the development of new anis an X-ray crystal structure of general anesthetic, 1-aminoesthetics centers on the injectable class. an ion channel from blue-green anthracene (Proc. Natl. Acad. That’s because the mechanistic picture is algae, a homolog of a channel thought to Sci. USA, DOI: 10.1073/pnas.0810590106). far more complicated for inhaled anesthetmediate anesthesia in humans, published The molecule immobilizes tadpoles reversics. Most physicians and researchers who earlier this year (Nature, DOI: 10.1038/ ibly, and with fluorescence microscopy, the spoke with C&EN say that between five nature09647; C&EN, Jan. 24, team can see its locations in a and 20 proteins may be targets of inhaled page 31). What’s more, most of tadpole body. What’s imporNH2 anesthetics. Those targets may include the ion channels that anesthettant for screening is that the GABA, but also certain ion channels that ics act on are tough to obtain molecule’s fluorescence goes respond to the neurotransmitter glutamate in purified form, which makes up dramatically when it binds and the amino acid glycine, as well as cerit challenging to develop the ferritin and goes down when 1-Aminoanthracene tain potassium ion channels. “Some people screens drug companies tradianother molecule, such as an will say they know what’s going on” with tionally use to find leads. anesthetic, outcompetes it for inhaled anesthetics, Eger says. “But I don’t But Eckenhoff pressed on anyway. In ferritin’s binding site, he adds. This means believe they really do.” Eger disclosed 2009 he reported that the horse version that by monitoring 1-aminoanthracene’s fluorescence level, researchers can tell when another molecule binds tightly to ferritin and can single it out for further testing. To make a truly high-throughput screen, the Penn team started working with the National Institutes of Health Chemical GeNow a Division of Evans Analytical Group! nomics Center (NCGC) in Rockville, Md. There, specialists miniaturized the screen so it would run on plates with 1,536 wells Custom Organic Synthesis apiece and made sure the screen worked Contract Research and Development as intended by testing it on a commercially Milligram to Kilogram Quantities available collection of 1,280 pharmacologiDEA Schedule I-V Analytical Samples cally active compounds (PLoS One, DOI: 10.1371/journal.pone.0007150). “That colLitigation Support for Chemical Processes lection is meant for validating screens,” www.chemir.com 866.470.9604 says David Maloney, a chemistry team lead©2011 CHEMIR ANALYTICAL SERVICES | CH0001 er at NCGC. “If you don’t get hits from that, WWW.CEN-ONLINE.ORG
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Pharmaceuticals, is a product of the serendipity school of anesthetic discovery, Raines says. Etomidate is an imidazole, and Janssen “made dozens of these compounds because many imidazoles are antifungal agents. “Of these dozens of compounds, several of them caused the rats to go off to sleep,” you’re not likely to Raines says. The company ultimately chose get hits at all.” to develop etomidate as an anesthetic The NCGC team because of its high therapeutic index, he has now hunted explains. for anesthetics Etomidate is part of the standard anesamong 375,000 thesia tool kit, but it’s not always the best compounds in NIH’s Molecular Libraries option because of a side effect: It interferes Small Molecule Repository. Maloney says with steroid synthesis, Miller says. “That’s one family of small molecules is under adbad news for certain patients, especially ditional investigation but declined to dispatients in intensive care,” who tend to be close the molecules’ structures. Promising on narcotic painkillers that exacerbate the compounds from NCGC’s medicinal chemsteroid imbalance, leading to complicaistry effort get shipped across the Capital tions or even deaths, he adds. Beltway and up Route 95 to Penn, where Miller, Raines, and their collaborators Eckenhoff’s team tests have studied etomidate them directly in tadpoles for years, with techPOCKET PEEK X-ray to verify that the hits niques including lightstructures of an ion channel truly mediate anesthetic activated analogs and from algae confirmed action. The best of those animal assays. “One of some researchers’ data on compounds go into mice. our hopes was to design how anesthetics such as “Our approach is unusuout that unfavorable acpropofol (green and red) and al in that we’re lacking tion while retaining acdesflurane (blue and red) the standard in-house tion at GABAA,” etomibind to their targets. structure-activity reladate’s main anesthetic tionship tests,” says Antarget, Miller says. ton Simeonov, NCGC’s Biochemists chief of biology. “Our knew that etomidate SAR assay is tadpoles.” blocks the action of “It’s a little like going 11β-hydroxylase, a cyback to the old-school tochrome P450 enzyme way of doing drug disthat’s critical for makcovery,” Maloney adds. ing multiple steroids. But old school Its interaction with doesn’t mean outdated, etomidate is not well Simeonov says. The asunderstood, “but there say has the potential to are X-ray structures uncover anesthetics with completely new of other members of that superfamily of chemical scaffolds, he says. “We might enzymes with imidazole-containing drugs even be able to find a target that’s been bound,” Raines says. overlooked,” he adds. Experts in computer modeling helped the Harvard-MGH team see what the SOMETIMES, it isn’t finding a target that etomidate-hydroxylase interaction might elicits anesthesia that leads to a new drug look like. They realized that the drug’s bacandidate. Biophysicist Keith W. Miller, sic nitrogen was likely coordinating to the anesthesiologist Douglas E. Raines, and hydroxylase’s heme group, which might colleagues at Harvard Medical School and have been leading to the blockade of steMassachusetts General Hospital (MGH) roid synthesis. learned that finding the target responThat result pointed to a clear course sible for a side effect is just as important. of action, Raines says. “We switched out They’ve developed a next-generation verthat nitrogen and put in a carbon. That sion of the intravenous anesthetic etomidramatically lowered the affinity for the date that is currently in preclinical studies. hydroxylase but maintained affinity for anEtomidate, first marketed by Janssen esthetic targets,” he says. The team called IN LIVING COLOR
COURTESY OF PIERRE-JEAN CORRINGER
1-Aminoanthracene lights up a tadpole’s brain and spinal cord.
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MORE ONLINE
Watch a video about the Penn anesthesia collaboration at C&EN Online, cenm.ag/anes. Read more anesthetic discovery vignettes on a CENtral Science blog, The Haystack, cenm.ag/blg32.
their resulting compound carboetomidate, and the molecule is now in preclinical studies (Anesthesiology, DOI: 10.1097/ ALN.0b013e3181cf40ed). Tweaking etomidate’s ester moiety to make it break down more quickly in the body also solves the steroid synthesis problem (Anesthesiology, DOI: 10.1097/ ALN.0b013e3181ae63d1). The resulting molecule is called MOC-etomidate, Miller says. To move carboetomidate or MOCetomidate to the point where drug companies might want to spend millions to get them to bedside, Raines has cofounded a company called Annovation BioPharma, together with the Partners Innovation Fund, a venture capital group launched by MGH and Brigham & Women’s Hospital. Raines thinks the lessons of the etomidate story could be widely applicable in anesthesia research. “As we learn more about what the targets are for side effects—for instance, the effect of suppressing breathing—we might be able to tweak molecules to reduce binding to those targets,” he says. Though the efforts at Harvard, Penn, and Washington University have uncov-
ered drug leads, the discovery programs are love to see more chemists working on this small. Few pharmaceutical or biotech comproblem,” she says. panies have anesthetics in their late-stage “The next big set of questions will be pipelines. “We’re not talking about the about what brain regions and networks of search for cancer drugs circuits are involved in here,” Franks says. producing anesthesia,” O “The drive to improve agrees Columbia’s anesthetics is nowhere Harrison. “The ‘how’ O N near as strong.” of anesthesia may N But some argue that be partially solved, Etomidate research in anesthetic but certainly not the discovery and target ‘where,’ ” he says. O O discovery remains imEventually, Weiser’s O OCH3 N portant because it could tadpoles were swimprovide more than new ming in their dish as N general anesthetics. though nothing had MOC-etomidate For example, it could happened. What’s lead to drugs for related most remarkable about O applications, including that is the fundamental O N memory drugs, sleep mystery that it exemaids, and sedatives, plifies, Dmochowski says Beverley Orser, an muses. All of biology, Carboetomidate anesthesiologist at the from plants to tadUniversity of Toronto. poles to people, can be New molecules that induce a state of anesreversibly immobilized with just about the thesia also have potential as tools to learn same concentration range of drug, he exmore about the mind, Orser adds. “I would plains. “It’s almost a defining characteristic of life that we can be anesthetized,” he says. Because of these similarities, anesthetic target discovery might someday point researchers to the proteins and processes behind consciousness itself. “It’s bigger than anesthesia,” he says. “It’s one of the last frontiers in pharmacological research.” ◾
“People in drug companies used to get some mice and inject them with whatever happened to be sitting on the shelf.”
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