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CHEMICAL & ENGINEERING NEWS1155—16th St., N.W., Washingt<strong>on</strong>, DC 20036(202) 872-4600 or (800) 227-5558EDITOR-IN-CHIEF: Rudy M. BaumDEPUTY EDITOR-IN-CHIEF: A. Maureen RouhiMANAGING EDITOR: Robin M. GirouxSENIOR ADMINISTRATIVE OFFICER: Marvel A. WillsNEWSWilliam G. Schulz, EditorBUSINESSMichael McCoy, Assistant Managing EditorNORTHEAST: (732) 906-8300 Lisa M. Jarvis (SeniorEditor), Rick Mullin (Senior Editor), Marc S. Reisch(Senior Corresp<strong>on</strong>dent), Alexander H. Tullo (SeniorEditor), Melody M. Bomgardner (Senior Editor),Rachel Eskenazi (Administrative Assistant). HONGKONG: 852 9093 8445 Jean-François Tremblay(Senior Corresp<strong>on</strong>dent). HOUSTON: (281) 486-3900 Ann M. Thayer (Senior Corresp<strong>on</strong>dent).GOVERNMENT & POLICYSusan R. Morrissey, Assistant Managing EditorBritt E. Ericks<strong>on</strong> (Senior Editor), Glenn Hess (SeniorEditor), Cheryl Hogue (Senior Corresp<strong>on</strong>dent),Jeff Johns<strong>on</strong> (Senior Corresp<strong>on</strong>dent),Andrea L. Widener (Associate Editor)SCIENCE/TECHNOLOGY/EDUCATIONBOSTON: (617) 395-4163 Amanda Yarnell, AssistantManaging Editor. WASHINGTON: Stuart A. Borman(Deputy Assistant Managing Editor), Celia Henry Arnaud(Senior Editor), Carmen Drahl (Associate Editor),Stephen K. Ritter (Senior Corresp<strong>on</strong>dent), Lauren K. Wolf(Associate Editor). BERLIN: 49 30 2123 3740Sarah Everts (Senior Editor). CHICAGO: (847) 679-1156 Mitch Jacoby (Senior Editor). NORTHEAST:(732) 906-8302 Bethany Halford (Senior Editor).WEST COAST: (925) 485-1034 Jyllian Kemsley(Associate Editor), (510) 870-1617 Elizabeth K. Wils<strong>on</strong>(Senior Editor). BEIJING: 150 1138 8372 Jessie Jiang(C<strong>on</strong>tributing Editor). JOURNAL NEWS & COMMUNITY:(202) 872-6039 Lila Guterman (Senior Editor), (626)765-6767 Michael Torrice (Associate Editor)ACS NEWS & SPECIAL FEATURESSophie L. Rovner, Assistant Managing EditorLinda Wang (Senior Editor). DALLAS:(972) 529-4351 Susan J. Ainsworth (Senior Editor)EDITING & PRODUCTIONKimberly R. Twambly, Assistant Managing EditorCraig Bettenhausen (Assistant Editor),Emily B<strong>on</strong>es (Assistant Editor), Sophia L. Cai(Assistant Editor), Nader Heidari (Assistant Editor),Arlene Goldberg-Gist, Senior EditorJeff A. Huber (Assistant Editor),Gail M. Mortens<strong>on</strong> (Associate Editor)ART & DESIGNRobert Brys<strong>on</strong>, Design DirectorRobin L. Braverman (Senior Art Director)Yang H. Ku (Associate Designer)C&EN ONLINERachel Sheremeta Pepling, EditorTchad K. Blair (Visual Designer), Luis A. Carrillo(Producti<strong>on</strong> Manager), Ty A. Finocchiaro (Web Associate),Pamela Rigden Snead (Web Products Manager)PRODUCTION & IMAGINGRenee L. Zerby, Lead Digital Producti<strong>on</strong> SpecialistTim Bauer, Richard C. Smith, andSteven J. Lovasz (Digital Producti<strong>on</strong> Associates)SALES & MARKETINGElise Swinehart, Assistant DirectorElaine Facciolli Jarrett (Marketing Manager)Wendy Wise (Marketing Manager)Angela Yeo (Associate Marketing Manager)ADVISORY BOARDPaul T. Anastas, Paul J. Bracher, Jean-Claude Bradley,Seth M. Cohen, Kendrew H. Colt<strong>on</strong>, Christopher C.Cummins, Joseph M. DeSim<strong>on</strong>e, D<strong>on</strong>ald Hilvert,Malika Jeffries-El, Rohit Khanna, Derek Lowe,Michael W. Major, Michael A. Marletta, Cheryl A. Martin,Harold Meckler, Stephen A. Munk, Nick Roelofs,Melanie Sanford, John M. Schwab, Francis X. Sherman,Linette M. Watkins, Christopher Welch, Vicki Wysocki,David Zimmermann, Dorothy ZolandzPublished by the AMERICAN CHEMICAL SOCIETYMadeleine Jacobs, Executive Director & CEOBrian Crawford, President, Publicati<strong>on</strong>s Divisi<strong>on</strong>EDITORIAL BOARD: Ned D. Heindel (Chair);ACS Board of Directors Chair: William F. Carroll Jr.;ACS President: Bassam Z. Shakhashiri; Stephanie L.Brock, John N. Russell Jr., Leah Solla, Peter J. StangCopyright 2012, <strong>American</strong> <strong>Chemical</strong> SocietyCanadian GST Reg. No. R127571347Volume 90, Number 14Letter From TheEditor-in-ChiefWELCOME TO the sec<strong>on</strong>d issue of “Focus On Life Sciences.” This editi<strong>on</strong> is acompilati<strong>on</strong> of news and feature stories that appeared in January and February2012 issues of C&EN, the weekly newsmagazine published by the <strong>American</strong><strong>Chemical</strong> Society, the world’s largest scientific society.We’re distributing the “Focus On Life Sciences”series because C&EN, like the chemistry enterpriseit is devoted to covering, is deeply involved in all aspectsof modern <strong>life</strong> <strong>sciences</strong>—from bench research<strong>on</strong> the fundamental chemistry of living organismsto breakthrough biopharmaceuticals, from the analyticalinstrumentati<strong>on</strong> that makes <strong>life</strong> <strong>sciences</strong> discoveriespossible to the tough policy choices someof those discoveries pose. Our audience of morethan 164,000 chemical professi<strong>on</strong>als knows that theinterface between chemistry and biology is <strong>on</strong>e ofthe most dynamic and important areas of modern science. It’s where many ofthem work, and C&EN is the magazine they rely <strong>on</strong> to keep them informed ofadvances in the field and of the products and services they use in their labs.For almost 90 years, C&EN’s editorial missi<strong>on</strong> has been to cover news,events, and trends in the chemistry enterprise in a timely, accurate, and balancedway. C&EN’s staff of 50 writers and editors based around the globeis the largest and most experienced team of journalists devoted to coveringchemistry, related <strong>sciences</strong>, and science-based industries. They go where thenews is, and these days, a lot of the news is in the <strong>life</strong> <strong>sciences</strong>. This compilati<strong>on</strong>of recent stories from our <strong>News</strong> of the Week, Business, Government &Policy, and Science & Technology Departments dem<strong>on</strong>strates clearly thatC&EN is right at the cutting edge of news in the <strong>life</strong> <strong>sciences</strong>.I hope you enjoy “Focus On Life Sciences.” With its large global circulati<strong>on</strong>and loyal readership, C&EN provides a tremendous opportunity for advertiserswho want to communicate with top scientists across many disciplines.C&EN reaches more than 164,000 readers each week, and C&EN Online(www.cen-<strong>on</strong>line.org) has grown to more than 12 milli<strong>on</strong> page views per year.In a new offering, C&EN Webinars is kicking off its Life Sciences Series <strong>on</strong>April 24, with “Next Generati<strong>on</strong> Human Genetics.” We think you will findC&EN, with its broad coverage, readership, and outstanding editorial quality,to be an ideal vehicle for reaching your customers.Thanks for reading.Rudy M. BaumWWW.CEN-ONLINE.ORG 1 MARCH 2012


news of the weekCLEARER PICTUREOF A PROTEASOMESTRUCTURAL BIOLOGY: Arrangementof proteasome’s comp<strong>on</strong>entsprovides clues to functi<strong>on</strong>ANEW STRUCTURAL analysis of the yeast proteasomeand <strong>on</strong>e of its comp<strong>on</strong>ents promisesto advance scientists’ understanding of thismacro molecular machine, which breaks down proteinsthat are no l<strong>on</strong>ger needed. The proteasome isa key regulator of the cell cycle and a target for anticancerdrugs.Larger than 1.5 milli<strong>on</strong> dalt<strong>on</strong>s, the proteasomec<strong>on</strong>sists of a barrel-shaped core, inside which proteolysisoccurs, capped at <strong>on</strong>e or both ends by structuresknown as regulatory particles, each of which is furtherdivided into a lid and base.Biochemist Andreas Martin and coworkers at theUniversity of California, Berkeley, report electr<strong>on</strong> microscopystructures of the complete proteasome andan isolated lid at 9- and 15-Å resoluti<strong>on</strong>, respectively( Nature, DOI: 10.1038/nature10774 ).The structures reveal that the lid sits <strong>on</strong> the side ofthe regulatory particle. In this orientati<strong>on</strong>, it can interactextensively with both the regulatory particle’sbase and the proteasome’s proteolytic core.“The lack of a detailed structural model for theregulatory particle has held back research into themechanism of how the proteasome operates,” saysRaym<strong>on</strong>d J. Deshaies , a biology professor at CaliforniaInstitute of Technology, who was not involved in theresearch.The researchers found that ubiquitinreceptors—which detect peptidesthat tag proteins for disposal—arelocated at the top of the complex, nearan enzyme that removes the tags asthe proteins are drawn into the core.The approximately 70–80 Å betweenthe receptors and enzyme may explainwhy tags need at least four ubiquitinunits, Martin says. Another group, ledby Wolfgang Baumeister of the MaxPlanck Institute for Biochemistry, inMartinsried, Germany, has also carriedout a structural study showing that thereceptors are located at the top of the“The lack ofa detailedstructural modelfor the regulatoryparticle hasheld backresearch intothe mechanismof how theproteasomeoperates.”complex ( Proc. Natl. Acad. Sci. USA,DOI: 10.1073/pnas.1119394109 ).The c<strong>on</strong>formati<strong>on</strong> of the lid subunitsin the completecomplex is significantlydifferentfrom their c<strong>on</strong>formati<strong>on</strong>inthe isolatedlid. Theserearrangementscouldexplain how thelid regulates the deubiquitinatingenzyme, Martinsays.Particularly surprisingin the structure isthe spiral-staircase arrangementof six subunitsin the base that unfoldthe protein substrate.These subunits, designatedRpt1–6 , are all different, andthe researchers were able todiscern the positi<strong>on</strong> of each<strong>on</strong>e. Martin says he and hiscoworkers expected a randomorganizati<strong>on</strong> of thesesubunits, but in every particle,Rpt3 was at the topof the staircase and Rpt2was at the bottom. “Wed<strong>on</strong>’t know yet what it meansmechanistically,” he says.The structures could pavethe way to developing inhibitorsthat target the regulatoryparticle insteadof the core, whichis currently amore comm<strong>on</strong>drug target. “Itmay be possibleWWW.CEN-ONLINE.ORG 3 MARCH 2012Structuralmodel shows theorientati<strong>on</strong> ofthe proteasomeregulatory particlebase (blue) and lid(gold) relative tothe proteasome’sproteolytic core(gray).to inhibit degradati<strong>on</strong> of certain substratesbut not others,” Martin says.“The regulatory particle has l<strong>on</strong>g resistedstructural dissecti<strong>on</strong>,” Deshaiessays. “One gets the feeling that this papermarks the beginning of the end, andit is now <strong>on</strong>ly a matter of time beforea high-resoluti<strong>on</strong> atomic model is inhand.” — CELIA ARNAUDReprinted fromC&EN, Jan. 16, 2012


NEWS OF THE WEEKRoche hopesto expand itsdiagnosticinstrumentati<strong>on</strong>business byacquiring Illumina.ROCHEROCHE BIDS TOBUY ILLUMINADIAGNOSTICS: Acquisiti<strong>on</strong> wouldcreate a leading manufacturer ofgene-sequencing instrumentsROCHE HAS MADE a hostile move to acquireIllumina for $5.7 billi<strong>on</strong> in cash. The Swissdrugmaker says it made the bid after Illumina ,a provider of gene-sequencing technology, refused toparticipate in “substantive discussi<strong>on</strong>s” about a friendlyacquisiti<strong>on</strong>.“We remain willing to engage in a c<strong>on</strong>structivedialogue with Illumina to jointly develop an optimalstrategy for maximizing the value of our combinedbusiness,” Roche CEO Severin Schwan said last weekwhen announcing Roche’s offer to buy shares fromstockholders without Illumina’s blessing. To allow timeto review the offer, Illumina’s board has resp<strong>on</strong>ded byadopting a shareholder rights plan, “designed to detercoercive or otherwise unfair takeover tactics.”Illumina dominates the market for next-generati<strong>on</strong>sequencing, followed by Life Technologies and RocheDiagnostics , according to the research firm Frost & Sullivan.After restructuring and cutting 200 jobs late lastyear, Illumina anticipates reporting 2011 revenues ofnearly $1.1 billi<strong>on</strong>.Roche Diagnostics wants to combine Illuminawith its Applied Science unit and move the business’sheadquarters from Germany to Illumina’s home in SanDiego. The acquisiti<strong>on</strong> would add to Roche’s positi<strong>on</strong> ingenetic and genome sequencing and microarrays. At thesame time, Roche envisi<strong>on</strong>s that it could help acceleratethe transiti<strong>on</strong> of DNA sequencing into clinical and routinediagnostic applicati<strong>on</strong>s.“The proposed acquisiti<strong>on</strong> will strengthen Roche’scurrent offering in the <strong>life</strong> science market by providingcomplementary soluti<strong>on</strong>s to our current portfolio,”says Daniel O’Day, chief operating officer of RocheDiagnostics. The combined offering, he adds, would“help enable the discovery of complex new biomarkers,improving drug discovery and the selecti<strong>on</strong> of patientsmost likely to resp<strong>on</strong>d to a targeted treatment.”Roche’s $44.50-per-share offer is a 64% premiumover Illumina’s stock price <strong>on</strong> Dec. 21, 2011, the daybefore rumors about a potential deal began. Since then,the company’s stock price has risen about 40%.Although Roche’s bid is a bit high, “a premium is justified,”Mizuho Securities USA stock analyst Peter Laws<strong>on</strong>told clients in a report. He bases his assessment <strong>on</strong>Illumina’s market dominance and its potential synergieswith Roche’s <strong>life</strong> science, diagnostic, and pharmaceuticalbusinesses, as well as prospects for “l<strong>on</strong>g-termsustainable growth.” —ANN THAYERNATUREStructure ofcytochromeP450 17A1, withprostate cancerdrug abirater<strong>on</strong>e(center, mostlyblack) bound to theenzyme’s heme ir<strong>on</strong>(sphere).PROSTATE CANCERTARGET ANALYZEDMOLECULAR STRUCTURE: Detailed viewof cancer-related cytochrome P450could aid drug designTO UNDERSTAND BETTER how two new anticanceragents work, researchers have obtainedthe first X-ray structures of a key cytochromeP450 enzyme to which they bind. Understanding howthe drugs inhibit the enzyme could aid the designof more effective medicati<strong>on</strong>s forprostate and breast cancer with fewerside effects.Cytochrome P450 17A1 (CYP17A1)is found in cell membranes in the humanreproductive tract and adrenal gland,where it catalyzes sequential hydroxylaseand lyase reacti<strong>on</strong>s in the biosynthesis oftestoster<strong>on</strong>e and other sex horm<strong>on</strong>es. Theapproved Janssen Biotech drug abirater<strong>on</strong>e( Zytiga ) and the Tokai Pharmaceuticalsagent TOK-001 (Galeter<strong>on</strong>e), which is inclinical trials, are prostate cancer treatmentsthat work by inhibiting both the enzyme’s lyaseactivity, accounting for most of their efficacy, and itshydroxylase activity, which causes side effects.Emily E. Scott , an associate professor of medicinalchemistry, and Natasha M. DeVore at the Universityof Kansas have obtained X-ray structures by gettingCYP17A1 to crystallize with each of these drugs bound( Nature, DOI: 10.1038/nature10743 ).The structures reveal that the drugs bind to theenzyme in a manner far different from what scientistsexpected. Modeling studies had suggested the drugswould orient parallel to the plane of the enzyme’sactive-site heme group; instead, the drugs are nearlyperpendicular to it.Other P450 enzymes have been analyzed structurally,says cytochrome P450 specialist Paul Ortiz deM<strong>on</strong>tellano of the University of California, San Francisco.But the new structures are “particularly important”in the search for prostate and breast cancer drugswith fewer side effects. For instance, they could leadto medicati<strong>on</strong>s highly selective for CYP17A1, <strong>on</strong>e of 57similar P450 enzymes found in humans.Scott says her group is collaborating with that ofUniversity of Kansas synthetic chemist Jeffrey Aubé “todesign new compounds we hope will be more selectivedrugs”—by inhibiting the enzyme’s lyase but not its hydroxylaseactivity, for example. — STU BORMANReprinted from C&EN, Jan. 30, 2012 (both)WWW.CEN-ONLINE.ORG 4 MARCH 2012


NEWS OF THE WEEKEASIER ROUTE TOGENOMIC CHANGESGENETIC ENGINEERING: Zinc fingernucleases induce duplicati<strong>on</strong>s,inversi<strong>on</strong>s of DNA sequencesARESEARCH GROUP in South Korea has shownfor the first time that zinc finger nucleases cantrigger sequence duplicati<strong>on</strong>s and inversi<strong>on</strong>sat specific sites in living cells’ chromosomes. The researchersalso engineered variati<strong>on</strong>s that correcteddisease-associated genetic defects.The technique could be used in gene therapy applicati<strong>on</strong>sand in studies of how genomic variati<strong>on</strong>s affectthe functi<strong>on</strong> of live cells or organisms.1 2 3 4Genomic variati<strong>on</strong>s such as deleted segments,duplicate sequences, and inversi<strong>on</strong>s (sequencereversals) often have significant effects <strong>on</strong> the1 2 3 4health of cells and organisms. For example, DNAinversi<strong>on</strong>s cause almost half of severe hemophilia1 2 3 3 4A cases.But in many instances the variati<strong>on</strong>s’ c<strong>on</strong>sequencesare poorly understood, in part because researchers lacksimple, effective techniques to engineer them into specificgenomic sites. Existing methods either introducechanges <strong>on</strong>ly at random sites or require laborious premanipulati<strong>on</strong>of genomic sequences in cells, which alsoleaves behind undesired foreign sequence elements inthe genome.Now, associate professor of chemistry Jin-Soo Kimand coworkers at Seoul Nati<strong>on</strong>al University have usedzinc finger nucleases—synthetic DNA-cleaving enzymesmade from zinc-coordinating protein motifs—to create such variati<strong>on</strong>s more easily ( Genome Res.,DOI: 10.1101/gr.129635.111 ).COURTESY OF JIN-SOO KIMThe researchers designed zinc finger nucleases thattarget specific genomic sequences in human embry<strong>on</strong>ickidney cells. The nucleases create DNA doublestrandbreaks that then induce cellular DNA-repairmechanisms to create duplicati<strong>on</strong>s or inversi<strong>on</strong>s at thesites. Sequence translocati<strong>on</strong>s have been achieved thisway before, but “we are the first to dem<strong>on</strong>strate thatzinc finger nucleases can be used to induce duplicati<strong>on</strong>sand inversi<strong>on</strong>s,” Kim says.He and his coworkers detected, isolated, and characterizedthose variati<strong>on</strong>s. They also dem<strong>on</strong>strated thattheir nucleases could reverse the abnormal genomicinversi<strong>on</strong> associated with hemophilia A.They believe the technique could eventually be usedto correct genomic aberrati<strong>on</strong>s in pluripotent stemcells of patients. Once corrected, the cells could thenbe differentiated into appropriate types of somaticcells and reintroduced into patients.“Perhaps the most intriguing aspect of this study isthe ability to generate individual structural variati<strong>on</strong>sat will so their biological c<strong>on</strong>sequences can be studied,”says biochemistry and molecular biology professor JohnH. Wils<strong>on</strong> of Baylor College of Medicine, who specializesin targeted genome modificati<strong>on</strong>. The new approach“should provide a useful gateway for such studies.”The technique also “potentially allows for the reversalof some [sequence] rearrangements in patients,” although“applicati<strong>on</strong> to patients will require surmountingother substantial obstacles,” Wils<strong>on</strong> notes. — STUBORMAN1 2 3 41 3 2 4Zinc fingernucleases (scissors)make breaks ingenomic doublestrandedDNAthat cells thenrepair to createduplicati<strong>on</strong>s (left)and inversi<strong>on</strong>s(right).Reprinted fromC&EN, Jan. 2, 2012(both)PHARMACEUTICALS Year ends with a flurry of <strong>on</strong>cology research pactsThe year 2011 ended with multiple acquisiti<strong>on</strong>sand research partnershipsthrough which the drug majors TakedaPharmaceutical , Merck & Co. , Eli Lilly &Co. , and AstraZeneca gain access to compoundsthat biotech firms are developingfor the treatment of cancer.Takeda announced that it will acquireIntellikine, a California-based smallmoleculedrug discovery firm that is developingtwo new compounds that targetthe cancer-c<strong>on</strong>nected PI3 kinase/mTORpathway. The Japanese firm will pay$190 milli<strong>on</strong> up fr<strong>on</strong>t and up to $120 milli<strong>on</strong>in milest<strong>on</strong>e payments.In a deal aimed at the same pathway,Merck and Exelixis signed a licensingagreement under which Merck will pay$12 milli<strong>on</strong> up fr<strong>on</strong>t for worldwide rightsto Exelixis’ PI3K-delta R&D program,including XL499, its most advanced preclinicalPI3K-delta inhibitor. Exelixis iseligible for up to $239 milli<strong>on</strong> in milest<strong>on</strong>epayments.Lilly, meanwhile, will obtain exclusivelicenses to ImmunoGen technology allowingLilly to develop antibody-drug c<strong>on</strong>jugatesthat link its own m<strong>on</strong>ocl<strong>on</strong>al antibodieswith cytotoxic maytansinoids. Lillywill pay $20 milli<strong>on</strong> up fr<strong>on</strong>t, with downstreampayments of up to $200 milli<strong>on</strong>.And AstraZeneca signed a pact withChina’s Hutchis<strong>on</strong> MediPharma, a subsidiaryof Chi-Med , to develop volitinib, aninhibitor of the c-Met receptor tyrosinekinase. The U.K. drug company will pay$20 milli<strong>on</strong> up fr<strong>on</strong>t and potential milest<strong>on</strong>epayments totaling $120 milli<strong>on</strong>.For AstraZeneca, the agreement withHutchis<strong>on</strong> follows an acknowledgmentthat its investigati<strong>on</strong>al compound olaparibwill not progress to Phase II developmentfor treatment of serous ovariancancer. The setback will result in a pretaxcharge of $285 milli<strong>on</strong>. — RICK MULLINWWW.CEN-ONLINE.ORG 5 MARCH 2012


NEWS OF THE WEEKChung’s team useda computer todock two differentc<strong>on</strong>formati<strong>on</strong>s ofresveratrol (pinkand turquoise)into the metalc<strong>on</strong>tainingcatalytic pocket of aphosphodiesterase.COURTESY OF JAY CHUNGHOW RESVERATROLWORKSBIOCHEMISTRY: Molecule’s metaboliceffects result from acting directly <strong>on</strong>phosphodiesterases, not sirtuinsHOCHALK UP the red wine compoundresveratrol’s presumedhealth benefits to a direct blockadeof phosphodiesterase enzymes, reports a OHmulti-instituti<strong>on</strong> team ( Cell, DOI: 10.1016/j.cell.2012.01.017). The researchers, led by NIHbiologist Jay H. Chung , say their study clarifies c<strong>on</strong>fusingevidence about the biochemistry of resveratrol.The study’s results point to new avenues of preventingage-associated metabolic diseases in people, saysUniversity of Wisc<strong>on</strong>sin, Madis<strong>on</strong>, calorie restricti<strong>on</strong>researcher Richard Weindruch , who was not involved inthe NIH research. In principle, preventing such diseasescould extend human <strong>life</strong> spans.Resveratrol is known to mimic the antidiabetic effectsof calorie restricti<strong>on</strong> in rodents, and it boosts <strong>life</strong>span in flies and worms. Whether resveratrol is beneficialto humans is not clear, but a recent study suggestsit also mimics the effects of calorie restricti<strong>on</strong> in obeseResveratrolpeople ( Cell Metab., DOI: 10.1016/j.cmet.2011.10.002 ).Resveratrol was thought to work in animals by directlyactivating sirtuin enzymes, which clip acetyl groupsfrom proteins, researchers say. From that idea grew Sirtris,a biotech company that GlaxoSmithKline acquiredfor more than $700 milli<strong>on</strong> in 2008. Since then the sirtuinclaim has come under heavy scrutiny as scientistsOHhave found that they can’t reproduce theearlier pro-sirtuin evidence.Chung and coworkers used cellular assaysand animal tests to show that resveratrolincreases levels of cyclic AMP. The cyclic AMPboost happens, they c<strong>on</strong>clude, because resveratrolblocks phosphodiesterase enzymes that break downcyclic AMP. Resveratrol does indeed activatesirtuin enzymes, Chung says, but indirectly, furtherdownstream in the phosphodiesterase-mediatedpathway.This work points to <strong>on</strong>e pathway resveratrol mayuse to activate sirtuins but doesn’t prove it’s the <strong>on</strong>lypathway, adds sirtuin expert, and Sirtris scientific advisoryboard cochair, Le<strong>on</strong>ard P. Guarente at MIT.Chung notes that it is impossible to prove thatno other mechanism exists but says when his teamblocked the key phosphodiesterase enzyme in muscle,it completely reproduced resveratrol’s effects. Thisstr<strong>on</strong>gly suggests that when it comes to metabolic effects,“the phosphodiesterase pathway is the majorpathway,” he says. — CARMEN DRAHLA computermodel depictsnanoclusters ofantibodies againstpertussis toxin;each disk in theclusters representsa single antibody.EASY-TO-USEPROTEIN DRUGSDRUG DELIVERY: Nanoclusters enableinjectable suspensi<strong>on</strong>s of proteinsTO IMPROVE DELIVERY of protein drugs, researchershave developed a way to make highlyc<strong>on</strong>centrated, injectable suspensi<strong>on</strong>s of proteins( ACS Nano , DOI: 10.1021/nn204166z .When patients need a high dose of a protein drug,they usually receive an intravenous drip.Some rheumatoid arthritis patients, for example,must go to a clinic for regular intravenoustreatment, which is expensive, stressful,and time-c<strong>on</strong>suming. Simple, quick injecti<strong>on</strong>sunder the skin often aren’t feasible, becausehighly c<strong>on</strong>centrated protein soluti<strong>on</strong>s becometoo viscous.In soluti<strong>on</strong>, proteins start to unfold andclump at c<strong>on</strong>centrati<strong>on</strong>s around 100 mg/mL.But inside cells, proteins abound at c<strong>on</strong>centrati<strong>on</strong>sfour times as high and d<strong>on</strong>’t normallyform clumps. By mimicking the crowdedenvir<strong>on</strong>ment inside cells, Keith P. Johnst<strong>on</strong>ACS NANOof the University of Texas, Austin , and colleagues canpack a high dose of protein drugs in a syringe.To re-create the crowding in a cell, the researchersencouraged the proteins to assemble into nanosizedclusters. First they adjusted the pH of the proteins’ soluti<strong>on</strong>so that the molecules had almost no net surfacecharge. Then they added trehalose, a n<strong>on</strong>toxic sugar,in sufficient quantities to push the proteins together.With no surface charge, the proteins d<strong>on</strong>’t repel <strong>on</strong>eanother, and they form nanoclusters measuring a fewhundred nanometers across.The scientists characterized nanocluster suspensi<strong>on</strong>sof three different proteins, including an antibodythat binds the pertussis toxin, at c<strong>on</strong>centrati<strong>on</strong>s as highas 700 mg/mL. When they injected the antibody nanoclustersunder the skin of mice, the nanoclusters tookl<strong>on</strong>ger to diffuse into the bloodstream than did c<strong>on</strong>venti<strong>on</strong>alprotein soluti<strong>on</strong>s, but <strong>on</strong>ce in the bloodstream,both protein formulati<strong>on</strong>s had similar activities and<strong>life</strong>times.Theodore W. Randolph of the University of Colorado,Boulder , says that to dem<strong>on</strong>strate that the nanoclustermethod is practical, the researchers will have to showthat the nanoclusters have at least a two-year shelf<strong>life</strong> and that the method works for other proteins aswell. The Texas group is working <strong>on</strong> further tests. —KATHERINE BOURZAC , Special to C&ENReprinted from C&EN, Feb. 6, 2012 (both)WWW.CEN-ONLINE.ORG 6 MARCH 2012


C&EN TALKS WITHG. STEVEN BURRILLLife <strong>sciences</strong> investment guru sees ‘WELLNESS CARE’ as the next fr<strong>on</strong>tierRICK M ULLIN , C&EN NO RTHEAST N EWS B UREAUG. STEVEN BURRILL reaches into his pocket and pulls out an electr<strong>on</strong>icdevice. “This is my iPh<strong>on</strong>e,” he says, scrolling <strong>on</strong> its screenand poking an applicati<strong>on</strong>. He holds it against his trademark pinkshirt. “And this is my EKG.” Sure enough, a pulsing fever graphmoves across the screen in a pattern very similar to an electrocardiogramreading. “This records it, sends it to the cloud, and downto my cardiologist.”Outside the window of his 27th-floor suite overlooking SanFrancisco’s Embarcadero, a small cluster of morning cloudsagainst the sunrise lends its effect as Burrill flips the ph<strong>on</strong>e backinto his suit jacket pocket, quite proud of the dem<strong>on</strong>strati<strong>on</strong>.And why shouldn’t he be? Thec<strong>on</strong>summate biotech insider,Burrill sits <strong>on</strong> the board of Alive-Cor, the San Francisco-basedtechnology start-up that developedthe iPh<strong>on</strong>e app. His investmentfirm, Burrill & Co ., is thelargest investor in AliveCor.Burrill’s enthusiasm over anapp developer may seem likenew behavior, given his record ofinvestment in traditi<strong>on</strong>al biotechfirms. The most recent coup forBurrill & Co. is the sale of the hepatitisC drug developer Pharmassetto Gilead Sciences for $11 billi<strong>on</strong>.Burrill & Co. was <strong>on</strong>e of the largest investors in Pharmasset, andBurrill was chairman of its board until January 2011. But Burrill nowviews traditi<strong>on</strong>al biotech in a broader health care perspective.His main talking point for some time now has been “The ThreeP s”: pers<strong>on</strong>al, preventive, and predictive. It’s an approach to medicinethat he says will amalgamate small and large molecules withdigital technology to define the future of health care—a future thatbig pharmaceutical companies are struggling with.The route to bringing drugs to market has changed fundamentally,he points out. “It used to be you basically had to prove safetyand efficacy, you threw it over the wall, and the system absorbedit,” he says. But insurance coverage is posing a heightened risk ashealth care legislati<strong>on</strong> evolves. “Now, the real questi<strong>on</strong> is, ‘What isthe pharmacoec<strong>on</strong>omic equati<strong>on</strong> that will determine that a drugwill be paid for?’ ” He adds that regulatory hurdles have“It is notapparent todaywho will bethe dominantplayers in2020, but youmight not bet<strong>on</strong> big pharma.”WWW.CEN-ONLINE.ORG 10 MARCH 2012also become higher as the Food & Drug Administrati<strong>on</strong>struggles to develop approval pathways for new scientificand therapeutic approaches.“The burden is <strong>on</strong> the innovator to prove a new drugis better and costs less than products <strong>on</strong> the market,”he says. “This puts an enormous barrier <strong>on</strong> innovati<strong>on</strong>and a protective layer <strong>on</strong> things already <strong>on</strong> the market.”Burrill believes that good technology will get financing,“but remember that the power is <strong>on</strong> the side of thepeople with the capital, not <strong>on</strong> the side of the entrepreneur.”And the hill is steep. As investors, “we would say no to 99 outof 100 things we see,” Burrill says.Moreover, investors are uneasy about staking companies withnew ideas that face ec<strong>on</strong>omic and regulatory uncertainties <strong>on</strong> topof the usual risk of a new drug’s failing. “Those things in the aggregatespook the investor,” Burrill says.Big drug companies, meanwhile, are acting strangely, Burrillsays. “There is clearly a kind of bipolar nature to big pharma,” hesays. “They are paying up for developed compounds where therehas been a substantial risk reducti<strong>on</strong> and they can see the marketopportunities. On the flip side, they are putting out some reas<strong>on</strong>ableamounts of m<strong>on</strong>ey <strong>on</strong> pureplaydiscovery hits. What is evenmore strange is how big pharmais reaching all the way back to theRICK MULLIN/C&ENacademic instituti<strong>on</strong>s.”What is obvious, he says, is thatmajor pharmaceutical companiesare well into the process of c<strong>on</strong>vertingfrom vertical enterprisesengaged in everything from discoveryto marketing, to far morehoriz<strong>on</strong>tal operati<strong>on</strong>s heavilyengaged in marketing and distributi<strong>on</strong>.But the shift is not happeningeasily. He points to formerPfizer chief executive officerJeffrey Kindler’s failed effort to “turn the ocean liner called Pfizeraround” with forays into biologics and digital health as an exampleof how large companies are resisting fundamental change.Others are also resisting change, he notes. “Twenty-two percentof our gross domestic product is in health care, and a lot of peopled<strong>on</strong>’t want to vote themselves off of the island,” Burrill says. “Thereis an enormous amount of perverse incentive to keep the systemthe way it is in the face of this <strong>on</strong>slaught of technologies that will totallydisenfranchise large amounts of the health care system today.”Still, change happens. “Health care is a funny label, because we areactually in a very dysfuncti<strong>on</strong>al ‘sickness care’ system,” Burrill says. “Butwe are moving to an increasingly effective ‘wellness care’ system, usinga c<strong>on</strong>fluence of technology to improve outcomes at the patient level.”The trick will be to identify the companies that best define thefuture.“I think we are going to see some strange bedfellows,”Burrill says. “ Samsung has a major biosimilarsdeal with Biogen Idec . Six m<strong>on</strong>ths ago, you would havesaid, ‘Samsung? What do they have to do with biotechnology?’” He points to Google, Facebook, and Twitter,which came from nowhere to become major ec<strong>on</strong>omicforces in just a few years. “It is not apparent today whowill be the dominant players in 2020,” Burrill says,“but you might not bet <strong>on</strong> big pharma.” ◾Reprinted from C&EN, Feb. 6, 2012


GOVERNMENT & POLICYNEW YORK ACADEMY OF SCIENCESA DUAL-USE DEBATEIn light of c<strong>on</strong>troversial H5N1 BIRD FLU experiments, expertshashed out myriad issues at a public forum in New York CityWILLIAM G . S CHULZ , C&EN WASHINGTONA SPIRITED—at times, bordering <strong>on</strong> nasty—publicforum <strong>on</strong> recent H5N1 avian fluresearch highlighted the diverse opini<strong>on</strong>swithin the scientific community <strong>on</strong> justhow to deal with dual-use research, whichis officially defined as research that canbe used for good or ill purposes. The experimentalwork discussed at the Feb. 2forum involved directed mutati<strong>on</strong>s of theH5N1 virus such that aerosol transmissi<strong>on</strong>between mammals (ferrets) was achievedfor the first time, a mutati<strong>on</strong> that has notoccurred in nature (C&EN, Feb. 6, page 6).The forum, hosted by the New YorkAcademy of Sciences (NYAS), capped anintense week of news about the yet-tobepublished research. Just days earlierthe Nati<strong>on</strong>al Science Advisory Board forBio security (NSABB) recommended thatwork d<strong>on</strong>e by independent groups in theU.S. and the Netherlands and reported intwo different papers be published <strong>on</strong>ly ingreatly redacted form—that is, with theexperimental methods and results excisedfrom the publicati<strong>on</strong>s. The <strong>on</strong>-hold papershave already been peer-reviewed and acceptedby the journals Science and Nature .The event also followed a call for a 60-day moratorium agreed to by <strong>on</strong>e of thetwo principal investigators involved, YoshihiroKawaoka of the University of Tokyoand University of Wisc<strong>on</strong>sin, Madis<strong>on</strong>( Nature, DOI: 10.1038/nature10884 ). Theother PI is R<strong>on</strong> A. M. Fouchier of ErasmusMedical Center, in the Netherlands, whois said to be in agreement. Neither researcherattended the NYAS forum.The H5N1 research brings to light a biggerquesti<strong>on</strong> about the broad definiti<strong>on</strong> ofdual-use research and the absence of anyagreed-up<strong>on</strong> protocols to deal with it. Thisambiguity, NYAS panelists noted, potentiallyraises c<strong>on</strong>cerns about a multitude offederally funded research projects acrossscientific disciplines.“The <str<strong>on</strong>g>focus</str<strong>on</strong>g> is <strong>on</strong> H5N1, but there arebroader issues at play—this is just an example,”said NYAS panel moderator IanW. Lipkin, a professor of epidemiology,neurology, and pathology at Columbia University.He pointed out that there is a needin the scientific community to <str<strong>on</strong>g>focus</str<strong>on</strong>g> <strong>on</strong> the“intellectual, emoti<strong>on</strong>al, and ethical” c<strong>on</strong>cernssurrounding dual-use research fromany discipline.THE FORUM ATTRACTED a packed auditoriumof students, academics, journalists,and members of the general public at NYASheadquarters in lower Manhattan, just afew blocks from the new 9/11 Memorial andthe rising towers of the city’s new WorldHOT ISSUE TheNew York Academyof Sciencesc<strong>on</strong>vened anexpert panelto discuss thec<strong>on</strong>troversysurroundingrecent H5N1 avianflu experiments.Trade Center. Their<strong>on</strong>y of holding thisdual-use discussi<strong>on</strong>so near Ground Zerowas noted by forumparticipants.The panelists includedprominentinfluenza researchersin the U.S., NSABBmembers, a former science journalistwho has written extensively about pandemichealth threats, and editors fromScience and Nature who were intimatelyinvolved with the handling of the papersin questi<strong>on</strong>.Lipkin in his opening remarks commented<strong>on</strong> the many stakeholders in influenzaresearch—the public, scientists,pharmaceutical companies, and people inthe developing world where such diseasesare often endemic. “Scientific investigati<strong>on</strong>of pathogenic diseases is essential,”he said, and H5N1 is no different. “If wed<strong>on</strong>’t make a plan ourselves,” he said,regarding the dual-use characteristics, “researchcritical to the public interest couldbe curtailed.”NSABB Acting Chair Paul Keim, a microbiologyprofessor at Northern Ariz<strong>on</strong>aUniversity who attended the forum viawebcast, and other members of the boardsaid the H5N1 papers were brought to theirattenti<strong>on</strong> in October 2011 from varioussources, including program officers at theNati<strong>on</strong>al Institutes of Health. Editors fromScience and Nature insist that they neverreferred either of the papers to NSABB—and never would. Science Deputy EditorBarbara R. Jasny, who was an NYAS forumpanelist, said that her journal did c<strong>on</strong>veneits own body of advisers when the difficultethical issues regarding publicati<strong>on</strong> of theresearch became apparent.Michael T. Osterhelm, NSABB memberand director of the Center for InfectiousDisease Research & Policy at the Universityof Minnesota, emphasized that the 23members of the advisory board were unanimousin their recommendati<strong>on</strong> that <strong>on</strong>lygreatly redacted versi<strong>on</strong>s of the Fouchierand Kawaoka papers be published. He wasalso a forum panelist.“I would hesitate to say that we areestablishing a protocol,” Osterhelm said.“We are not the final arbiter.” NSABBmembers, he said, are recommending astep back because <strong>on</strong>ce the papers’ researchmethodologies and results becomewidely available, “you can’t unring theWWW.CEN-ONLINE.ORG 11 MARCH 2012


GOVERNMENT & POLICYbell.” Any<strong>on</strong>e seeking the informati<strong>on</strong>,for any purpose, could readily find it, hepointed out.And while research journals and federalagencies are under no obligati<strong>on</strong> to followNSABB’s recommendati<strong>on</strong>s, the advisoryboard and its decisi<strong>on</strong>s do carry weight inthe scientific community. The board wascreated in light of a recommendati<strong>on</strong> inthe 2004 Nati<strong>on</strong>al Research Council report,“Biotechnology Research in an Age ofTerrorism.”NSABB members came to the tableto discuss the H5N1 papers withdiffering opini<strong>on</strong>s, recalled memberArturo Casadevall, a professor ofmicrobiology at Albert Einstein Collegeof Medicine. Initially, he said,he was opposed to the recommendati<strong>on</strong>for publishing <strong>on</strong>ly redactedversi<strong>on</strong>s of the research papers.But “the process is deliberative andpeople can change their minds,” hesaid. “I changed mine.”But other NYAS panel membersare alarmed by the NSABB recommendati<strong>on</strong>,which they see as aneffort to shut down productive linesof research and censor experimentalresults.“WE NEED TO specifically address the situati<strong>on</strong>of H5N1,” said Peter Palese, chair ofthe department of microbiology at MountSinai School of Medicine. The Fouchier andKawaoka research groups, he said, “are passagingan H5N1 virus in ferrets. This is perfectlylegitimate,” and many other groupshave d<strong>on</strong>e similar work, he noted, referringto the experimental method in which viraladaptati<strong>on</strong> is achieved by repeated passagein cells or in naive animal hosts. He said theexperiments are necessary because theyhelp researchers identify the very mutati<strong>on</strong>sthey should be m<strong>on</strong>itoring in wildstrains of the virus that could signal <strong>on</strong>setof a human pandemic.Palese went <strong>on</strong> to questi<strong>on</strong> the casefatality rate of H5N1 infecti<strong>on</strong> in humans,which is estimated to be in the range of50 to 80%. He explained that this rate iscalculated <strong>on</strong>ly from documented cases ofhuman H5N1 infecti<strong>on</strong> as defined by WorldCYNTHIA GOLDSMITH/CDCHealth Organizati<strong>on</strong> (WHO) guidelines.The overwhelming majority of infecti<strong>on</strong>s,including those that do not result in deathor that create asymptomatic H5N1 carriers,are not likely to be reported to WHO, hesaid. These undocumented infecti<strong>on</strong>s occurin poor, rural areas where medical care,including access to advanced laboratory diagnostics,is not likely. Because the numberof n<strong>on</strong>lethal cases is being underreported,Palese said, “the case fatality rate is muchlower than what WHO is putting out.”The questi<strong>on</strong>ing of thecase fatality rate by Paleseled to a testy exchangewith NSABB memberOsterhelm, who accusedPalese of putting forwardfalse informati<strong>on</strong>. “Thestudy you c<strong>on</strong>tinue to cite is the worst<strong>on</strong>e of all,” Osterhelm said, referring to aJanuary Proceedings of the Nati<strong>on</strong>al Academyof Sciences article cowritten by Palese andMount Sinai School of Medicine colleagueTaia T. Wang, titled “H5N1 influenzaViruses: Facts, Not Fear” (DOI: 10.1073/pnas.1121297109 ).Casadevall chimed in <strong>on</strong> the exchangeand said, “The [fatality] numbers areunbelievable no matter any way you lookat it. When these things [the experimentalviruses] get out and combine with existingstrains, the situati<strong>on</strong> becomes completelyunpredictable.”Palese c<strong>on</strong>tinued to draw fire when heDUAL USE A colorizedTEM image showsH5N1 (gold) avian fluvirus growing am<strong>on</strong>gkidney cells (green).questi<strong>on</strong>ed the use of ferrets in the experiments.“People are equating transmissi<strong>on</strong>in ferrets with ease of transmissi<strong>on</strong> inhumans,” he said. The animals are indeeda model, he c<strong>on</strong>tinued, but the implicati<strong>on</strong>sfor human disease are unknowableat this time. “In fact, the ferret is much toosensitive” to make good predicti<strong>on</strong>s abouthuman infecti<strong>on</strong>, Palese said. “An animalmodel is just that—a model.”“But then what would be a good model,sir?” fellow panelist Laurie Garret, a seniorfellow at the Council <strong>on</strong> ForeignRelati<strong>on</strong>s and a former journalistwho has written extensively <strong>on</strong>emerging diseases, shouted acrossthe table.A visibly shaken and angry Paleseresp<strong>on</strong>ded: “These are not c<strong>on</strong>clusiveexperiments, they are notcompelling, and we are using them toshut down a lot of science.”Other panelists also raised questi<strong>on</strong>sabout how to advance importantbut risky research. Jasny tried to get ata root questi<strong>on</strong>: “What is it that causesvirus to jump from species to species—to become more virulent?” Getting theanswer to this and other importantquesti<strong>on</strong>s in virology requires dualuseresearch, she said. “Any kind ofresearch can have risk associated withit—it doesn’t mean we ignore the risk.How do we move ahead?”Panel member Alan S. Rudolph,director of the joint science and technologyoffice for the government’s<strong>Chemical</strong> & Biological Technologies Directorate,noted c<strong>on</strong>cerns about including internati<strong>on</strong>alperspectives <strong>on</strong> the research—some of which did occur outside the U.S.“The problem extends well bey<strong>on</strong>dour borders,” Rudolph said. He notedthat quite a lot of activity <strong>on</strong> the H5N1experiments is starting to happen abroad,including a closed-door meeting at WHOheadquarters in Geneva scheduled for Feb.16 and 17. No agenda has been posted, butthe invitati<strong>on</strong>-<strong>on</strong>ly c<strong>on</strong>ference is likely totouch <strong>on</strong> data sharing and other issues,including the possibility of distributingthe mutated experimental viruses to otherresearchers.“Any kind of research can haverisk associated with it—it doesn’tmean we ignore the risk.”WWW.CEN-ONLINE.ORG 12 MARCH 2012GARRETT N OTED that internati<strong>on</strong>al views<strong>on</strong> how to handle this research may be verydifferent than the c<strong>on</strong>sensus in the U.S.She said it is important to note that, whenpeople discuss what is ultimately a healthcare issue, the U.S. is often criticized in the


internati<strong>on</strong>al community. The U.S. insistence<strong>on</strong> intellectual property protecti<strong>on</strong>sfor prescripti<strong>on</strong> drugs, she said, is oftenseen as an uncaring obstacle to health carefor people in the developing world.As for H5N1, Garrett pointed out anewspaper editorial in Ind<strong>on</strong>esia that hailsFouchier as a hero for achieving breakthroughs<strong>on</strong> H5N1 research that could leadto new protecti<strong>on</strong>s for people in a countrywith potentially huge reservoirs of the virusin domesticated birds.NSABB’S RECOMMENDATION to redactinformati<strong>on</strong> prior to publicati<strong>on</strong> of thepapers, however, comes a bit late, severalpanelists noted. This is because theFouchier and Kawaoka papers—includingmethodology and results—have alreadybeen discussed at a meeting in Malta lastyear. What’s more, the eyes of many expertsin influenza research have seen thepapers during peer review. Some panelistsestimated that about 1,000 people aroundthe world have read the papers or have directknowledge of the work, leading to furtherspeculati<strong>on</strong> about the value of at least“We need to maintain the integrityof the scientific process.”some limited censorship of the data now.If the papers do appear in Science andNature in redacted form, which seems allbut certain, the panelists agreed that sharingthe full data sets and deciding whichresearchers are qualified to receive themwill be fraught with difficulty.“We need to maintain the integrity ofthe scientific process,” said panelist Vér<strong>on</strong>iqueKiermer, executive editor for Natureand the Nature journals. She said the rolesof serendipity, interdisciplinary attenti<strong>on</strong>,and other factors in scientific research cannotbe underestimated for sparking “thingsthat can be field-changing.” In any systemto limit distributi<strong>on</strong> of the redacted datasets, “we need to be very careful about howto establish these criteria and decide whothese people will be,” she said.So far Nature has made no public statementabout its plan for the H5N1 paperunder its c<strong>on</strong>trol. Science Editor-in-ChiefBruce M. Alberts has stated his supportfor the NSABB recommendati<strong>on</strong> but hasalso suggested that the journal wait for acredible plan for data sharing from the U.S.government before publishing the paper itc<strong>on</strong>trols.As the forum c<strong>on</strong>cluded, the panelistsremained split in their opini<strong>on</strong>s. Allseemed to agree that biology was at anothercrossroads, especially with the growingbody of research in synthetic biology,and that resp<strong>on</strong>sibility for the safety andwelfare of the general public should be ofparamount c<strong>on</strong>cern.“But we can’t be wr<strong>on</strong>g,” said Osterhelmof the H5N1 research. “There’s no goingback. We have no choice but to act <strong>on</strong> this<strong>on</strong>e.”Palese asked perhaps the ultimate questi<strong>on</strong>about H5N1 and any dual-use research:“Where do you stop being afraid?” ◾Reprinted from C&EN, Feb. 20, 2012D<strong>on</strong>’t Miss C&EN’s Life Sciences Focused IssuesC&EN Editorial CalendarFrom display advertising to cover tips, deliveryour message al<strong>on</strong>gside our <strong>life</strong> <strong>sciences</strong>-specificeditorial c<strong>on</strong>tent for high-impact exposure.June 4June 18July 23August 6September 3September 17October 22November 5December 3NanotechnologyCancer Drug ResearchDiagnosticsBiobased <strong>Chemical</strong>sPharmaceutical ChemistryBiofuels & EnergyLife SciencesPharmaceutical PartneringProteomics & GenomicsInterested in receiving a printcopy of C&EN? C<strong>on</strong>tact ElaineJarrett at jarrett@acs.org toreceive a complimentary copy.Visit cen-<strong>on</strong>line.org/advertisefor informati<strong>on</strong> about C&EN’sadvertising opportunities.WWW.CEN-ONLINE.ORG 13 MARCH 2012


SHUTTERSTOCKTHE U.S. SUPREME COURT heard argumentslast m<strong>on</strong>th in a highly complex patentdispute between two medical testinglaboratories that could affect the future ofmolecular diagnostics and the emergingfield of pers<strong>on</strong>alized medicine, which involvesm<strong>on</strong>itoring an individual’s resp<strong>on</strong>seto a drug to tailor treatment.The decisi<strong>on</strong>, which is expected thisspring, will likely have important implicati<strong>on</strong>sfor the biotechnology and pharmaceuticalindustries because the high courtis being asked to determine whether certaintypes of medical treatment methodsare eligible for patent protecti<strong>on</strong>.“Some fundamental issues around patenteligibility in the <strong>life</strong> <strong>sciences</strong> area arefr<strong>on</strong>t and center before the court,” saysPaul M. Rivard, a patent attorney in theWashingt<strong>on</strong>, D.C., office of Banner & Witcoff, an intellectual property (IP) law firm.“The court is expected to lay down somebasic principles in the area, such as whetherprocesses involving administering a syntheticdrug are always patent-eligible, andwhether the lack of novelty of individualmethod steps should be taken into accountwhen determining patent eligibility,” heremarks.The ruling will impact players in the pharmaand <strong>life</strong> <strong>sciences</strong> fields because thereare thousands of issued patents relating todiagnostic methods and tests, says KendrewH. Colt<strong>on</strong>, a partner at Chicago-based IP lawfirm Fitch, Even, Tabin & Flannery .GOVERNMENT & POLICYSUPREME COURT LOOKSAT MEDICAL PATENTSPatent eligibility of PERSONALIZED MEDICINEis at stake in closely watched caseGLENN HE SS , C&EN WASHINGTON“The Supreme Court’s decisi<strong>on</strong> willlikely refine precedent generally for whatis patent-eligible subject matter,” Colt<strong>on</strong>says. “While not limited to <strong>life</strong> <strong>sciences</strong>, thedecisi<strong>on</strong> will affect <strong>life</strong> <strong>sciences</strong> and the stillnascent field of pers<strong>on</strong>alized medicine.”In the case before the high court, MayoCollaborative Services, part of the Rochester,Minn.-based Mayo Clinic , is appealinga judgment that it infringed patents held byPrometheus Laboratories . The patents involvea blood test that helps doctors determineoptimal drug dosages for patients withgastrointestinal disorders such as Crohn’sdisease and other autoimmune ailments.THE TEST MEASURES metabolite levelsand the efficacy and toxicity of thiopurinedrugs, the use of which is often accompaniedby serious side effects. Becausepatients metabolize the compounds differently,it had been difficult for doctors tocalibrate the proper dosage, and some doctorswere reluctant to prescribe the drugsat all, fearing harmful side effects.The Mayo Clinic used the Prometheustest until 2004, when its doctors announcedthat they had created a cheaper and fasterWWW.CEN-ONLINE.ORG 14 MARCH 2012MEDICALPROCEDURESThe S upremeCourt isc<strong>on</strong>sideringwhether thecorrelati<strong>on</strong>between bloodtest results andpatient health ispatent-eligiblesubject matter.test to determine a patient’soptimal dosage.At that point, San DiegobasedPrometheus—aunit of Switzerland’sNestlé Health Science—sued for patent infringement,setting off thelegal fight now before thenati<strong>on</strong>’s highest court.Mayo c<strong>on</strong>tends thatPrometheus is seekingto protect an abstract idea based <strong>on</strong> naturalphenomena: the observed correlati<strong>on</strong>sbetween blood test results and patienthealth. By law, natural phenomena may notbe patented. But Prometheus asserts thatits patents describe a specific method forimproving the treatment of certain diseasesthrough a series of c<strong>on</strong>crete and transformativesteps, and should be allowed.In March 2008, the U.S. District Courtfor the Southern District of California, SanDiego, agreed with Mayo’s positi<strong>on</strong> andinvalidated the patents, finding that Prometheus’inventi<strong>on</strong> was no more than “anatural body process … preexisting in thepatient populati<strong>on</strong>.”But in September 2009, the U.S. Courtof Appeals for the Federal Circuit, in Washingt<strong>on</strong>,D.C., reversed the lower court ruling,saying the claims are patent-eligiblebecause they involve a physical transformati<strong>on</strong>and thus are not merely an abstractidea or law of nature.Trade associati<strong>on</strong>s representing biotechand research-based drug companies, as wellas the Associati<strong>on</strong> of University TechnologyManagers, are siding with Prometheusand have filed briefs urging the SupremeCourt to uphold the appellate ruling.In its filing, the Biotechnology IndustryOrganizati<strong>on</strong>, which represents more than1,100 biotech companies, warns that excludingbiomarker-assisted therapeutic methodsfrom patent eligibility would be “devastatingto pers<strong>on</strong>alized medicine” because itwould discourage investment in the field.Also called targeted therapy, pers<strong>on</strong>alizedmedicine entails the use of a patient’sgenetic informati<strong>on</strong> to select medicinesand treatments that precisely match theneeds of the individual.“The Supreme Court’s decisi<strong>on</strong> willlikely refine precedent generally forwhat is patent-eligible subject matter.”


Mayo’s view is backed by a coaliti<strong>on</strong> ofphysicians and health care groups, includingthe <strong>American</strong> Medical Associati<strong>on</strong>. In a jointbrief, the medical establishment argues that“health care will be undermined if c<strong>on</strong>venti<strong>on</strong>almedical applicati<strong>on</strong>s of naturally occurringbodily processes can be patented.”DURING ARGUMENTS BEFORE the SupremeCourt <strong>on</strong> Dec. 7, 2011, Mayo Clinicattorney Stephen M. Shapiro said that barringMayo and others from using tests similarto Prometheus’ would be detrimental topatients’ health.“The problem with the Prometheus patentis its broad preempti<strong>on</strong> of a physicalphenomen<strong>on</strong>, which prevents others likeMayo Clinic from offering a better metabolitetest with more accurate numbers. Thisis a huge practical problem for patients,”Shapiro said.Several justices also raised questi<strong>on</strong>sabout Prometheus’ diagnostic patentclaims. “This is not a treatment protocol;it’s not a treatment regimen,” Justice ElenaKagan told Prometheus’ attorney, RichardP. Bress. “All you have d<strong>on</strong>e is pointed outa set of facts that exist in the world and areclaiming protecti<strong>on</strong> for something thatanybody can try to make use of in any way,and you are saying ‘you have to pay us,’ ”she remarked.Bress acknowledged that the lab’s patentsbuild <strong>on</strong> a known process. “Peopleknew that you could administer thiopurinesfor these particular diseases” andmeasure the resulting metabolites, he said.There have been efforts to “come up withwhat [Prometheus] came up with—a newtreatment method, a new way of calibratingthe right dose for each individual patientbased <strong>on</strong> their metabolism.”But, Bress explained, the mere fact thatothers had previously combined administrati<strong>on</strong>of thiopurines with measurementof the metabolites in laboratory experimentsdoes not, under the court’s precedents,negate the novelty of Prometheus’use of the same steps as integral parts of asuccessful working treatment method.Rivard says it is not clear exactly wherethe court will draw the line between processesinvolving mere abstract ideas andthose meriting patent protecti<strong>on</strong>. But thejustices, he observes, “seem to be wellaware of the need to tread carefully, sinceany significant limitati<strong>on</strong> <strong>on</strong> patent eligibilityin this area could have a chilling effect<strong>on</strong> research.”Justice Stephen G. Breyer, for example,expressed skepticism over the patent eligibilityof diagnostic method claims, buthe also took note of the significant investmentsthat companies have made. “Discoveringnatural laws is often a very expensiveprocess,” he said. “There’s lots of investmentto be protected.”If the Supreme Court c<strong>on</strong>cludes thatthe Prometheus patents are invalid, someindustry players may encounter challengesin the short term <strong>on</strong> their patents relatingto diagnostic methods and tests, saysColt<strong>on</strong>, a past chair and current executivecommittee member of the <strong>American</strong><strong>Chemical</strong> Society’s Divisi<strong>on</strong> of Chemistry& the Law.Over the l<strong>on</strong>g term, he adds, differentapproaches to drafting patent claims toprotect investments in diagnostic methodsand tests will emerge. ◾Reprinted from C&EN, Jan. 16, 2012AQO\bVSQ]RSb]dWSebVSdWRS]2]e\Z]ORbVSO^^b]OQQSaa(13


SCIENCE & TECHNOLOGYCOVANCEDRUG DILIGENCEAnalysts p erformbioassays in cell-culturehoods at Covance’s NorthAmerica BiotechnologyServices facility.ASSAYING ANTIBODIESDrug manufacturers pinpoint TECHNIQUES TO ANALYZEgrowing and diverse class of therapeuticsJYLLIAN KE MSLEY , C&EN WEST C OAST NE WS B UREAUMONOCLONAL-ANTIBODY-based drugsare a large and growing segment of pharmaceuticals.Since murom<strong>on</strong>ab-CD3(Orthocl<strong>on</strong>e OKT3) was first approved bythe Food & Drug Administrati<strong>on</strong> in 1986 asan antirejecti<strong>on</strong> drug for organ transplantpatients, more than 30 antibody-baseddrugs have entered the market, and manymore are in the pipeline. In 2011 al<strong>on</strong>e,FDA approved belimumab (Benlysta) forlupus, ipilimumab (Yervoy) for metastaticmelanoma, and brentuximab vedotin (Adcetris)for two types of lymphoma. Marketresearch company Datam<strong>on</strong>itor estimatesthat sales of antibody therapeutics willgrow by 8.2% from 2010 to 2016, the fastestof any therapeutic class, with sales expectedto surpass $65 billi<strong>on</strong> by 2016.Although drugs based <strong>on</strong> m<strong>on</strong>ocl<strong>on</strong>alantibodies—MAbs—have widely variedtherapeutic acti<strong>on</strong>, they are all based <strong>on</strong> thesame protein, immunoglobulin G (IgG).But this shared attribute does not meanthat these drugs are simple to assay. MAbsare composed of more than 1,000 aminoacids in four peptide chains, and they bearsugars and other chemical modificati<strong>on</strong>s.As the MAb class grows, scientists aresettling <strong>on</strong> some standard critical proteinqualities and analytical testing approachesfor drug identity, quality, and purity forthese large, complex molecules.Al<strong>on</strong>g those lines, the U.S. PharmacopeialC<strong>on</strong>venti<strong>on</strong> (USP), the pharmaceuticalstandard-setting organizati<strong>on</strong>in the U.S., is in the midst of developing“best practice” guidelines and regulatorystandards for MAb drug developers. “Theindustry has more than 20 years of manufacturingexperience with these types ofmolecules, and we’re seeing products thatare extremely pure and really well characterized,”says Tina S. Morris , USP’s vicepresident for biologics and biotechnology.“Every antibody has slightly differentpurificati<strong>on</strong> and analytical characteristics,but you’re never starting from zero with acompletely unknown protein.”One set of standards, to be known asChapter 1260, will <str<strong>on</strong>g>focus</str<strong>on</strong>g> <strong>on</strong> how to develop,define quality attributes for, and manufactureMAbs.These standards will not be legallybinding <strong>on</strong> MAb manufacturers but insteadare intended to provide worldwidebest practice guidance, says Anth<strong>on</strong>yMire-Sluis, corporate vice president forproduct and device quality at Amgen andchair of the USP Recombinant TherapeuticM<strong>on</strong>ocl<strong>on</strong>al Antibodies Expert Panel,which is developing the standards. Theexpert panel includes 13 industry members,as well as seven representatives fromregulatory and other standard-settingagencies.The other set of standards, Chapter 129,will be legally binding <strong>on</strong> MAb manufacturersand outlines the minimal quality attributescomm<strong>on</strong> to all antibodies and thecorresp<strong>on</strong>ding recommended analyticaltests for MAb therapeutics. Several companieswill validate the proposed methodsWWW.CEN-ONLINE.ORG 16 MARCH 2012


to ensure that they work, and the standardswill also include limits derived frommanufacturer data <strong>on</strong> actual products. But,Mire-Sluis notes, companies will be ableto justify using different tests or limits aspart of their drug applicati<strong>on</strong> package ifthey think their product merits somethingatypical.STRUCTURALLY, an antibody,or immunoglobulin, is a large,Y-shaped protein composedof two “heavy” and two “light”peptide chains. The heavychains come together at thebottom half of the Y, also calledthe crystallizable fragment, orFc. Disulfide b<strong>on</strong>ds and n<strong>on</strong>covalentinteracti<strong>on</strong>s hold thechains together. This part ofthe antibody has a c<strong>on</strong>servedamino acid sequence and bindsto receptors to activate partsof the immune system. Differentantibody classes—IgA,IgD, IgE, IgG, and IgM—havedifferent heavy chains that determinespecific immunologicalroles. IgG provides most of thebody’s immunity to invadingpathogens.At the top half of the antibodyY, the heavy chains branchout and each associates witha light chain, also through disulfideb<strong>on</strong>ds and n<strong>on</strong>covalentinteracti<strong>on</strong>s. This regi<strong>on</strong> of the antibodyis called the antigen-binding fragment,or Fab, and it is here that the amino acidsequence varies to bind to assorted foreignmolecules: Both arms of a specific antibodyhave the same amino acid sequence, whilethe variable porti<strong>on</strong> differs between differentantibody molecules.But because MAbs are made by cl<strong>on</strong>edcell lines with identical cells, all of the antibodiesproduced by a cell line engineeredfor producing a particular therapeuticshould be identical. M<strong>on</strong>ocl<strong>on</strong>al IgG therapeuticswere originally based <strong>on</strong> mouseantibodies, but they now can be chimeric,which means a part-mouse/part-humansequence, or “fully” human. Structurally,the three types are very similar, but theymay provoke different immune resp<strong>on</strong>sesin patients.In additi<strong>on</strong> to their basic structurallikeness, MAb therapeutics also tend tobe purified similarly, USP’s Morris notes.Purificati<strong>on</strong> typically starts with affinitychromatography using a solid phase thatanchors protein A, a bacterial proteinthat selectively binds to the antibodies’c<strong>on</strong>served Fc regi<strong>on</strong>. After that, the drug islikely treated to inactivate any c<strong>on</strong>taminatingviruses and may undergo fine-tuningpurificati<strong>on</strong> steps. Most antibodies arealso currently produced from two popularcell lines, which means that their impurityCONFIGURATION The basic IgG structure involvestwo heavy chains and two light chains held together bydisulfide b<strong>on</strong>ds and n<strong>on</strong>covalent interacti<strong>on</strong>s. The proteinmay be glycosylated at a c<strong>on</strong>served site (shown) orelsewhere <strong>on</strong> the structure.SSSSSSOligosaccharideHeavy chainVariable ◼C<strong>on</strong>served ◼Light chainVariable ◼C<strong>on</strong>served ◼SSSSSSSSprofiles are similar. The combinati<strong>on</strong> ofsimilar cell culture and purificati<strong>on</strong>, <strong>on</strong> topof targeting the same protein, further lendsanalysis of MAb therapeutics to similarprotocols, Morris says.But despite these similarities, MAbsmay show heterogeneity depending <strong>on</strong>how they were produced and processedby their originating cells. Problems withDNA translati<strong>on</strong> or transcripti<strong>on</strong> may leadto different protein sequences. Proteinsmay misfold or mismatch their disulfideb<strong>on</strong>ds. Posttranslati<strong>on</strong>al modificati<strong>on</strong> mayproduce different glycosylati<strong>on</strong> patterns.Side chains may also be subject to chemicalreacti<strong>on</strong>s, such as oxidati<strong>on</strong> or deamidati<strong>on</strong>.Proteolytic enzymes may clip theprotein, especially the lysines at the ends ofthe heavy chains in the Fc. MAbs may alsodenature or aggregate as they go throughpurificati<strong>on</strong> or formulati<strong>on</strong>.Because of these effects, even a perfectlyprepared MAb therapeutic will includeIgG proteins with varied structuresWWW.CEN-ONLINE.ORG 17 MARCH 2012SSSSSSSand properties, says William Whitford,senior market manager for cell culture andbioprocessing at Thermo Fisher Scientific .“Unlike small-molecule drugs, biologicsshow a distributi<strong>on</strong> of slightly differentmolecules,” he says. The analytical challengeis therefore to ensure that the populati<strong>on</strong>distributi<strong>on</strong> doesn’t vary significantlyfrom the drug that was developed,tested clinically, and approved.SOligosaccharideSSSOME PROPERTIES are aprocess red flag. Oxidati<strong>on</strong> ordeamidati<strong>on</strong>, for example, byitself generally doesn’t affectan antibody’s clinical functi<strong>on</strong>,says Raym<strong>on</strong>d Kaiser,global science leader and vicepresident for biotechnologyservices at Covance , a c<strong>on</strong>tractdrug development company.But variability in those categoriesis usually a sign thatthe manufacturing process isnot well c<strong>on</strong>trolled, and otherprotein characteristics, such asaggregati<strong>on</strong>, may be affected,Kaiser says.Other modificati<strong>on</strong>s are aclinical c<strong>on</strong>cern. A misfoldedprotein or <strong>on</strong>e with abnormaldisulfide b<strong>on</strong>ds probably w<strong>on</strong>’thave the structure needed tofuncti<strong>on</strong> properly. Additi<strong>on</strong>ally,IgGs have a c<strong>on</strong>servedglycosylati<strong>on</strong> site <strong>on</strong> the Fc,but variati<strong>on</strong>s in those sugars and glycosylati<strong>on</strong>elsewhere <strong>on</strong> the protein may elicitan unwanted immunological resp<strong>on</strong>se,such as anaphylaxis. Protein aggregatesmay also provoke an immune resp<strong>on</strong>se.And so a suite of analytical tests is necessaryto ensure that a MAb therapeuticis within the bounds of the drug that wasoriginally prepared, tested, and approved.“You live and die by your analyticals,” Kaisersays. “If you d<strong>on</strong>’t have your analyticals,you d<strong>on</strong>’t know anything.”The first order of business is often toidentify whether a manufacturer has madethe correct protein. One comm<strong>on</strong> approachis to do an immunoassay, such asan enzyme-linked immunosorbent assay(ELISA). In these types of assays, a stati<strong>on</strong>arysolid phase is loaded with a moleculethat will bind specifically to the target antibody.A drug sample is loaded, incubated,and washed, and some sort of label is usedto assay whether and how much of the targetantibody is bound.


“The industry has more than 20 years of manufacturingexperience with these types of molecules, and we’re seeingproducts that are extremely pure and really well characterized.”Another opti<strong>on</strong> for identifying proteinsis peptide mapping, which involves usinga protease enzyme to cleave an antibodyinto characteristic segments that can thenbe separated by gel electrophoresis or columnchromatography to yield a particularfingerprint.ON THE QUALITY fr<strong>on</strong>t, tests such asi<strong>on</strong>-exchange chromatography, isoelectric<str<strong>on</strong>g>focus</str<strong>on</strong>g>ing, or capillary electrophoresis willpick up variati<strong>on</strong>s in charge within theprotein structure that might indicate oxidati<strong>on</strong>,deamidati<strong>on</strong>, or protein clipping.In i<strong>on</strong>-exchange chromatography, howproteins interact with a charged stati<strong>on</strong>aryphase determines how quickly they elute.Isoelectric <str<strong>on</strong>g>focus</str<strong>on</strong>g>ing takes advantage of thefact that protein charges are a functi<strong>on</strong> ofpH and separates proteins using a gel witha pH gradient. Capillary electrophoresisseparates proteins by applying an electricfield to a capillary tube and can be d<strong>on</strong>eusing either free soluti<strong>on</strong> (capillary z<strong>on</strong>eelectrophoresis) or a gel (capillary gelelectrophoresis).For glycosylati<strong>on</strong>, manufacturers oftencreate a glycan map similar to a proteinmap. The antibody is enzymatically orchemically treated to remove the sugars,which are then given a fluorescent tag andseparated using high-performance or ultraperformanceliquid chromatography. Theglycans’ identities determine when theyelute from the column. Sialic acids, however,need a different approach. Because theyare destroyed by the standard acid treatmentto remove sugars, they are insteadremoved using mild acid and then analyzedby high-pH ani<strong>on</strong>-exchange chromatographywith electrochemical detecti<strong>on</strong>.To further pin down the specifics ofchemical modificati<strong>on</strong>s or changes inglycosylati<strong>on</strong> patterns, liquid chromatography-massspectrometry (LC/MS) has becomethe standard approach. An antibodyor its sugars are digested and separated asin other tests, but the resulting fragmentsare analyzed using mass spectrometry.LC/MS provides exact mass identificati<strong>on</strong>of the fragments but requires a highlyskilled operator, so it’s mostly used in developmentor problem solving, says TaegenClary, marketing manager for biopharmaceuticalsat Agilent .Nevertheless, LC/MS data have becomea necessary part of drug applicati<strong>on</strong> packagesto dem<strong>on</strong>strate that manufacturersunderstand what they’re seeing in theirquality-c<strong>on</strong>trol suite of assays. “I believethat if you d<strong>on</strong>’t supply some LC/MS datain a package, you’ll be asked for it because itis so comm<strong>on</strong> now,” Covance’s Kaiser says.To pick up aggregati<strong>on</strong>, <strong>on</strong>e approach issimply to look at a protein soluti<strong>on</strong>. If theaggregates are big enough, the soluti<strong>on</strong> willappear hazy rather than clear. To check forsmaller particles, size-exclusi<strong>on</strong> chromatography,also known as gel-filtrati<strong>on</strong> chromatography,is the standard method, withdetecti<strong>on</strong> by either ultraviolet light absorpti<strong>on</strong>or multiangle laser light scattering.Other techniques to look for aggregati<strong>on</strong>include light obscurati<strong>on</strong> and microflowimaging. Microflow imaging is amicroscopy technique that allows scientiststo distinguish, for example, whether aparticle is composed of protein or of silic<strong>on</strong>oil, which is used in drug formulati<strong>on</strong>s as alubricant for syringes. Those techniques aswell as analytical ultracentrifugati<strong>on</strong> mightbe used in development to help understandantibody aggregati<strong>on</strong> or in producti<strong>on</strong> todiagnose a problem, Kaiser says, but theprocess workhorse is high-throughputsize-exclusi<strong>on</strong> chromatography.ASIDE FROM THE QUALITY of the IgGmolecule, manufacturers must also keepan eye <strong>on</strong> the purity of the drug soluti<strong>on</strong>.Comm<strong>on</strong> impurities that manufacturerswatch for include residual DNA or otherproteins from the host cells, plus anyprotein A that might have carried throughpurificati<strong>on</strong>. Quantitative real-time polymerasechain reacti<strong>on</strong> (Q-PCR) methodsare used to amplify and quantify DNA,while ELISA targets protein A and host-cellproteins. In early development, manufacturersmight use cell-line-specific ELISAkits that c<strong>on</strong>tain antibodies to comm<strong>on</strong>cellular proteins. In late-stage development,however, companies often developprocess-specific assays by taking cells thatd<strong>on</strong>’t produce the therapeutic through thepurificati<strong>on</strong> process and using the outputto develop antibodies to whatever proteinsremain, Kaiser says.One additi<strong>on</strong>al area of MAb analysis isassays for potency. Potency tests, however,cannot readily be standardized across theclass. Different clinical acti<strong>on</strong>s by necessityrequire different potency tests, which areoften cell-based assays in which adding thedrug product elicits some kind of measurableresp<strong>on</strong>se.What’s being looked at is also a c<strong>on</strong>siderati<strong>on</strong>,whether it’s IgG proteins by themselves;antibody-drug c<strong>on</strong>jugates, whichtether a small-molecule drug to an IgG; orantibody fusi<strong>on</strong> proteins, which typicallycombine the IgG Fc comp<strong>on</strong>ent with anotherprotein. For antibody-drug c<strong>on</strong>jugates,most assays would be similar to those forunc<strong>on</strong>jugated IgGs, but scientists might paymore attenti<strong>on</strong> to a peptide mapping assayto make sure they know where and how successfullythe small molecule attached, Kaisersays. For fusi<strong>on</strong> proteins, manufacturerswould probably turn to the same analyticaltools as for intact IgGs, but they would haveto do more development work to optimizeassays and understand what the data show.Many of the analytical methods generallyused <strong>on</strong> MAbs, such as ELISA, chargeseparati<strong>on</strong> procedures, or size-exclusi<strong>on</strong>chromatography, are time-h<strong>on</strong>ored biochemicaltechniques, says Martina Bielefeld-Sevigny, vice president and generalmanager of drug discovery and researchreagents at PerkinElmer . The push now isto make those methods faster and easier,with better precisi<strong>on</strong>, accuracy, reproducibility,and sensitivity. In many cases, thatmeans going to microfluidic, lab-<strong>on</strong>-a-chipplatforms that use smaller amounts of materials,provide faster resp<strong>on</strong>se times, andenable high-throughput analysis.“I think that the m<strong>on</strong>ocl<strong>on</strong>al industry isa trailblazer,” USP’s Morris says. Pointingas an example to comm<strong>on</strong> use of capillarytechniques instead of plain gel electrophoresis,she adds, “I think they use fairly sophisticatedanalytical techniques comparedto plasma or peptide industry people.”That’s something that c<strong>on</strong>sumers canappreciate, for the extra assurance that adrug will work as advertised. ◾Reprinted from C&EN, Jan. 16, 2012WWW.CEN-ONLINE.ORG 18 MARCH 2012


SCIENCE & TECHNOLOGYCOURTESY OF MARSHALL REAVESTHE ARSENIC-BASED-LIFE AFTERMATHRESEARCHERS CHALLENGE a sensati<strong>on</strong>al claim,while others revisit arsenic biochemistryCARMEN DR AHL , C&EN WASHINGTONJUST AFTER THANKSGIVING, MarshallL. Reaves got a package in the mail. He’dbeen anticipating it for m<strong>on</strong>ths. And scientistsworldwide were anticipating theanswers that package might bring.The special delivery for Reaves, a graduatestudent in chemist Joshua D. Rabinowitz’ lab at Princet<strong>on</strong> University, c<strong>on</strong>tainedseveral small plastic tubes holding DNA.The DNA came from GFAJ-1, the microbethat’s lived in infamy ever since researchersplucked it from California’s arsenicrichM<strong>on</strong>o Lake and claimed it has arsenicin place of phosphorus in its biomolecules,including DNA ( C&EN, Dec. 6, 2010, page36 ; Science, DOI: 10.1126/science.1197258 ).The package came from University of BritishColumbia microbiologist Rosemary J.Redfield , Reaves’s collaborator and <strong>on</strong>e ofthe work’s many critics ( C&EN, Dec. 13,2010, page 7 ).This m<strong>on</strong>th, Redfield posted <strong>on</strong>linemass spectrometry data Reaves obtainedMORE ONLINEby analyzing the package’s c<strong>on</strong>tents al<strong>on</strong>gwith the c<strong>on</strong>clusi<strong>on</strong>, based in part <strong>on</strong>those results, that she and her colleaguesreached: The DNA from GFAJ-1 c<strong>on</strong>tainsno arsenic. Compared with the high-profilepress c<strong>on</strong>ference, fr<strong>on</strong>t-page headlines,and scientific backlash the original reportgenerated, which included eight rebuttalspublished in Science ( C&EN, June 6, 2011,page 7 ), the resp<strong>on</strong>se to Reaves’s preliminarydata has thus far been sedate. But theso-called arsenic-<strong>life</strong> paper has left a noticeablefootprint <strong>on</strong> science and sciencecommunicati<strong>on</strong> just over a year after itspublicati<strong>on</strong>.The lead researcher <strong>on</strong> the originalreport, Felisa Wolfe-Sim<strong>on</strong> , has beendelving deeper into GFAJ-1’s biology andchemistry. Other researchers are alsostudying GFAJ-1, which stands for “ GiveFelisa a Job ,” a name designed to highlightWolfe-Sim<strong>on</strong>’s quest to trade temporaryscientific positi<strong>on</strong>s for a permanent post.For direct links to the Redfield-Princet<strong>on</strong> data, visit C&ENOnline at cenm.ag/rrblog.PRIORITY MAILReaves r eceivedthis set of GFAJ-1DNA samplesfrom Redfield.Still other scientistsare looking into arsenicbiochemistry ingeneral.Meanwhile, Redfield,who goes byRosie, has beenkeeping scientists and journalists alikeengaged with updates <strong>on</strong> her efforts toreplicate Wolfe-Sim<strong>on</strong>’s work, all postedto her blog RRResearch . The mainstreampress c<strong>on</strong>tinues to cover the unfoldingstory, and news and commentary relatedto the saga abound <strong>on</strong> the social Web ,often labeled with the Twitter hashtag#arsenic<strong>life</strong>.If arsenic-based DNA really existed,it would fundamentally alter scientists’understanding of the chemistry of <strong>life</strong><strong>on</strong> Earth and point to chemistry that, atleast in principle, could be used to sustain<strong>life</strong> elsewhere in the universe. For thatclaim to be right, however, 50 to 100 yearsof chemical precedents about arsenictoxicity and arsenic biomolecule stabilitywould have to be wr<strong>on</strong>g, says chemistSteven A. Benner of the Foundati<strong>on</strong> forApplied Molecular Evoluti<strong>on</strong> in Florida,who has questi<strong>on</strong>ed Wolfe-Sim<strong>on</strong>’s findingssince their publicati<strong>on</strong>. “No chemistis going to let that go down with <strong>on</strong>ly theexperiments that appeared in the originalpaper,” he says.Redfield wasted no time putting theclaims to the test. To some extent shethinks it’s a waste of scientific time to tryto replicate the work. But “given that thework had that big a splash, it should betested, not just discarded,” she says. Andas a l<strong>on</strong>gtime practiti<strong>on</strong>er of open science,she also saw in GFAJ-1 an opportunity toshowcase openness in research “under circumstanceswhere people would be reallyexcited to see the results.”REDFIELD’S BL OG chr<strong>on</strong>icles how at firstshe had a tough time getting GFAJ-1 togrow reproducibly in media that is arsenicrichbut is low in phosphorus, the key c<strong>on</strong>diti<strong>on</strong>sin the Science paper. In her blog’scomments secti<strong>on</strong>, others left suggesti<strong>on</strong>sfor experiments and c<strong>on</strong>trols. Last November,the bacteria finally started growingc<strong>on</strong>sistently. Redfield isolated DNA fromGFAJ-1 grown in arsenic-rich mediumas well as in several types of c<strong>on</strong>trol media.Then she shipped everything off toPrincet<strong>on</strong>, where Reaves, Rabinowitz, andgenomics professor Le<strong>on</strong>id Kruglyak werewaiting.WWW.CEN-ONLINE.ORG 19 MARCH 2012


SCIENCE & TECHNOLOGYReaves further purifiedRedfield’s GFAJ-1 DNAsamples with a techniquenot used in the originalwork—cesium chloridedensity-gradient centrifugati<strong>on</strong>,which separatesDNA from impurities <strong>on</strong>the basis of density. “It canremove a lot of other thingsthat might want to stick toDNA because of charge,” heexplains. Then he removedexcess salts left over fromthe cesium chloride stepand used a pair of enzymesto chew up the DNA to itsindividual nucleotide buildingblocks. After <strong>on</strong>e lastcleanup to remove any residualprotein, he examinedthe nucleotides with liquidchromatography/massspectrometry (LC/MS),– Owhich physically separates nucleotides<strong>on</strong> the basis of polarity and then analyzestheir mass.N<strong>on</strong>e of the purified DNA samplesshowed any signs of c<strong>on</strong>taining an arsenicc<strong>on</strong>tainingDNA building block, Reavessays. He looked for masses that would beindicative of individual arsenic deoxynucleotides,as well as for free arsenatethat might result from arsenic-basedDNA falling apart in water. Work byother labs suggests that DNA with weirdnucleotides might not get completelychewed up by normal enzymes ( Nat. Chem.Biol. , DOI: 10.1038/nchembio.2007.39 ;Proc. Natl. Acad. Sci. USA, DOI: 10.1073/pnas.1017261108 ). So he also searched forpairs or small stretches of nucleotides thatmight occur if that were the case. He cameup empty time and again.PO– OOOPOBaseOOOBaseOAT ISSUE Wolfe-Sim<strong>on</strong> and coworkers’report claims GFAJ-1replaces phosphorus(red) in its DNA witharsenic.“Most scientists hedgetheir bets until all the datais presented,” says PatrickA. Limbach of the Universityof Cincinnati, an expertin nucleic acid MS. On thebasis of what he’s read, hethinks the team’s LC/MSapproach is a solid way tocheck for arsenic incorporati<strong>on</strong>into individual DNAbuilding blocks. “It wouldbe speculati<strong>on</strong> to extrapolatethese few data piecestoward the entire picture,”but given the caveat oflimited data “I would becomfortable with theirc<strong>on</strong>clusi<strong>on</strong>,” he says. “Theirdata str<strong>on</strong>gly suggest thatit is unlikely that arsenic isbeing integrated into thegenomic DNA of the samplethis group isolated, purified,and analyzed.”The Princet<strong>on</strong> team’s process for preparingthe DNA for LC/MS analysis “is exactlywhat I would’ve tried,” says MichaelG. Bartlett , whose work at the Universityof Georgia Center for Drug Discoveryincludes MS of olig<strong>on</strong>ucleotides. But, headds, it’s tough for him to evaluate theteam’s c<strong>on</strong>clusi<strong>on</strong>s <strong>on</strong> the basis of what’sbeen posted <strong>on</strong>line.He recommends a complementary experiment—performingi<strong>on</strong> chromatographyof the intact DNA, without chopping itup, followed by inductively coupled plasmaSim<strong>on</strong>’s GFAJ-1 growth c<strong>on</strong>diti<strong>on</strong>s—Redfieldadded a tiny amount of phosphorus,an amino acid, and a source of potassium.It’s not clear how much phosphorus waspresent in Wolfe-Sim<strong>on</strong>’s original c<strong>on</strong>diti<strong>on</strong>s,so prop<strong>on</strong>ents could say that Redfield’sGFAJ-1 had enough phosphorus tosurvive and so wouldn’t bother to incorporatearsenic.But Kruglyak doesn’t think those adjustmentsinvalidate their study. Redfieldadded <strong>on</strong>ly enough phosphorus to matchlevels of c<strong>on</strong>taminati<strong>on</strong> she estimatedfrom the original report, and potassium isabundant in M<strong>on</strong>o Lake and present in adifferent form in some of Wolfe-Sim<strong>on</strong>’sbacterial growth media. “It’s alwayssomething <strong>on</strong>e can claim, that nobody’sreproduced c<strong>on</strong>diti<strong>on</strong>s exactly,” Kruglyaksays. The <strong>on</strong>ly real resp<strong>on</strong>se to that, hesays, would be to request samples fromWolfe-Sim<strong>on</strong> and her collaborators andtest those.THE REDFIELD-PRINCETON team plansto replicate their results and submit themto Science for peer review. “We thought thatthe record should be tested and correctedif needed,” Kruglyak says. “And we thoughtit would be kind of fun to do it ourselvesinstead of waiting around for somebody elseto do it.”The Redfield-Princet<strong>on</strong> team’s workcomes <strong>on</strong> the heels of the first genomesequence for GFAJ-1. Last December, amulti-instituti<strong>on</strong> team deposited the draftsequence into GenBank, the freely availableNati<strong>on</strong>al Institutes of Health genetic“What’s nice about science isthe truth will reveal itself.”REAVES ALSO examined samples ofDNA from GFAJ-1 that had been grown inarsenic-rich media, but without doing anyof the purificati<strong>on</strong> steps. That’s when hedetected arsenate. “It’s kind of stuck to theDNA” n<strong>on</strong>covalently, he says, but he canremove most of it by washing the DNA withwater.Given the limited amount of data presentedso far, experts in nucleic acid MSare cautious about judging the Redfield-Princet<strong>on</strong> team’s results. But they are c<strong>on</strong>fidentthe study’s methods are sound, and<strong>on</strong>e expert told C&EN he is reas<strong>on</strong>ably inagreement with the study’s c<strong>on</strong>clusi<strong>on</strong>s.MS. In that method, “you would combustthe entire DNA and test for arsenic,” hesays. The plasma method tends to producea less noisy baseline than LC/MS, so vanishinglysmall amounts of arsenic specieswould be more easily detectable if they arepresent. Defenders of the arsenic-based<strong>life</strong>claim might argue that using an enzymedegradati<strong>on</strong> method with arsenic-basedDNA would affect the experiment’s outcome,Bartlett notes. The plasma method,he says, would make enzyme degradati<strong>on</strong>unnecessary.Prop<strong>on</strong>ents of arsenic-based <strong>life</strong> mightalso c<strong>on</strong>tend that Redfield tweaked Wolfe-sequence database. Leading the effort wasUniversity of Illinois, Chicago, microbiologistSim<strong>on</strong> Silver , who early in 2011 publisheda scathing critique of the arsenicbased-<strong>life</strong>work ( FEMS Microbiol. Lett.,DOI: 10.1111/j.1574-6968.2010.02202.x ).Silver says that like Redfield, his team hada hard time getting GFAJ-1 to grow c<strong>on</strong>sistently.Once his team determined the genomesequence, though, he was surprisedby how few of GFAJ-1’s genes were knownto be involved with arsenate resistance,given that the microbe managed to ekeout an existence in an envir<strong>on</strong>ment teemingwith normally toxic arsenic. Even theWWW.CEN-ONLINE.ORG 20 MARCH 2012


workhorse lab bacterium Escherichia colihas more arsenate-resistance genes, hesays.The genome al<strong>on</strong>e doesn’t answer thequesti<strong>on</strong> of whether arsenic replaces phosphorusin the backb<strong>on</strong>es of GFAJ-1’s nucleicacids, Silver says. He’s not sure that any resultswould c<strong>on</strong>vince Wolfe-Sim<strong>on</strong> and hercoauthors to change their minds about that.However, the genome might provide cluesabout how the microbe copes with an abundanceof an element that should be lethal.His team is planning to see how GFAJ-1’sgenome compares with genomes of threeother microbes that grow in arsenic-richenvir<strong>on</strong>ments.R<strong>on</strong>ald S. Oremland , a coauthor of thec<strong>on</strong>troversial Science paper and Wolfe-Sim<strong>on</strong>’s postdoctoral adviser at the time,declined to comment <strong>on</strong> Reaves’s results.“That is not a reflecti<strong>on</strong> <strong>on</strong> my views c<strong>on</strong>cerningthe validity of the work,” he says.“I am old-school in following the scientificpublicati<strong>on</strong> review process, reservingcommentary for later.” His lab isn’tworking <strong>on</strong> GFAJ-1 right now, but theyhave cultures of the microbe and may domore work in the future. He did not renewWolfe-Sim<strong>on</strong>’s postdoctoral appointmentbut says he helped her make arrangementsto find a new home base for her arsenicresearch.Oremland, a senior scientist with theU.S. Geological Survey, says he’s receivedplenty of criticism about the GFAJ-1 work,some of it c<strong>on</strong>structive but much of itvenomous. “My fear is that scientists willbe afraid to test radical ideas” because ofthe arsenic-based-<strong>life</strong> saga, he says. “Ifyou d<strong>on</strong>’t think, and you d<strong>on</strong>’t try, and youd<strong>on</strong>’t test, then every<strong>on</strong>e just keeps doingthe same incremental stuff.” The Sciencepaper’s c<strong>on</strong>clusi<strong>on</strong>s accurately reflect whatthe data looked like to him and his coauthorsat the time, he says. “We put this ideaout there so it could be tested.“Even if we are dead wr<strong>on</strong>g with thisarsenic-DNA business, with a bit morework this bug could shed light <strong>on</strong> the limitsof what microbes can and can’t do.”Asked to comment <strong>on</strong> whether he isopen to changing his mind about whetherGFAJ-1 has arsenic DNA, Oremland said,“I’m not going to go down with the ship.”If other groups’ work makes it clear that heand his coauthors overreached, “I’ll say Iwas wr<strong>on</strong>g. I make mistakes. That’s the wayscience works,” he says. “But you mightfind Felisa holding <strong>on</strong> a bit tighter.”“We are thrilled that our results arestimulating more experiments from othersas well as ourselves,” Wolfe-Sim<strong>on</strong> wrotein an e-mail. She is now a Nati<strong>on</strong>al Aer<strong>on</strong>autics& Space Administrati<strong>on</strong> AstrobiologyResearch Fellow in structural biologistJohn A. Tainer ’s lab at Lawrence BerkeleyNati<strong>on</strong>al Laboratory . “We do not expect tosee arsenate in cesium chloride gradientexperiments—although this is a great thingto check.” Once GFAJ-1 cells are crackedopen, any arsenic linkages in DNA are liableto fall apart, she writes. As a result,“arsenate-c<strong>on</strong>taining bands may then alsobe shifted in the gradient—so that theexpected amounts associated with DNAwould be undetected because the band isso faint.”“That c<strong>on</strong>cern doesn’t make muchsense,” Redfield says. She performed a gelanalysis to ensure that GFAJ-1 DNA fromcells grown in arsenate-rich c<strong>on</strong>diti<strong>on</strong>swas not falling apart prior to Reaves’spurificati<strong>on</strong>. She posted those results toher blog <strong>on</strong> Jan. 14. The gel “shows thatthe DNA from arsenate-grown cells isno more degraded than DNA from phosphate-growncells, both immediately afterisolati<strong>on</strong> and after two m<strong>on</strong>ths’ storageat 4 °C,” Redfield says. Combined withthe LC/MS data, Redfield’s evidence givesarsenic-based DNA “no place to hide,”Reaves adds.IN TAINER’S L AB, Wolfe-Sim<strong>on</strong> is tryingmultiple tactics to learn more about whathappens to arsenic inside GFAJ-1. “We are<str<strong>on</strong>g>focus</str<strong>on</strong>g>ed <strong>on</strong> isolating and characterizingthe ribosome via X-ray crystallography.Additi<strong>on</strong>ally, we are looking at the DNA/CONTROVERSYIn t hisSeptember2010 photo,Wolfe-Sim<strong>on</strong>collects samplesat arsenic-richM<strong>on</strong>o Lake, theplace where shefound GFAJ-1.RNA and other cellularmetabolites with quadrupoletime-of-flightLC/MS,” she says. “Wewill be purifying DNA,RNA, and proteins andseeing whether or notthe arsenic tracks withthese comp<strong>on</strong>ents.”In a poster presentedat last December’s<strong>American</strong> Geophysical Uni<strong>on</strong> meeting ,Wolfe-Sim<strong>on</strong> reported progress <strong>on</strong> optimizingGFAJ-1’s growth with a combinati<strong>on</strong>of carb<strong>on</strong> sources and amino acids,as well as progress in preparing samplesfor nucleic acid stability testing, ribosomecrystallizati<strong>on</strong>, and MS analyses. “Wethink structure is the way to go,” Wolfe-Sim<strong>on</strong> says. “We want to solve the structureof the ribosome and see what it’sgoing to tell us,” she adds. “Is the arsenicthere? Is it randomly distributed? Is it inspecific places?” she says. “We’re going tolook at the RNA to see if [arsenic] is in thebackb<strong>on</strong>e.“We’re super excited about our nextexperiments,” she says. “What’s nice aboutscience is the truth will reveal itself.”To some researchers, however, an attemptto crystallize GFAJ-1’s ribosome tocheck whether it c<strong>on</strong>tains arsenic is likeleaving the low-hanging fruit behind andclambering straight for the treetops. The<str<strong>on</strong>g>focus</str<strong>on</strong>g> at first should be <strong>on</strong> more straightforwardtests that get at the heart of the isthere-or-isn’t-there-arsenic-in-the-DNAquesti<strong>on</strong>, Benner says. He’d like to seeWolfe-Sim<strong>on</strong> grow GFAJ-1 in the presenceHENRY BORTMANWWW.CEN-ONLINE.ORG 21 MARCH 2012


COURTESY OF ROSIE REDFIELDof radioactive arsenate andthen perform multiple fracti<strong>on</strong>ati<strong>on</strong>s—separati<strong>on</strong>sof thevarious cellular comp<strong>on</strong>ents—while tracking the radioactivityto see where the arsenate goes.In the original report, “she did<strong>on</strong>e fracti<strong>on</strong>ati<strong>on</strong>. She starteddown the road a chemist understands,”Benner says. Butshe didn’t do enough rounds offracti<strong>on</strong>ati<strong>on</strong> to see whether radiolabeledarsenate could end up in specific molecules,such as DNA, he adds.“Crystals of bacterial ribosomes havethe resoluti<strong>on</strong> to yield atomic modelsthese days,” says Venkatraman Ramakrishnanof the Medical Research Council Laboratoryof Molecular Biology in Cambridge,England, who shared the 2009 Nobel Prizein Chemistry for ribosome crystallizati<strong>on</strong>work. But it’s not necessary to crystallizeGFAJ-1’s ribosome to put the arsenicbased-<strong>life</strong>c<strong>on</strong>troversy to rest, he says.“Crystallizati<strong>on</strong> of ribosomes from a newspecies is a n<strong>on</strong>trivial undertaking that cantake many years, and possibly never succeed,”he says. He suggests that the teamtry to isolate ribosomal RNA, or nucleotidesfrom ribosomal RNA, and determine,with radioactivity or other tools, whetherarsenic ends up there.Even if GFAJ-1 turns out to be a microbethat can tolerate arsenic while scavengingwhat phosphorus it can from phosphoruspoorsurroundings, that would still befascinating, Ramakrishnan adds, “althoughcertainly not as dramatic as incorporatingarsenic for phosphorus in its nucleicacids.”Other labs besides Redfield’s andSilver’s now have access to GFAJ-1, butGROWING AT LASTAfter m<strong>on</strong>ths ofsearching, Redfieldfound reliablec<strong>on</strong>diti<strong>on</strong>s toreproducibly growcultures of GFAJ-1.Each tube andbottle c<strong>on</strong>tains aGFAJ-1 culture.Oremland did not disclose towhom he’s sent samples. Oremlandadds that he doesn’t havean accurate count of how manysamples he’s given out but saysthat the cultures are availablefrom collecti<strong>on</strong>s in the U.S. andGermany.Even as follow-up <strong>on</strong> Wolfe-Sim<strong>on</strong>’s report c<strong>on</strong>tinues, thework has spurred studies ofarsenate chemistry and biochemistry inother labs. At Harvard Medical School, A.Michael Sismour is trying to make arsenatedinucleotides—the chemical motifthat would appear in arsenic DNA, if itwere to exist. Sismour, who earned hisPh.D. with Benner and is now a postdoc inGeorge M. Church ’s lab, says that after thearsenic-based-<strong>life</strong> paper came out he tooka look at the literature <strong>on</strong> arsenic biomolecules,which largely dates to the 1960sthrough the 1980s, and says he found smallc<strong>on</strong>tradicti<strong>on</strong>s when it came to how l<strong>on</strong>gthe molecules could last. He figured moderntechniques and instrumentati<strong>on</strong> mightprovide new insights, so he decided to examinethe matter as a side project.So far, Sismour has made arsenate dinucleotidesjoined by a 5ʹ-5ʹ linkage, whichdoes not occur in standard DNA biochemistry.Making arsenate dinucleotides withthe 5ʹ-3ʹ linkage that occurs in DNA hasproven challenging. If he does manage toget there, he says he’ll determine the compounds’half-lives in water.<strong>Chemical</strong> precedent suggests thatthe type of arsenic diesters that wouldexist in arsenic DNA would fall apart ina fracti<strong>on</strong> of a sec<strong>on</strong>d under biologicalc<strong>on</strong>diti<strong>on</strong>s ( ACS Chem. Biol., DOI: 10.1021/cb2000023 ). But Benner says that thearsenic-<strong>life</strong> paper at least lets scientistsentertain the possibility that in a verycold envir<strong>on</strong>ment where reacti<strong>on</strong> ratesslow down, perhaps <strong>on</strong> a distant planetsomewhere, arsenic-c<strong>on</strong>taining DNA’s liabilityof falling apart easily might be lessof a problem. “If we understand what thehydrolysis rates are,” Sismour says, “wemight be able to understand what temperatureranges might allow arsenic <strong>life</strong>to exist.”Half a world away in Israel, WeizmannInstitute of Science biochemist Dan S.Tawfik is thinking about how enzymesthat have evolved to work with phosphatemight deal with arsenate. In resp<strong>on</strong>se tothe original GFAJ-1 report, Tawfik teamedwith R<strong>on</strong>ald E. Viola of the University ofToledo to publish a summary of researchefforts in that area ( Biochemistry, DOI:10.1021/bi200002a ).SINCE THEN, Tawfik’s team has set out tolearn about arsenic-survival measures thatorganisms could possibly have developedin arsenic-rich envir<strong>on</strong>ments.Work by Viola and others suggestsmany enzymes that bind to or use phosphatehave a hard time telling the differencebetween phosphate and arsenate ( Inorg.Chem., DOI: 10.1021/ic981082j ; Acta.Cryst., DOI: 10.1107/S0907444904026411 ;J. Biol. Chem. 1981 , 256, 5981 ). Tawfik is<str<strong>on</strong>g>focus</str<strong>on</strong>g>ing <strong>on</strong> a bacterial transport proteinthat he says binds phosphate <strong>on</strong>e thousandtimes more str<strong>on</strong>gly than arsenate.In preliminary work, his team used X-raycrystallography to obtain molecular-levelclose-ups of the protein bound to both arsenateand phosphate. It’s not uncomm<strong>on</strong>for different substrate analogs to bind toa protein with a highly different affinitythan the natural substrate. But it may be,Tawfik speculates, that at some point <strong>on</strong>the primitive Earth, some proteins had toevolve to distinguish between phosphorusand arsenic, an adaptati<strong>on</strong> that wouldrarely be necessary today.Few sci entists believe GFAJ-1 hasarsenic-c<strong>on</strong>taining DNA, Tawfik says. “Butthat doesn’t mean there w<strong>on</strong>’t be otherintriguing findings related to this microbe”or related to arsenic biochemistry.“This is the way science works,” saysViola. “You have some<strong>on</strong>e that comes upwith an unexpected result, some peopleimmediately believe it and some disbelieveit, and then comes the follow-up,” he says.“Science moves <strong>on</strong>.” ◾Reprinted from C&EN, Jan. 30, 2012WWW.CEN-ONLINE.ORG 22 MARCH 2012


SCIENCE & TECHNOLOGYPICTURING BIOLOGYMulti-isotope imaging MASS SPECTROMETRYanswers biological questi<strong>on</strong>sCELIA HE NRY ARNAUD , C&EN WASHINGTONCOURTESY OF CLAUDE LECHENEMULTI-ISOTOPE IMAGING mass spectrometry,or MIMS, is proving to beabout more than just pretty pictures. It’salso a powerful way to answer biologicalquesti<strong>on</strong>s.The method, developed by Claude Lecheneand coworkers at Harvard MedicalSchool over the past two decades, is allowingscientists to track the synthesis of DNAand proteins in whole organisms—bacteria,fruit flies, mice, and even humans.“The three-dimensi<strong>on</strong>al images are trulymind-blowing,” says Nicholas Winograd ,a mass spectrometry imaging expert atPennsylvania State University.Lechene and his coworkers build theseimages from samples that have underg<strong>on</strong>enormal metabolic processes in the presenceof stable-isotope tracers. They gathersuch samples by feeding organisms metabolitessuch as amino acids or nucleosidesenriched in stable isotopes such as nitrogen-15.Organisms use these metabolites asbuilding blocks as they replenish c<strong>on</strong>stituentsin their cells. The researchers then calculateincorporati<strong>on</strong> of the stable isotopesby comparing the isotopes’ abundance insamples to their natural abundance.15N is particularly useful for tracingthe synthesis of new DNA and proteins.Researchers can use other stable isotopes,such as oxygen-18, carb<strong>on</strong>-13, or deuterium,to track biological molecules, but 13 Cand deuterium are less useful for whole-organismstudies because of their ubiquity inbiological molecules. In additi<strong>on</strong>, more 15 Nand 13 C compounds are commercially availablethan 18 O compounds, Lechene notes.Although the method uses nanometerscalesec<strong>on</strong>dary i<strong>on</strong> mass spectrometry(SIMS) to detect these stable isotopes,Lechene hesitates to call it mass spectrometry.The instrument detects <strong>on</strong>ly up toseven masses simultaneously. “The methodis more i<strong>on</strong> microscopy,” he says.Those i<strong>on</strong>s are generated by bombardinga sample with abeam of cesium i<strong>on</strong>s.The cesium i<strong>on</strong>s breakmolecules into atomicand polyatomic fragments,which evaporateand are i<strong>on</strong>izedto form so-calledGUT REACTION A15N- 14 N image ofthe mouse smallintestine showsthe incorporati<strong>on</strong>of 15 N-labeledthymidine in DNA.sec<strong>on</strong>dary i<strong>on</strong>s. The sec<strong>on</strong>dary i<strong>on</strong>s arethen guided through a series of i<strong>on</strong> opticsto the mass spectrometer. In his biologicalapplicati<strong>on</strong>s, Lechene detects nitrogen,both as 14 N and 15 N, with the cyanide i<strong>on</strong>,CN – . By moving the tightly <str<strong>on</strong>g>focus</str<strong>on</strong>g>ed cesiumi<strong>on</strong> beam from point to point and drillingthrough the sample layer by layer, Lechenecan c<strong>on</strong>struct 3-D maps of 15 N incorporati<strong>on</strong>.He has achieved spatial resoluti<strong>on</strong> of30 nm and depth resoluti<strong>on</strong> of 10 nm.“Since 1995, Lechene has produced thehighest quality images in the field,” saysWinograd, who uses c<strong>on</strong>venti<strong>on</strong>al time-offlight(TOF) SIMS for biological imaging.“Trouble is, he almost never publisheshis work. In 2007, he published a Sciencepaper <strong>on</strong> nitrogen fixati<strong>on</strong> which blewevery<strong>on</strong>e in the field away [DOI: 10.1126/science.1145557 ]. Many of us in the communitysure wish we could see more of it.”NOW THE COMMUNITY is getting itschance: Lechene published two highprofileMIMS papers last m<strong>on</strong>th. In <strong>on</strong>eexample, he and his colleagues used MIMSto reveal slow protein turnover in the stereociliaof mouse hair cells, which despitethe name are actually found in the innerear ( Nature, DOI: 10.1038/nature10745 ).These stereocilia help hair cells c<strong>on</strong>vertsound waves into nerve impulses. Earlierexperiments by other researchers used actinlabeled with green fluorescent proteinand suggested that all the actin in hair cellsis replaced within two or three days. Thosestudies were d<strong>on</strong>e with hair cells removedfrom infant mice and then grown in cultureswith high levels of actin.The MIMS analysis, in c<strong>on</strong>trast, wasperformed with adult mice and revealedsomething very different. “We could seethat hair cells themselves were turningover relatively fast,” Lechene says, “but thestereocilia were lagging for a l<strong>on</strong>g periodof time.” The <strong>on</strong>ly part with high turnoverwas the tip, the porti<strong>on</strong> of the stereociliaresp<strong>on</strong>sible for transducing mechanicalmoti<strong>on</strong> into auditory nerve impulses. “Thisfinding was c<strong>on</strong>trary to the dogma that stereociliaturn over very fast,” he says.The use of stable-isotope labeling allowedLechene to study the cells undernormal c<strong>on</strong>diti<strong>on</strong>s. “We have dem<strong>on</strong>stratedthat we can study protein turnoverin volumes that are smaller than anythingthat has ever been d<strong>on</strong>e, in c<strong>on</strong>diti<strong>on</strong>s thatd<strong>on</strong>’t disturb the system,” he says. “Thereis nothing that approaches this kind ofmeasurement in biology.”Lechene has also used MIMS to investigatea l<strong>on</strong>g-standing questi<strong>on</strong> aboutstem cell divisi<strong>on</strong> ( Nature, DOI: 10.1038/“Since 1995, Lechene has produced the highest quality imagesin the field. Trouble is, he almost never publishes his work.”WWW.CEN-ONLINE.ORG 23 MARCH 2012


SCIENCE & TECHNOLOGYCOURTESY OF CLAUDE LECHENEFIRST AIRBORNEFROG PHEROMONEMadagascar frogs use volatile SMALL MOLECULES to communicatePROTEIN TURNOVER The stereocilia (thinprojecti<strong>on</strong>s) in adult mouse hair cells havehigh incorporati<strong>on</strong> of 15 N (red) at the tips, asshown in this map of the ratio of 15 N to 14 N.nature10734 ). The immortal strand hypothesisproposes that when stem cells divide, theolder DNA strand stays with the daughtercell, which is destined to remain a stem cell.TO TEST THIS HYPOTHESIS, Lecheneused MIMS to track the incorporati<strong>on</strong> of15N-labeled thymidine in the DNA of cryptcells, a type of stem cell in the small intestine.His team labeled the stem cell DNAby administering 15 N-thymidine to youngmice during their first eight weeks of <strong>life</strong>,when intestinal stem cells are still forming.They then fed the mice 14 N-thymidinefor four more weeks. At the end of those 12weeks, if the immortal strand hypothesiswere true, 15 N-labeled DNA would still befound in the stem cells. Lechene’s teamfound that no dividing cells, including stemcells, retained the label.In the same paper, Lechene showed thatMIMS can be used to study human metabolism.His team administered 15 N-thymidineintravenously to a healthy volunteer andthen analyzed smears of peripheral whiteblood cells with MIMS. As expected, theydidn’t find any 15 N-labeled white bloodcells immediately after the infusi<strong>on</strong>, butthey detected some four weeks later.Others in the mass spectrometry imagingcommunity can’t wait to see more of these3-D MIMS images. “How he makes thatwork, I just d<strong>on</strong>’t know,” Winograd says.“We’ve been trying to do this sort of thingwith TOF-SIMS, and it is really difficult.” ◾ANYBODY WHO SPENDS time near aswamp can easily hear that frogs use theirvoices to chitchat, but it wasn’t until abouttwo decades ago that researchers announcedthat these animals also c<strong>on</strong>versewith water-transported protein pherom<strong>on</strong>es.Now, research shows that frogsbanter with airborne chemicals too.“It’s the first proof that frogs use volatilepherom<strong>on</strong>es” to communicate, says StefanSchulz, a chemical ecologist at the TechnicalUniversity of Braunschweig (TU), inGermany. In fact, it’s the first proof thatany amphibians communicate using chemicalsin the air, he adds ( Angew. Chem. Int.Ed., DOI: 10.1002/anie.201106592 ).“So few pherom<strong>on</strong>es have beenchemically identified in vertebrates, sothis is really exciting news,” commentsSarah Woodley , an amphibian biologist atDuquesne University. She points out thatbiologists had d<strong>on</strong>e behavioral studies suggestingfrogs used airborne pherom<strong>on</strong>es,but n<strong>on</strong>e had been identified until now.In the new study, Schulzcollaborated with TU Braunschweigzoologist Miguel Vencesand Katharina Wollenberg, nowat Harvard University, who wentto Madagascar to study a localfamily of frogs called Mantellidae.Male Mantellidae frogs havebulbous organs <strong>on</strong> their innerthighs called femoral glands,and it’s from these sacs that theteam isolated two moleculesthat waft through the air aspherom<strong>on</strong>es, namely 8-methyl-2-n<strong>on</strong>anol and a macrolidecalled phoracantholide J.The team discovered thatWWW.CEN-ONLINE.ORG 24 MARCH 2012OHSTEFAN SCHULZ8-Methyl-2-n<strong>on</strong>anol(S)-Phoracantholide JOOMantellidae frogs will hop toward a mixtureof these two molecules and that differentspecies have different ratios of them in theirfemoral glands. Precisely what these frogsare saying with the molecules is up in the air,but Schulz can speculate.“Frogs occur in high species diversity inthese swampy areas—there are about 100 ecules are pherom<strong>on</strong>es. — SARAH EVERTSReprinted from C&EN, Feb. 13, 2012 species,” Schulz says. Although the differ-Reprinted from C&EN, Feb. 6, 2012ent species croak uniquely, the frog densityis so high that “it can be hard to find a mateof the correct species.” Perhaps the odorshelp with species recogniti<strong>on</strong>, he suggests.The new research also c<strong>on</strong>firms the resultsof frog genome sequencing, Woodleysays. Frog DNA has all sorts of genes forvolatile chemical receptors, but nobodyknew whether they were functi<strong>on</strong>al genesor just an artifact of evoluti<strong>on</strong>. “It turns outOOGephyromantolide AAIRBORNE Researchersdiscovered that Mantellidaefrogs (shown) produce tw<strong>on</strong>ew airborne pherom<strong>on</strong>es,8-methyl-2-n<strong>on</strong>anol andphoracantholide J, which aremade in their femoral glands.Some also produce a previouslyunreported natural productcalled gephyromantolide A.they may be functi<strong>on</strong>al,” sheadds.Schulz’s team isolated ahandful of other alcohols andmacrolides from the frogs’femoral glands, including a newnatural product called gephyromantolideA. The team also devised a new syntheticroute for building the ringed moleculesthat uses a reacti<strong>on</strong> called Corey–Nicolaoumacro lact<strong>on</strong>izati<strong>on</strong>. The route, the shortestsuch path ever reported, provided enoughsample to test which of the additi<strong>on</strong>al mol-


BEHIND AMUSHROOMSCOURGEScientists search for the compoundsresp<strong>on</strong>sible for UNEXPLAINED DEATHSFOR THE PAST 30 YEARS apparently healthy villagers in southwesternChina’s Yunnan province have been mysteriously dying.More than 260 people have been claimed by what has come to beknown as Yunnan Sudden Unexpected Death.Recent epidemiological studies have implicated a previously undescribedmushroom, named Trogia venenata Zhu L. Yang for its discoverer,in the deaths. Seeking further evidence that this mushroomis in fact resp<strong>on</strong>sible, a team of scientists in China have identifiedthree toxic amino acids that they believe give T. venenata its pois<strong>on</strong>ousproperties ( Angew. Chem. Int. Ed., DOI: 10.1002/anie.201106502 ).Extracts of T. venenata c<strong>on</strong>tain γ-guanidinobutyric acid, a compoundknown to cause seizures, as well as two previously unidentifiedamino acids that are derivatives of hexynoic acid, reportsa team led by Ji-Kai Liu, of the Kunming Institute of Botany, andGuang Zeng, of the ChineseCenter for DiseaseC<strong>on</strong>trol & Preventi<strong>on</strong>.The researcherssynthesized the newlydiscovered aminoacids and found thecompounds to be lethalin studies with mice.They also used liquidchromatography andmass spectrometry toexamine blood takenOHONH 2OHNew toxic amino acidsH 2 NNHNHOOHγ-Guanidinobutyric acidONH 2OHCOURTESY OF JI-KAI LIUfrom a victim of Yunnan Sudden Unexpected Death. They foundthat the blood and <strong>on</strong>e of the newly discovered amino acids sharedsimilar fragment i<strong>on</strong>s.“We are 90% certain we have solved the mystery—that thismushroom is the cause of the deaths,” Liu tells C&EN. “And we arealmost 100% certain that these three toxins are the cause of themushrooms being fatal.”But not every<strong>on</strong>e is as certain. “The c<strong>on</strong>tents of those toxins inthe mushroom are amazingly high,” comments Hirokazu Kawagishi,an expert <strong>on</strong> toxic mushrooms at Japan’s Shizuoka University.Therefore, he says, it is possible that these compounds are thecause of the deaths. However, Kawagishi admits to some skepticismbecause, <strong>on</strong> the basis of Liu and Zeng’s results, a pers<strong>on</strong>would still have to eat about 4 kg of the fresh mushroom to c<strong>on</strong>sumea lethal amount of the new amino acids.“That is a staggering amount,” agrees Michael W. Beug, a retiredenvir<strong>on</strong>mental chemistry professor and mycologist at EvergreenState College, in Olympia, Wash. “The fact that there have been nodeaths since 2009 when the villagers were warned not to eat thesemushrooms is pretty str<strong>on</strong>g evidence that the mushrooms are involvedin the deaths,”he says, but “the toxinor combinati<strong>on</strong> oftoxins is yet to be adequatelyexplained.”Liu tells C&EN thathe believes a synergisticcombinati<strong>on</strong> of thenew amino acids withγ-guanidinobutyricacid might make themushrooms morepotent than the currentstudy suggests. Adetailed mechanismof the compounds’toxicity is still not clear, Lui says. He notes, however, that the groupis doing further research and believes that the amino acids mightinduce hypoglycemia that results in death. — BETHANY HALFORDReprinted from C&EN, Feb. 13, 2012WEBINARSLIFE SCIENCES SERIESSynthesis & Analysis ofMilli<strong>on</strong>s of Genomes &Nanostructures for NovelTherapies and MaterialsFATAL FUNGUS Thedeadly mushroomTrogia venenata Zhu L.Yang may have claimedhundreds of lives.Speaker:George ChurchProfessor of Genetics, Harvard MedicalSchool, Director of the Center forComputati<strong>on</strong>al GeneticsStr<strong>on</strong>gerb<strong>on</strong>ds.Date: July 12th, 2012Time: 11:00am EDTAdditi<strong>on</strong>al Details Available So<strong>on</strong>!Please email CENWebinars@pubs-acs.orgfor more informati<strong>on</strong>.WWW.CEN-ONLINE.ORG 25 MARCH 2012


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