focus on life sciences - american chemical society

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WELCOME TO “Focus On Life Sciences,” a compilation of news and feature stories that appeared in October, November, and December 2011 issues of C&EN, the weekly newsmagazine published by the American Chemical Soci-ety, the world’s largest scientific society. It is the first in a series of such com-pilations that we will produce in 2012.

We’re distributing the “Focus On Life Sciences” series because C&EN, like the chemistry enterprise it is devoted to covering, is deeply involved in all as-pects of modern life sciences—from bench research on the fundamental chemistry of living organisms to breakthrough biopharmaceuticals, from the ana-lytical instrumentation that makes life sciences dis-coveries possible to the tough policy choices some of those discoveries pose. Our audience of more than 164,000 chemical professionals knows that the interface between chemistry and biology is one of the most dynamic and im-portant areas of modern science. It’s where many of them work, and C&EN is the magazine they rely on to keep them informed of advances in the field and of the products and services they use in their labs.

For almost 90 years, C&EN’s editorial mission has been to cover news, events, and trends in the chemistry enterprise in a timely, accurate, and bal-anced way. C&EN’s staff of 50 writers and editors based around the globe is the largest and most experienced team of journalists devoted to covering chemistry, related sciences, and science-based industries. They go where the news is, and these days, a lot of the news is in the life sciences. This compila-tion of recent stories from our News of the Week, Business, Government & Policy, and Science & Technology Departments demonstrates clearly that C&EN is right at the cutting edge of news in the life sciences.

I hope you enjoy “Focus On Life Sciences.” With its large global circulation and loyal readership, C&EN provides a tremendous opportunity for advertis-ers who 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-online.org) has grown to more than 12 million page views per year. We think you will find C&EN, with its broad coverage, readership, and out-standing editorial quality, to be an ideal vehicle for reaching your customers.

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Serving the chemical, life sciences, and laboratory worlds

JANUARY 2012

NEWS OF THE WEEK 3 ENZYME MIMIC IS STELLAR

For an artificial enzyme, three-helix, two-metal assembly has unprecedented catalytic activity.

4 BOOSTER SHOT FOR U.K. SCIENCE Prime Minister David Cameron lays out a plan to support life sciences research.

4 NIH BROADENS GENOME RESEARCH Health agency will shift funds from genome sequencing to medical applications.

5 SWITCHABLE FLUORESCENCE Fluorophore-bearing particles remain dark until they enter cells, then they shine at full intensity.

5 ADVANCING PERSONALIZED MEDICINE Major research institutions partner to establish genomics centers in Connecticut, New York City.

BUSINESS 6 CONSUMABLES STRATEGY

Instrumentation companies take different approaches toward the repeat-sale product market.

GOVERNMENT & POLICY 10 TESTING FOR PESTICIDE EXPOSURE

Researchers are on a hunt for biomarkers to help develop diagnostic tests for pesticide exposure.

SCIENCE & TECHNOLOGY 12 BRYOSTATINS RETAIN

PROMISE ▶

New results in total synthesis reinvigorate a 40-year-old field of research.

18 INVESTIGATING METALLOPROTEOMES Innovative methods help scientists understand complexity of proteins that interact with metals.

21 MICHAEL MARLETTA C&EN talks with the incoming president of Scripps Research Institute.

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news of the week

IN WORK that could lead to a new generation of tai-lored protein catalysts, a research team has prepared an artificial enzyme that works uniquely well. The

simple assembly, of three helices and two different metal ions, catalyzes reactions with efficiencies that approach those of the corresponding natural enzyme more closely than ever before ( Nat. Chem., DOI: 10.1038/nchem.1201 ).

Melissa L. Zastrow, Anna F. A. Peacock, Jeanne A. Stuckey, and chemistry professor Vincent L. Pecoraro of the University of Michigan, Ann Arbor, designed, created, and tested the artificial enzyme, which acts as a hydrolase. The work could help the design of catalysts for many applications.

Structurally much simpler than natural hydrolases, the artificial enzyme includes just three linear α-helices in an arrangement called a “three-helix bundle,” a Hg(II) ion for structural stability, and an active-site Zn(II) ion. Yet its efficiency in catalyzing the hydration of CO 2 is 0.2% that of human carbonic anhydrase II, one of the fastest hydrolases. And in catalyzing the hydrolysis of an acetate, its efficiency is 1% that of the natural enzyme.

Such levels of efficiency are considered stellar in the field of artificial enzyme design.

In 2009, for example, protein designers William DeGrado of the University of Pennsylvania (now at the University of California, San Francisco), Angela Lombardi of the University of Naples, and coworkers developed a four-helix-bundle enzyme with a di-iron active site that catalyzes a phenol oxidase reaction. The achievement was considered a major breakthrough in protein design, but the synthetic enzyme’s activity was only 0.01 to 0.1% that of natural oxidases.

The new artificial enzyme, DeGrado says, is “a mon-umental piece of work.” It represents, he adds, “the culmination of a large body of data from Vince’s lab, relating to the fine interplay between protein stability, folding, and the structure of metal-binding sites” and is “the beginning of a new chapter in what should prove to be an exciting and rapidly expanding area of research.”

The artificial hydrolase is “the first really good exam-ple where both structure and reactivity have been effec-tively designed,” says computational protein designer Vikas Nanda of Robert Wood Johnson Medical School, in Piscataway, N.J. “There’s also a lot here for the pro-

tein design gearheads. To have two different metals and have them go to their specific sites is exciting.” The way Pecoraro and coworkers achieved this result is instruc-tive for other researchers in the field, he says.

The work is “exciting,” says metalloprotein special-ist Yi Lu of the University of Illinois, Urbana-Cham-

paign. “Although de novo-designed α-helical bundle proteins have been reported before,” he says, “design-ing functional activities into them has been very chal-lenging,” and the efficiencies of the artificial hydrolase are “quite impressive.” — STU BORMAN

PROTEIN DESIGN: Mimic narrows efficiency gap with natural enzyme

MAKING A BETTER ENZYME

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Reprinted from C&EN, Dec. 5, 2011

COPYCAT Three-helix bundle (top), a mimic of human carbonic anhydrase II (bottom), catalyzes hydration and hydrolysis with unprecedented e� ciency for an artifi cial enzyme. Hg(II) ion is yellow and Zn(II) ions are red.


NEWS OF THE WEEK

Reprinted from C&EN, Dec. 12, 2011 (both)

BRITISH PRIME MINISTER David Cameron out-lined his government’s strategy for the U.K.’s life sciences industry at a conference in London last

week. The plan, spelled out in two reports , includes a $282 million fund to support medical research as well as changes to the delivery of new therapies through the National Health Service.

Britain’s ambition is not just to retain a foothold but to take a bigger share of the global life sciences market, Cameron said. “I want the great discoveries of the next decade happening in British labs, the new technologies born in British start-ups,” he said. New funding will target the gap between idea generation in the lab and market investment in a new drug or technology.

With more than 4,500 companies, 165,000 em-ployees, and $78 billion in annual revenues, the life sciences sector has been growing faster than the U.K. economy as a whole, according to the U.K. govern-

ment. Still, officials acknowledge that rapid changes occurring in the industry need to be addressed.

“We need to create the right environment for sci-entists and business to work together and translate research into new, cutting-edge technologies and medi-cines,” Minister of State for Universities & Science Da-vid Willetts said. “This will boost our economy, create new jobs, and lead to better treatments for patients.”

Through its Medical Research Council , the U.K. government is also investing close to $16 million in a collaboration with AstraZeneca . Under the agreement, the U.K. drug firm will make 22 compounds available free of charge to academic researchers, who will study the compounds’ efficacy against various diseases. Separately, AstraZeneca has added $100 million to its venture capital arm, MedImmune Ventures, to invest in biopharmaceutical companies.

Leaders of U.K.-based health care, pharmaceutical, and biotechnology industry associations welcomed the strategy and initiatives. GlaxoSmithKline called the plan “a very important next step on the journey to make the U.K. the best place in the world to locate pharmaceutical investment.”

Stating its commitment to work with the govern-ment to deliver on the promises, GSK said it is “posi-tive about Britain’s future prospects as a place to re-search, develop, manufacture, and commercialize our medicines.” — ANN THAYER

FUNDING: British government looks to support the life sciences industry

U.K. RESEARCH GETS A SHOT IN THE ARM

“I want the great discoveries of the next decade happening in British labs, the new technologies born in British start-ups.”

—BRITISH PRIME MINISTER DAVID

CAMERON

THE NATIONAL Institutes of Health is broaden-ing its genome-sequencing program to focus more on medical applications. Although most of

the program’s budget will fund basic research at three large-scale sequencing centers, nearly one-quarter of

the money will be redirected to help push genomics into clinical care.

“There have been some remarkable medical successes for genomics, but genome sequencing has yet to find its way into standard medical practice,” Eric D. Green, direc-tor of NIH’s National Human Genome Research Institute ,

said at a Dec. 6 briefing. NHGRI, which runs the federal sequencing program, hopes its future investments in the program will accelerate the realization of genomic medi-cine, Green noted.

NHGRI plans to maintain its current level of funding for the program and invest $416 million over the next four years, Green said. The bulk of the funding, some 77%, will continue to support basic research at three sequencing centers: the Broad Institute of Harvard Uni-versity and MIT, the Genome Institute at Washington University in St. Louis, and the Human Genome Se-quencing Center at Baylor College of Medicine.

The remaining 23%, or about $100 million, will be redirected to support three new priority areas aimed at bringing genome sequencing into routine medical prac-tice. These areas are finding causes of rare, inherited disorders; evaluating the medical, ethical, and societal impacts of using genome sequencing in clinical care; and addressing the bioinformatics bottleneck created by the deluge of sequencing data.

The shift in funds will cut the budgets of the three sequencing centers, but the reductions won’t hit all at once. NHGRI plans to reduce the base funding of each center by about 5% each year over the next four years , NHGRI Deputy Director Mark S. Guyer noted at the briefing. That reduction in funding is expected to coin-cide with a drop in cost of DNA sequencing.

“We believe the cost of sequencing will continue to decline,” Guyer said. As a result, NHGRI’s sequencing program can maintain its high level of productivity at even lower costs, he noted. As costs drop, money will be redirected to other priorities, he said. — BRITT ERICKSON

RESEARCH: Federal sequencing effort shifts funds to clinical applications

NIH EXPANDS GENOME PROGRAM

Large-scale sequencing centers face less NIH funding for basic research.

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NEWS OF THE WEEK

Fluorophoredeaggregation

Fluorophore o�

Nonfluorescent Fluorescent

Fluorophore onSurfactantWater

Watch fluorescent nanoparticles turn on in kidney cells at cenm.ag/nanofluor.VIDEO ONLINE

Several major research organizations are joining forces to establish two institu-tions that will explore the genetic under-pinnings of human disease. The centers, in Connecticut and New York City, both aim to use personal genetic information to advance drug development and cre-ate opportunities for company spin-offs while improving health care.

The Connecticut project, the Jackson Laboratory for Genomic Medicine , will link Jackson Laboratory, a Maine-based non-profit, with the University of Connecticut Health Center and Yale University. It will

be located at UConn’s campus in Far-mington. Funding over the next 10 years will include $291 million from the state and more than $800 million from Jackson Laboratory, the project’s backers say.

The center will open new R&D oppor-tunities in a state where drug firm Pfizer is cutting more than 1,000 jobs (C&EN, Feb. 7, page 5). The 173,500-sq-ft lab is expected to be completed in 2014 and ul-timately house more than 660 people.

The second project, the New York Ge-nome Center , will link 11 academic medi-cal centers and research universities in-

cluding Rockefeller University , Memorial Sloan-Kettering Cancer Center , and Cold Spring Harbor Laboratory at a yet-to-be-disclosed location in New York City. Gene-sequencing instrument firm Illumina and drugmaker Roche will be collaborators.

About $125 million in private and pub-lic money will pay for the 125,000-sq-ft facility, which is set to open next year. Executive Director Nancy Kelley says the center “will allow us to support the world’s premier research and medical in-stitutions, as well as their diagnostic and pharmaceutical partners.” — MARC REISCH

PERSONALIZED MEDICINE Organizations plan genomics centers in Connecticut, New York City

A RESEARCH TEAM based at Ireland’s Uni-versity College Dublin has demonstrated fluorescence-switchable polymer nanopar-

ticles in action. Bearing functional groups that turn on fluorescence for imaging when captured by cells, these particles are not subject to the interfering background fluorescence common with fluorophores that are al-ways turned on.

According to Donal O’Shea , who spearheaded the work, the unique “off ” to “on” switching “allows us to use the nanoparticles for real-time, continuous imag-ing of their uptake into live cells for the first time. Some of the movies we have recorded are quite dramatic.”

Near-infrared fluorescence imaging using molecular fluorophores is a popular method for investigating biological processes, such as the cellular uptake of mol-ecules, including drugs. An often-encountered problem is background fluorescence from fluorophores not inside the cells. The extrane-ous light can mask imaging of events scientists want to see or limit the imaging to snapshots in time when the background fluorescence has been removed.

O’Shea’s team circumvented this prob-lem by designing poly(styrene- co -methacrylic acid) nanoparticles cov-ered with hydrophobic BF 2 -chelated azadipyrromethene groups ( J. Am. Chem. Soc., DOI: 10.1021/ja208086e).

These groups shy away from water, which causes them to aggregate, thereby compressing the fluorophores and quenching their fluorescence. Common surfactants or interactions with cellular components such as mem-brane phospholipids cause deaggregation. When the groups are apart, the fluorophores are free to cut loose and shine with their full fluorescence intensity.

To demonstrate the power of the fluorescence switching, the re-searchers tracked fluorescence after cellular uptake of the nanoparticles by human breast cancer and kidney cells. It takes about 15 minutes for a diffuse pattern of red fluo-rescence to emerge from the dark back-ground as the particles enter cells and switch on. By 100 minutes, strong red fluorescence concentrates in individual cells. The particles don’t enter the nucleus, so that area in each cell remains dark.

Turn-on nanoparticles are “indeed a cool tool to follow fluorescence within living cells,”

comments Wendelin J. Stark , a func-tional nanomaterials expert at the Swiss

Federal Institute of Technology, Zurich (ETH). The inherent tendency of the new nanoparticles to quench when close to one

another “is a different kind of switch for fluorophores,” Stark notes.

Besides biological imaging and drug delivery, the method “may also find

interesting use in low-cost, portable detection of surfactants, maybe in water analysis,” Stark adds. — STEVE RITTER

BIOLOGICAL IMAGING: Functionalized nanoparticles

light up upon entering cells

SWITCH-ON FLUORESCENCE

Aggregation and deaggregation enable fluorophore-bearing nanoparticles to switch from “off” to “on.”

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Reprinted from C&EN, Nov. 7, 2011



BUSINESS

BUILD IT and they will come—again and again. Having sold a big-ticket piece of equipment, laboratory instrumentation manufacturers hope customers will return repeatedly for the consumable products they’ll need to run it.

In the instrumentation business, con-sumables typically include anything be-yond the instrument itself, which industry managers like to call “the box.” Ranging from sample-prep items and chromatogra-phy columns to life sciences reagents and assays, these high-margin repeat-sale prod-ucts can be a steady revenue source.

In 2011, worldwide sales of consumables are expected to reach roughly $8.5 billion, or about 20% of instrument industry sales, and are to grow by 4% over 2010, according

to Strategic Directions International (SDI), a Los Angeles-based research firm.

Each instrumentation firm approaches the market differently, with consumables that mirror its instrument types and cus-tomer needs. Life sciences research, for example, uses large quantities of reagents, assays, and test kits. Mass spectrometry (MS) consumes less, because samples are usually processed beforehand via chro-matography, which requires columns and sample extraction methods. Some suppli-ers offer products that work only with their

own equipment, whereas others provide generic consumables.

Although strategies vary, instrument makers consider consumables a strate-gic business. In tough economic times, repeat-sale products can help sustain an instrument supplier’s business. Customers need consumables “no matter what” to run their existing instruments, even though their capital purchases may fluctuate, says Franco Spoldi, director for consumable products and business development at PerkinElmer . “In general, the number of

REPEAT SALES FOR STABLE REVENUES

Instrumentation firms view CONSUMABLE PRODUCTS as a strategic business ANN M. THAYER , C&EN HOUSTON

“We would like to be instrument agnostic in our development of new products.”

OPTION BLOCK Life Technologies’ QuantStudio system can accommodate five different assay formats.

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samples that customers are running is only increasing.”

Economic conditions in some of instru-mentation’s biggest markets are dampen-ing sales of both equipment and consum-ables. Stock analysts point out that spend-ing by the pharmaceutical and biotech industries, which accounts for about 30% of the life sciences tools market, has been declining. In the government and academic arenas, which make up another 30% of the market, budgets are at best uncertain, if not down, and economic stimulus monies are running out.

NOT SURPRISINGLY, high-end equip-ment and infrastructure are the most likely laggards along with basic lab equipment, all down 3–4%, says Peter Lawson, executive director at investment firm Mizuho Securi-ties USA, about academic spending plans over the next 12 months. “Reagents appear

better positioned,” he adds, “but still down approximately 2%.” In Mizuho’s academic spending survey, 25–30% of respondents think purchases of consumables will be down, compared with 42% who say spend-ing on instruments will drop.

Lawson reports similar trends in the market for gene sequencing, with funding expected to be down and 29% of genom-ics labs cutting back. These large-volume users have been delaying purchases of both instruments and consumables. When reporting third-quarter sales and earnings, Illumina , which leads in this market, high-lighted negative factors that it expects to continue at least through 2011.

“We saw what we believe to be an un-precedented slowdown in purchasing due to uncertainties in research funding and overall economic conditions, as well as a temporary excess of sequencing capacity in the market,” Illumina Chief Executive

Officer Jay T. Flatley said when reporting results in October. The excess capacity has decreased the consumables revenue per instrument owing to fewer runs.

Overall, consumables continue to fare better than boxes. “Companies with high consumables flow, such as Qiagen and Life Technologies , appear more insulated versus more instrumentation-exposed companies like Bruker and Waters ,” Law-son says.

Lawson ranks Thermo Fisher Scientific among relatively well positioned firms, with a product mix skewed toward con-sumables, service, and lower cost instru-ments. About $5.8 billion, or more than half, of its annual sales are in lab supplies and consumables. Within its $4.6 billion analytical technologies segment, 44% of sales are in consumables, 41% in instru-ments, and the rest in services.

The creation of Thermo Fisher in

ELECTRONIC CHANNELS

Companies Offer Many Ways To Learn About, Use, And Purchase Consumables Mobile technology is starting to put product information in researchers’ hands literally through smartphones and tablets that are now appear-ing in labs. And instrument suppliers are creating soft-ware applications, or apps, to help customers search for and use reagents and con-sumable products.

Agilent Technologies has apps for calculating liquid and gas chromatography param-eters to determine equipment setups. Likewise, Thermo Sci-entific has a GC column se-lector tool. And Waters has a part selector app that allows users to select ultra-perfor-mance LC sample plates, vi-als, filters, and columns. Once users find a desired configu-ration, they can save or e-mail it, or place an order.

In biosciences, Life Tech-nologies has mobile apps for cell imaging and viewing; plot-ting and comparing spectra; and calculating common sci-

entific parameters. Merck Mil-lipore has an app for finding filters and another for finding data and research reagents for histone modification and epigenetics research. Another app allows customers to view its EMD Chemicals catalog, with supporting product documents and pricing.

Aggregated information on commercially available chem-icals is accessible through Eidogen-Sertanty ’s MObile REagents app, which inte-grates with the firm’s other chemistry apps. The MORE app covers about 6 million compounds from more than 50 suppliers. Chemical struc-ture searches are possible through a mobile device’s camera and the app’s optical structure recognition capabil-ity. And by using mobile de-vices’ ability to scan and print bar codes, Eidogen-Sertanty is trying to enable local inven-tory management.

Although ubiquitous in

everyday life, mobile devices are just be-ginning to be adopted in the lab. “More and more compa-nies are looking into bringing these devices into the labs,” says Maurizio Bronzetti, Eidogen-Sertanty’s business development director. Cost may be an issue and secu-rity a concern, especially in regulated industries such as pharmaceuticals.

Even so, companies “rec-ognize that bringing the de-vice closer to the experiment has its advantages,” Eidogen-Sertanty CEO Steven Muskal adds. And for scientists, “it is really important to be close to a device that gives them access to applications and content that helps them ex-plore their ideas.”

Online ordering remains popular, and suppliers contin-ue to improve their websites to make it simpler for custom-ers to find what they need. The “find and decide experience,” which may happen on a mo-bile device, is often apart from the “buy experience,” says Larry Milocco, senior market development manager at Life Technologies. Many research-ers shop from their desks, but an agent completes the pur-chase. “We are trying to look at how our customers want to connect with us,” he adds.

EID

OG

EN

-SE

RT

AN

TY

AT HAND A researcher uses the MORE app to search for chemicals.


BUSINESS

2006 was one of several recent mergers motivated at least in part by the desire to strike a good balance between instruments and consumables. The nearly $13 billion deal combined Thermo’s strength in lab equipment and instruments with Fisher’s broad range of reagents, consumables, and services. One expected result was more overlap in R&D between the hardware and consumables sides of the company.

Similarly, with its recent $2.1 billion acquisition of Dionex, Thermo anticipates linking its MS and lab information software systems with chromatography units and consumables from Dionex, rather than those from other vendors. In 2011, Ther-mo’s combined chromatography business will have $650 million in sales, with 27% in consumables and 50% in instruments.

In another big consumables deal, Merck KGaA , of Germany, acquired Millipore in 2010 for $7.0 billion. The resulting Merck Millipore division, which is called EMD Millipore in North America, is on track to have about $3.2 billion in sales this year. Targeting bioresearch and bioproduction markets, the division has three business units: bioscience, lab solutions, and pro-cess solutions.

Damien Tuleu, head of biomonitoring for the company’s R&D group says the bio-monitoring business model “is to design and develop instruments which can be used only with our consumables.” When a customer buys a piece of equipment, “it means you are going to capture the stream of consumables,” he adds.

WEIGHTED TOWARD consumables, the biomonitoring group focuses on kits, sys-tems, and services for quality control and assurance. Its products are used to detect microbial contaminants in pharmaceutical samples or pathogens in food and bever-ages. The group falls within Merck’s lab solutions unit, which provides reagents, solvents, chromatography products, and high-purity lab water systems.

Many of the biomonitoring group’s products are sold to regulated markets, where an instrument and the related consumables may be incorporated into an approved protocol. Because custom-ers tend to be conservative about changes that would require revalidating methods, Tuleu explains, a supplier can retain con-sumables sales long after it sells a piece of equipment.

Without large amounts of consumables, biosciences instruments would be idle.

About 80% of Life Technologies’ nearly $3.7 billion in annual sales are in consum-ables associated with its instruments. Even when its customers’ R&D budgets are shrinking, the company’s “consumables product mix is positioned to grow faster than the market,” said Morgan Stanley stock analyst Marshall Urist in an early 2011 outlook report to clients. The compa-ny has a strong position in segments poised for growth, including the leading area of sample prep for RNA, DNA, and protein analysis.

Another growth area is real-time poly-merase chain reaction technology, also called quantitative PCR. According to Urist, “qPCR’s growth looks set to con-tinue with the second-highest growth indicator among consumables segments.”

Life Technologies itself has predicted high-single- to low-double-digit growth for the qPCR market.

In October, the company launched the QuantStudio 12K Flex qPCR system. The high-end unit avoids the need for mul-tiple PCR systems because it can conduct various postsequencing gene expression, genotyping, biomarker, pharmacogenomic, and other experiments. Users can also run digital PCR experiments using nanoflu-idic consumables and dedicated analysis software.

Because of the huge growth in next-generation sequencing, a lot of researchers are moving toward qPCR to confirm their sequencing results. The trend is leading to an uptick in both academic and com-mercial markets, says Larry Milocco, se-

nior market development manager at Life Technologies.

Target users include time-crunched screening facilities, service providers, and contract research organizations that want high throughput and flexibility in the type and number of assays they can run. “We are trying to build products that are going to scale with the research needs,” Senior Prod-uct Manager Ricardo Mancebo says.

CONSUMABLES ARE INTEGRAL to the system’s capabilities because the system can accommodate five interchangeable blocks—including OpenArray plates, Taq-Man array cards, and multiwell plates—to match the size and type of experiment. “Consumables are key to our product strategy,” Mancebo says. In particular, he calls the nanofluidic OpenArray plates a “unique type of consumable” that can be run on only two Life Technologies instruments.

“A large part of our business was cus-tomer-configurable products,” Mancebo says, with customers specifying the assays they want in a given format for a specific application. “More recently we have been building fixed-content panels.” Drawing on more than 8 million collected assays, which Life Technologies manufactures itself, the company is developing panels with cus-tomer input.

Similarly, significant advances in another consumable segment, chromatography col-umns, have contributed to the emergence of the ultra-performance liquid chromatog-raphy (UPLC) business. These systems use stationary phases with particles of less than 2 μm in diameter for high resolution, speed, and sensitivity. “The science happens in the consumable,” says Michael Yelle, senior di-rector for chemistry commercial operations at separations specialist Waters.

The company launched its first Acquity UPLC instrument in 2004. Innovations in the materials science and the hardware of the columns led to “a holistic instrumenta-tion design,” Yelle says about the develop-ment process. The UPLC columns can be connected through an eCord chip that contains quality-control data and tracks use and performance.

Although designed to work with its own instruments, Waters’ columns can be used with other manufacturers’ equipment. In practice, however, the “attachment rate” is usually high in UPLC, with customers typi-cally preferring to use columns and instru-ments from the same company.

–3

–2

–1

0

0

1

2

3

4

Change in budget from previous year, %

09 10 11 122008

◼ Consumables

◼ Instruments

SEGMENTS Spending on life sciences instruments and consumbles is expected to decline this year.

SOURCE: Morgan Stanley Research


The adoption of UPLC has helped drive growth at Waters, according to Morgan Stanley’s Urist. But the shift from HPLC to UPLC is expected to be “gradual rather than abrupt,” at least in research settings, he adds. Quality control and other regu-lated testing settings offer faster growth opportunities.

Waters has a new line of columns, called XP, that can be used on both HPLC and UPLC systems. “If customers have an installed base of HPLC, they can develop methodology and run it on HPLC, and in the future when they migrate to UPLC they can use the same physical columns,” Yelle says.

Consumables are about a $300 million business for Waters, or about 18% of its overall sales. “We regard the consumable products as being key differen-tiators and drivers of perfor-mance, and so the research and development of these products takes a lot of focus,” Yelle says. “One thing that differentiates us in the consumables space is our grounding in basic materials science.”

BESIDES MAKING the materi-als, Waters designs hardware and assembles columns. Controlling the synthesis and being verti-cally integrated is “all-around optimizing performance and maintaining quality and consis-tency,” Yelle says. These aspects, he adds, are important to the company’s customers in regu-lated markets, such as pharma-ceuticals, where LC columns are frequently needed for the life span of a drug.

“Once we have launched a product and it is used in a regulated method, we will continue to manufacture and support our customers that are using it,” Yelle says. And these long product life cycles can help even out the peaks and valleys in demand, he adds.

Consumables for sample preparation is another long-standing market for most firms. Even though this business is mature, it touches almost all analytical work and continues to grow.

For example, Waters’ solid-phase ex-traction products (SPE) are widely used for bioanalytical sample prep, Yelle says. Its SPE devices and vials can be used in front of most analyses. The move toward lower analytical detection limits has led to advances such as vials with optimized

surface chemistry to prevent interference with samples.

As instrumentation has advanced with faster processing times and greater sensitivity, “sample prep has become the bottleneck,” says Rebecca Duguid, seg-ment manager for analytical sample prepa-ration in Millipore’s biosciences group. For the HPLC market, Millipore recently introduced the Samplicity filtration sys-tem, which simultaneously prepares eight samples.

PerkinElmer’s Spoldi calls sample prep a “major pain point” for customers. In some of the company’s focus markets, such as environmental and food analysis, up to 60% of a lab’s workload can be consumed by sample preparation. In addition to

greater efficiency, these customers want consistency, reliability, and ease of use.

“Without great sample preparation, you can certainly compromise your results and analysis,” says Brian J. Kerslake, director of aftermarket solutions at PerkinElmer. The company also sells consumables for a vari-ety of chromatographic and spectroscopic methods. Its strategy is to “follow the path of the sample,” he says, from collection and preparation to analysis on the instrument and ultimately to data handling.

Consumables are part of PerkinElmer’s broader aftermarket business, which in-cludes anything other than the box, such as parts, accessories, and services. Although the company doesn’t report its consum-ables sales, Spoldi says its business is “in line with the industry.” PerkinElmer also offers a service and equipment manage-ment program called OneSource that

covers any vendor’s instruments in a cus-tomer’s lab.

“We have been keen on trying to create a relationship with customers, not only at the acquisition of the box, but also with what comes after the acquisition,” he says. “We have a multivendor approach to all our accessories and consumables, and we would like to be instrument agnostic in our development of new products.”

So even as the company develops com-plete systems around its own product offer-ings, it also provides protocols and methods to allow customers to operate seamlessly on whatever instrument platform they are using, Kerslake explains. Customers “don’t want to be tied into a whole work flow from one supplier when they can look at the best

of breed, even if they already have a competitor’s box or in-strument in the lab.”

TO OFFER the range of prod-ucts that it does, PerkinElmer conducts product development and manufacturing in-house or through partners. And it invests substantially “in new consum-ables and newer technologies, whether it’s advanced materials, different lamp sources, or other pieces that allow an instrument to perform to the detection lim-its required,” Kerslake says.

Similarly, Agilent Technolo-gies is looking to consumables to help expand business, not only with existing customers, but also with users of non-Agilent instru-

ments. In March, the company launched its CrossLab supplies program for gas chro-matography, which supplies consumables for several companies’ GC systems.

The firm’s strategy is to sell products across work flows, explained Life Sciences Group President Nick H. Roelofs in a Sep-tember presentation for analysts. In addi-tion to GC supplies, Agilent’s other consum-ables include lab reagents and microarrays, as well as LC and sample-prep products. Consumables make up about 20% of $1.3 bil-lion in combined sales for Agilent’s chemical analysis and life sciences groups and grew 22% for the 12 months ending on Oct. 31.

“It is a really nice renewable revenue stream,” Roelofs said. “With our new moves and portfolio expansions from recent acquisitions, we are seeing a lot of opportunity here.” ◾

% of total sales

0 20 40 60 80 100

TOTAL INDUSTRY

Mettler ToledoWaters

BrukercAgilent Technologiesb

Thermo FisherIllumina

Pall Life Technologies

QiagenMerck KGaAa

◼ Consumables ◼ Instruments ◼ Services & other

a For Millipore business. b For life sciences and chemical analysis groups. c Consumables percentage includes services. SOURCES: Company information, Strategic Directions International

BUSINESS MIX Instrumentation companies di� er in amount of consumables they sell.

Reprinted from C&EN, Nov. 28, 2011


GOVERNMENT & POLICY

THE ONLY DIAGNOSTIC routinely used by clinicians to confirm a pesticide poi-soning case is a test that measures inhibi-tion of the enzyme cholinesterase. The assay works well for diagnosing patients exposed to organophosphate pesticides, which dominated the pesticide market in the 1990s. But the use of organophosphate pesticides has been declining over the past decade as less toxic alternatives, such as pyrethroids, have become available. There are no diagnostic tests for these increas-ingly common alternatives.

The key reason for the absence of di-agnostic tests for pesticide exposure is a lack of biomarkers. This makes it difficult for physicians, who typically have little training in environmental health, to diag-nose acute cases of pesticide poisoning. Epidemiological studies and pesticide risk assessments are also being hobbled by this lack of biomarkers.

These problems came to light last month at a stakeholder workshop hosted by the Environmental Protection Agency’s Office of Pesticide Programs (OPP). Experts from the pesticide community congregated at the meeting to discuss opportunities and chal-lenges associated with advancing toxicology in the 21st century and developing biomark-er-based tests for pesticide exposure.

Workshop participants agreed that new tools for monitoring pesticide exposure are needed, and they grappled with how to pri-oritize which pesticides to study. They also questioned how to deal with the variability of biomarkers over time and the instability of biomarkers in blood and urine samples.

The test for cholinesterase inhibition, the gold standard in pesticide exposure, is old and nonspecific. It measures exposure to pesticides that bind the cholinesterase enzyme—any of the organophosphate and carbamate pesticides—not exposure to one particular pesticide.

“Unfortunately, cho-linesterase inhibition is the only test we have for pesticide exposure,” said Matthew C. Keifer , a senior research scien-tist with the Marshfield Clinic Research Foun-dation’s National Farm Medicine Center in Wisconsin. Physicians regularly order the test when it isn’t appropri-ate, such as in the cases of herbicide poisoning, he said.

Keifer highlighted

the importance of pesti-cide biomarkers for iden-tifying worker protec-tion practices that have failed. He and others also pointed out the need for biomarkers to confirm pesticide exposures in worker compensation claims.

“In mild to moderate pesticide overex-posure, a nonspecific clinical presentation is common,” said Amy K. Liebman, director of environmental and occupational health at the Migrant Clinicians Network , a group dedicated to health care for migrant farm-workers. The availability of a diagnostic biomarker could provide objective confir-mation of a work-related illness, she said.

Biomarkers and diagnostics are also needed for pesticide risk assessments and to help interpret and design epidemiological studies, OPP Director Steven Bradbury said. Biomonitoring tools are a critical part of EPA’s long-term vision to integrate molecu-lar and exposure science into its pesticide risk assessments, but EPA doesn’t have the tools to get an in-depth understanding of what exposure information means, he said.

In terms of where to start developing those tools, Dana Boyd Barr, a researcher at Emory University’s Rollins School of Public Health, recommended that EPA focus on pesticides that are the most toxic and have the highest potential for human exposure. Pyrethroids and pyrethrins are some of the most widely used pesticides, yet no clinical test is available for them, said James R. Roberts , associate professor of pediatrics at the Medical University of South Carolina. Many other commonly used pesticides, including organochlo-

rines, neonicotinoids, chlorophenoxy her-bicides, and chloro-picrin, also don’t have diagnostics, he noted.

Other people sug-gested a more holistic approach. “Individuals are not exposed to a single compound. We want to come up with a comprehensive view of the individual envi-ronment,” said David M. Balshaw , a program administrator at the National Institutes of Health’s National

DETECTING PESTICIDE EXPOSURE

Researchers, clinicians search for NEW BIOMARKERS to keep up with changes in product usage

BRITT E. ERICKSON , C&EN WASHINGTON

SH

UT

TE

RS

TO

CK

SPRAY ZONE Clinicians have no way of knowing when farmworkers have been overexposed to many common pesticides.

1990 98 0294 06

Millions of lb

0

20

40

60

80

100

◼ Organophosphates ◼ Other

SOURCE: Environmental Protection Agency estimates based on Department of Agriculture/National Agricultural Statistics Service and EPA proprietary data

TRADING PLACES Use of organophosphate pesticides has dropped, leaving other pesticides to fi ll the void.


Institute of Environmental Health Sciences. Balshaw described a program on exposure biology led by NIEHS to develop new tech-nologies and biomarkers to characterize the entire personal environment, including chemical exposures as well as dietary intake, physical activity, and psychosocial stress.

Balshaw also highlighted an NIEHS ef-fort to improve the way biomarkers are measured. “Biomarkers in this case are not a single gene or protein,” he said. NIEHS is coming up with new ways of detecting biomarkers, focusing on arrays and other techniques to understand how the entire bi-ological pathway responds, Balshaw noted.

Along similar lines, Dean P. Jones , a med-ical biochemist and professor at Emory Uni-versity, pointed out that his lab can measure approximately 10,000 chemicals—includ-ing 1,500 to 2,000 metabolites—in a drop of blood in 20 minutes using high-resolution mass spectrometry. The analysis provides a “relatively complete understanding of metabolic pathways,” he said.

So far, high-resolution mass spectrom-etry hasn’t found its way into the clinic for

routine medical exams. But Jones predicts that it might not be too long before that happens. “The methodology has been around for about 30 years,” he said, adding that it took about that long for nuclear mag-netic resonance—NMR—spectrometry to go from being a basic lab research tool to the clinical imaging technique called MRI.

Jones referred to the concept of the exposome—all of the exposures of an indi-vidual over a lifetime and how those expo-sures relate to disease. “If we had a system where we could collect samples and collect exposure information throughout life, then we would really have a new opportunity in terms of epidemiology to be able to look at disease associations,” he said. The biggest limitations right now are in the informatics side, he noted.

IN ADDITION to debating what to measure, workshop participants pondered how often to take samples. Biomarker concentrations vary over time, so a single spot sample doesn’t provide the whole picture, said Lesa L. Aylward, principal at the Colorado-

based consulting firm Summit Toxicology . Variation is often significant within one person and within one day, she noted.

For biomarkers related to chemical exposures, interpretation of the results “is not as simple as higher concentration equals higher exposure,” Aylward said. “If you understand the pharmacokinetics of the compound, you can improve how you use biomonitoring for patients,” she added.

In the end, there are many different needs for pesticide-specific diagnostic tools. Public health advocates are push-ing EPA to require the pesticide industry to develop such tests for their products as part of the approval process. Without such requirements, diagnostic tests will not be created, the advocates say.

“If industry can’t come up with a test for pyrethroids, surely they are not going to come up with a test for the newer nicoti-noids or fipronil,” a chemical used on dogs to control fleas, South Carolina’s Roberts said. “Coming up with it on their own,” he warned, “is just not going to happen.” ◾

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Reprinted from C&EN, Oct. 31, 2011


SCIENCE & TECHNOLOGY

ONE JUNE DAY in 1968 marine biologist Jack Rudloe went down to collect speci-mens from the docks at his local marina on the northern Florida coast of the Gulf of Mexico. As the man behind Gulf Speci-men Co ., Rudloe was used to catching some of the Gulf ’s more interesting crea-tures—electric rays, bonnethead sharks, and live jellyfish—for aquariums and research centers throughout the country.

But on this day, his mission was sim-pler: Gather some marine organisms

that were abundant and easy to collect (inexpensive, in other words) and send them to Jonathan L. Hartwell’s anticancer drug discovery group at the National Can-cer Institute (NCI).

Among the dozen organisms Rudloe col-lected “shotgun style” that day, was a small brownish spray that

looked like seaweed. Despite its appearance,

the material was not a plant but rather a colony of the bryo-

zoan Bugula neritina , tiny filter-feeding crit-ters, each about a mil-

limeter long that clump together in a branching

structure. B. neritina is, in fact, a pest

that fouls floating docks

and boats in waters worldwide.

Rudloe put a few handfuls of

the bryozoan through a meat grind-er, packed it

in isopro-pyl alcohol, and sent it

to Freder-ick, Md. “It

was just sheer luck,” he says,

that he had picked an organism armed with mol-

ecules that could fight cancer, Alzheimer’s disease, and HIV.

Those compounds are the bryostatins, a family of 20 macrolide lactones, 18

of which have been structurally charac-terized. Since they were first plucked from obscurity more than 40 years ago, the compounds have had a colorful history. They were hailed as key compounds in the fight against cancer, but over the years, bryostatin 1, the most-studied member of the family, failed to

impress. In more than three dozen clinical trials to fight various forms of the disease, it gave mostly mediocre results, both on its own and in combinations with other cancer-fighting drugs.

THE COMPOUNDS have also fallen out of favor as drug candidates for a more practi-cal reason: Harvesting them from the natu-rally occurring bryozoan is impractical, and their long chemical syntheses were too unwieldy for drugmakers.

Recently, however, the cloud that was hanging over the bryostatins has begun to lift. Animal tests show that bryostatin 1 enhances memory and could be used to treat Alzheimer’s disease and strokes. And some preliminary studies show it could help eradicate HIV. What’s more, chem-ists have dramatically whittled down the number of steps it takes to make these molecules. This year, three total syntheses of bryostatin natural products were pub-lished, with the shortest being just 36 steps. Finally, as chemists have found a way make bryostatins faster, there’s been a push to make analogs of these compounds so that scientists might get a better handle on how they operate biologically and make simpler molecules that would be more practical drug candidates.

George R. (Bob) Pettit , a natural prod-ucts expert and chemistry professor at Arizona State University, was one of the people driving research to find cancer-

THE BRYOSTATINS’ TALE With the promise of treating CANCER, ALZHEIMER’S, AND HIV, this family of marine natural

products continues to intrigue scientists more than four decades after its discovery BETHANY HALFORD , C&EN NORTHEAST NEWS BUREAU

BRYOSTATIN BOUND A hypothetical model, based on computational studies, of bryostatin 1 binding to PKC.

AD

AM

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ER

& B

RIA

N L

OY

/S

TA

NF

OR

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CH3O

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Bryostatin 1

B A

C


fighting agents from marine or-ganisms back when Rudloe scooped those first handfuls of B. neritina . In the early 1970s Pettit began collecting bryozoans from the Gulf of Califor-nia, in Mexico, and the Sagami Gulf, in Japan. But it was extracts from a sample of B. ne-ritina taken from the California coast that most interested Pettit and the folks at NCI.

PETTIT’S GROUP spent most of the late 1970s trying to isolate the compounds re-sponsible for the antineoplastic activity in bryozoan extracts. By 1981, Cherry L. Her-ald, a scientist working in Pettit’s lab, had isolated the first milligram of what would be known as bryostatin 1 from that Cali-fornia collection of B. neritina . “I dashed it off to the National Cancer Institute and the activity was tremendous,” Pettit re-calls. “It was clear we had to determine the structure.”

Using 500 kg of B. neritina , Pettit’s research team isolated 120 mg of bryo-statin 1. They crystallized the material and determined the compound’s structure, which they reported in 1982 ( J. Am. Chem. Soc., DOI: 10.1021/ja00388a092 ). “We were blessed,” Pettit says of the ease with which they crystallized the compound.

The structures of 17 other bryostatins would follow (extracts from Rudloe’s samples became known as bryostatins 4 through 8). But it was bryostatin 1 that NCI began to focus on as a potential drug. In 1991 the institute undertook a massive isolation of bryostatin 1 from B. neritina off the California coast, collecting some 14 tons of the animal, which Pettit recalls shipping to NCI in 120 55-gal drums.

From those 14 tons, researchers isolated 18 g of bryostatin 1 ( J. Nat. Prod., DOI: 10.1021/np50077a004 ). That’s enough to fill a typical salt shaker up just a quarter of an inch, Pettit estimates. Nevertheless,

because bryostatin 1 is so potent, those 18 g have been enough to supply all the clinical trials using the compound.

BRYOSTATIN 1 works by modulating the activities of a family of enzymes known as protein kinase C, or PKCs. Once acti-vated, these enzymes phosphorylate certain proteins and play an im-portant role in intracellu-

lar signaling cascades. PKCs first attracted the attention of biologists because they are the target of phorbol esters, the archetypal tumor promoters.

But while phorbol esters make tumors grow like crazy, bryostatin 1 suppresses tu-mor growth—even though they both bind to the same part of PKCs. It’s a phe-nomenon that still puzzles biologists. “Of the known activators of PKC, bryo-statin 1 is the only known agent that is a functional antagonist of most phorbol ester functions,” says Gary E. Keck , a chem-istry professor at the University of Utah who has been studying the bryostatins for the past decade.

In fact, a number of natural products activate PKCs. Like bryostatin 1, they bind to a region of the enzymes known as the C1 domain. When a small molecule fills this C1 cleft, a PKC enzyme opens to receive its substrates. The binding also makes the C1 region hydrophobic, enabling PKC to move from the cytosol, where it resides in the absence of activation, to a membrane. That membrane could be the cell membrane, the nuclear membrane, or membranes of other cell organelles. Once stuck to the

Bryostatin time line 1968 First samples of Bugula neritina screened for anticancer activity

1976 A compound that would come to be known as bryostatin 1 identified for the first time in extracts from B. neritina collected from the California coast

1982 Structure of bryostatin 1 reported

1990 First total synthesis of bryostatin 7 in 79 steps by Satoru Masamune and coworkers at Massachusetts Institute of Technology

1991 18 g of bryostatin 1 ex-tracted from 14 tons of B. neritina collected off the California coast

1998 First total synthesis of bryostatin 2 in 72 steps by David A. Evans and cowork-ers at Harvard University

2000 First total synthesis of

bryostatin 3 in 88 steps by Shigeru Nishiyama, Shosuke Yamamura, and coworkers at Japan’s Keio University

2008 First total synthesis of bryostatin 16 in 42 steps by Barry M. Trost and Guang-bin Dong of Stanford University

2011 ◾ First total synthesis of bryostatin 1

in 58 steps by Gary E. Keck and co-workers of the University of Utah

◾ First total synthesis of bryostatin 9 in 43 steps by Paul A. Wender and Adam J. Schrier of Stanford University

◾ Total synthesis of bryostatin 7 in 36 steps by Michael J. Krische and coworkers at the University of Texas, Austin

CH3O

OCH3

O O

O

O

O

O

O

O

OH

O

HO

OH

OH

OH

H

Bryostatin 2

Bryostatin 3

CH3O

O O O

O

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O

O

O

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O

HO

OH

OH

O

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H

This year, three total syntheses of bryostatin natural products were published,

with the shortest being just 36 steps.



membrane, PKC finds its protein targets and phosphorylates them, setting off the signaling cascade.

PKC-acti-vated proteins are involved in some of the most important cellular functions, Keck points out. They make cells grow. They make cells morph into different kinds of cells. And they are involved in apoptosis, or programmed cell death. “The most critical processes of the cell turn out to be heavily regulated by this family of enzymes,” Keck notes. “That’s why bryostatin 1 can have such a wide range of biological effects. It’s not like a lot of agents that target one specific site in an enzyme and inhibit its activity. This is very different.”

Despite promising results as a cancer treatment in animal studies, bryostatin 1 has stalled in Phase II clinical trials. It has failed to show significant activity against tumors either on its own or in combination with other chemotherapeutic agents. “The bryo-statins still haven’t quite found the right niche,” says Peter M. Blumberg , chief of the Molecular Mechanisms of Tumor Promo-tion Section at NCI. “By understanding the mechanism of the bryostatins we might be better able to pinpoint which are the specific subclasses of cancers in which this would represent the rational therapy.”

ALTHOUGH ITS STATUS as a cancer-fighting agent may have taken a hit, bryo-statin 1 has started to gain some traction in treating other diseases, particularly in ill-nesses associated with memory loss, such as Alzheimer’s disease and strokes.

Researchers led by Daniel Alkon , scientific director and pro-fessor at Blanch-ette Rockefeller Neurosciences Institute at West Virginia Univer-sity, were trying to work out how memories are stored on the molecular level when they dis-

covered that PKC plays a critical role in the pro-cess. “It’s a very powerful regulator of molecular switches that send signals, especially at the most important junctions in the brain called synaptic junctions—the connec-tions in the brain between neurons,” he says. “We discovered when we form

memories we actually induce the forma-tion of new synapses, and that’s regulated by protein kinase C and a whole host of other molecular players in the orchestra that protein kinase C regulates.”

With this understanding, Alkon’s team wondered whether PKC might be relevant to the memory loss associated with Alzheimer’s disease. “It turns out that the central molecular pathways of the pathophysiology of Alzheimer’s disease all involve protein kinase C,” Alkon explains. This led Alkon to sev-eral compounds that activate PKC, of which bryostatin 1 was the most potent.

“We found that PKC activators are remark-ably effective in animal models of Alzheimer’s disease in addressing virtually all of the as-pects of Alzheimer’s disease,” Alkon says.

These compounds “enhance memory. They correct memory deficits. They restore lost synapses and prevent the loss of syn-apses. They prevent the death of neurons. They prevent the amyloid plaques. And they prevent the neurofibrillary tangles. All of those are hallmarks of Alzheimer’s dis-

ease,” Alkon continues. “There’s no one therapy except activators of protein kinase C that does that.” These find-ings, he argues, suggest a new way of looking at Alzheimer’s disease.

Animal tests with bryostatin 1 have also shown that it restores memory after strokes and traumatic brain in-juries. “Essentially what it’s doing is building new

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connections in the brain and preventing the death of neurons,” Alkon says. “It also has the potential of enhancing memory in normal patients or aging patients or de-pressed patients. We believe that there is a tremendous potential here.”

Alkon recently received approval to be-gin a Phase II clinical trial using bryostatin 1 to treat Alzheimer’s. He wants to partner with a private-sector company before mov-ing forward, however.

BRYOSTATIN 1’S ability to activate PKC has also recently gotten attention for treating another disease—HIV. Patients with HIV who take the antiviral drug cocktails still retain latent reservoirs of the virus in their cells. That’s why the cocktails don’t cure the disease but merely treat it. Once a patient goes off the therapy the virus reawakens.

But PKC “can activate transcription fac-tors that can rouse slumbering HIV provi-

ruses,” according to Warner C. Greene , who directs virology and immunology research at the J. David Gladstone Institutes in San Francisco. “So bryostatin 1 is a drug that’s under active investigation for an eradication treat-ment,” he says, al-though he’s quick to point out that such

therapy is still in the early stages. No animal testing has been done with bryostatin 1 and HIV, Greene notes.

Even if clinical tests prove the medicinal potential of bryostatins, treatments based on the compounds will have to grapple with supply. In the late 1990s, the now-defunct company CalBioMarine Technolo-gies tried aquaculture, growing B. neritina on what Dominick Mendola , the company’s former president, describes as a giant “un-dersea box kite” off the California coast. Although they succeeded in growing the bryozoan, the company eventually went under as postponed clinical trials demol-ished demand for the bryostatin 1 it was ready to supply, and the firm was unable to secure venture capital funding in the early 2000s to stay afloat.

The bryostatins, current research sug-gests, don’t actually come directly from B. neritina , but rather from a bacterial sym-biont that lives within the bryozoan. The

Bryostatin 16

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compound appears to protect the organ-ism’s larvae from being eaten by predators. Scientists have tried to isolate the symbi-ont so that they might create bryostatins in a petri dish.

To date, however, no one has been able to culture the bacterium. “It may be miss-ing some capabilities it needs to live out-side of its host,” says Margo G. Haygood , a professor at Oregon Health & Science University who has been studying how the symbiont makes the bryostatin skeleton.

She adds that, despite efforts, no one has been able to transfer enough of the symbi-ont’s biosynthetic machinery into another organism, such as Escherichia coli, to make the bryostatin skeleton.

And that has left the bryostatins’ fate in the hands of chemists. With their complex skeleton and multiple stereocenters, the bryostatins are a trophy for any synthetic organic chemist up to the challenge. Until recently, however, total syntheses of bryo-statin natural products weighed in at more

than 70 steps—too unwieldy to make large amounts of the molecules.

In 2008, Stanford University chemists Barry M. Trost and Guangbin Dong report-ed the synthesis of bryostatin 16 in only 42 steps ( Nature, DOI: 10.1038/nature07543 ). And there’s been a flurry of activity in the field in 2011. A team led by the University of Utah’s Keck and graduate student Yam B. Poudel reported the first total synthesis of bryostatin 1—the one that’s been used in all the clinical trials—in 58 steps ( J.

PIONEER

Undersea Treasure Hunter The bryostatins, and many other natural products, might never have been mined from the sea for their disease-fighting compounds if not for the efforts of George R. Pettit . Known to his friends as Bob, Pettit has been searching the sea for cancer-fighting compounds for more than 40 years, as a chemistry professor at Ari-zona State University (ASU) and director of the ASU Cancer Research Insti-tute, a position he still holds.

It was a convergence of events in his childhood and teenage years, Pettit tells C&EN, that led him to cast his eye toward the ocean to seek cancer cures. Pet-tit grew up on the Jersey Shore, just half a mile from the ocean, and spent much of his childhood exploring the interesting inverte-brates that lived there.

At age 15, Pettit began work-ing in a medical center lab in Monmouth County, N.J. As part of his duties, he had to assist the hospital pathologist in doing postmor-tem exams, where he saw the ravages of cancer firsthand. “It made such a shock-ing impression on me that I still haven’t recovered from it,” he says.

Pettit began thinking of all those squishy creatures he had played with at the beach as a child. Physically, they seemed so vulnerable. And yet they’d managed to survive and evolve over billions of years without being gobbled up by predators. Pettit reckoned their chemical defenses must therefore be highly evolved, and he wondered wheth-er those same defenses might also fight cancer. “I always thought, even when I

was a kid, that naturally occurring mate-rial—plants, animals, microorganisms—would really be the best place to look for drugs,” he recalls.

In 1965, Pettit was lured away from his position at the University of Maine, where he’d explored natural products from fungi, plants, and arthropods, and joined the faculty at ASU. Working in

collaboration with the National Cancer Institute (NCI), Pettit began what he describes as the first worldwide explo-ration of marine invertebrates as new sources for anticancer drugs.

In the first 25 years of this research effort, he never once took a vacation, Pet-tit says. All his family trips were to sites where he could collect specimens. And he often enlisted his five children to help him collect underwater creatures. “They were our expeditionary force,” he jokes.

Over the years, Pettit and his col-leagues have collected some 14,000 specimens from the sea and elsewhere. And from them he has identified myriad

tumor-fighting natural products—the bryostatins, the combretastatins, the spongistatins, and the dolastatins, to name just a few. A synthetic dolostatin analog, named monomethyl auristatin E, recently received approval from the Food & Drug Administration to treat two types of lymphoma as part of a drug-an-tibody conjugate, named Adcetris, which

is marketed by Seattle Genet-ics and Takeda Pharmaceutical ( C&EN, July 25, page 10 ).

“Pettit has been an outstand-ing leader in natural products research, particularly in the search for new drugs for the treatment of various types of cancer,” says longtime Journal of Natural Products Associate Editor Richard G. Powell. “The contributions of Pettit and his research group to this area of research since the early 1960s have been quite numerous, the research meticulous, and under

his leadership the group has been out-standingly successful in the discovery of new compounds useful, or potentially useful, for cancer chemotherapy.”

“He can truly be regarded as one of the great pioneers in natural products drug discovery who was among the first to explore the realm of marine organisms as a source of potential an-titumor agents,” adds Gordon M. Cragg, a National Institutes of Health special volunteer with NCI’s Natural Products Branch . “Pettit is an outstanding and re-sourceful scientist totally committed to improving the treatment and quality of life of cancer patients worldwide.”

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Am. Chem. Soc., DOI: 10.1021/ja110198y ). Paul A. Wender and graduate student Adam J. Schrier, also at Stanford, prepared bryostatin 9 in 43 steps ( J. Am. Chem. Soc., DOI: 10.1021/ja203034k ). And Michael J. Krische and coworkers at the University of Texas, Austin, set a new record for mak-ing the molecules when they prepared bryostatin 7 in just 36 steps ( J. Am. Chem. Soc., DOI: 10.1021/ja205673e ).

The recent syn-theses are also highly convergent, with the longest linear sequence clock-ing in at 31 steps for Keck’s synthesis, 28 for Trost’s, 25 for Wender’s, and just 20 for Krische’s. And each shows off a different use of chemis-try. Trost takes advantage of a palladium-catalyzed union of two alkynes to create the bryostatins’ macrocyclic structure. Keck makes use of a pyran annulation method to unite the A-ring and C-ring subunits with simultaneous formation of the B-ring. Wender uses a similar mac-rocyclization strategy, employing the Prins reaction to wed an aldehyde with a hydroxyallylsilane. Krische uses Keck’s as-sembly strategy but decreases the number of steps to make each fragment by employ-ing hydrogenative carbon-carbon bond formation. Each strategy gives chemists flexibility to make a range of analogs.

“In natural product synthesis, I feel that it’s really important to select targets that represent authentic unmet challenges in terms of the chemistry and biology,” Krische says. “With sufficient resources, it’s pretty clear that one can complete the total synthesis of nearly any natural product. So I think now it’s in-cumbent upon synthetic organic chemists not only to make the target but to focus on how the target is made,” with an eye toward flexibility, he says. “It’s important to select natural products where the syn-thesis of the target is not an end point but a beginning.”

To that end, Krische says, his group is now aiming to use the synthetic methods they developed to make analogs of bryo-statin in as few as 12 steps. He’s in good company in the bryostatin analog game. Wender has been making simplified ver-sions of the bryostatins for 25 years, and

Keck has been creating bryostatin analogs for the better part of the past decade.

“We need to un-derstand collectively as a community that natural products are not made in nature to do what we ask of them. Bryostatin is not made in B. neritina for the purposes of addressing HIV or

cancer or Alzheimer’s,” Wender says. “The natural product traditionally has been often perceived as being the drug, when in fact an emerging emphasis is that it’s a tre-

mendous lead. And if we could learn what nature is teaching us in this lead, we could then, using modern science, translate that into molecules that would be more effec-tive than what nature has produced.”

More than 100 bryostatin analogs—dubbed bryologs—have come out of Wender’s research group over the years. “We’ve been trying to understand the lesson of bryostatin and then to use what we have learned to come up with agents that are superior to the natural product,” he says.

For example, they have learned that an alkoxy group at a cer-tain position in the bryostatin backbone is critical. They’ve determined the structure of the C-ring and its surrounding func-tional groups are also important, as that’s the portion of the molecule thought to bind to PKC. Finally, they’ve figured out how

to simplify bryostatin’s A- and B-rings , so the analogs maintain the same shape as the bryostatins but are easier to make.

WENDER POINTS to the analog from his lab known as “picolog” as one of the most promising. It can be made in fewer than 30 steps. It’s 100 times more potent than bryostatin 1 in some in vitro anticancer tests, and it’s shown promise in treating mice with leukemia.

While Wender sees potential new therapies from bryostatin analogs, Keck is more restrained. “Any talk of drugs based on bryostatin at this juncture is really premature because we don’t know yet what kinds of structures you need to elicit a particular kind of response,” he says. “What we’re doing is making a tool-box of compounds that vary in structure, and then going in and finding out in great and gory detail what those compounds do biologically. The goal is to link specific structural features with specific biological responses.”

The analogs made in Keck’s lab are known as Merle compounds, named after country music legend Merle Haggard, of whom Keck is a friend and “probably the world’s greatest fan.” Keck says his collabo-rator, NCI’s Blumberg, told him he needed succinct identifiers for his analogs that wouldn’t change from publication to publi-cation. “I said, ‘I know just the thing. We’ll give them Merle numbers because nothing lifts my spirit like a Merle number,’” Keck recalls.

Keck believes the substitution around bryostatin 1’s A-ring is critical. His group, in collaboration with NCI’s Blumberg, compared analogs that were simplified around either both the A- and B-rings (Merle 23) or just the B-ring (Merle 28). Those that were simplified around the A-ring did not behave like bryostatin but instead behaved like the tumor-promoting phorbol esters. “This was a big surprise because these things look very much like bryostatin. They look nothing like phorbol esters, and yet to the cell, well, I guess the cell does not have ChemDraw,” Keck says .

“There’s a great opportunity to make im-

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“The goal is to link specific structural features with specific biological responses.”


portant findings in biol-ogy just from looking at analogs that people are making,” Keck adds. “If nothing else, there’s a great deal to be learned about the fundamental biology that’s relevant to cancer, Alzhei mer’s disease, and other dis-eases through this kind of research.”

So will one of the bryostatins or their analogs ever become a drug? It’s tough to say. “In my view there are two key things in advancing a natural product into drug development,” says Guy T. Carter, a con-sultant with natural products discovery consulting firm Carter-Bernan Consulting . “One is making enough material to start with, and the other is the ability to make a broad range of analogs in sufficient quanti-ties in order to address whatever issues you

encounter as you go through the development process,” such as problems with solubility, perme-ability, or metabolic stability.

Having a synthesis that lends itself to modifications makes the bryostatins attractive, but “it’s still going to be a hard sell,” Cart-er says. “I think the dogma in big pharma has always been that we

don’t do total synthesis. It’s just not practi-cal.” Still, he notes that some companies are challenging that dogma. He points to Eisai ’s drug Halaven, which is an analog of a natu-ral product made via a 62-step synthesis.

For the bryostatins ever to make it to patients will require a tremendous devotion of resources and a strong willingness on the part of those in charge to stick with such a project, Carter notes. “That bit of wisdom that’s required to see it through to the end product is something that is in short sup-

ply,” he says. “It’s much easier to say ‘no’ to something like that than to say ‘this is something special and therefore we need to devote the resources to make it happen.’ Pursuit of challenging targets like the bryo-statins, while risky, has great potential for creating major breakthroughs in medicine and eventually profits for the company.”

John A. Lowe, a medicinal chemistry ex-pert with the consulting firm JL3Pharma , doesn’t see pharma executives running out to make bryostatin analogs just yet, but notes: “It certainly is intriguing how much more approachable the bryostatins or their analogs are when you start talking about potential commercialization. It’s now competitive with the other things that are going on, and I don’t believe anybody be-lieved that would be the case 20 years ago when the structures were elucidated. That in itself is pretty impressive.” ◾

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DRUG DEVELOPMENT

Taking The Long Route

Drugmakers have been known to shy away from molecules that must be made via lengthy multistep syntheses. But taking the long route, chemically speaking, to make a medicine is not unheard of. Late last year the phar-maceutical company Eisai introduced Halaven (eribulin mesylate), a compound that requires 62 chemical trans-formations to make.

Halaven is used to treat patients with late-stage metastatic breast cancer. It is an analog of halichondrin B, a natural product first isolated from the sea sponge Halichondria okadai in 1986 by researchers in Japan ( Pure Appl. Chem., DOI: 10.1351/pac198658050701 ). The molecule is a beast, with a 54-carbon backbone and 32 stereogenic centers. A team led by Harvard University’s Yoshito Kishi completed the first total synthesis of ha-lichondrin B in 1992 ( J. Am. Chem. Soc., DOI: 10.1021/

ja00034a086 ). Kishi’s team—working with collaborators at Eisai , where Kishi was a scien-tific adviser—began making analogs of the compound soon after.

An intermediate in the Harvard synthesis contain-ing only the macrolide sector was screened at Eisai and found to retain bioactivity. Eisai chemists then created a range of analogs based on the structure, settling on Halaven as the most promising by the late 1990s. Although simpler than its parent structure, Halaven is “a very challenging target by anybody’s assessment,” ac-cording to Frank Fang, vice president of U.S. process research and development at Eisai. The structure features a complex ring system and 19 stereocenters.

Fang says that Hala-ven’s potency and unique biological profile made it too attractive a target to pass up simply because it could be

made only through a lengthy synthesis. “The perception wasn’t so much that this was an obstacle but rather it was a challenge that we knew how to deal with,” he says.

And even though it takes

a total of 62 steps to make Halaven, Fang points out that the synthesis is fairly convergent; the longest lin-ear sequence is 30 steps. “The number of steps of a synthesis is one feature that people tend to focus on be-cause it’s easy to remember,” he says. “But what really is critical to the successful implementation of a process in commercial manufacturing

is not so much the number of steps but the types of pu-rifications that are employed during the processing of the material.”

Chromatography, for example, takes a lot more

time and generates a lot more waste than crystallization, Fang notes. “If you can take a 60-step synthesis and get rid of most of the chro-matographies and replace them with crystallizations, then it’s a much more manageable process than even a 10- or 15-step synthesis that has entirely

chromatographic purifica-tions,” he says.

Step count, Fang says, is nothing to be scared of. “Our feeling at Eisai is that natural products represent a large space of untapped potential new medicines,” he says. “We’re not deterred by a chemical obstacle. If the biological activity warrants it, we’re more than happy to go after a compound.”

Halaven (eribulin mesylate,counterion not shown)

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Reprinted from C&EN, Oct. 24, 2011



TO UNDERSTAND how proteins work in living organ-isms, more and more researchers are probing the complex interactions of huge ensembles of proteins as they act together in cells. Many studies involve all the proteins found in cells, tissues, or organisms, collections known as proteomes. The systematic study of those proteins—their structure, function, local-ization, and modifications, as well as how they change in response to different stimu-li—is called proteomics.

An essential subset of the proteome is the metalloproteome, con-sisting of all the proteins that contain or interact with metals when they do their jobs. Estimates indicate that 9% of eukaryotic proteins bind zinc, 33% of all proteins bind various metals, and 40% of all enzyme-catalyzed reactions involve metals, including Mg, Zn, Fe, Mn, Ca, Co, Cu, Ni, Mo, W, Na, K, and V.

Because the metalloproteome is so im-portant, scientists have devised innovative techniques to better understand its com-plexities, including not only conventional ex-perimental strategies but also bioinformatics techniques. Using those methods, they have begun to systematically catalog the roles of various metals in protein function.

Bioinformatics is necessary because traditional experimental techniques to purify and investigate individual enzymes are slow and difficult, says Ivano Bertini , a chemistry professor at the University of Florence , in Italy. And just as biologists generally now take advantage of gene and protein databases for insights into how biological systems work, he adds, “we should have something to target metal ions in biology.”

One of the challenges in using infor-mation technology to study metallopro-teomes, however, is that metal-binding sites in proteins are three-dimensional and don’t always follow a set linear amino

acid sequence. Instead, ligands may come from different loops of a protein, different subunits, or even two different proteins, making it difficult to use simple sequence searches to look for protein homology.

IN SOME CASES the problem can be ad-dressed by looking for what Bertini calls metal-binding patterns. By mining the Protein Data Bank for structures of known metalloproteins, Bertini, University of Florence chemistry researcher Claudia Andreini, and colleagues determine ar-rangements of metal ligands along peptide chains and how the ligands bind to metal-binding domains. For a metal bound to three histidine (H) ligands, for example, a metal-binding domain might look some-thing like HXHX 60 H, where X is amino acids other than histidine.

Bertini, Andreini, and coworkers used that approach to look for zinc-binding proteins in the proteomes of bacteria, ar-

chaea, and eukaryotes. From the Protein Data Bank, they found 744 zinc-binding patterns, which they then used to search the proteomes. They found that, on aver-age, 4.9% of bacterial, 6.0% of archaeal, and 8.8% of eukaryotic proteomes are com-posed of zinc proteins ( J. Proteome Res., DOI: 10.1021/pr0603699 ).

Examining the function of those pro-teins, the researchers found that most pro-karyotic zinc proteins perform some kind of enzymatic catalysis, and two-thirds of them have eukaryotic homologs. Eukaryotic proteomes add to those catalytic enzymes a cohort of zinc proteins that regulate DNA

transcription—principally zinc-finger proteins, which likely

evolved to meet the complex needs of cell compartmental-

ization and differentiation in eukaryotes. More recently, Bertini and

Andreini have expanded their database-mining approach to look

at 3-D representations of metal-binding sites, focusing specifi-cally on a “minimal functional

site” that includes not just a metal and its ligands but everything within a 5-Å radius of the metal ligands. The reactions catalyzed by metalloenzymes are not gov-erned by metal and ligand identity alone, but also by the geometry and environment of the active site, Bertini notes. The additional structure information is therefore critical for predicting function. Two proteins with different overall structures but the same metal functional sites likely do similar chemistry, possibly through convergent evolution of unrelated proteins. In contrast, different metal functional sites found in oth-erwise similar proteins may have divergently evolved to do different things.

The researchers used minimal func-tional site analysis to examine nonheme iron proteins, which are proteins that bind iron without using a porphyrin ring. The researchers found that they could group the sites into five structural clusters ( J. Mol. Biol., DOI: 10.1016/j.jmb.2009.02.052 ). A similar effort focusing on zinc sites in pro-teins yielded 10 clusters of highly similar structures and seven “pseudoclusters” with broadly similar features ( PLoS One, DOI: 10.1371/journal.pone.0026325). Comparing and contrasting the active site structures within a cluster can yield clues about how the proteins evolved or how they catalyze their respective reactions, Bertini says. The structural clusters can also help predict the

MERGING METALS INTO PROTEOMICS

Tackling the systemic study of METALLOPROTEINS JYLLIAN KEMSLEY , C&EN WEST COAST NEWS BUREAU

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ESSENTIAL ELEMENTS The zinc-finger protein Zif268 binds to DNA to regulate transcription.


function of uncharacterized proteins. Bertini and Andreini are also working

with Janet M. Thornton of the European Bioinformatics Institute , located in Eng-land, to develop a database of the properties and roles of metals in metalloenzyme ca-talysis. The database is called Metal MACiE ; MACiE stands for Mechanism, Annotation & Classification in Enzymes. Metal MACiE pulls together information on the 3-D struc-ture of metalloenzyme active sites as well as a step-by-step description of the reactions they catalyze ( Bioinformatics, DOI: 10.1093/bioinformatics/btp256 ). Although the da-tabase has only 188 entries so far, it will be a useful resource for additional systemic studies of metalloenzymes, Bertini says.

BUT FOR ALL THAT can be learned from mining databases and comparing structural and functional information, the databases are only as good as the information they house, which must originate in experiment. Michael W. W. Adams , a biochemistry pro-fessor at the University of Georgia , divides genomes into thirds: We know for certain what proteins are encoded and what the proteins do for about one-third of any particular genome, we have a good idea about another third, and “we have no idea whatsoever” about the remaining third, he says. Of the unknown third, “potentially a lot of them are metal-containing proteins,” Adams says.

Historically, determining the metal con-tent of a protein has often been an experi-mental afterthought. Scientists purified a protein and then perhaps discovered that it needed a metal to be structurally sound or perform a catalytic function. Some researchers, Adams included, are now reversing that approach by looking for metal content first, by separating proteins on ion-exchange columns or gels and then assaying for metal content. In all cases, researchers note, it is important to purify and characterize proteins in their native forms because proteins that are unfolded or denatured will lose their metal cofactors.

In one set of studies, Ad-ams and colleagues used a combination of inductively coupled plasma mass spec-trometry (ICP-MS) to look for metals and high-through-

put tandem electrospray ionization mass spectrometry (ESI-MS) to identify proteins in Pyrococcus furiosus, a species of archaea that optimally grows at 100 °C ( Nature, DOI: 10.1038/nature09265 ). After separating P. furiosus biomass through 2-D liquid chroma-tography—with fractions from one column further separated on a second—Adams and coworkers turned up 343 metal peaks for proteins that variously bound Zn, Fe, Mn, Co, Ni, Mo, W, V, U, and Pb.

Comparing the sequences of the pro-teins underlying those peaks, they found that 158 of the peaks contained proteins without a previously identified metal-bind-ing domain. Following up on some of those unknowns, Adams and coworkers identi-fied some novel metalloproteins, including one with a new type of Mo site. They also found that the U and Pb peaks most likely came from the organism misincorporat-ing those elements into proteins. Further study of proteins vulnerable to metal mis-incorporation could reveal mechanisms of metal toxicity, Adams notes.

Nigel J. Robinson , a biology professor at Durham University , in England, used a similar approach to determine which proteins predominantly bind Mn 2+ or Cu 2+ in the periplasm, the space between the cell wall and inner membrane, of the cyanobacterium Synechocystis PCC 6803. Although the microbe requires both metals for photosynthesis, Mn 2+ binds weakly and Cu 2+ binds strongly to proteins, raising the question of how Mn 2+ manages to compete successfully for binding sites.

Analyzing periplasm proteins, Robin-son and colleagues turned up something completely unexpected: two proteins with similar structure and metal-binding ligands,

one of which contained Mn 2+ and the other Cu 2+ , although both preferred Cu 2+ ( Nature, DOI: 10.1038/nature07340 ). Further inves-tigation revealed that the proteins fold and incorporate their metals in different parts of the organism. The Mn-binding protein folds in the cytosol, where researchers believe all copper atoms are already tightly bound to proteins, then gets exported to the peri-plasm. The Cu-binding protein, in contrast, gets sent to the periplasm unfolded and picks up Cu 2+ there. “This highlights the fact that which metals bind to which proteins in vivo is determined by the cell controlling the availability of metals,” Robinson says.

METALLOPROTEINS are not limited to organisms. Viruses, too, may include metals in their proteins. University of Cincinnati chemistry professor Joseph A. Caruso and colleagues have used chromatography and MS techniques to look at bacteriophage λ, a virus that infects and replicates in bacte-ria. Although the genome and proteome of the virus have been well studied, its metal complement has not. The virus encases its genetic material in a protein cage and then injects it into a bacterial cell to replicate. The protein cage contains a number of cysteine and methionine residues, which are common metal ligands. Caruso and colleagues found that bacteriophage λ in-corporates Zn, Fe, Mn, Co, Ni, and Cu, most likely in two proteins in the virus’s cage ( Metallomics, DOI: 10.1039/c0mt00104j ). Scientists are interested in engineering bacteriophage λ to produce nanoparticles or deliver drugs, and understanding the role that metals play in the virus may further those efforts, Caruso says.

In other application-oriented work, Caruso and colleagues have done metalloproteomic screens for biomarkers to predict disease. In one case, they looked for a particular form of the iron-transport protein transferrin as a signal for leaking cerebrospinal flu-id after head injury ( Analyst, DOI: 10.1039/c0an00207k ). Another study involved identifying possible markers for narrowing of brain arter-ies after hemorrhagic stroke ( Metallomics, DOI: 10.1039/c0mt00005a).

Chromatography com-bined with MS techniques to study metalloproteins

NOTE: Data are based on an analysis of all ligands bound to iron in 86 nonheme iron sites.SOURCE: J. Mol. Biol.

STRUCTURE-FUNCTION LINK The most common ligands in nonheme iron sites vary depending on protein function.

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Sensors

Structural centers

Nonredox catalysis

Redox catalysis

Electron transfer

◼ Cysteine◼ Histidine◼ Aspartic acid/glutamic acid◼ Tyrosine◼ Asparagine◼ Other N donors◼ Other O donors



could be improved, says David W. Koppenaal , chief technology officer of the William R. Wiley Environmental Molecular Sci-ences Laboratory at Pacific Northwest National Labora-tory . Right now, combining techniques such as ICP-MS to get metal content and ESI-MS to get protein sequences means doing them separately and cor-relating the data. Koppenaal’s lab and others are working on technology to use one or more chromatography columns to separate samples, then split the eluent into two streams: one for elemental analysis through ICP-MS and the other for molecular mass analysis through ESI-MS or other techniques.

A simultaneous, integrated approach enables unequivocal and global determinations of metal-protein associations, allowing the researchers to collect “richer and more precise informa-tion,” Koppenaal says. One of the projects that Koppenaal and colleagues are work-ing on is investigating the photosynthetic machinery of cyanobacteria. “We’ve done a lot of proteomics work on the system, and now we want to complement that work with better characterization of the protein-associated metals, metabolite-associated metals, and free metals,” Koppenaal says. “Understanding the dynamic among those metal pools is an important part of the whole picture.”

A slightly different approach to metal-loproteomics is to separate proteins by 2-D gel electrophoresis and then analyze the gel bands for metals. Norbert Jakubowski , a sci-entist at BAM Federal Institute for Materials Research & Testing , in Germany, has used gel electrophoresis plus laser ablation ICP-MS to analyze Se enrichment in proteins and to profile cytochrome P450 expression in rat livers ( J. Anal. At. Spectrom., DOI: 10.1039/c003889j and 10.1039/c0ja00077a ), as well as to look for Cd in spinach.

Plants can take up Cd and other toxic metals from soil, potentially reducing plant growth and contaminating the food supply. Researchers suspected that Cd was displac-ing other metals in enzymes, rendering them nonfunctional, but they didn’t know which proteins Cd affected. Jakubowski and colleagues analyzed protein extracts of spinach leaves and found that Cd mainly substitutes for Mg in the active site of ribu-

lose-1,5-bisphosphate carboxylase oxygen-ase, commonly known as RuBisCO, a criti-cal enzyme in carbon fixation and the most abundant protein in leaves ( Metallomics, DOI: 10.1039/c1mt00051a ).

OTHER WAYS to detect metalloproteins in gels include X-ray fluorescence mapping, which allows simultaneous imaging of mul-tiple metals to get identity and quantity. Adding X-ray absorption near-edge struc-ture analysis can also reveal metal oxida-tion states. Argonne National Laboratory scientist Lydia Finney and coworkers used the X-ray techniques to study the effects of Cr 3+ and Cr 6+ spiked into blood serum, demonstrating that Cr 3+ compounds pro-moted as nutritional supplements bind to serum proteins and form some Cr 6+ spe-cies, contrary to marketing claims ( ACS Chem. Biol., DOI: 10.1021/cb1000263 ).

Finney is now collaborating with Worces-ter Polytechnic Institute biochemistry pro-fessor José M. Argüello to investigate metal-trafficking pathways in bacteria by mutating known copper transport proteins. The ability to simultaneously image multiple metals is critically important to the study, she says. “We’re specifically interested in what happens with copper, but we also see big changes in other metals,” Finney says. “Their homeostasis is very intertwined.”

In yet another approach to metal de-tection, researchers at Delft University of Technology , in the Netherlands, take advantage of an on-site nuclear reactor to enrich protein samples with rare nuclear

isotopes, such as 69m Zn, 59 Fe, 64 Cu, 99 Mo, and 187 W. Biotech-nology professor Peter-Leon Hagedoorn and colleagues have dosed Escherichia coli cultures with the isotopes and tracked where the metals go: More than 99% of Cu is locat-ed in the protein CueO, a mul-ticopper oxidase involved in Cu homeostasis ( J. Biol. Inorg. Chem., DOI: 10.1007/s00775-009-0477-9 ), and 90% of Fe resides in superoxide dismutase, ferritin, and bacte-rioferritin ( Metallomics, DOI: 10.1039/c1mt00154j ). Under Zn stress, E. coli shuttles Zn to ZraP, which Hagedoorn pro-poses is a novel prokaryotic Zn storage protein that scav-enges Zn in the periplasm. His group has also looked at how

P. furiosus distinguishes between chemically similar Mo and W in its metalloproteome ( J. Bacteriol., DOI: 10.1128/JB.00270-10 ).

One additional method to study me-talloproteomes originated with the U.S. Protein Structure Initiative (PSI) to de-termine the 3-D structures of all proteins. The initiative organizes sequences into families and then solves the structures of selected representatives of each family. At Brookhaven National Laboratory , Case Western Reserve University proteomics professor Wuxian Shi and colleagues devel-oped a high-throughput X-ray absorption spectroscopy (HTXAS) technique to iden-tify and quantify metals in nearly 4,000 proteins selected for PSI analysis.

The combination of crystal structure, HTXAS information, sequence homology comparisons, and bioinformatics mining of protein sequences for likely metal ligands helped clarify the metal-binding sites in the proteins ( Genome Res., DOI: 10.1101/gr.115097.110 ). Although the project previ-ously screened all proteins by HTXAS, in the next phase the scientists will analyze only those proteins that have been crystallized.

For all the work that has gone into study-ing metalloproteins, there is still much to learn, Adams says, pointing again to the one-third of the genome about which “we don’t have much of a clue.” It took roughly 100 years to get the knowledge that we have now, he notes. How long it will take to close the gap, even with new, high-through-put proteomics, remains to be seen. ◾

ALIGHT X-ray fl uorescence reveals metals in proteins separated on a gel. MW = protein standard, CA = carbonic anhydrase, Tyr = tyrosinase, Hb = hemoglobin, Mix = mix of CA, Tyr, and Hb.

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C&EN TALKS WITH


ON JAN. 1, 2012, Michael A. Marletta will take the reins as president of Scripps Research Institute , a private, nonprofit re-search organization focused on biomedical research. Excited to lead the organization, Marletta nonetheless will be challenged to maintain funding for the institute as its tra-ditional sources of money tighten their belts.

Marletta, 60, moves to Scripps after a 30-year academic career that saw him serve as a professor at several universities, most recently in the chemistry department at the University of California, Berkeley.

At Scripps, Marletta succeeds Richard A. Lerner , who served as president for more than two decades. Lerner tried to recruit Marletta to join Scripps’s faculty several times over many years, “but I truly liked being at a big, complicated university,” Marletta says. “I enjoyed the full spectrum of things I was doing, including teaching undergraduates.”

And when he chaired the Berkeley chemistry department, Marletta found that he also enjoyed leading people beyond his lab group. “When I think I know what every day is going to look like, when the challenges in front of me are only ones that I create, that is when I get a little bored,” he says.

After stepping down as department chair in 2010, Marletta started exploring job options in administration, but he had some requirements. One was that he could continue to maintain his lab group and research into the catalytic and biological properties of redox enzymes. “That’s an exciting piece that I can’t imagine doing without,” Marletta says.

The other involved family. His son is in 11th grade, and Marletta didn’t want to move him during his final years of high school. Any organization hiring Marletta had to agree to allow him to commute between work and Berkeley.

Scripps accepted those conditions; Lerner, in fact, has kept a successful research program going while serving as president, and the president’s office connects to adjacent lab space.

Marletta started at Scripps part-time as president-elect on July 1, spending a couple of days a week at Scripps headquarters in La Jolla, Calif., or its second campus in Jupiter, Fla. Come January, his re-search group will move to La Jolla. Marletta plans to spend weekdays at one of Scripps’s locations and weekends in Berkeley.

As he has gotten to know the Scripps organization and people over the past few months, Marletta says, what has surprised him is how enthusiastic the faculty and staff all seem to be about his arrival. Given Lerner’s long tenure, there has been some uncertainty about what the leader-ship change may bring, “but it hasn’t taken on any tone of negativity that I’ve seen,” Marletta says.

Marletta’s leadership style is one of inclusion without bureaucracy. He appreci-ates Scripps’s flat organization and likes to treat meetings “kind of like a group meet-ing,” he says, with people presenting their latest results or problems and getting input from the crowd on the next steps to take. “I don’t have the market cornered on good ideas,” Marletta says, but “there’s still one person that’s going to make decisions,” he adds.

Looking ahead, Scripps must keep its emphasis on fundamental research to un-derstand biology, with a focus on human health and disease, Marletta says. His chal-lenge will be to ensure the institute has the funding to retain current faculty members and recruit new ones, as well as to provide the instruments and other infrastructure that enable Scripps scientists to do top-notch research.

Scripps has historically had two main streams of funding: federal grant money, principally from the Na-tional Institutes of Health, and agreements with pharmaceutical companies that gave Scripps unrestricted funds in exchange for first rights to capitalize on Scripps’s discoveries. Scripps’s budget for 2012 includes about $317 million from grants, $30 million from pharmaceu-tical companies, and $44 million from other sources, Marletta says.

JUST LIKE other federally funded scientists, however, Scripps researchers are affected by government budget cuts, Marletta says. And pharmaceutical companies now want agreements that are more targeted to specific research, he says.

Going forward, Marletta has his eyes on other sources of fund-ing. “Private philanthropy is clearly one of the most important and immediate solutions to our financial stability,” he says, and he has already been meeting with potential donors.

He also aims to focus on improving the return on Scripps’s intel-lectual property. As agreements with pharmaceutical companies expire and Scripps regains control over when, how, and with what return its discoveries are translated into medical therapies, licens-ing income “will be an important part of our financial picture,” Mar-letta says. He acknowledges that the effort will require institutional investments in technology transfer and translational infrastructure.

Marletta says that he’s more excited now about the opportunity to lead Scripps than he was when he learned that he would get the job. “It’s not like every-thing is wonderful. There are challenges, but that’s why I took the job,” he says. “But the people that I’ve met and how they feel about the place and what they’re do-ing, that makes my excitement level higher.” ◾

JYLLIAN KEMSLEY , C&EN WEST COAST NEWS BUREAU

Incoming SCRIPPS PRESIDENT discusses move to administration, challenges for research institute

MICHAEL MARLETTA

Research is “an exciting piece that I can’t imagine doing without.”

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