From Viper's Venom to Drug Design - Federation of American

Developed by the Federation of American Societies for Experimental Biology (FASEB) to educate the general public about the benefits of fundamental biomedical research.

INSIDEthis issueFrom Viper’s Venom

to Drug Design:Treating Hypertension

Hazards of High BloodPressure

2Blood Pressure Basics

3The Search for a Diuretic

3Beta and Calcium Channel

Blockers

4The Kidney Connection

6The Hunt for Renin

7Snake Venom Leads to ACE

Inhibitors

9Enzyme Research Shows the

Way

10Designing an Enzyme

Inhibitor

11

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AcknowledgmentsFROM VIPER’S VENOM TO DRUG DESIGN:TREATING HYPERTENSION

Author, Margie Patlak

Scientific Advisor, Robert M. Carey, M.D., MACP, David

A. Harrison III Distinguished Professor of Medicine, Dean

Emeritus, University of Virginia School of Medicine

Scientific Reviewer, Edward D. Frohlich, M.D., Alton

Oschner Medical Foundation,

BREAKTHROUGHS IN BIOSCIENCE COMMITTEE

Fred R. Naider, Ph.D., Chair, College of Staten Island,

CUNY

David L. Brautigan, Ph.D., University of Virginia School of

Medicine

John Grossman, M.D., Ph.D., MPH, George Washington

University

Tony T. Hugli, Ph.D., Puracyp Research Institute for

Toxicology

Richard G. Lynch, M.D., University of Iowa College of

Medicine

S. Walter Englander, Ph.D., University of Pennsylvania

J. Leslie Redpath, Ph.D., University of California, Irvine

BREAKTHROUGHS IN BIOSCIENCE PRODUCTION STAFF

Director, Carrie D. Golash, Ph.D. Science Policy Analyst,

FASEB Office of Public Affairs

Breakthroughs in Bioscience C2

COVER IMAGE: Isolation of compounds

from the venom of the deadly Brazilian

pit viper (Bothrops jararaca) was critical

in development of certain drugs to treat

hypertension� Snake photo courtesy of Dr� Ivan Sazima� Universidade Estadual de Campinas; graphic design by Corporate Press�

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Breakthroughs in Bioscience 1Breakthroughs in Bioscience 1

HYPERTENSIONFrom Viper’s Venom to Drug Design:Treating HypertensionBy Margie Patlak

Figure �: When Franklin D� Roosevelt was president� there were no effective treatmentsfor the high blood pressure that made him excessively tired� and plagued him withsevere headaches� Some historians believe Roosevelt’s weakened condition gave Stalinthe advantage during the negotiations over post�war Europe—an advantage that putthe fate of Eastern Europe in Stalin’s hands� Photo from the Roosevelt PresidentialLibrary�

During the dozen yearsthat he was president,Franklin D. Roosevelt’s

blood pressure steadily climbed.By the start of World War II, hisconsistently high blood pressurebegan to take its toll. It made himexcessively tired, and plaguedhim with severe headaches. In hisradio addresses at the time of theYalta Conference, FDR was hav-ing such trouble breathing he wasunable to complete sentences

(Figure 1). Some historiansbelieve Roosevelt’s weakenedcondition gave Stalin the advan-tage during the negotiations overpost-war Europe—an advantagethat put the fate of EasternEurope in Stalin’s hands andchanged the course of the 20th

century.

There were no effective drugsfor high blood pressure during the1940s. Consequently, Roosevelt’s

cardiologist was unable to controlthe condition and it eventually ledFDR to his death at age 63 from astroke. Writing about his experi-ences treating the president’s highblood pressure, (which he terms“hypertension”), Howard Bruennlamented in the Annals ofInternal Medicine, “I have oftenwondered what turn the subse-quent course of history mighthave taken if the modern methodsfor the control of hypertensionhad been available.”

In the last 50 years, an impres-sive arsenal of medications thatbring blood pressure down to normal levels in more than 95 per-cent of all patients has been devel-oped. These drugs include dozensof medicines whose names arebased on the different ways theywork in the body. Diuretics, calci-um channel and beta blockers,angiotensin-converting enzyme(ACE) inhibitors and angiotensinII-receptor blockers (ARBs) haveextended the lives of millions ofAmericans and contributed to themore than 50 percent drop inheart attacks and strokes that hasoccurred in the last two decadeswithin the United States.

Surprisingly, the successfuldevelopment of such blood pres-sure medications hinged not juston the efforts of pharmacologists

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whose sole objective was to cre-ate them, but rather on a diversearray of scientists including bio-chemists, pathologists, physiolo-gists and chemists. Many of thesecurious investigators were notdirectly involved in hypertensionresearch, but instead were receiv-ing government funding to pur-sue answers to such basic ques-tions as how does a digestiveenzyme do its job?, what roledoes the kidney play in salt andwater excretion?, and why issnake venom so deadly? Thanksto their efforts, high blood pres-

sure has been transformed froman untreatable, often fatal diseaseto a manageable condition thatdoes not hamper the daily func-tioning of the two-thirds of olderadults who have it in this country.

Hazards of High BloodPressureA major step on the way to con-

trolling hypertension was the dis-covery, in the middle of the 20th

century, that elevated blood pres-sure is a serious health problemand not a benign consequence of

aging. By tracking the long-termhealth of thousands of individu-als, government-funded studies,including the well-knownFramingham Heart Study, uncov-ered that hypertension often fosters heart attacks, strokes,heart and kidney failure, and adeadly rupture of blood vessels(aneurysm). The risk of deathfrom these conditions rises consistently as blood pressureincreases into the abnormal range (Figure 2). These findingshighlighted the need for medi-cines that adequately treated


Figure �: The damage chronic hypertension wreaks on arteries can be seen in these photographs of the internal surface of the aortafrom a normal young adult� (left panel) and that from a patient with long�standing hypertension (right panel)� Unlike the smooth surface of the artery in the left panel� the one on the right is studded with plaque deposits comprised of cholesterol and lipoproteins(irregular shaped yellow and brown areas indicated by the arrows)� Hypertension injures the cells that line the inner surfaces of arter�ies such that blood cholesterol� lipoproteins and calcium are deposited in the vessel walls� Over years the vessels become narrow andrigid thereby impeding the flow of blood to vital organs� The end result is a greater likelihood of suffering a heart attack or stroke�Courtesy of Dr� Richard Lynch� University of Iowa College of Medicine�

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hypertension. But progress in thisregard could not be made untilscientists gained a better under-standing of how the body con-trols blood pressure.

Because blood pressure is sovital to maintaining normal func-tion (see box – “Blood PressureBasics”), the body has manyoverlapping systems for control-ling blood pressure that involvethe heart, kidneys, nervous sys-tem and other organs or tissues.When one means for changingblood pressure is ineffective,another backup system kicks in,thereby ensuring that the bodycontinues to function properly.Although it is reassuring thatthese backup systems exist, mak-ing sense of the many facets ofblood pressure control was diffi-cult, to say the least.

It took more than two centuriesfrom the time when blood pres-sure was first measured in 1733(in the horse) by Steven Hales towhen doctors began to use thefirst effective blood pressuremedicines after World War II.Discovery and development ofdrugs that effectively took thereins of blood pressure were notpossible until scientists painstak-ingly teased out the strings con-trolling blood pressure one byone. The end result is a fascinat-ing story of discovery that starsdeadly snakes in the tropical rain-forests of Brazil, a researcher try-ing to find a cure for menstrualcramps, and four major organs,including a hardworking pairknown as the kidneys.

The Search for a DiureticBasic research done in the early

20th century revealed that the kid-neys filter one quarter of all theblood that leaves the heart eachminute. Part of that filteringprocess determines how muchsalt is left in the blood. The moresalt, the more fluid the bodyretains to dilute it. Excessive

fluid retention to dilute salt raisesblood pressure and can eventuallycause congestive heart failure. Inpatients with this condition, theexcess fluid in the blood leaksout into the spaces between cells,causing swelling and fluid in thelungs that can disrupt breathing.During Roosevelt’s time, the onlytreatment for hypertension andcongestive heart failure was a

Blood Pressure Basics Not much bigger than a fist, the human heart beats 100,000 times each

day, sending about 2,000 gallons of blood coursing through vessels, which,laid end-to-end, would be long enough to circle the earth more than twice.Adequate blood pressure is key to ensuring that all parts of the body aresupplied with the vital fluid that sustains them. To be adequate, not onlydoes blood pressure have to be high enough to flow rapidly throughout the body, but it needs to change quickly to accommodate the body’s needs.For example, rising in the morning after sleep requires a rapid rise in bloodpressure so that a person’s boosted activity is supported by the increasedsupply of energy that the blood provides. A major loss of blood or waterfrom the body also requires a compensatory rise in blood pressure.

The body has three main ways to raise blood pressure:

• Increase how frequently the heart contracts or make each contraction stronger so a greater volume of blood is propelled through vessels;

• Constrict blood vessels so that blood flows more quickly through them; and

• Retain more salt and water in the blood to boost its volume.

High blood pressure (hypertension) is currently defined as being above140/90 mmHg (millimeters of mercury; a way to measure pressure). Stressoften causes blood pressure to rise above this level as the body gears up for the “fight or flight” response that was needed in prehistoric times, whenthe major stressors were carnivorous animals and not overbearing bosses!Blood pressure is thought to stay elevated due to chronic stress and/ormany other factors. These might include a high salt diet that causes waterretention, kidney disease and excessive weight gain. Most people withhypertension inherit a tendency to be particularly sensitive to environmentalfactors, such as dietary salt, that can raise blood pressure.

More than 50 million adults in this country have high blood pressure.Many do not know it because early stages of the condition have no symp-toms other than frequent headaches. Nine out of every 10 people willdevelop hypertension during their lifetimes, and more than half of all middle-aged and elderly adults need to take blood pressure medication.

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stringently low-salt diet to whichfew patients were able to adhere.

Building on this knowledge,physiologist Karl Beyer, Jr. andhis pharmacologist and chemistcolleagues at Merck Sharp andDohme Research Laboratoriesreasoned that rather than requirehigh blood pressure patients torestrict the salt in their diets, whynot give them a drug that makestheir kidneys expel more salt intotheir urine? A way of doing thissurfaced with the serendipitousobservations of clinicalresearchers. They noticed thatwhen they gave patients a newantibiotic called sulfanilamide,their urine became more acidicand had greater than normal concentrations of various saltcomponents. Intrigued by thisfinding, British physiologists T.Mann and David Keilin set out to discover why. They uncoveredthat such boosted salt excretionstemmed from the antibioticdisrupting the work of a particular enzyme called carbonic anhydrase.

This finding led Beyer and hiscolleagues to try creating a drugthat blocked carbonic anhydrasein the kidney. Fortunately, univer-sity-based researchers, interestedin exactly how certain enzymeswork, had already learned quite a bit about carbonic anhydrase,thanks to federal governmentsupport of their endeavors. Theseresearchers showed, in paperspublished in scientific journals,that the enzyme-inhibitingactions of sulfanilamide stemmed from a specific

chemical constituent. With thistarget in mind, the Merckresearchers made a number ofchemical changes to a sulfanil-amide drug they had in hand.Because of the complexity of the physical changes they neededto measure and the precisionrequired, they chose to test theirnew drug on dogs, rather than on rats or other smaller animals.Their first experiment on a dog showed, to the researchers’delight, that their compoundselectively boosted the amount of salt components expelled intothe animal’s urine.

This compound was the diureticknown as chlorothiazide (Diuril).In 1957, investigators demon-strated diuril dramatically low-ered blood pressure in patients,making it the first highly effec-tive oral drug for hypertension.Since then, over a dozen addi-tional diuretics have been devel-oped. These drugs have becomemainstay treatments for hyperten-sion, and slash the risk of suffer-ing a stroke, congestive heartfailure or heart attack by about athird. But diuretics boost the lev-els of sugar and artery-cloggingfats in the blood, which could belife-threatening in the long term.They also frequently cause impo-tence, a major deterrent to their useby men. So the quest for betterblood pressure-lowering drugs con-tinued after the debut of diuretics.

Beta and CalciumChannel Blockers The next two groups of drugs to

enter the market for the treatment

of hypertension were actuallydeveloped to treat chest pain, orangina. This pain comes from alack of oxygen reaching heart tis-sue, often because of partiallyblocked arteries. To stem the oxy-gen needs of the heart and thusthe angina, researchers strived tocreate a drug that could lower thefrequency of heart contractions(heart rate). That way, the heartwould not work as hard and needas much oxygen.

To accomplish this feat, drugdevelopers relied on a century’sworth of basic research showingthat the hormone epinephrineaffected how frequently the heartbeats. But this hormone controlsa number of vital functions in thebody so attempts to block itsactions in the heart could not beaccomplished without causingserious side effects. A break-through came in 1948, whenpharmacologist RaymondAhlquist (Figure 3) of theMedical College of Georgia, who was searching for a drug to relieve menstrual cramps, accidentally discovered that the actions of epinephrine inthe body varied depending on

the type of cell receptor the hormone used to affect tissues.The heart rate was largely con-trolled by the actions of epineph-rine on “beta” adrenergic recep-tors, Ahlquist discovered.

This discovery opened up thepossibility of selectively slowingheart rate without hamperingother vital functions that involved“alpha” receptors, and eventuallyled to a hunt for drugs that would

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Figure �: Pharmacologist Raymond Ahlquist was searching for a drug to relieve men�strual cramps when he discovered that the heart rate was largely controlled by theactions of epinephrine on "beta" adrenergic cell receptors� This discovery opened up thepossibility of selectively slowing heart rate without hampering other vital functions thatinvolved "alpha" receptors� and eventually led to the development of the first beta block�er drug in the ��s� Photo from the National Library of Medicine

block beta receptors. In 1964, the first beta blocker drug to be marketed, propranolol(Inderal) was shown to be effective at relieving angina.Clinical researchers also noticedthat propranolol lowered theblood pressure of many of theirpatients, and the drug was sub-sequently approved for the

treatment of hypertension in the

early 1970s.

The success of propranolol at

relieving angina triggered a quest

for similar drugs and led to the

development of the drug vera-

pamil. Later studies revealed that

verapamil was not actually a beta

blocker at all, but rather lowered

the heart rate by hindering theentry of calcium into heart cells.This observation rang bells in theresearch community, because acentury’s worth of bench workhad revealed that the flow of cal-cium into heart muscle cells andblood vessels prompts them tocontract. The discovery that vera-pamil hindered such calcium flowled to efforts to create similar cal-cium channel blockers, of whicheight are now used to treat angi-na. Studies in the 1970s revealedthat calcium channel blockersalso effectively lowered bloodpressure by relaxing blood vesselsand led to their use to treat thiscondition by the 1980s.

Clinical research reveals that calcium channel and beta blockersreduce the risk of heart attack andstroke by about one-third. But theireffectiveness is often marred bytheir frequent side effects becausetheir actions are not specificenough in the body. Beta blockersoften cause fatigue, dizziness,insomnia and impotence. Calciumchannel blockers frequently causeconstipation, headaches and aswelling of the ankles.

By the late 1980s, despite thearray of drugs available to treathypertension, this condition still could not be adequately overcome in many patients who had to discontinue their bloodpressure medicines because ofside effects. In patients withextremely high blood pressures,even a combination of diuretic,beta and calcium channel block-ers was not enough to lower theirblood pressure levels to normal.

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Help came once more from basicresearch on the kidney. Theseexplorations led to the discoveryof an amazingly powerful andhighly specific control system for blood pressure that went farbeyond the kidney’s effect on salt excretion.

The KidneyConnectionScientists began to suspect that

the kidney acts a major guardianof blood pressure in the mid 19th

century. Several doctors notedthat their patients with kidneydisease also had the enlargedhearts that are seen in peoplewho have experienced high bloodpressure for long periods of time.At that time, an easy and accu-rate way of determining bloodpressure wasn’t available. But in1872, an Indian doctor FrederickAkbar Mahomed, who was work-ing in London, was able to makecrude blood pressure measure-ments to show that his patientshad high blood pressure in addi-tion to kidney disease andenlarged hearts.

How the kidney might affectblood pressure remained a mystery until the Scandinavianphysiologist, Robert Tigerstedt(Figure 4), of the KarolinskaInstitute, ground up the kidneysof rabbits. He tested various com-ponents of this rabbit kidneymash to see if they triggered arise in blood pressure when theywere injected back into rabbits.After dozens of experiments, hefound a kidney extract that madethe animals’ blood pressure shoot

up. Tigerstedt also established that blood taken from the veinleading out of the kidney raisedthe blood pressure of rabbitswhose kidneys have beenremoved. From this evidence, he concluded, in 1897, that thekidney released into the blood-stream a substance that raisedblood pressure. He called thatelusive substance renin, in honorof its kidney (renal) origins.

Unfortunately, because fewother scientists were able toduplicate his results, Tigerstedt’sfindings were essentially shelvedfor years. One of the scientists tonext pursue how the kidneyaffected blood pressure wasHarry Goldblatt (Figure 5) of

Case Western Reserve Universityin Cleveland. As a pathologistwho frequently conducted autopsies, Goldblatt noticed thatthe kidney blood vessels in manypatients with hypertension wereoften abnormally narrowed. Such narrowing was rarely seen in patients without highblood pressure.

Intrigued, Goldblatt wanted to better understand this linkbetween high blood pressure andthe constriction of blood vesselsin the kidney. Did one cause theother?, he wondered. To seek ananswer to this question, in 1934Goldblatt conducted a simpleexperiment that some sayunleashed more research than

Figure : Experiments on rabbits done by the Scandinavian physiologist RobertTigerstedt revealed� in �� that the kidney releases into the bloodstream a substancethat raises blood pressure� Photo from the National Library of Medicine�

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any other, single biomedicalexperiment in recent history.Using an adjustable silver clampthat he had fashioned himself, he partially constricted the arteryto the kidney of a dog. He thenmonitored the dog’s blood pres-sure and was surprised to see that even though the artery to the other kidney was unimpeded, the dog’s blood pressure rose

in response to the clamping. (He didn’t clamp both kidneyarteries because he was afraidthat would have killed the dog.)For the first time, researchersnow had a hypertension animalmodel—a highly useful tool withwhich to further explore bloodpressure control. Large animals,such as dogs, have proven invalu-able to biomedical research

because their physiology oftenclosely mimics that of humans.

Goldblatt suspected that theconstriction of the kidney arterytriggered excess production ofrenin, which not only caused high blood pressure in the experimental dogs, but also might be responsible for thehypertension that commonlyafflicts patients without kidneydisease. Researchers couldn’tclarify the role of renin in causing high blood pressure,however, until they pinpointedthe biochemical nature of renin.

The Hunt for ReninGoldblatt’s animal model of

hypertension fostered an interna-tional hunt for this mythical substance called renin thatTigerstedt had hypothesized but no one had yet identified. But researchers kept hitting apuzzling impasse—the purer they made their renin-containingextracts, the less these extractsraised blood pressure. However,if blood was added to the purifiedextracts, they regained theirpotency and caused blood pressure to rapidly elevate.

By 1940, two research labsworking independently—one based in Buenos Aires,Argentina, led by EduardoBraun-Menendez, and one inIndianapolis, led by IrvinePage—discovered why these confusing findings reoccurred.These scientists uncovered thatrenin was an enzyme that forms a blood pressure-raising peptide

Figure : Pathologist Harry Goldblatt quickened the pace of hypertension research bydeveloping an animal model for high blood pressure in �� Photo from the NationalLibrary of Medicine�

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Figure �: The Renin–Angiotensin–Aldosterone System� Angiotensinogen (� amino acids)� the precursor of all angiotensin peptides� issynthesized by the liver� In the circulation� it is cleaved by renin� forming angiotensin I� In turn� angiotensin I (� amino acids) is cleavedby angiotensin�converting enzyme (ACE)� which is bound to the cells lining the blood vessel� to form angiotensin II (� amino acids)� Inthe adrenal glands� angiotensin II stimulates the production of aldosterone� Aldosterone raises blood pressure by prompting the kidneysto retain more salt� Used with permission from the New England Journal of Medicine (��; � :��)�

Liver

Kidney

AdrenalGland

(a chain of amino acids, thebuilding blocks of proteins)called angiotensin from a largepeptide (angiotensinogen) thatcirculates in blood. The purifiedrenin extracts didn’t raise bloodpressure because they lacked theprecursor, angiotensinogen, thatrenin makes into angiotensin.Later studies revealed that thisprecursor peptide is made by theliver, adding yet another organ tothe control of blood pressure.

Serendipity then played a role in uncovering an additional miss-ing piece to the puzzle of how the kidney affects blood pressure. In 1956, while trying to purifyangiotensin, biochemist LeonardSkeggs, Jr. of Case WesternReserve University and his colleagues discovered that notone, but two substances in theirextracts raised blood pressure.This led them to hypothesize thatthe fashioning of angiotensin is a

two-step process and renin canonly accomplish the first step.The second step is triggered by a different enzyme, which waschristened angiotensin-convertingenzyme (ACE), fully described in1955 by Ervine Erdos. Reninmakes angiotensin I, a compound which has no effect on bloodpressure until it makes contactwith ACE. This enzyme snips off two amino acids ofangiotensin I to convert it to

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Figure �: Basic research on the venom of this Brazilian pit viper (Bothrops jararaca) ledto the development of the first ACE inhibitor drugs to treat hypertension� These drugscause the fewest side effects of all blood pressure drugs� and have additional benefitsbeyond lowering blood pressure� such as stemming the risk of developing diabetes andkidney disease� Photo courtesy of Dr� Ivan Sazima� Universidade Estadual de Campinas�

Figure �: British pharmacologist Sir JohnVane’s studies of the venom of aBrazilian pit viper led to the develop�ment of the first ACE inhibitor to selec�tively control high blood pressure� Photofrom the National Library of Medicine�

angiotensin II, which researcherslater found was a powerful blood-pressure raising peptidehormone in animals.

Despite all these exciting laboratory discoveries, manywere skeptical that renin playedany role in governing the bloodpressure of people in the clinic.Feeding that skepticism wereconflicting findings on whetherpeople with hypertension alsohad high renin levels. Progresswould not be made untilresearchers working in a totallyunrelated area—the study of hormones released by tiny organscalled adrenal glands—unveiledyet another renin-related trick the body uses to control bloodpressure.

During the first half of the 20th

century, fundamental research onthe adrenal glands (sometimes

called adrenals), which rest atopthe kidneys, revealed that theseorgans release a hormone calledaldosterone. This hormone raisesblood pressure by prompting the kidneys to retain more salt. In 1960, James Davis and otherresearchers at the National HeartInstitute (now the National Heart, Lung and Blood Institute), discovered that the kidneyssecrete a substance into the blood that triggers the adrenals torelease aldosterone. Investigatorssoon discovered that the aldos-terone-prompting substance wasangiotensin II, suggesting oncemore that the renin-angiotensin-aldosterone system played a key role in high blood pressure(Figure 6.) The proof of the pudding, though, came from an unusual and unexpectedsource—the Brazilian pit viper.

Snake Venom Leads toACE Inhibitors Workers in the banana planta-

tions of southwestern Brazil wereknown to collapse suddenly afterbeing bitten by a pit viper due toa drastic drop in blood pressure.Curious about what exactly thisvenom does to the body, bio-chemist Mauricio Rocha e Silvaof a Brazilian research institutemade extracts of the venom andbegan to study their effects indogs and guinea pigs. In the mid1960s, one of Silva’s postdoctoralstudents, Sergio Ferreira, traveled to London to work in the lab ofpharmacologist, Sir John Vane (Figure 8) of the Royal College ofSurgeons. In Ferreira’s pocket was avial full of the venom of the notori-ous Brazilian pit viper (Figure 7).

Although Vane eventually wonthe Nobel Prize for basic research

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explaining how aspirin works, atthe time Ferreira arrived, Vanewas heavily immersed in studiesof angiotensin I and II, and ACE.He had recently shown that much of ACE is generated by the blood vessels in the lungs,and was curious to see if thesnake venom’s blood pressure-lowering effect could be due to an effect on ACE.

Unable to interest Ferreira into studying this further, Vaneconvinced one of his colleagues,Mick Bakhle to pursue it. Theresearchers were thrilled to dis-cover, in 1970, that something in the snake venom was a potentinhibitor of ACE’s ability to generate angiotensin II. Vane was quick to realize that withsuch an inhibitor in hand,researchers could easily showexactly what effect angiotensin IIhas on blood pressure and put torest the uncertainty over whetherrenin and angiotensin were rele-vant to blood pressure control.

During this period, Vane became a consultant of the pharmaceutical company Squibbin New Jersey. He suggested toresearchers there that they studythe snake venom and use it toshow whether angiotensin II was important in regulating blood pressure. If it was, thenthey had a starting point for anew hypertension drug, Vaneargued. But it wasn’t easy con-vincing management at the company that researchers mightbe able to dish up a useful drugfrom an extract of poisonoussnake venom. Any drugs that

might evolve from these studieswould have to be injected, theSquibb marketing people argued,and only oral drugs are practicalto control high blood pressure.

But eventually an enzymologistand peptide chemist at Squibb—David Cushman and MiguelOndetti—were put to work iden-tifying, purifying and testing the blood-pressure lowering substance in the snake venom.After making a synthetic versionof the substance, they then passedit on to Vane. He injected it intohuman volunteers and showedthat it inhibited their conversionof angiotensin I to angiotensin II.Squibb also sent the substance to John Laragh, a hypertensionexpert at Cornell Medical Centerin New York. When he injected it into some of his patients withhigh blood pressure, he wasdelighted to find their blood pressure dropped significantly(but not as dramatically as in those bitten by the Brazilianpit viper!) At this point, few people doubted that renin andangiotensin II played an impor-tant role in regulating blood pressure, and that an ACEinhibitor would have some value in treating hypertension.

But there was one major problem—the ACE inhibitorCushman and Ondetti had created could not be taken in pill form because it was too big to be absorbed by the gut,and was likely to be torn apart by digestive enzymes. Theresearchers tried standard chemi-cal approaches to convert the

inhibitor into one that could betaken orally, but such tinkeringfailed abysmally and frustratedthe scientists, not to mention thehigher-ups in Squibb who pre-dicted this would be a problem.In the meantime, Cushman andOndetti screened thousands ofchemicals on Squibb’s laboratoryshelves to see if they too mightinhibit ACE, but came up empty-handed.

At this point, attempts to createan ACE inhibitor were abandonedat Squibb for a few years and theCushman-Ondetti research teamsplit up. But in their spare time,these men still continued theirdiscussions on how they couldconvert the venom-based ACEinhibitor into a compound thatcould be taken orally. A break-through in their thinking aroseafter publication of basic researchresults from biochemists at theUniversity of North Carolina,Chapel Hill and HarvardUniversity pursuing a totallyunrelated topic—a digestiveenzyme made in the pancreascalled carboxypeptidase A.

Enzyme ResearchShows the WayTo figure out how this digestive

enzyme worked, WilliamLipscomb of Harvard Universityused X-ray crystallography, animaging technique, to visualizethe three-dimensional structure of the enzyme. To gain furtherinsight into how carboxypepti-dase A worked in the body, Larry Byers and RichardWolfenden of the University of

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North Carolina used Lipscomb’sfindings to create a simple yethighly potent chemical inhibitorof the enzyme by mimicking thestructure of the portion of thecompound to which the enzymeusually attaches. They publishedthe structure of the inhibitor in aMay 1973 issue of the scientific

journal Biochemistry. Cushmanhad previously noticed howremarkably similar in structureACE was to the pancreaticenzyme. So the discovery of a small yet powerful inhibitor of carboxypeptidase A ignited awhole new approach to creatingan inhibitor to ACE.

Designing an EnzymeInhibitor

A key workhorse for raisingblood pressure is angiotensin-converting enzyme (ACE).Angiotensin I is a peptide—a chain of eight protein buildingblocks called amino acids—and acts as a substrate for thisenzyme. A portion of angiotensinI has just the right shape, andpositive or negative charges atjust the right spots, to slip intothe crevices of ACE. Like a handinto a glove, it can bind tightly to the enzyme. (See top draw-ing.) ACE then snips off two ofangiotensin I’s amino acids toform the peptide angiotensin II, a powerful blood-pressure raisingagent. Image design byCorporate Press.

To design a drug that couldblock ACE’s actions, researchershad to create an angiotensin I mimic—a molecule that alsowas the right shape, with positiveor negative charges in the rightplaces, to bind to ACE. But thismimic cannot be converted intoangiotensin II. Instead, by bind-ing to ACE, it blocks the enzymefrom making contact with its real target, angiotensin I. Thebottom drawing shows one of thefirst “ACE inhibitors,” the drugCaptopril, binding to ACE. It isremarkably similar to a portionof angiotensin I that binds toACE. This approach to makingdrugs has been called rational or structure-based drug design.Bottom image courtesy of Dr.Cherie Koch, Merck & Co., Inc.;adapted by Corporate Press.

+

+SUBSTRATEENZYME

ENZYME INHIBITOR

ACTIVE SITE OF ENZYME IS BLOCKED SUBSTRATE CAN NO LONGER FIT

SUBSTRATE FITS INTO ACTIVE SITE ON

Hand�Glove Analogy

S

CH2 CH C N C

O-

CH3

Structure�Based Drug Design

Angiotensin Converting Enzyme (ACE)

Angiotensin Converting Enzyme (ACE)PORTION OF ANGIOTENSIN I (SUBSTRATE) THAT INTERACTS WITH ACE

CAPTOPRIL

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Breakthroughs in Bioscience 12

In the spring of 1974, Cushmanand Ondetti began manipulatingthe pancreatic enzyme inhibitorso that it more closely resembledthe snake venom-ACE inhibitorprototype that they had created.After trying out nearly 60 differ-ent changes to the molecule, they finally struck pay dirt—acompound that was amazinglyeffective at inhibiting ACE, butunlike their previous syntheticinhibitor, this one was smallenough to be absorbed by the gut. They then modified thiscompound so that it wouldn’t be torn apart by enzymes in thestomach and bloodstream. Theend result, in the autumn of 1975,was the drug we now know ascaptopril—the first ACE inhibitorthat could be taken by mouth.(See Side Bar – “Designing anEnzyme Inhibitor.”)

Captopril was also one of thefirst drugs created with an inno-vative “structure-based design”approach. Prior to this techniqueof designing new drugs based ona specific desired chemical struc-ture, drug companies relied onluck to find new medicines. Theytypically screened thousands ofnatural and synthetic compoundsuntil they found one that had theeffect they desired. This processis extremely laborious and time-consuming, and often can befruitless, as was Squibb’s initialscreening search for an ACEinhibitor.

Structure-based drug design is much more efficient and hasbeen used successfully to makedrugs for AIDS, cancer, and other

disorders. This technique oftenrelies heavily on the findings of government-supported basicresearchers, who use X-ray crystallography and other tech-niques to pinpoint the portions of enzymes and other compoundsthat are responsible for theiractions in the body. Researchershave used structure-based designto make other versions of capto-pril, ten of which are now on themarket. Scientists also used thistechnique in the 1990s to makefive effective angiotensin II-receptor blockers (ARBs). Thesedrugs lower blood pressure bypreventing angiotensin II fromreaching its target (receptor) in the body.

ACE inhibitors and ARBs areremarkably effective at loweringblood pressure because they offera two-punch impact—not only do they prevent the blood pres-sure-raising constriction of bloodvessels, but they also lower blood pressure by restrictingwater retention in the blood.Recent studies have also foundthat ACE inhibitors and ARBshave additional benefits.Laboratory research has revealedthat angiotensin II appears to foster a number of processes thatunderlie cardiovascular disease,including the build-up of plaqueon blood vessel walls, and theexcessive growth of heart tissuethat often occurs in response to a heart attack, leading to conges-tive heart failure. ACE inhibitorsand ARBs appear to counterthose effects. The end result, clinical studies show, is not only

do these drugs decrease the riskof developing a heart attack bymore than 20 percent, but theyalso boost the heart attack sur-vival rate by nearly the sameamount, as well as significantlypreventing a dangerous buildupof fluid in the lungs and else-where caused by congestive heartfailure. ACE inhibitors have alsobeen shown to cut the risk ofstroke by about a third.

Surprisingly, ACE inhibitors and ARBs may alleviate condi-tions beyond cardiovascular disease. By lessening inflamma-tion and the buildup of fibroustissues, these drugs are thought to stall the progress of kidneydisease. One study found that an ACE inhibitor cut in half thenumber of patients with hyper-tension who progressed to serious kidney disease during athree-year period. Another ACEinhibitor reduced by one-third the number of patients over age 55 who developed diabetesduring a nearly 5-year period. It is not known how exactly the ACE inhibitors and ARBsprevent diabetes, although itcould be by hampering inflam-mation and increasing blood flow to the pancreas. Diabeticsare particularly prone to kidneydisease, but ACE inhibitors and ARBs significantly stem the decline of their kidney function, as well.

Although ACE inhibitors and ARBs are more expensivethan diuretics and other bloodpressure-lowering drugs, none of these other drugs have been

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shown to possess their added, far-flung benefits. In addition,because they are highly specificin the way they act in the body,ACE inhibitors and ARBs causethe fewest side effects of anyblood pressure medication. Themost common side effect of anACE inhibitor is a benign drycough, tolerated by most patients.ARBs rarely cause side effects.Without a doubt, these drugs,along with diuretics, beta andcalcium channel blockers, have

helped extend the lives of mil-lions, allowing parents to becomegrandparents, and presidents toserve out their terms. None ofthese drugs would have been possible if it weren’t for thecuriosity and diligence of a num-ber of government-supported sci-entists, from biochemists tophysicians, who were bent on fig-uring out the basic mechanismsof the body, including the exquis-itely intricate maneuvers it usesto control blood pressure.

Breakthroughs in Bioscience 13

Biographies Margie Patlak writes about biomedical research and health from thePhiladelphia region. She has written for Discover, Glamour, Physician’sWeekly, Consumer Reports on Health, The Washington Post, Los AngelesTimes, Dallas Morning News and numerous other publications. She alsowrites frequently for the National Institutes of Health and the NationalAcademy of Sciences. This is her fourth article for the Breakthroughs inBioscience series.

Robert M. Carey, M.D., MACP is the David A. Harrison, III Professor of Medical Science, Dean, Emeritus and University Professor at theUniversity of Virginia School of Medicine. His research interests include:the hormonal control of blood pressure and kidney function; the renin-angiotensin-aldosterone system; the dopominergic system; renal and vascular paracrine systems; and cell and molecular biology of vascular hormones. Dr. Carey’s research is directed to understanding the function of dopamine and angiotensin receptors in the kidney, heart and peripheralblood vessels. He was the 2001 recipient of the prestigious Irvine Page-Alva Bradley Lifetime Achievement Award given by the American HeartAssociation for outstanding achievements in the field of hypertension. Dr. Carey is a member of the Institute of Medicine within the NationalAcademies of Science.

Selected PublicationsBeyer, KH. 1977. Discovery of the Thiazides: Where Biology andChemistry Meet, Perspectives in Biology and Medicine. 20(3):410–420.

Dustan HP, Roccella EJ, and Garrison HH. 1996. Controlling Hypertension:A research success story. Archives of Internal Medicine. 156: 1926–1935.

Vane, JR. 1999. The History of Inhibitors of Angiotensin- ConvertingEnzyme, Journal of Physiology and Pharmacology. 50 (4), 489–498.

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