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Inhibiting by Design |
As might be expected, many proteases have natural inhibitors; a cell could not endure for long if these powerful enzymes were not regulated. Although the first natural protease inhibitors were identified by Northrop and Kunitz as part of their protease studies in the 1930s, the era of designing protease inhibitors in the laboratory did not get under way until a decade or so later, and even then progress came piecemeal. In 1949 Arnold Kent Balls, of the Western Regional Research Laboratory in Albany, California, discovered that a synthetic substance--a nerve gas in fact--could inactivate acetylcholine esterase, an enzyme involved in the processing of the neurochemical acetylcholine. This inhibitor worked by reacting with the amino acid serine in the enzyme's active site. It turned out that the proteases trypsin, chymotrypsin, and elastase also were inactivated by the nerve gas, suggesting that they too possessed the amino acid serine in their active sites. Scientists concluded that all these proteins formed a family related by mechanism, a concept of relatedness that would, in another 20 years, prove useful time and time again.
The first test of this concept came in the 1970s, in the course of efforts by David W. Cushman and Miguel A. Ondetti, of the Squibb Institute for Medical Research in Princeton, New Jersey, to find a means to control high blood pressure. Cushman and Ondetti were investigating ways to inhibit the angiotensin-converting enzyme (ACE), one of two proteases required to make angiotensin, a hormone found in the kidney that raises blood pressure. No crystallographic image existed of ACE, but scientists understood something of how it worked. For one thing, they knew of at least one inhibitor--a snake venom protein--that worked on ACE. For another, they knew that ACE required charged metal atoms, or ions, to function, making it a member of a family of proteases called metalloproteases because they contain metal ions in their active sites. Looking around for similar proteins whose structures were already known, Cushman and Ondetti turned to carboxypeptidase A, another metalloprotease whose structure had been solved a few years earlier by W. Lipscomb and his colleagues at Harvard University. Cushman and Ondetti extrapolated from the shape of carboxypeptidase A a prediction as to which chemicals might alter the activity of ACE. The result of their effort was the development in 1979 of the ACE inhibitor Captopril, the first protease inhibitor developed through the process of "drug design," meaning a drug is fine-tuned by a kind of step-by-step modification and retesting. Captopril has been in medical use ever since.
More solutions by analogy followed, as scientists interested in finding other substances to lower blood pressure looked to solve the structure of renin, a protein that works in concert with ACE to make angiotensin. Renin was difficult to crystallize, but clues to its structure came, albeit in roundabout fashion, through investigators who were looking into the structure of the digestive enzyme pepsin. Both renin and pepsin are members of a family known as aspartic proteases for the amino acid aspartate in their active sites. Although scientists took the first x-ray crystallography images of pepsin in the 1930s, they had a difficult time unraveling the structure of pepsin. It was not until the 1970s that research on the structure of fungal acid proteases served as a guide for solving the structure of pig pepsin.
The pepsin structure in turn was a leg up for scientists searching for a renin inhibitor. During the 1980s researchers used an ingenious technique to develop inhibitors that effectively halted renin's enzymatic reaction. In essence, scientists focused on the instant in the reaction process when the enzyme binds most tightly to its substrate. They examined the structure of the substrate during this critical transition state and designed an inhibitor that would mimic not the final form of the substrate-enzyme reaction but the substrate's structure during this highly attractive transition. As effective as these renin inhibitors were in the laboratory, though, they were metabolized in the liver before they could reach the kidneys, where renin is made. The renin inhibitors thus were not useful as blood pressure drugs and were shelved--at least for the moment. |
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