Synthetic Compounds Mimic Peptide β Pleated Sheet Motif

• Novel structures that simulate zigzag shape of bioactive peptides could lead to drugs that act as enzyme inhibitors

Stu Borman, C&EN Washington

Anew family of synthetic organic / % compounds that mimic the

JL J L ß-pleated sheet conformation of certain peptides and proteins have been developed as potential enzyme inhibitors. By inhibiting key biological processes catalyzed by proteolytic en­zymes, the novel compounds could po­tentially lead to medicines that lower blood pressure and fight AIDS, among other possible applications.

The designed compounds are the first that overlay precisely the zigzag shape of a peptide ß-pleated sheet, and yet they don't have any amide bonds, the type of bonds that normally link amino acids in peptides. Rather, they contain an alternative scaffolding.

Elimination of the amide bonds pro­tects the compounds from cleavage by peptide- and protein-cleaving enzymes in the body (peptidases and proteases). Such cleavage reactions are believed to contribute to the poor oral bioavailabil­ity of peptides.

The ß-pleated sheet mimics were de­signed and synthesized in a collabora­tive effort by two research groups at the University of Pennsylvania. One of the groups is headed by Ralph Hirsch­mann, a distinguished scientist and ex­ecutive who entered academia at the age of 65 to teach and do research. Hirsch­mann has shown that a scientific career need not be impeded by a little thing like retirement.

Hirschmann spent 37 years at Merck & Co., finally as senior vice president of chemistry. One of his key scientific achievements came in the late 1960s,

when, with then-vice president for ex­ploratory research Robert G. Denke-walter, he led a Merck group to the first total synthesis of an enzyme (ri-bonuclease) in solution, exploiting a new approach for the synthesis of pep­tides.

'Tn 1987 Ralph retired from Merck/' says Rhodes-Thompson Professor of Chemistry Amos B. Smith III, chemis­try department chairman at Penn and Hirschmann's collaborator on the pep-tidomimetics study. "During one of my consulting trips to Merck, Ralph asked me if there would be any opportunity for him to join the faculty of the chem­istry department here at Penn. In some ways this was not a trivial operation, given that Ralph Hirschmann, al­though a household word in the bioor-ganic, organic, and biomedical arenas, was not necessarily as well known to all of my colleagues/7

On Smith's recommendation and that of chemistry professor K. C. Nicolaou (now at Scripps Research In­stitute, but then at Penn), the univer­sity brought Hirschmann on board as

a research professor, the first in the chemistry department. Hirschmann has all rights and privileges of a ten­ured faculty member, although he's not tenured.

"The result has been a most remark­able association," says Smith. Upon his arrival at Penn, Hirschmann initiated collaborative research projects with Smith's and Nicolaou's groups to elim­inate the peptide backbone in peptide hormone and neurotransmitter ago­nists and antagonists, and in inhibitors of various enzymes. "The work has been successful in both areas," says Smith. "Ralph is having a marvelous second career."

One part of this program is the de­sign of mimics of ß-pleated sheets as potential inhibitors of protease en­zymes like renin and human immuno­deficiency virus (HIV) protease. The work was done by Smith; postdoctoral associates Terence P. Keenan and Ryan C. Holcomb; graduate students Paul A. Sprengeler, Mark C. Guzman, and John L. Wood; x-ray facility director Patrick J. Carroll; and Hirschmann [/. Am.

Hirschmann (left) and Smith discuss research in Smith's office at Penn

FEBRUARY 8,1993 C&EN 27

SCIENCE/TECHNOLOGY

SCIENCE/TECHNOLOGY

Chem. Soc, 114, 10672 (1992) and Γ Tetrahedron Lett., 34, 63 (1993)]. It was supported by the National In­stitutes of Health, Bachern CA, Merck Research Laboratories, and Sterling Winthrop.

Using computer models, the re­searchers predicted that a series of 3,5-linked pyrrolin-4-ones would adopt a backbone conformation mimicking a peptide ß-strand. For example, they compared the pro­posed pyrrolinone structure with the x-ray structure of an angioten-sinogen fragment, a tetrapeptide with a ß-pleated sheet conforma­tion. Based on this comparison, they predicted that carbonyl groups and side chains in the pyrrolinone would be oriented similarly to cor­responding structures in the tet­rapeptide.

Furthermore, because pyrrolin­one NH groups are comparable to amide NH groups in basicity, it appeared they might stabilize a ß-pleated sheet conformation through interstrand hydrogen bonding. This and other evidence L suggested that the pyrrolinones would effectively mimic ß-pleated sheet conformations. Synthesis of the pyrrolinones and analysis of their x-ray crystal structures showed that the compounds do mimic ß-pleated sheets, just as the researchers had pre­dicted.

The ß-pleated sheet motif is of inter­est in part because it constitutes a rec­ognition element for enzymes such as renin and HIV protease. Hence, mimics of the ß-pleated strand motif can po­tentially inhibit those enzymes. Inhibit­ing HIV protease is a strategy for fight­ing AIDS, and inhibiting renin could potentially lower blood pressure in persons at risk for heart disease.

In the case of renin inhibitors, the idea is to shut down one of two enzy­matic reactions that transform an-giotensinogen to angiotensin II, the physiological substance that raises blood pressure in the body. The first reaction, catalyzed by renin, cleaves an-giotensinogen to generate the biologi­cally inactive decapeptide, angiotensin I. In the second step, angiotensin-converting enzyme (ACE) catalyzes the conversion of angiotensin I to the bio­logically active octapeptide, angioten­sin II.

Until now, approved antihyperten-

Linked pyrrolinone compound mimics shape of ß-pleated sheet

Pyrrolinone-based peptidomimetic

ί ί Χ Μ Η j? LH Η2Ν^ΝΧ^Ν>(ΛΝΧ^ΟΟΗ3

ΓΗ Η Ο/\Η Η Ο

Angiotensinogen fragment

Ar = Aromatic group Pyrrolinone-based peptidomimetic simu­lates ß-pleated sheet conformation of tet-rapeptidyl angiotensinogen fragment. Dashed lines show alignment of carbonyl groups in the two structures. Side chains are also aligned

1 sive drugs have been ACE inhibitors. 2 Examples include blockbuster drugs r such as Squibb's Captopril and Merck's t enalapril. However, says Hirschmann, ϊ "from animal and clinical data ob­

tained by many laboratories, there is very good reason to think that an

- orally bioavailable, long-acting renin - inhibitor will also prove highly effec-s tive." s In the past, drug researchers have - been able to obtain peptides that are

very potent inhibitors of renin and HIV protease when tested in vitro (outside

ϊ live organisms). However, in vivo (in ι living organisms), peptides are poorly

absorbed orally, and to the extent they e are absorbed they don't last very long - in the bloodstream. Both problems re­

late in part to the tendency of peptides s to be chewed up by enzymes in the s body. t "If you have a peptidomimetic that - cannot be degraded by enzymes in the - gastrointestinal tract, then there is a ι possibility that the oral bioavailability - will be better," says Hirschmann. "And e obviously you have a very good chance - that the biological half-life will also be

improved." "We now have some unpublished

- experimental evidence," says Smith,

Ί "that one of our pyrrolinones is a renin inhibitor and that another in­hibits HIV protease. These two compounds are only intermediates on the road toward our ultimate synthetic targets. Although these compounds possess only modest potency, the results suggest that we are on the right track. The bioavail­ability and biological half-lives of our pyrrolinones have yet to be studied."

The new peptidomimetic com­pounds are also expected to have other biomedical and basic re­search applications. For example, Daniel H. Rich, professor of or­ganic chemistry and medicinal chemistry at the University of Wisconsin, Madison, points out that, in addition to their potential ability to inhibit HIV protease at the enzyme's active site, ß-pleated sheet mimics might also inhibit formation of the active enzyme. Catalytically active HIV protease is formed from two identical mono­mers by a dimerization process,

J which involves the pairing of two extended ß-sheet structures. Bio­

chemist Roger A. Poorman and co­workers at Upjohn, says Rich, "recent­ly reported low molecular weight mol­ecules that bind to the monomer at that extended β structure and prevent dimerization. They are proposing that these types of compounds might ulti­mately be useful as anti-HIV drugs." The ß-pleated sheet mimics developed at Penn could also possibly be used in this way, he says.

In addition, says Rich, chemistry professor Peter Lansbury's group at Massachusetts Institute of Technology has found that the amyloid protein as­sociated with Alzheimer's disease is characterized by a repeating extended ß-like structure. "One could immedi­ately see how low molecular weight mimics of this β structure might have some effect on the formation of amy­loid," he says.

Inhibiting HIV protease dimeriza­tion and amyloid protein formation are but two examples of possible ap­plications of ß-pleated sheet mimics that come immediately to mind, says Rich, but "there would probably be many other potential targets that would behave in much the same way. This work is likely to stimulate a lot of other research." •

28 FEBRUARY 8,1993 C&EN