Protein targets of bioactive natural products probed

Β , iologically active natural products .carry out their roles in the body by interacting with receptor pro­

teins inside or on surfaces of cells. Now, in independent studies, two research groups have advanced the art and sci­ence of identifying such protein targets.

The studies mirror each other, in a sense. In one study, researchers devel­oped a new technique to screen large protein libraries in a streamlined man­ner, demonstrating it with a known tar­get. In the other, scientists applied es­tablished methodology to identify a pre­viously unknown protein target—the target of ilimaquinone, a natural product that inhibits cellular secretion.

The new technique for identifying protein targets of natural products and other small molecules is display clon­ing, developed by assistant professor of chemistry David J. Austin and cowork­ers at Yale University [Chem. Biol, 6, 707 (1999)]. The technique's main ad­vantage over previous strategies is that it identifies a protein target and the gene for that protein all in one step. These tasks have traditionally required two separate and often quite involved experimental routines.

"If people wanted to discover what binds to a natural product or small mol­ecule they were studying, they had to go through a painfully laborious pro­cess," says chemistry professor and Howard Hughes Medical Institute in­vestigator Stuart L. Schreiber of Har­vard University's Institute of Chemistry & Cell Biology. "It's a process that's been reasonably well developed over the years, but it still is a lot of work."

Austin and coworkers now report "the very interesting trick of getting vi­ruses to put on their surfaces the entire collection of human proteins in a recom­binant form," Schreiber says. "They then used a natural product to find the virus that had the target protein of that natural product on its surface. The beau­ty of the technique is that the virus has got within it the DNA encoding that pro­tein. So the act of discovering a protein leads to its cloning directly."

Display cloning is a variant of phage display—a procedure in which large col­lections of peptides or proteins are ar­rayed on surfaces of bacteriophage virus­es after genetic modification of the phage

with randomized DNA sequences. The viruses express surface peptides or pro­teins based on those added sequences, and the resulting phage libraries can be screened for bioactivity.

Phage display with M13 virus—the most common experimental phage system by far—has been used to study protein-protein binding interactions. But it hasn't been possible to use it to identify binding between small molecules and large col­lections of proteins generated by incorpo­rating complementary DNA (cDNA) li­braries into phage. Such libraries contain sequences from all the transcribed genes (actively expressed proteins) in a cell.

M13 phage display of cDNA libraries is inefficient and problematic, but Aus­tin and coworkers realized that a much more rarely used virus called T7 is bet­ter at it. "They took advantage of the T7 system by using it in conjunction with small-molecule affinity chromatography techniques," Schreiber says. "Nobody recognized that this one peculiar virus had the feature required to make this combination of techniques work."

Nevertheless, Austin says, "Our major advance here was showing that cDNA phage display could be utilized with a small-molecule epitope. There are a few examples where people had used this

technique with proteins and antibodies, but these are huge molecules with large surface areas. People have been trying to extend this method to small molecules without success. We had been trying for over two years before we identified an ap­propriate phage system. Then we had to incorporate several modifications over traditional biopanning [phage screening] in order to see results."

The researchers demonstrate dis­play cloning by identifying FK binding protein (FKBP) as the molecular target of the immunosuppressive natural prod­uct FK506—a well-known interaction. Because the phage found to participate in the interaction not only had FKBP on its surface but also contained the cDNA for FKBP, the act of isolating the protein to which FK506 binds also pulls out the DNA encoding that protein.

Schreiber's group, and, independent­ly, that of molecular biologist and immu-nologist John Siekierka at Merck Re­search laboratories, Rahway, N.J., dis­covered more than a decade ago that FKBP was the receptor protein for FK506. But these researchers had to first identify the interaction and then, in a la­bor-intensive second step, clone and characterize the protein.

Display cloning combines those two steps into one. But so far, it's been dem­onstrated only on the FK506-FKBP test system. The real test for the technique will be if people can use it to discover something new.

Display cloning uses T7 phage Natural product

probe Proteins displayed on T7 Dhaae surfaces

Adapted with permission from Chemistry & Biology, © Elsevier Science Ltd.

OCTOBER 4, 1999 C&KN 3 3

Infect E. coli

Amplify phage

Incubate cDNA phage with affinity column

Additional rounds / of selection /

Elute

Column wash

Affinity column

s c i e n c e / t e c h n o l o g y

"We are very optimistic about the pos­sibilities because this system has the po­tential to provide functional access to ev­ery protein in the genome," Austin says. "But we are also very cautious because not all types of proteins are amenable to prokaryotic expression."

It's worth noting, Austin says, "that when we first used this system, you had to clone your own cDNA, but now Nova-gen [Madison, Wis.] is selling the cDNA phage libraries, so no cloning is neces­sary. Anyone can utilize this system im­mediately if they have access to an incu­bator. Our future goals are to define the limits of this phage and then reach fur­ther into more advanced phage systems, such as baculovirus. This is an insect-based expression system that has proven quite useful for the preparation of pro­teins that do not express well in Es­cherichia coli."

The compound ilimaquinone is the subject of a second study in which a pro­tein target of a natural product was identi­fied. Ilimaquinone, a natural product from a marine sponge, made a splash sev­eral years ago when researchers discov­ered that it was a hotbed of biological activity. Ifs a potent inhibitor of cellular secretion and also has antimicrobial, anti-human immunodeficiency virus, anti­inflammatory, and antimitotic properties.

"From the cell biologist's viewpoint, that compound was terribly exciting and promised to be a very power­ful probe of vesicle traffick­ing," Schreiber says. Vesicle trafficking is the process by which vesicles containing hormones and other sub­stances are induced to move to cell surfaces to secrete their payloads.

"Cell biologists are studying the whole process of how a mo­lecular signal can cause a vesicle to move," Schreiber says. "Because ilima­quinone has a particular effect on the ves­icle-trafficking process, it was put forward several years ago as possibly a key to un­derstanding it."

Now, chemistry professor Marc L. Snapper and coworkers at Boston College have identified for the first time the direct molecular targets of ilimaquinone [Chem. Biol, 6, 639 (1999)]. They report that the natural product interacts with enzymes of the activated methyl cycle, an enzyme-catalyzed process involved in amino acid metabolism. Ilimaquinone inhibits cellu­lar methylation processes, and hence se­cretion, by interacting with (5)-adenosyl-homocysteinase and other enzymes in the activated methyl cycle.

H3CO

H3C (-)-llimaquinone

The study can be traced back to the first total synthe­sis of (-)-ilimaquinone by Snapper and coworkers in 1995. That synthesis provid­ed an efficient route to the construction of a wide range of ilimaquinone analogs. Snapper's group has now used these analogs in pho-toaffinity labeling and affini­ty chromatography experi­ments to successfully tease

out the compound's cellular targets—the activated methyl cycle enzymes.

'These observations provide a clearer understanding of ilimaquinone's influ­ence on vesicle-mediated processes of the cell," the researchers note. "More­over, new opportunities for confronting viral infection and atherothrombosis might also be possible, considering the in­volvement of (5)-adenosylhomocysteine in these diseases."

The results are "a big surprise," Schrei­ber says, "because these targets previ­ously had not been known to be involved in the vesicle-trafficking process. The work is a very nice illustration of how syn­thesis can be used to make probe re­agents that uncover a mystery in a funda­mental biologic process."

Stu Barman

R = n-hexyl

Porphyrin trimer boasts/hosts trinity of metals A molecule made from zinc(II), ruthenium(II), and tin(IV) porphyrins has been assembled by chemists in England [J. Am. Chem. Soc, 121, 8120 (1999)]. In fashioning the oli-goporphyrin, in which divalent ruthenium is stabilized as Ru-CO, Hee-Joon Kim, Nick Bampos, and Jeremy Κ. Μ. Sanders at the University of Cambridge exploited the coor­dination chemistry of the individual porphyrins, equipping each with the appropriate donor ligand for the metal to be bound. Knowing that Sn(IV) porphyrins prefer oxygen-donor ligands while the two other metal porphyrins gravi­tate toward ligands that donate nitrogen, the group deriva-tized the zinc-containing porphyrin with a carboxylate ligand that binds Sn(IV) and the tin-containing porphyrin with a pyridyl group that binds both Zn(II) and Ru(II). Coordina­tion of the Zn(II) pyridyl and Sn(IV) carboxylate ligands is highly synergistic. In the heterodimer, the Sn(IV) porphyrin rotates about the Sn(IV)-carboxylate bond in the presence of excess pyridine. Addition of the ruthenium porphyrin to the heterodimer efiFectively locks the whole structure into a rig­id conformation. "Most remarkably," the researchers say, the same heterotrimer assembles when the individual com­ponents are mixed in solution. The aimer's rigidity and the geometry of its component porphyrins make it ideal for characterization by two-dimensional nuclear magnetic reso­nance spectroscopy, they note. They suggest that comple­mentary coordination properties might be employed to pro­vide a new way to prepare complex porphyrin assemblies such as catenanes and rotaxanes.

Μεάήη Brennan

34 OCTOBER 4, 1999 C&KN