Synthetic Molecule Blocks Exit from Cell Organelle

Inhibition of GTPase Disrupts Traffic out of Golgi, Offers Tool to Analyze Vesicle Transport

Images courtesy of Tom Kirchhausen Vesicular traffic jam. Secramine (left), a synthetic inhibitor of Cdc42, traps a fluorescent protein (shown above in green) in the Golgi apparatus (top). The protein, a fusion of enhanced green fluorescent protein and a viral glycoprotein, is normally transported to the cell membrane (bottom).

History shows that serendipity can play a huge role in scientific discovery. Not that this takes away from achievement—one skill shared by all good scientists is the ability to recognize that they have observed something important. After all, many baths were taken before Archimedes established his principle.

Recently, Tom Kirchhausen, Matt Shair, Henry Pelish, and colleagues had a “Eureka” moment of their own. They found that a small synthetic molecule called secramine inhibits protein transport out of the Golgi apparatus. In the Nov. 20 online edition of Nature Chemical Biology, they report that secramine acts by blocking the Rho GTPase Cdc42. More specifically, they reveal that the inhibitor works by preventing Cdc42 from fusing with lipid membranes. The finding offers a new mechanism for probing vesicular transport from the Golgi apparatus without disturbing the morphology of the organelle—something that has been lacking until now—and it reveals a new way to inhibit GTPases, which, unlike their ATPase paralogs, kinases, have proven very difficult to restrain.

Lights ... Actin
This work began as a collaboration between Kirchhausen, HMS professor of cell biology and principal investigator at the CBR Institute for Biomedical Research, and Shair, professor in Harvard University’s Department of Chemistry and Chemical Biology. The two joined forces to test the hypothesis that synthetic small molecules could perturb complex biological processes, such as vesicular transport, and in so doing identify mechanisms or molecules that have been missed by more traditional genetic approaches. The beauty of using small molecules to perturb cellular processes is that they act almost as soon as the molecule diffuses into the cell. This offers a great advantage over random mutagenesis, for example, which usually works very slowly and gives systems that have built-in redundancy time to compensate. Kirchhausen compares it to a traffic jam: “A road closure may affect traffic initially, but drivers soon learn to take another route,” he said.

Secramine, however, acts rapidly. When Pelish, the lead author on the paper and a graduate student in Shair’s lab at the time, used it to probe transport of a green fluorescent protein through the Golgi apparatus in cultured cells, he found that export of the protein stopped dead. “That would have been a great achievement in and of itself,” said Kirchhausen, but Pelish, a synthetic chemist by training, went on to learn enough biology that he could tweeze apart exactly how secramine was perturbing the Golgi—no easy feat, given that it has taken dozens of labs nearly 20 years and about 1,000 research papers before they uncovered the mechanism of action of the natural Golgi inhibitor, brefeldin A.

Pelish noticed that secramine seemed to have very similar effects on the Golgi as another well-known molecule, cytochalasin B, an inhibitor of actin polymerization. When Kirchhausen heard this idea he was skeptical at first, but Pelish went ahead, with the help of Jeffrey Peterson, then a postdoc in the laboratory of Systems Biology head Marc Kirschner and tested secramine in a cell-free assay for actin polymerization. They found that the molecule had indeed prevented the growth of actin chains. But what component was it affecting? That question would be almost impossible to answer using the cell-free system, which is not well defined, being made up of Xenopus laevis cytoplasmic egg extract supplemented with liposomes. Over the next year or so Pelish purified all the components necessary to reconstitute the actin polymerization assay so he could determine the precise step blocked by secramine. “This was crucially important, to have pure actin, pure Cdc42, pure components,” said Kirchhausen.

Rho vs. Wait
Actin polymerization is induced by an evolutionarily conserved pathway that begins with activation of -membrane-bound Cdc42. The GTPase then activates effector molecules, such as the protein N-WASP, leading to nucleation and growth of actin chains by another protein called Arp2/3. But Pelish found that secramine cannot block actin polymerization induced by a constitutively active form of N-WASP, indicating that the inhibitor acts somewhere upstream of this step.

Photo by Graham Ramsay; inset courtesy of Matthew Shair

Tom Kirchhausen (below right), Matthew Shair (left), and Henry Pelish (inset) have discovered that the small synthetic compound secramine inhibits the GTPase Cdc42, which is crucial for vesicular transport. The finding offers a new means to study Golgi transport and offers hope that unique GTPase inhibitors might one day be used to treat disease.

So he began to investigate Cdc42 itself. This GTPase can bind to cell membranes by virtue of a post-translational modification—the addition of a lipophilic prenyl chain. But this side chain makes the Cdc42 insoluble in the cytosol unless it is bound to another protein called Rho guanine dissociation inhibitor (RhoGDI). Without RhoGDI, Cdc42 fails to translocate between the cytosol and cell membranes, where it can activate effector molecules.

Cdc42 also undergoes conversion from an off state to an on state. Pelish found that secramine inhibited this activation, but only when Cdc42 was prenylated. Activation of the non--prenylated form of the GTPase, which does not bind RhoGDI, was unaffected by the inhibitor. The finding indicates that secramine inhibits activation of Cdc42 in a RhoGDI-dependent manner. This was supported by the finding that secramine also prevents the translocation of prenylated Cdc42 into membranes. “This work is a huge testament to Henry because it is highly unusual for someone who’s trained as a synthetic chemist to delve into hard-core cell biology—that’s very rare,” Shair said.

Exactly how secramine interacts with Cdc42/RhoGDI is not clear—that is currently under investigation. But because GTPases are crucially important for many biological processes, the discovery of the molecule and its target has the potential to spark a new class of therapeutic molecules. “While we don’t yet have the complete molecular picture, what secramine shows us is that it is possible to find a small molecule that can inhibit these GTPases in unique and perhaps specific ways,” said Shair. The researchers did, for example, test secramine in assays for Rab GTPases and found it did not affect them.

The molecule is also proof that synthetic small molecules discovered by phenotypic screens can yield biological insights. It has been relatively easy to achieve some sort of effect on cells with small molecules, but what has proven much harder is to find out how the small molecule works. “That’s when you really begin to learn something,” Shair said.

Kirchhausen agrees: “What we are only beginning to appreciate is the concept of chemical space—spaces that exist at the interfaces between different molecules. One day we may have a catalog describing those surfaces and chemistries, which will allow us to fine-tune small molecules or drugs. For me, that is why this work is so exciting.”

—Tom Fagan

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