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Biological Chemistry

Full Accounting of Drug Effects

Profiling technique enables complete assessment of real effects of an important class of drugs

by A. MAUREEN ROUHI, C&EN WASHINGTON
March 21, 2005 | A version of this story appeared in Volume 83, Issue 12

GATEWAY
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Credit: COURTESY OF CHARLES KUNG
In the wild-type kinase, a bulky residue (surface shown in red) in the ATP-binding site precludes the binding of the small molecule 1-NA-PP1 (shown) to this and other wild-type kinases. Mutation of that residue to glycine (surface shown in green) opens the ATP-binding site. The small molecule binds tightly because it had been designed to perfectly complement the mutated kinase.
Credit: COURTESY OF CHARLES KUNG
In the wild-type kinase, a bulky residue (surface shown in red) in the ATP-binding site precludes the binding of the small molecule 1-NA-PP1 (shown) to this and other wild-type kinases. Mutation of that residue to glycine (surface shown in green) opens the ATP-binding site. The small molecule binds tightly because it had been designed to perfectly complement the mutated kinase.

The pharmaceutical industry is still reeling from doubts cast on the safety of drugs such as the COX-2 inhibitor Vioxx. At the heart of the drug safety issue is incomplete understanding of the full effects of drugs now in the market. These drugs were developed at least a decade ago, when tools to understand drug effects were limited. Now, through methods combining chemistry, biochemistry, biology, and genetics, scientists are better able to identify the full spectrum of a drug's cellular effects. With such tools, it is hoped that the safety, risks, and benefits of candidate drugs still in the development pipeline could be better assessed.

One such tool comes from the lab of Kevan M. Shokat, professor of chemistry at the University of California, Berkeley, and of cellular and molecular pharmacology at the University of California, San Francisco. Recently, he and his coworkers used the tool to develop the first complete picture of the effects of a kinase inhibitor in a cell. They pinpointed the specific kinases that are targeted by the inhibitor. More important, they showed that the cellular effects of kinase inhibitors with multiple targets--or multiplex inhibitors--cannot be understood simply by assessing the individual effects on single kinases.

Kinases catalyze the transfer of a phosphate group from ATP to another molecule. They regulate many cellular signaling pathways, and they are important drug targets in many disease areas. "Kinases are rapidly becoming the largest class of drug targets," Shokat points out. "Kinase inhibitors are being developed in drug discovery companies and in academia, yet methods for understanding how these inhibitors truly work are lacking."

Two facts complicate discovery of kinase inhibitors: First, the human genome codes for a large number of kinases, about half of which still are not characterized. Second, the active site in kinases is highly conserved, which means that it is extremely difficult to find inhibitors that target only one kinase. It also means that drugs designed to hit a particular kinase very likely inhibit multiple kinases.

Shokat and coworkers turn nature's conservation of the kinase binding site on its head as follows: To pinpoint the effect of a single kinase, they enlarge its ATP-binding site in a manner that does not alter the function of the enzyme. Next, they design a small molecule that will perfectly complement the enlarged site. Because this molecule will not fit into any wild-type ATP-binding site, it is specific to the kinase of interest.

The technology, called analog-sensitive kinase alleles (ASKA), is now licensed to Cellular Genomics, a drug discovery company based in Branford, Conn.

Because of where the mutation is made, the mutant behaves like an ordinary kinase. When it is incorporated in a cell (or in a whole animal), the cell lives and functions just like a normal cell. When this cell is exposed to the complementary small-molecule inhibitor, the molecule will bind only to the mutant kinase. The effects of that binding can then be tracked to the gene level through microarray analysis. The result is a reference profile of cellular effects caused by inhibiting this kinase alone.

MASTERMINDS
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Credit: COURTESY OF KEVAN SHOKAT
Shokat (left) and Kung have demonstrated how analog-sensitive kinase alleles can fully account for the effects of kinase inhibitor drugs.
Credit: COURTESY OF KEVAN SHOKAT
Shokat (left) and Kung have demonstrated how analog-sensitive kinase alleles can fully account for the effects of kinase inhibitor drugs.

WITH SUCH a system, it becomes possible to identify all the targets of a kinase inhibitor with unknown or incompletely characterized specificity, in effect mapping the full effects of that inhibitor. It also becomes possible to know whether the effects of a multiplex inhibitor are simply the sum of the effects on each individual kinase.

Shokat, with graduate student Charles Kung and other colleagues, recently demonstrated the technique on the kinase inhibitor called GW400426. The kinase targets of this inhibitor are unknown. Shokat and coworkers suspected that its targets are two cell-cycle kinases, Cdk1 and Pho85.

They made mutants and perfectly complementary small molecules and produced reference profiles of cellular effects for each. They expressed the mutants in yeast cells and then compared the effects of inhibiting Cdk1 and Pho85 with the effects of GW400426 (Proc. Natl. Acad. Sci. USA 2005, 102, 3587).

"We saw whole groups of genes that were regulated by inhibition of each kinase that matched with the effects of GW400426," Shokat says. The results support the hypothesis that the small molecules hit both Cdk1 and Pho85. "Then we asked, 'Are there any other effects of GW400426 that we don't see in our reference profiles?' " Analysis of the microarray data revealed a set of genes that could not be explained by the reference profiles.

The unexplained gene set could be due either to a third kinase that GW400426 is inhibiting or to an effect that arises only when both kinases are inhibited at the same time, Shokat says. He and coworkers tested the hypothesis by expressing both mutant kinases in a cell. This time, the profiles matched completely.

The key result of this work is identifying gene clusters that are affected by simultaneous inhibition of the two kinases, comments Henrik Daub, group leader for proteomics at Axxima Pharmaceuticals, a Munich-based company that tries to develop kinase inhibitor drugs. More specifically, the work identifies that only coinhibition of the two kinases regulates certain genes involved in polarized cell growth, he explains. The approach used by Shokat and colleagues to reach this conclusion is "elegant," he adds.

REVELATION
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Credit: COURTESY OF CHARLES KUNG
The cellular targets of GW400426 (molecule shown at left) were determined from reference profiles generated by inhibition of analog-sensitive kinase alleles Cdk1-as1 and Pho85-as1 by the small molecule 1-NA-PP1. The profiles reveal that some cellular effects of GW400426 (green dots) are due to simultaneous inhibition of Cdk1 and Pho85.
Credit: COURTESY OF CHARLES KUNG
The cellular targets of GW400426 (molecule shown at left) were determined from reference profiles generated by inhibition of analog-sensitive kinase alleles Cdk1-as1 and Pho85-as1 by the small molecule 1-NA-PP1. The profiles reveal that some cellular effects of GW400426 (green dots) are due to simultaneous inhibition of Cdk1 and Pho85.

THE FINDING is particularly important in light of the resistance developing against relatively specific drugs such as Gleevec, Daub says. Gleevec is the first kinase inhibitor drug approved by the Food & Drug Administration and is the leading treatment for chronic myelogenous leukemia. "More-promiscuous kinase inhibitors targeting more than one essential oncogenic factor might become important in avoiding the development of resistance to targeted anticancer therapy," he adds.

Andrew Hopkins, head of knowledge discovery at Pfizer Global R&D, based in Kent, England, echoes those comments in a broader sense. He points out that a key assumption of rational drug design is the need to develop selective compounds against single proteins. Shokat's results challenge the "one gene/one phenotype/one drug dogma," he says. "The implication is [that] to effect many biological phenotypes we may need drugs that can simultaneously hit several targets."

In theory, Shokat's approach could be applied to targets beyond kinases. Commenting on the current woes of COX-2 inhibitors, Shokat points out that if it were possible to prepare a mutant COX-2 that would be sensitive only to a perfectly complementary small-molecule inhibitor but would be completely functional otherwise, that system could tell if a COX-2 inhibitor drug only affects pain or, in addition, produces subtle effects on heart condition. "That's why we think this approach is so powerful," he says.

Shokat points out that a potential criticism of the approach is that the profiling is done with yeast cells. "Yet the same principles we uncover apply to mammalian systems," he adds. Furthermore, mice with ASKA versions of particular kinases are available from Cellular Genomics.

"The methods we describe provide a breakthrough in our ability to deconvolute and understand the effects of kinase inhibitors in complex biology and drug discovery," Shokat says. "Biologists are constantly telling us that genetics is the benchmark for understanding the system. Actually, small molecules are better to work with. The caveat is understanding their specificity. We show that we can do that by putting the kinase of interest under chemical control."

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