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Varying Approaches to Inhibiting Kinases

Advanced tools can help researchers address acquired resistance to kinase

blockers for cancers and expand to other disease targets

Lori Valigra

Valigra is a freelance writer based in Cambridge, Mass.

The future looks bright for kinase inhibitors, therapeutic compounds that

block kinase enzymes from catalyzing the transfer of a phosphate group from

a donor such as ATP to another molecule. So far, much of the work on the 600

or so different kinases in mammals has been focused on cancers. More than 30

protein kinase inhibitors for cancer are in clinical testing or approved by

the US Food and Drug Administration (FDA), including the blockbuster drugs

Gleevec (imatinib mesylate), Iressa (gefitinib), and Tarceva (erlotinib).

Those drugs have proven effective in blocking the action of their kinase

target without causing the negative side effects of traditional

chemotherapy.

Although recent reports indicate some patients have developed mutations that

cause resistance to these drugs, scientists are not discouraged. Advanced

tools such as high-throughput screening, single nucleotide polymorphism

(SNP) arrays, exon resequencing, and structural analysis are being used to

help better understand the targets, the mutations, and which patients will

most likely respond to more potent, second-generation compounds. Kinase

targets are expected to be broadened in the future to inflammatory,

autoimmune, central nervous system, and cardiovascular diseases.

Kinases are now where G-protein-coupled receptors (GPCRs) were 20 years ago,

says Andy Barker, PhD, head of chemistry and oncology at AstraZeneca

Pharmaceuticals, Alderley Park, UK. " We're at the early stage of a group of

proteins that could supply some very important drugs in cancer and other

diseases in the next 10 to 20 years, " says Barker. He says the majority of

drugs on the market now are GPCR agonists and antagonists.

Barker and other researchers are not very surprised about the emergence of

somatic mutations. " In a disease like cancer, it is a classic process where

a protein changes and mutates to provide survival advantage. " Daley,

MD, PhD, associate professor of biological chemistry and molecular

pharmacology at Harvard Medical School and Children's Hospital, Boston, has

developed a method to profile a drug's activity against various mutants and

the way a target might mutate to evade the drug. " I don't believe there is a

[cancer] target that is immune to mutation, " says Daley. " If we can make a

drug against a target, the cancer cell will figure out a way to mutate away

from the drug. "

<http://www.dddmag.com/images/0504/CEL1_lrg.jpg>

click the image to enlarge

Diagram shows several strategies to inhibit EGFR signaling. [Adapted from S.

B. Noonberg and C. C. Benz, Drugs, vol. 59, pp. 753–767 (2000)] (Source:

AstraZeneca)

Gleevec, a BRC-ABL inhibitor for chronic myelogenous leukemia (CML) from

Novartis International AG, Basel, Switzerland, is probably the best known of

the three big cancer kinase inhibitors, having been heralded by some as a

" magic bullet " when it came to market. AstraZeneca makes an epidermal growth

factor receptor (EGFR)-tyrosine kinase inhibitor for non-small-cell lung

carcinoma (NSCLC) called Iressa, as does OSI Pharmaceuticals Inc., Melville,

N.Y., with Tarceva.

The Gleevec mutations were the first to be heavily publicized, with point

mutations detected in the ATP-binding domain of the ABL gene, a factor which

disturbs the binding of Gleevec to its target [Z. Iqbal et al., Biol.

Proced. Online, vol. 6, pp. 144-148 (2004)]. More recent papers cite

mutations that cause resistance to Iressa or Tarceva [s. Kobayashi et al.,

N. Engl. J. Med., vol. 352, pp. 786-792 (2005); W. Pao et al., PLoS

Medicine, vol. 2 (no. 3), p. e73 (2005)]. Some second-generation compounds

already are in development, including a " super Gleevec " molecule called

AMN107 by Novartis, which is in phase II clinical trials.

Overcoming Initial Skepticism

Initial development of kinases was fraught with naysayers, many of whom

didn't

<http://www.dddmag.com/images/0504/CEL2.jpg>

The structure of ABL kinase (light blue) in complex with NVP-AMN107 (yellow

spheres). The compound binds near the hinge region (green) between the N-

and C-terminal lobes of the kinase. The glycine-rich loop (red) and the

activation loop (magenta) adopt inactive conformations, as in the complex

with Gleevec. (Source: Novartis Institutes for Biomedical Research)

believe it was possible to inhibit kinases, especially at the ATP binding

site, says Sasha Kamb, PhD, vice president and global head of oncology at

Novartis Institutes for Biomedical Research Inc., Cambridge, Mass. ATP is

used broadly in cells as a source of energy and phosphate, which is used by

kinases. It is present in high millimolar concentrations in cells, so any

drug would have to compete with ATP. " There was much skepticism about

whether that was possible, " says Kamb. " Scientists in the predecessor

company of Novartis [Ciba-Geigy] took a gamble and invested in what at that

time was considered to be an extremely high-risk project. "

The original work on Gleevec (also known as Glivec in Europe) started in the

early 1960s, when it was discovered that certain leukemias such as chronic

myelogenous leukemia (CML) had a telltale chromosome abnormality, the

so-called Philadelphia chromosome. With the advent of cloning technology, it

was possible to identify the molecule, which had a chromosomal reciprocal

translocation that exchanged two arms of two human chromosomes that fused

the Abelson kinase with the BCR gene. This became known as the BCR-ABL

protein. Kamb says the reasonable assumption was that because BCR-ABL was

present in the leukemias, it was somehow essential to the tumor growth of

those leukemic cells, and it made sense to try to inhibit it. That

assumption wasn't proven correct until Gleevec was developed.

Analyzing Mutations, Resistance

Anyone developing anticancer drugs will have to face resistance as a

challenge, says Daley, MD, PhD, associate professor of biological

chemistry and molecular pharmacology at Harvard Medical School and

Children’s Hospital, Boston. Two years ago, he published a paper [M. Azam et

al., Cell, vol. 112, pp. 831-843 (2003)] on a new technique he and his

colleagues, then at the Whitehead Institute for Biomedical Research,

Cambridge, Mass., devised to identify mutations that will cause resistance

to targeted anticancer drugs.

Focusing on patients who had relapsed because of resistance to Gleevec,

Daley’s group used recombinant DNA methods to randomly mutate the BCR-ABL

gene and mimic potential variations that might be found in patients with

chronic myelogenous leukemia (CML), and then test those mutations when

exposed to Gleevec. They cataloged a group of more than 100 mutations that

could help doctors discover which mutation caused drug resistance or detect

the presence of a mutation before a patient relapses.

“We are trying to take a given drug that a company has screened and give

them as much information as possible about the sensitivity of various

mutants, the activity of that drug against various mutants, and the way the

target will mutate to evade the drug directly,” Daley says. He adds that it

is best for his group to get involved in the lead identification or lead

optimization stages. “Drug companies want to use us if they have a new

target and want to have a follow-on compound right behind it. If they choose

another lead that they think is drug-like and then choose a variant on that

lead, that would also cover the resistance pattern of the first drug.” The

group, as well as researchers at the Dana-Farber Cancer Institute, Boston,

recently characterized Novartis’ follow-on drug to Gleevec, AMN107, in a

paper [E. Weisberg et al., Cancer Cell, vol. 7, pp. 129-141 (2005)].

Although AMN107 does catch most of the mutations, one mutation remains

elusive: the threonine 315 isoleucine mutation. A structural feature of all

the established kinase inhibitors is that part of their chemical structure

inserts into a hydrophobic pocket that the threonine 315 isoleucine

regulates like a gatekeeper. So a common Achilles heel of the different

inhibitors developed to date is that they anchor themselves on this

hydrophobic pocket, says Daley. “This will be a vexing problem for a whole

series of kinases, but mTOR isn’t homologous in this particular susceptible

amino acid residue.”

The researchers were looking for a drug that looked like ATP. At the time,

there was no high-throughput screening available to help cull through

potential compounds, so they used what Kamb says were low-throughput screens

and assays. " A number of molecules looked at were basically eyeballed, and

they looked a bit like ATP. That's how Gleevec was first discovered, " he

says. Some modifications were then made based on screening and medicinal

chemistry decorating of the original molecule in different ways to see which

modifications would provide higher potency. It was then screened for safety

using standard techniques. " The fortunate and key thing is that ABL is a

normal kinase that's present in the body. Very few, if any, cells depend on

it for survival, so it turns out to be a pretty well-tolerated cancer drug, "

Kamb says.

Work on kinases can be a challenge, because they make up a large gene

family. Because kinases bind to ATP, which doesn't evolve very quickly,

chemists are under pressure to come up with selective inhibitors. " If you

inhibit all the kinases, obviously you've got a very toxic compound, " Kamb

says. " That's one of the challenges in this area, to produce compounds that

have reasonable selectivity for a specific target or targets. " But that's

where all the tools of the drug discovery profession come into play, he

says. It is extremely helpful to have crystals, or high-resolution

structures of the targets, to help guide compound design to get affinity and

selectivity. It also helps to have many of the kinases in a screening format

so they can be screened against the target and counterscreened against other

kinases that are not desired. Kamb says that having cellular assays as well

as biochemical assays is very important when studying kinases. " It's often

seen that inhibitors that work nicely in a biochemical assay actually don't

work well in a cellular assay, and sometimes vice versa. So there's

something about the cellular context that changes the chemical or

biochemical properties of the protein of target. It isn't clear why. "

Finding New Mutations

Meyerson, PhD, assistant professor of pathology at the Dana-Farber

Cancer Institute and Harvard Medical School, Boston, and his colleagues are

trying to discover new mutations, primarily somatic ones, in protein

kinases.

The researchers are systematically using single-nucleotide polymorphism

(SNP) arrays to find copy number alterations in the genome and

high-throughput exon resequencing to find coactivating point mutations. The

group is taking cancer samples and sequencing all of the protein tyrosine

kinase genes. They perform genotyping to determine if mutations exist, then

repeat the sequencing in larger samples to validate the results.

Meyerson says a major discovery of his group is EGFR mutations. EGFR

inhibitors such as Iressa are good candidates for treating lung-cancer

patients, and a significant number of patients showed responses, but it

wasn’t clear who those patients were or why they responded, he says. “Using

the systematic exon resequencing approach, we discovered mutations in lung

cancer and found that those mutations correlated with response to Iressa or

Tarceva. It was a surprise.”

Gleevec initially was targeted at CML patients, but Novartis researchers

determined it also hits other kinases, including KIT, with reasonably high

affinity. KIT is activated in gastrointestinal stromal tumors (GIST), and

Gleevec is approved for use in GIST as well. Phase II trials of a

second-generation Gleevec-like compound called AMN107, which hits the same

target but has a different chemical structure, will begin soon. AMN107 has

higher potency and a sufficiently different binding mechanism, so it can

actually hit many of the resistant mutants. In retrospect, Kamb says,

Novartis was lucky with Gleevec because it was a first molecule that had

very few problems. Novartis used the toolkit initially developed for Gleevec

and some newer technologies such as high-throughput screens to develop the

follow-on compound. They also used more biochemical and cellular assays,

larger chemical libraries, and more crystal structures.

Honing Activity, Selectivity

Although some people think selectivity can be a problem with kinases,

AstraZeneca's Barker doesn't agree. " I don't think it's any more of a

problem than with any other large class of biochemical targets. " A typical

drug program aimed at a GPCR would look similar in its steps to one aimed at

a kinase, he says, but the levels of knowledge are different.

When Iressa was developed about 10 years ago, the company used standard

biochemical assays. AstraZeneca incorporated radiolabels into its substrate

or used colorimetric detection to look for changes in a protein or

substrate. But some of the newer tools AstraZeneca finds helpful include the

use of more structural information on kinases to design directed libraries,

refine structural activity relationships, and develop selectivity between

different kinases. Barker says that recent steps forward include a better

understanding of some of the signal transduction pathways that kinases are

involved in and their relative importance in various disease states. " A lot

of that has been teased apart by new specific antibodies that recognize

activated and nonactivated kinases. Those sorts of tools have proven very

useful not only in understanding the fundamental bioscience, but in

understanding how that bioscience relates to overall effects in a disease

downstream. "

An advantage with kinases is that it is possible to model many of the

mutations in structural terms and generate proteins to test against. " The

bigger challenge is to know which patients have which mutation and when to

treat with a drug, " Barker says. " With some cancers, there are very specific

mutations that seem to occur regularly and in others there are a variety of

mutations in different parts of the protein, none of which is dominant, and

that makes targeting specific mutations quite difficult. " Like other

researchers, he thinks cancer treatment may evolve to be like HIV treatment,

where a cocktail of two or three inhibitors can cover 99% of the kinases and

their mutations and stabilize the disease for a period.

" I suspect it's possible that one molecule may not serve all needs in this

field [kinases], " says Tomi Sawyer, PhD, senior vice president of drug

discovery, Ariad Pharmaceuticals Inc., Cambridge, Mass.

mTORs Pose Other Challenges

To Ariad's Sawyer, there are two different extremes of developing protein

kinase inhibitors. One is the story of Iressa and Gleevec, and having to

figure out a way to override the mutations. The other is Ariad's experience

with mammalian target of rapamycin (mTOR), which did not require starting

development work with a dozen or more different templates to optimize into

potent inhibitors. The mTOR inhibitors appear to not have the problem of

kinase mutations. " We started with nature's optimized inhibitor, rapamycin, "

he says. The challenge to Ariad, and other companies, is to develop their

own creative ways to advance a proprietary rapamycin analog. Ariad developed

AP23464, a proof-of-concept molecule that is a potent inhibitor of SRC, ABL,

and KIT, which it is now optimizing in analogs. The company is in phase II

tests with AP23573, an mTOR inhibitor of hematologic malignancies and

various solid tumors.

Sawyer says there is very little wiggle room from a chemistry and

drug-design standpoint to provide an active rapamycin analog. In the case of

rapamycin optimization to make proprietary second-generation analogs,

chemists have focused on one site in the rapamycin molecule, a hydroxyl

group on carbon 43 that is unstable because it is a site of metabolism.

Ariad used its proprietary phosphorus chemistry to develop a potent and

effective in vitro and in vivo mTOR inhibitor. " We knew exactly where to

modify the molecule. We had proprietary chemistry that only had to be

exploited within the framework of the rapamycin molecule, and it was a

straightforward process to evaluate the initial process in cells and then go

right to in vivo [testing], " Sawyer says of AP23573.

Even though Ariad hasn't had to deal with the issue of mutations, Sawyer

thinks new tools like the mutational cell analysis approach being used at

Children's Hospital in Boston will be important in future kinase work. " This

is an important emerging tool to deal with these mutant kinases, " says

Sawyer. " It will become more and more important for people to determine

whether or not they have a novel and potentially very effective new molecule

that can escape some of these key mutants. "

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