R E S E A R C H H I G H L I G H T S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 4 | APRIL 2005 | 279

Sequencing of the human genome has so farrevealed more than 500 genes encoding proteinkinases. Many of these enzymes are directlyinvolved in diseases such as cancer andinflammation, making them excellent targets fordrug development. In the March issue of NatureBiotechnology, Lockhart, Zarrinkar and colleaguesgo into uncharted protein kinase territory bydeveloping a new type of assay to determine thespecificity of a number of kinase inhibitorsagainst a panel of 119 protein kinases.

Kinase inhibitors are an important new classof anticancer drugs, and have clinical activity intumours in which the target kinase is activated bymutation, such as the mutant kinase BCR–ABL inchronic myeloid leukaemia (CML). The successof small-molecule inhibitors such as imatinib(Gleevec; Novartis) to treat CML, and gefitinib(Iressa; AstraZeneca) and erlotinib (Tarceva;Genentech/OSIP), both of which target theepidermal growth factor receptor (EGFR) to treatlung cancer, has demonstrated clear proof-of-principle that this strategy is effective.

Most kinase inhibitors in clinical developmenttarget the ATP-binding site common to allkinases, and bind multiple kinases. However, it isnot possible to predict binding specificity andaffinity on the basis of available sequence orstructural information. Conventional profilingmethods that use the measurement of in vitroactivity are limited by the difficulty of buildingand running large numbers of kinase activityassays. Such information is very valuable, bothfor finding new clinical uses for inhibitors andfor predicting or explaining toxicity.

Fabian et al. have developed a quantitativecompetitive assay for measuring the binding of small molecules to the ATP-binding site ofkinases. Human kinase domains are firstexpressed as a fusion attached to T7 bacteriophagecapsid protein. Then, the test compound insolution competes with an immobilized ‘bait’ligand to bind the phage expressing the kinasedomain. The amount of phage bound to the baitis quantified to determine the affinity of the testcompound for each kinase. If the free test com-pound binds the kinase and directly orindirectly blocks the ATP site, fewer proteinmolecules bind to the immobilized bait; if thefree test compound does not bind to the kinase,the fusion proteins are able to bind to theimmobilized bait.

For most of the test compounds, the tightestinteraction is with the kinase that the drug wasoptimized to inhibit, but the difference inaffinity between the primary target and otherkinases varies substantially. For the inhibitorsBIRB-796, VX-745, erlotinib, GW-2016 andSU11248 there is at least a tenfold difference inaffinity between the intended target and off-targets, whereas for SP600125, EKB-569 andZD-6474 there is less than a twofold difference.This indicates that optimization efforts aregenerally successful, but there is room forimproved discrimination if necessary.

A number of CML patients develop resistanceto imatinib. The authors developed similarassays for imatinib-resistant ABL kinases. Themost dramatic finding is that the p38 inhibitorBIRB-796 binds tightly to an imatinib-resistantABL mutant. The authors also uncovered a newtarget for imatinib, the SRC-family tyrosinekinase LCK, to which imatinib binds tightly.Finally, the authors showed that clinicallyobserved mutations in EGFR do not affect thebinding affinity of gefitinib or erlotinib.

The authors show that their kinase-bindingassay provides results consistent with morestandard in vitro results, although whether thisreflects the capability of a compound to inhibit akinase in a cell remains to be determined.Certainly, this assay is a useful tool and it willprobably accelerate drug discovery anddevelopment efforts for kinase inhibitors.

Melanie Brazil

References and linksORIGINAL RESEARCH PAPER Fabian, M. A. et al.A small molecule–kinase interaction map for clinical kinase inhibitors.Nature Biotechnol. 13 Feb 2005 (doi:10.1038/nbt1068)FURTHER READING Daub, H., Specht, S. & Ullrich, A. Strategies toovercome resistance to targeted protein kinase inhibitors. Nature Rev.Drug Discov. 3, 1001–1010 (2004) | Yingling, J. M., Blanchard, K. L. &Sawyer, J. S. Development of TGF-β signalling inhibitors for cancertherapy. Nature Rev. Drug Discov. 3, 1011–1022 (2004)

Surveying the kinome

K I N A S E I N H I B I TO R SThe transgenic rat expresses humanproteins that provoke a misdirectedimmune response, the first stage ofwhich is chronic intestinal inflamma-tion. This phenotype serves as a modelof IBD in which low doses of EE havebeen shown to be effective, and solends itself to studies of WAY-169916.

Treatment with WAY-169916 hada positive outcome on the diseasephenotype: it rapidly halted chronicdiarrhoea and significantly reducedall histological parameters of intestinalinflammation to an extent compara-ble with EE. Co-administration of anER antagonist showed that theseeffects resulted from ER activity. Inaddition to its potential developmentas an anti-inflammatory drug, WAY-169916 could provide insight into themolecular mechanism of the divergentroles of the ER.

Joanna Owens

References and linksORIGINAL RESEARCH PAPERChadwick, C. C. et al. Identification of pathway-selective estrogen receptor ligands that inhibitNF-κB transcriptional activity. Proc. Natl Acad.Sci. USA 102, 2543–2548 (2005)FURTHER READINGGronemeyer, H. et al. Principles for modulation ofthe steroid hormone receptor superfamily. NatureRev. Drug Discov. 3, 950–964 (2004)