Nuclear receptors: Choosing the right path
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278 | APRIL 2005 | VOLUME 4 www.nature.com/reviews/drugdisc RESEARCH HIGHLIGHTS A compound that acts via the oestro- gen receptor (ER) to selectively inhibit nuclear factor-κB (NF-κB) without displaying classical oestrogenic side effects could be used to treat chronic inflammatory disorders such as inflammatory bowel disease (IBD), according to a recent paper in the Proceedings of the National Academy of Sciences. Oestrogens have a wide variety of biological effects and although many of them are benefi- cial, oestrogen therapy has been associated with side effects such as uterine bleeding, increased risk of endometrial cancer and venous thrombosis. This has led to a search for ER ligands that retain beneficial properties but without the side effects. Non-selective oestrogens (those that bind both ERα and ERβ with equal affinity) are known to have anti-inflammatory activity that is thought to result from interference with the NF-κB signalling pathway. Douglas C. Harnish and colleagues therefore set out to identify ER lig- ands that inhibited NF-κB without displaying any conventional oestro- genic functions and identified a first-in-class pathway-selective group of ER ligands. The authors used cells transiently expressing ERα or ERβ and an NF-κB–luciferase reporter gene that was induced by interleukin-1β (IL-1β) to identify compounds that inhibited NF-κB activity. The first compound they discovered was WAY-169916, a non-steroidal, orally active molecule that inhibited 50% of IL-1β-stimu- lated NF-κB activity, without affecting a marker of conventional oestrogenic activity. They then studied the path- way-selectivity of this compound in vivo using a high-fat-diet mouse model in which NF-κB and other inflammatory genes are upregulated in the liver. Daily treatment of ovariectomized mice with either 17α-ethynyloestradiol (EE) or WAY- 169916 significantly inhibited the high-fat-diet-induced expression of these genes, but, significantly, WAY- 169916 did so without the accompa- nying oestrogenic effects observed with EE. The potential of WAY-169916 as an anti-inflammatory therapy for IBD was then demonstrated using the HLA-B27 transgenic rat model. A computational approach that allows a quantitative assessment of the ability of a given binding site of a protein to bind small ‘leadlike’ molecules has been reported by Hadjuk and colleagues in the Journal of Medicinal Chemistry. Their studies, which are based on nuclear magnetic resonance (NMR) screening data, also illustrate how ‘hot spots’ on protein surfaces — regions that make major contributions to the binding energy in protein–ligand complexes — can be identified. Such approaches should be particularly useful for novel genomics-derived targets, for which information on small-molecule-binding sites is typically limited. NMR-based screening methods such as that described by Hadjuk et al. have a number of strengths. First, the perturbations to NMR signals that are indicative of small-molecule binding can be monitored for the whole protein, and so small-molecule binding to any region can in principle be detected. Consequently, both the affinity and the site of small-molecule–protein interactions can be characterized. Second, NMR-based screening approaches can detect small-molecule ligands that have considerably weaker affinities than would be detectable in standard high- throughput screens. These advantages make such approaches ideal for identifying and characterizing hot spots on protein surfaces. To investigate both the capacity of proteins to bind small molecules and the nature of the binding sites, Hadjuk and colleagues analysed NMR-based screening data from 23 diverse protein targets. Across all targets, the vast majority of the hits identified in the screens bind to a known small-molecule-binding site, demonstrating the selectivity of protein surfaces to bind to ligands at only very specific locations. Interestingly, a small number of novel potential hot spots were also identified. Furthermore, their data indicated that NMR- based screens can be used as a reliable guide to the ‘druggability’ of a given protein target before investing in the development of complex biochemical assays, or even before the function of the protein is known. As an alternative to performing an NMR- based screen against every potential protein target, it would be of great value to be able to confidently predict that high-affinity leads can be identified for a particular target. With this in mind, the authors analysed the protein pockets and NMR screening data to try to understand the factors that influence the observed hit rate. Using this analysis, they developed a simple computational model that included parameters describing the protein binding pocket, such as surface complexity, that could be used to predict with high accuracy the druggability of protein targets not used to construct the model. As the authors conclude, the relationships derived between hit rate and binding-pocket parameters have important implications for the understanding of fundamental principles of molecular recognition, and should facilitate quantitative comparative analyses of binding pockets for use in target assessment and validation, virtual screening and structure-based drug design. Peter Kirkpatrick References and links ORIGINAL RESEARCH PAPER Hajduk, P. J., Huth, J. R. & Fesik, S. W. Druggability indices for protein targets derived from NMR-based screening data. J. Med. Chem. 24 Feb 2005 (doi:10.1021/jm049131r) FURTHER READING Rees, D. C. et al. Fragment-based lead discovery. Nature Rev. Drug Discov. 3, 660–672 (2004) | Pellecchia, M. et al. NMR in drug discovery. Nature Rev. Drug Discov. 1, 211–219 (2002) Hitting the hot spots SCREENING Choosing the right path NUCLEAR RECEPTORS
Transcript of Nuclear receptors: Choosing the right path
278 | APRIL 2005 | VOLUME 4 www.nature.com/reviews/drugdisc
R E S E A R C H H I G H L I G H T S
A compound that acts via the oestro-gen receptor (ER) to selectively inhibitnuclear factor-κB (NF-κB) withoutdisplaying classical oestrogenic sideeffects could be used to treat chronicinflammatory disorders such asinflammatory bowel disease (IBD),according to a recent paper in theProceedings of the National Academyof Sciences. Oestrogens have a widevariety of biological effects andalthough many of them are benefi-cial, oestrogen therapy has beenassociated with side effects such asuterine bleeding, increased risk ofendometrial cancer and venousthrombosis. This has led to a searchfor ER ligands that retain beneficialproperties but without the sideeffects.
Non-selective oestrogens (thosethat bind both ERα and ERβ withequal affinity) are known to haveanti-inflammatory activity that isthought to result from interferencewith the NF-κB signalling pathway.Douglas C. Harnish and colleaguestherefore set out to identify ER lig-ands that inhibited NF-κB withoutdisplaying any conventional oestro-genic functions and identified a
first-in-class pathway-selective groupof ER ligands.
The authors used cells transientlyexpressing ERα or ERβ and an NF-κB–luciferase reporter gene thatwas induced by interleukin-1β (IL-1β)to identify compounds that inhibitedNF-κB activity. The first compoundthey discovered was WAY-169916, anon-steroidal, orally active moleculethat inhibited 50% of IL-1β-stimu-lated NF-κB activity, without affectinga marker of conventional oestrogenicactivity. They then studied the path-way-selectivity of this compound invivo using a high-fat-diet mousemodel in which NF-κB and otherinflammatory genes are upregulatedin the liver. Daily treatment ofovariectomized mice with either17α-ethynyloestradiol (EE) or WAY-169916 significantly inhibited thehigh-fat-diet-induced expression ofthese genes, but, significantly, WAY-169916 did so without the accompa-nying oestrogenic effects observedwith EE.
The potential of WAY-169916 asan anti-inflammatory therapy forIBD was then demonstrated usingthe HLA-B27 transgenic rat model.
A computational approach that allows aquantitative assessment of the ability of agiven binding site of a protein to bind small‘leadlike’ molecules has been reported byHadjuk and colleagues in the Journal ofMedicinal Chemistry. Their studies, which arebased on nuclear magnetic resonance (NMR)screening data, also illustrate how ‘hot spots’on protein surfaces — regions that makemajor contributions to the binding energy inprotein–ligand complexes — can be identified.Such approaches should be particularly usefulfor novel genomics-derived targets, for whichinformation on small-molecule-binding sitesis typically limited.
NMR-based screening methods such as thatdescribed by Hadjuk et al. have a number ofstrengths. First, the perturbations to NMRsignals that are indicative of small-moleculebinding can be monitored for the wholeprotein, and so small-molecule binding to anyregion can in principle be detected.Consequently, both the affinity and the site of
small-molecule–protein interactions can becharacterized. Second, NMR-based screeningapproaches can detect small-molecule ligandsthat have considerably weaker affinities thanwould be detectable in standard high-throughput screens. These advantages makesuch approaches ideal for identifying andcharacterizing hot spots on protein surfaces.
To investigate both the capacity of proteinsto bind small molecules and the nature of thebinding sites, Hadjuk and colleagues analysedNMR-based screening data from 23 diverseprotein targets.Across all targets, the vastmajority of the hits identified in the screensbind to a known small-molecule-binding site,demonstrating the selectivity of proteinsurfaces to bind to ligands at only very specificlocations. Interestingly, a small number ofnovel potential hot spots were also identified.Furthermore, their data indicated that NMR-based screens can be used as a reliable guide tothe ‘druggability’ of a given protein targetbefore investing in the development ofcomplex biochemical assays, or even beforethe function of the protein is known.
As an alternative to performing an NMR-based screen against every potential proteintarget, it would be of great value to be able to
confidently predict that high-affinity leads canbe identified for a particular target.With this inmind, the authors analysed the protein pocketsand NMR screening data to try to understandthe factors that influence the observed hit rate.Using this analysis, they developed a simplecomputational model that included parametersdescribing the protein binding pocket, such assurface complexity, that could be used topredict with high accuracy the druggability ofprotein targets not used to construct the model.
As the authors conclude, the relationshipsderived between hit rate and binding-pocketparameters have important implications forthe understanding of fundamentalprinciples of molecular recognition, andshould facilitate quantitative comparativeanalyses of binding pockets for use in targetassessment and validation, virtual screeningand structure-based drug design.
Peter KirkpatrickReferences and links
ORIGINAL RESEARCH PAPER Hajduk, P. J., Huth, J. R. &Fesik, S. W. Druggability indices for protein targets derivedfrom NMR-based screening data. J. Med. Chem. 24 Feb2005 (doi:10.1021/jm049131r)FURTHER READING Rees, D. C. et al. Fragment-basedlead discovery. Nature Rev. Drug Discov. 3, 660–672 (2004) |Pellecchia, M. et al. NMR in drug discovery. Nature Rev. DrugDiscov. 1, 211–219 (2002)
Hitting the hot spots
S C R E E N I N G
Choosing the right path
N U C L E A R R E C E P TO R S
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)
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