Drug discovery and the human kinome: Recent trends

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<ul><li><p>Drug discovery and the human kinome: Re</p><p>Richard Eglen , Terry Reisine 1</p><p>Bio-discovery, 940 Winter St., Waltham, MA 02451-1457, United States</p><p>AllosterismProteinprotein iKinase translocatGrowth factor ag</p><p>g to the catalyticprovide a numberre selective, sinceeveloped to drugsctors that inhibiturthermore, theytivity. One hurdlet proteinproteinemsof identifying</p><p>allostericmodulators,major gains havebeenmade in identifying allosteric inhibitors and activators of the growtholuble tyrosine and serine/threonine kinases and some of these drugs are now in</p><p>Contents</p><p>1. Introduction . . . . . .2. Discovering small molecu3. Allosteric modulators of s</p><p>novel a. . .. . .. . .</p><p>Pharmacology &amp; Therapeutics 130 (2011) 144156</p><p>Contents lists available at ScienceDirect</p><p>Pharmacology &amp;</p><p>j ourna l homepage: www.e lsev1. Introduction</p><p>Protein kinases are a family of enzymes involved in signaltransduction in every human cell. The enzymes detect both externaland internal stimuli to cells and produce their functions by phosphor-ylating proteins. This process initiates and propagates information owto allow cells to respond to their changing environment. This family of</p><p>Abbreviations: ATP, adenosine triphosphate; AKT, v-akt murine thymoma viraloncogene homolog; BRAF, v-raf murine sarcoma viral oncogene homolog B1; BDNF,</p><p>brain-derived neurotrophic factor; CNS, central nervgrowth factor; EPO, erythropoietin; ERK, extracellular-Frster resonance energy transfer; IL, interleukin; Jtransducer and activator of transcription; MAP kinakinase; NGF, nerve growth factor; NMR, nuclear magnephosphoinositide-dependent kinase-1. Corresponding author at: Bio-discovery, PerkinElme</p><p>02451-1457, United States. Tel.: 781 663 5599; fax: 781E-mail address: richard.eglen@perkinelmer.com (R.</p><p>1 Terry Reisine, PhD is an independent consultant.</p><p>0163-7258/$ see front matter 2011 Elsevier Inc. Aldoi:10.1016/j.pharmthera.2011.01.007References . . . . . .4. Assays to discover5. Summary . . . .Acknowledgments . . 2011 Elsevier Inc. All rights reserved.</p><p>. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144les that act like large proteins . . . . . . . . . . . . . . . . . . . . . . . . . 145oluble protein kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146llosteric modulators of protein kinases . . . . . . . . . . . . . . . . . . . . . 148. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150</p><p>144145149153154154154proteins is essenleads to abnorma</p><p>The protein kthe pharmaceut2009, 2010). A laclinical developmto treat a widediabetes, immun</p><p>ous system; EGF, epidermalsignal-regulated kinase; FRET,AK-STAT, Janus kinase-signalse, mitogen-activated proteintic resonance; PDK1, PDK1, 3-</p><p>r, 940Winter St., Waltham, MA663 5984.Eglen).</p><p>l rights reserved.een employed to identify such compounds.</p><p>various stages of clinical trials. This review will focus on the discovery of novel allosteric modulators of proteinkinases and drug discovery approaches that have bfactor receptors as well as sto overcome in discovering tinteractions; generally difcunteractionsiononists/antagonists</p><p>compounds differ from ATP-centric drugs in that they do not compete with ATP for bindindomain, generally acting by inducing conformational changes tomodulate activity. They couldof advantages over more classical protein kinase drugs. For example, they are likely to be mothey bind to unique regions of the kinase andmay be useful in overcoming resistance that has dthat compete with ATP. They offer the ability of activating the kinases either by removing fakinase activity or by simply producing changes to the enzyme to foster catalytic activity. Fprovidemore subtlemodulation of kinase activity than simply blocking ATP access to inhibit ac</p><p>hese compounds is that allosteric modulators may need to inhibilt to accomplishwith smallmolecules. Despite the technical problA major new trend in drugs targeted at protein kinases is the discovery of allosteric ma b s t r a c ta r t i c l e i n f o</p><p>Keywords: odulators. Thesecent trends</p><p>Therapeutics</p><p>i e r.com/ locate /pharmtheratial for normal physiology and when dysfunctionall cellular activity and disease.inase family is a major target for drug discovery byical industry (Simpson et al., 2009; Eglen &amp; Reisine,rge number of protein kinase inhibitors are either inent or have been approved for marketing by the FDAvariety of diseases including cancer, inammation,odeciency and CNS disorders (see Eglen &amp; Reisine,</p></li><li><p>145R. Eglen, T. Reisine / Pharmacology &amp; Therapeutics 130 (2011) 1441562009, 2010). Many of these drugs have improved survival and qualityof life of cancer patients as well as in individuals suffering from othercomplications.</p><p>In general, kinase inhibitors have been classied into four differenttypes based on their mechanisms of action (see Zhang et al., 2009;Eglen &amp; Reisine, 2010). Type 1 inhibitors work via a classicalmechanism of action to block the access of ATP to the catalyticdomain of the kinase in a competitive manner. Type 2 inhibitorsinteract with kinases in a somewhat different manner, specicallybinding to the inactive form of the kinase to prevent the activationprocess, much in the manner of Gleevec. Type 3 inhibitors, which willbe the major focus of this review act via allosteric sites to block theactivity. As dened by Zhang et al. (2009) and others, allostericmodulators interact with A site distinct from the enzyme active site...[to] regulate[s] enzyme activity. This can mean interacting with sitesnear the active site but not within the catalytic domain or more distalsites such as regions involved in the regulatory subunit interactionwith the catalytic domain of the cAMP dependent protein kinase, oreven the growth factor binding sites on the N-terminal transmem-brane domain receptors that affect conformational changes in the C-terminal catalytic domain to affect enzyme activity. Thus, allosterismencompasses a large gamut of mechanisms of kinase regulation.Finally, type 4 inhibitors are primarily covalent inhibitors of kinasesthat target active sites.</p><p>While much of the classical discovery of the protein kinase drugshas targeted regions around the ATP binding sites to identifyinhibitors, emerging trends have focused on identifying allostericmodulators. The interest in developing allosteric modulators is thatsuch drugs may provide unique advantages over more classicallydeveloped compounds (Li et al., 2004; Noble et al., 2004; Zhang et al.,2009; Eglen &amp; Reisine, 2009, 2010).</p><p>First, they offer the possibility of greater selectivity because theytarget sites in kinases more unique in sequence and structure thanthose compounds that bind to the regions near the ATP bindingdomain, which are generally more conserved amongst proteinkinases. Greater selectivity might be expected to reduce the side-effects compared to the more pervasive kinase inhibitors. Theselectivity of the allosteric modulators can also provide approachesto differentially regulate the subtypes of a kinase within a subfamily,which may have similar or the same substrates and high overallsequence similarity.</p><p>Secondly, allosteric modulators hold a promise in selectivelytargeting mutant forms of disease causing protein kinases and inovercoming resistance of the kinases to the ATP binding competitivedrugs. As described elsewhere (Cohen, 2002; Bardelli et al., 2003;Dancy &amp; Sausville, 2003; Noble et al., 2004; Pearson et al., 2006; Zhanget al., 2009), many diseases, notably proliferative diseases such ascancer, are caused in part or completely by mutations that generateconstitutive kinase activity. The mutations can change the conforma-tion of the kinase and drugs that selectively interact with the mutantform of the kinase may block the activity of the disease causingenzyme while having less or no effect on the natural form of enzymepreserving normal function.</p><p>For example, this has been shown to be the case for the serine/threonine kinase BRAFV600E which causes almost half of the malignantmelanomas and is the most common disease causing mutant kinase(Tsai et al., 2008). The mutation causes constitutive activity of thekinase and continuous stimulation of the downstream MAPkinase/ERK signaling pathway. The small molecule drug PLX4032 (see Fig. 1for structure) selectively inhibits BRAFV600E and is much less potentagainst the wild type kinase or any other kinase and blocks theMAPkinase/ERK signaling pathway only in cells expressing themutant kinase both in vitro and in vivo. This drug selectively targetsthe disease causing kinase providing an incredible level of specicityover any other protein in the body and this drug. PLX4032 is currently</p><p>being tested in the clinic and has shown great promise in treatingmelanomas which have previously not been effectively treated byother drugs (Flaherty et al., 2009). While this drug is not allosteric inaction, the allosteric inhibitors are more likely to show this prolebecause in general they are much more selective in targeting kinasespecic conformations than the more classical ATP competitiveinhibitors.</p><p>Thirdly, while most of the drug discovery activities against proteinkinases have focused on identifying the inhibitors of kinase activityand ATP binding, allosterism allows for the identication ofcompounds that could result in activators of kinases. This may beparticularly relevant for the family of receptor kinases such as thegrowth factor receptors, where binding of large proteins to theextracellular domains induces conformational changes that activatethe intracellular catalytic activity of the kinase. Some of these growthfactors, such as BDNF and NGF, have an important therapeutic value inabrogating neurodegeneration due to a supporting role in neuronalsurvival and blocking disease progression in Alzheimer's andParkinson's diseases. Large growth factors are not generally gooddrug candidates and are difcult to optimize for CNS penetration.Novel technologies have now been developed to allow for theidentication of small molecules that bind to similar regions of thegrowth factors on their receptors and cause kinase activation,providing approaches to identify new growth factor receptormodulators with optimal pharmacokinetic properties.</p><p>Finally, small molecule allosteric modulators can provide subtleregulation of kinases controlled by multiple endogenous factors. Forexample, the cyclin dependent kinases (CDK) are regulated by bothendogenous protein activators (cyclins) and inhibitors (CDKI) (Roy &amp;Sausville, 2001). Small molecules could affect the balance of CDKcontrol by these endogenous factors to cause cell apoptosis,something not easily done with the ATP-centric drugs.</p><p>Developing small molecule regulators of the endogenous factorscontrolling protein kinases can require the use of approaches toidentify compounds that inhibit proteinprotein interaction. Onceconsidered a difcult, if not impossible approach, numerous exampleshave in fact become available (White et al., 2008; Arkin &amp; Whitty,2009). New technologies have been adapted to discover proteinprotein inhibitors (PPI) in a high throughput screening (HTS) format,as discussed below. Furthermore, there is good evidence thatallosteric sites are druggable (Hajduk et al., 2005; Fuller et al.,2009) and that some of the same structurefunction analysis theindustry has employed to discover ATP binding site inhibitors canactually be used to develop the allosteric regulators.</p><p>The focus of this review, rather than discussing protein kinase drugdiscovery as a whole, will attempt to describe the innovations thatprovide the basis of drug development that targets allostericregulators. We will be liberal in the use of the term allostericmodulator to encompass factors that affect kinase activity throughmechanisms independent of a direct ATP binding site competition toinclude molecules binding to sites outside of the catalytic domain,such as the growth factors that affect kinase conformation or dimerformation to regulate activity. Importantly, using the knowledge ofkinase function and its regulation, new technologies have beendeveloped to exploit the utility of these advances to foster a newgeneration of drugs for the future that may not only provideadvantages over the drugs developed to date, but may lead to newcompounds as tools for dening their biological function.</p><p>2. Discovering small molecules that act like large proteins</p><p>Receptor tyrosine kinases (RTKs) are a major subfamily of kinasesthat mediate the biological effects of many growth factors. Unlike thesoluble kinases, this family consists of the integral membrane proteinscontaining an extracellular domain that binds the growth factors andintracellular domains which contain the tyrosine kinase catalytic</p><p>activity. The binding of the growth factor to the allosteric regions in</p></li><li><p>the extracellular domain induces oligomerization of the receptor andconformational changes to increase catalytic activity to induce auto-phosphorylation of the receptor itself to heighten the catalytic activityas well as the phosphorylation of downstream substrates includingtranscription factors to affect long term cell activity.</p><p>The allosteric regions can be employed as targets for the discoveryof therapeutics to block activity. In fact, most drug discoveriestargeting the RTKs have focused on identifying inhibitors since theover-expression or over-activity of these receptors is associated witha number of proliferative diseases. This is most clearly seen with theEGF receptor (EGFR) which is linked to breast cancer (DiGiovannaet al., 2005).</p><p>Antibodies have been developed to target the allosteric regions ofthe extracellular domains of EGFR as therapeutics to block the growthfactor activation and in the case of EGFR, antibodies have beendeveloped to block other allosteric sites to prevent the interaction ofmonomeric forms of the EGFR with other subunits (Dancy &amp; Sausville,2003; Piccart-Gebhart et al., 2005). Small molecule inhibitors havealso been developed such as Iressa (Barker et al., 2001) and Tarceva,(Perez-Soler et al., 2004) but these focus on the catalytic domain ofthe kinase, not binding to the allosteric sites. Generally, smallmolecule allosteric inhibitors or activators of these receptor-kinaseshave not been identied.</p><p>2.1. Allosteric activators of growth factor receptors</p><p>While most therapeutic approaches have attempted to identify</p><p>brain permeability. Clearly, the development of small moleculeagonists of BDNF could overcome hurdles in using this protein as aCNS therapeutic to treat brain diseases.</p><p>BDNF induces its action via the receptor kinase tropomyosin-related kinase (Trk) receptor B (TrkB)(Chao, 2003). While littleinformation existed on the regions of TrkB involved in BDNF bindingand activation, structural information is available on the regions ofBDNF involved in TrkB activation. In fact,...</p></li></ul>