drug discovery and the human kinome: recent trends

13
Drug discovery and the human kinome: Recent trends Richard Eglen , Terry Reisine 1 Bio-discovery, 940 Winter St., Waltham, MA 02451-1457, United States abstract article info Keywords: Allosterism Proteinprotein interactions Kinase translocation Growth factor agonists/antagonists A major new trend in drugs targeted at protein kinases is the discovery of allosteric modulators. These compounds differ from ATP-centric drugs in that they do not compete with ATP for binding to the catalytic domain, generally acting by inducing conformational changes to modulate activity. They could provide a number of advantages over more classical protein kinase drugs. For example, they are likely to be more selective, since they bind to unique regions of the kinase and may be useful in overcoming resistance that has developed to drugs that compete with ATP. They offer the ability of activating the kinases either by removing factors that inhibit kinase activity or by simply producing changes to the enzyme to foster catalytic activity. Furthermore, they provide more subtle modulation of kinase activity than simply blocking ATP access to inhibit activity. One hurdle to overcome in discovering these compounds is that allosteric modulators may need to inhibit proteinprotein interactions; generally difcult to accomplish with small molecules. Despite the technical problems of identifying allosteric modulators, major gains have been made in identifying allosteric inhibitors and activators of the growth factor receptors as well as soluble tyrosine and serine/threonine kinases and some of these drugs are now in various stages of clinical trials. This review will focus on the discovery of novel allosteric modulators of protein kinases and drug discovery approaches that have been employed to identify such compounds. © 2011 Elsevier Inc. All rights reserved. Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Discovering small molecules that act like large proteins . . . . . . . . . . . . . . . . . . . . . . . . . 3. Allosteric modulators of soluble protein kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Assays to discover novel allosteric modulators of protein kinases . . . . . . . . . . . . . . . . . . . . . 5. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction Protein kinases are a family of enzymes involved in signal transduction in every human cell. The enzymes detect both external and internal stimuli to cells and produce their functions by phosphor- ylating proteins. This process initiates and propagates information ow to allow cells to respond to their changing environment. This family of proteins is essential for normal physiology and when dysfunctional leads to abnormal cellular activity and disease. The protein kinase family is a major target for drug discovery by the pharmaceutical industry (Simpson et al., 2009; Eglen & Reisine, 2009, 2010). A large number of protein kinase inhibitors are either in clinical development or have been approved for marketing by the FDA to treat a wide variety of diseases including cancer, inammation, diabetes, immunodeciency and CNS disorders (see Eglen & Reisine, Pharmacology & Therapeutics 130 (2011) 144156 Abbreviations: ATP, adenosine triphosphate; AKT, v-akt murine thymoma viral oncogene homolog; BRAF, v-raf murine sarcoma viral oncogene homolog B1; BDNF, brain-derived neurotrophic factor; CNS, central nervous system; EGF, epidermal growth factor; EPO, erythropoietin; ERK, extracellular-signal-regulated kinase; FRET, Förster resonance energy transfer; IL, interleukin; JAK-STAT, Janus kinase-signal transducer and activator of transcription; MAP kinase, mitogen-activated protein kinase; NGF, nerve growth factor; NMR, nuclear magnetic resonance; PDK1, PDK1, 3- phosphoinositide-dependent kinase-1. Corresponding author at: Bio-discovery, PerkinElmer, 940 Winter St., Waltham, MA 02451-1457, United States. Tel.: 781 663 5599; fax: 781 663 5984. E-mail address: [email protected] (R. Eglen). 1 Terry Reisine, PhD is an independent consultant. 144 145 149 153 154 154 154 0163-7258/$ see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.pharmthera.2011.01.007 Contents lists available at ScienceDirect Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/pharmthera

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Pharmacology & Therapeutics 130 (2011) 144–156

Contents lists available at ScienceDirect

Pharmacology & Therapeutics

j ourna l homepage: www.e lsev ie r.com/ locate /pharmthera

Drug discovery and the human kinome: Recent trends

Richard Eglen ⁎, Terry Reisine 1

Bio-discovery, 940 Winter St., Waltham, MA 02451-1457, United States

Abbreviations: ATP, adenosine triphosphate; AKT,oncogene homolog; BRAF, v-raf murine sarcoma viralbrain-derived neurotrophic factor; CNS, central nervgrowth factor; EPO, erythropoietin; ERK, extracellular-Förster 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

02451-1457, United States. Tel.: 781 663 5599; fax: 781E-mail address: [email protected] (R.

1 Terry Reisine, PhD is an independent consultant.

0163-7258/$ – see front matter © 2011 Elsevier Inc. Aldoi:10.1016/j.pharmthera.2011.01.007

a b s t r a c t

a r t i c l e i n f o

Keywords:

AllosterismProtein–protein interactionsKinase translocationGrowth factor agonists/antagonists

A major new trend in drugs targeted at protein kinases is the discovery of allosteric modulators. Thesecompounds differ from ATP-centric drugs in that they do not compete with ATP for binding to the catalyticdomain, generally acting by inducing conformational changes tomodulate activity. They could provide a numberof advantages over more classical protein kinase drugs. For example, they are likely to be more selective, sincethey bind to unique regions of the kinase andmay be useful in overcoming resistance that has developed to drugsthat compete with ATP. They offer the ability of activating the kinases either by removing factors that inhibitkinase activity or by simply producing changes to the enzyme to foster catalytic activity. Furthermore, theyprovidemore subtlemodulation of kinase activity than simply blocking ATP access to inhibit activity. One hurdleto overcome in discovering these compounds is that allosteric modulators may need to inhibit protein–proteininteractions; generally difficult to accomplishwith smallmolecules. Despite the technical problemsof identifyingallostericmodulators,major gains havebeenmade in identifying allosteric inhibitors and activators of the growthfactor receptors as well as soluble tyrosine and serine/threonine kinases and some of these drugs are now invarious stages of clinical trials. This review will focus on the discovery of novel allosteric modulators of proteinkinases and drug discovery approaches that have been employed to identify such compounds.

v-akt murine thymoma viraloncogene homolog B1; BDNF,ous system; EGF, epidermalsignal-regulated kinase; FRET,AK-STAT, Janus kinase-signalse, mitogen-activated proteintic resonance; PDK1, PDK1, 3-

r, 940Winter St., Waltham, MA663 5984.

Eglen).

l rights reserved.

© 2011 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1442. Discovering small molecules that act like large proteins . . . . . . . . . . . . . . . . . . . . . . . . . 1453. Allosteric modulators of soluble protein kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1464. Assays to discover novel allosteric modulators of protein kinases . . . . . . . . . . . . . . . . . . . . . 1485. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

144145149153154154

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150154

1. Introduction

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 flowto allow cells to respond to their changing environment. This family ofproteins is essential for normal physiology and when dysfunctionalleads to abnormal cellular activity and disease.

The protein kinase family is a major target for drug discovery bythe pharmaceutical industry (Simpson et al., 2009; Eglen & Reisine,2009, 2010). A large number of protein kinase inhibitors are either inclinical development or have been approved for marketing by the FDAto treat a wide variety of diseases including cancer, inflammation,diabetes, immunodeficiency and CNS disorders (see Eglen & Reisine,

145R. Eglen, T. Reisine / Pharmacology & Therapeutics 130 (2011) 144–156

2009, 2010). Many of these drugs have improved survival and qualityof life of cancer patients as well as in individuals suffering from othercomplications.

In general, kinase inhibitors have been classified into four differenttypes based on their mechanisms of action (see Zhang et al., 2009;Eglen & 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, specificallybinding 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 defined 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.

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 & Reisine, 2009, 2010).

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.

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 & 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.

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 specificityover any other protein in the body and this drug. PLX4032 is currentlybeing tested in the clinic and has shown great promise in treating

melanomas 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 profilebecause in general they are much more selective in targeting kinasespecific conformations than the more classical ATP competitiveinhibitors.

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 identification 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 difficult to optimize for CNS penetration.Novel technologies have now been developed to allow for theidentification 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.

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 &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.

Developing small molecule regulators of the endogenous factorscontrolling protein kinases can require the use of approaches toidentify compounds that inhibit protein–protein interaction. Onceconsidered a difficult, if not impossible approach, numerous exampleshave in fact become available (White et al., 2008; Arkin & Whitty,2009). New technologies have been adapted to discover protein–protein 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 structure–function analysis theindustry has employed to discover ATP binding site inhibitors canactually be used to develop the allosteric regulators.

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 defining their biological function.

2. Discovering small molecules that act like large proteins

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 catalyticactivity. The binding of the growth factor to the allosteric regions in

146 R. Eglen, T. Reisine / Pharmacology & Therapeutics 130 (2011) 144–156

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.

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).

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 & 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 identified.

2.1. Allosteric activators of growth factor receptors

While most therapeutic approaches have attempted to identifydrugs that inhibit RTKs, there is a large family of the growth factorreceptor kinases for which allosteric activators could have importanttherapeutic uses. Specifically, BDNF is known to support and facilitateneuronal growth and survival (Kaplan & Miller, 2000; Chao, 2003;Huang & Reichardt, 2003). Loss of BDNF in the brain has been linked toneurodegeneration in a number of diseases including Alzheimer's,Huntington's and Parkinson's diseases (Fumagalli et al., 2006; Zuccato& Cattaneo, 2007; Schindowski et al., 2008). The reversal orattenuation of neuronal loss has been found in the animal models ofthese diseases after the BDNF treatment. BDNF is not easy to employas a therapeutic to treat CNS diseases because of the proteins' limited

N NH

O

F

FNH

S

Cl

PLX 4032 - CID42611257

NH

OO

NH

OOH

NHO

OH

LM22A-4

Fig. 1. Structures of allosteric modulators.Structures of a number of allosteric modulators areare included. Others are taken from the articles from which they are published and the ref

brain permeability. Clearly, the development of small moleculeagonists of BDNF could overcome hurdles in using this protein as aCNS therapeutic to treat brain diseases.

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, the peptides modeled tosome of the loop regions of BDNF have been made that mimic BDNFactivity (O'Leary & Hughes, 2003; Williams et al., 2005). Furthermorechimeric BDNF–NGF proteins revealed discrete regions of BDNFinvolved in the activation of TrkB, in contrast to TrkA, the receptorthat mediates the NGF effects (Ibanez et al., 1991, 1993).

Using this information, Massa et al. (2010) developed a pharmaco-phore model of the regions of BDNF needed for activity, and employedvirtual high throughput screening to identify small molecules withstructural similarities of the BDNF pharmacophore. They then screenedsmall sets of compounds in low throughput screening for the BDNFactivity, first by their ability to promote the survival of primary brainneurons in culture, then by their ability to stimulate the TrkB receptor toinduce the phosphorylation of its activation loop. They also tested smallmolecule allosteric agonists for their selectivity in stimulating TrkBsignaling pathways to promote cell survival in vitro.

From these studies, a highly effective small molecule allosteric agonistat TrkB was identified, LM22A-4 (see Fig. 1 for structure). It was tested inmultiple disease models of neuronal protection and shown to be aseffective as BDNF. In particular, LM22A-4 protected animals in a model oftraumatic brain injury (TBI), and significantly improved motor perfor-mance. This is important because presently there is no treatment of TBInor is there any effective therapeutic to block neurodegeneration in a hostof CNS diseases.

Interestingly, TrkB, like other RTKs requires dimerization for fullactivation, both for the autophosphorylation and optimal catalyticactivity. Dimers of TrkB form when two BDNF molecules are boundand crosslinked to the receptor complex. In fact, the requirement fordimer formation is one characteristic of the growth factor receptorsignaling that would be expected to present a hurdle in use of thesmall molecule ligands as growth factor agonists, since howwould the

NH

NH

O

O

OHOH

L-783,281 CID:3013166

O2

H

described. Some structures were taken from the Pubmed database and the CID numberserences included.

NH

NH

O

O

OHOH

L-783,281 CID:3013166

NH

CO2CH3

O

N

NH NH2

CF3CO2H

Ro26-4550 CID16760522

OCl

ClOHO2C

NNN

O

NH

OCH3 NH

NH2

NH

SP4206

Fig. 1 (continued).

147R. Eglen, T. Reisine / Pharmacology & Therapeutics 130 (2011) 144–156

small molecules physically bridge the TrkB subunits. The finding that asmall molecule can activate TrkB and induce phosphorylation anddownstream signaling either implies that the small molecule caninduce conformational changes in the receptor to activate the kinasecatalytic domain independent of the dimer formation or somehow thesmall molecule agonist induces dimerization to activate signaling. Infact, studies by Tian et al. (1998) have indicated that small moleculescan induce the dimerization of the growth factor receptors such as thegranulocyte colony stimulating factor (G-CSF) receptor to increasedownstream signaling pathways.

Importantly, the studies of Massa et al. (2010) suggest that it ispossible to discover small molecule allosteric activators of RTKs usinga combination of pharmacophore modeling, 3D database and virtualhigh throughput screening and low throughput functional screening.The work also may provide the foundation for the further develop-ment of small molecule BDNF ligands that could have widespreadutility in treating neurological disorders. The study implies that thereis no reason that the approach could not be employed for othergrowth factors to identify agonists to provide the development of anew generation of pharmacological agents both to study the function

N

N

N

NH

O

OH

O

Ro 08-2750

N

N

NH

CONH2

OCF3

GNF-2 CID5311510

NH2N

NH N

NO

MK-2206

CH3

CH3

N

NN

N

NH

NCH3 CH3

Akt-I-1

CH3

N

N

CH3

NH2

Akt-I-1,2

Fig. 1 (continued).

148 R. Eglen, T. Reisine / Pharmacology & Therapeutics 130 (2011) 144–156

of these receptors in vivo and potentially to the transition into newpotential therapeutics.

While studies with BDNF focused on developing ligands based onmodeling of the growth factor, Zhang and associates (Zhang et al., 1999;Qureshi et al., 2000; Salituro et al., 2001) identified allosteric activatorsof the insulin receptor kinase using more conventional receptorscreening approaches testing natural product libraries for agonists.The insulin receptor, like other RTKs consists of an extracellular domain

that binds insulin and intracellular domains with the kinase activity. Toidentify the small molecule demethylasterriquinone B-1 (L-783,281)(see Fig. 1 for structure) and its analogs as allosteric activators of thereceptor, Zhang et al. (1999) expressed the recombinant receptor inCHO cells, screened natural products and measured insulin receptorkinase activity as a response. L-783,281 stimulated the activity of thecloned insulin receptor with EC50 values of 3–6 uM. Furthermore,L-783,281 effectively stimulated insulin receptor activity in the liver andactivated downstream signaling pathways of the insulin receptorincluding PI-3-kinase and phosphorylation of the Akt kinase. Theallosteric activator had limitedor noeffect on the IGF-I receptor, EGFR orPDGF receptor kinase activity, suggesting it was selective for insulinreceptor activation.

Like insulin, L-783,281 stimulated insulin uptake into adipocytesand muscle cells in vitro. More importantly, it lowered glucose in thediabetic db/db mice and corrected the hyperinsulinemia. Also, in theob/ob mice, a model of obesity in which the mice exhibit extremehyperinsulinemia and hyperglycemia, L-783,281 suppressed the ele-vated insulin levels, suggesting that the compoundmay be effective intreating type 2 diabetes.

Mechanism studies suggest that L-783,281 binds to the betasubunit of the insulin receptor containing the tyrosine kinase domain,rather than acting via the extracellular insulin binding domain. Thiswas suggested by studies showing that the small molecule did notdisplace insulin binding to the receptor, could activate the kinasedomain in cells, and activated a receptor chimera consisting of thetyrosine kinase domain linked to an insulin receptor-related receptorthat does not respond to insulin. Studies by Qureshi et al. (2000)indicated that these insulin allosteric activators are likely to bind tothe inactivate insulin receptor kinase domain to induce conforma-tional changes to remove auto-inhibition of the kinase activity toallow ATP access to the active site.

These studies were important because they were one of the firstevidences of the small molecule allosteric activators of an RTK.Furthermore, L-783,281 and some of its analogs are orally availableproviding a potential insulin substitute for the treatment of diabetes.In addition, because of their mode of action in bypassing theextracellular insulin binding domain, these small molecule allostericactivators may be useful in treating some forms of insulin resistancewhich are known to occur due to either mutations in the extracellularinsulin binding domain that prevent insulin binding or because of thereduced expression of the insulin binding domain. In fact, studies byLi et al. (2001) showed that these compounds effectively stimulatedmutated forms of the insulin receptor kinase which were no longerresponsive to insulin. Lastly, while the method employed waslaborious and costly, these studies provide an approach to possiblyidentify small molecular allosteric activators of other receptor kinasesand indicate that direct interaction with the same sites as the growthfactor are not necessary for activating these receptor kinases.

Thus, in summary, despite the large size of the protein growthfactors and potentially large number of sites on the receptorextracellular domains to which they may bind to activate kinases, itis possible to design and discover small molecule agonists at thesereceptors to mimic the actions of the growth factors. This has shownto be the case for BDNF (Massa et al., 2010), EPO (Qureshi et al., 1999),growth hormone receptor (Guo et al., 2000) and G-CSF (Tian et al.,1998) receptors. This suggests that it could be possible to use theallosteric sites to discover small molecule drugs that stimulate thesepathways which could provide important therapeutic advantagesover the use of protein therapeutics.

2.2. Allosteric inhibitors of growth factor receptors

One approach to inhibit the growth factor receptors via allostericmechanisms is to prevent the activation either by blocking the dimerformation and/or preventing the conformational changes needed for

149R. Eglen, T. Reisine / Pharmacology & Therapeutics 130 (2011) 144–156

catalytic activation. Antibodies can block the dimer formation bypreventing growth factor binding to the receptors. Generally, this ismore difficult to accomplish with the small molecules because thegrowth factors are generally large polypeptides and have multiplepoints of contact with the extracellular domains of RTKs, making itdifficult to design small molecules that could block enough sites todiminish protein–protein interaction.

One approach that can be used to design the inhibitors of growthfactor binding to RTKs employs pharmacophore modeling, much likethat described above in studies byMassa et al. (2010). A technology thatwas first employed on IL-2 receptors, but which can be applied to RTKsto design allosteric inhibitors was described by Arkin andWells (2004).IL-2 receptors are not RTKs. However, cytokines, like IL-2 are proteinsand theybind to theN-terminal regionsof the transmembrane spanningIL-2 receptors. Furthermore, the binding of IL-2 induces oligomerizationto activate the cytokine receptor,much like that found in RTKs. Thus, thebasics of IL-2 binding and activation of its receptor is similar to thatfound withmany RTKs and therefore approaches used to develop smallmolecule inhibitors of IL-2 binding could have applications indeveloping small molecule inhibitors of RTKs.

Arkin and Wells (2004) reasoned that because structural informa-tion identified “hot spots”, of limited regions of contact between IL-2and its receptor, one could employ the structural information of thisinteraction to design pharmacophores of IL-2 that could lead to smallmolecule inhibitors of the protein–protein interaction to block thebinding of IL-2 to its receptor to prevent dimer formation. In fact,Emerson et al. (2003) employedNMR to identify residues in IL-2 neededfor binding to the IL-2 receptor (IL-2R). Using this information, thisgroup designed a rigid peptidomimetic that simulated the IL-õ2pharmacophore and showed that it blocked the binding of IL-2 to itsreceptor. They (Tilley et al., 1997; Emerson et al., 2003) also designed asmall molecule acylphenylalanine derivative corresponding to pharma-cophore of IL-2 and identified a smallmolecule compound (Ro26-4550)(see Fig. 1 for structure) that blocked IL-2 binding to the IL-2R with anIC50 of 3 uM. Interestingly, the NMR studies showed that this relativelypotent IL-2 inhibitor is bound to IL-2 itself, rather than directlyinteracting with the receptor, suggesting the molecule masked siteson the IL-2 needed for binding to its receptor.

Based on these findings, Wells and associates developed a tetheringtechnology to identify potent small molecule inhibitors of IL-2. They(Arkin et al., 2003) mutated IL-2 by substituting cysteine residuesaround the Ro26-4550 binding site in IL-2. They then screened themutant IL-2 with a library of small molecules tethered to disulfidelinkers. Compounds in the library that bound to the Ro26-4550 sitewithreasonable affinitywere able to form disulfide bridgeswith the cysteineresidues incorporated into the receptor and bound ligands weredetermined by mass spectroscopy. In follow up studies using thistechnology, Raimundo et al. (2004) and Thanos et al. (2006) identified asmallmolecule compound (SP4206) (see Fig. 1 for structure) thatboundto IL-2with a 60 nMaffinity.Molecular dynamic studies suggest that thecompound binds to the low-energy conformations of IL-2 (Thanos et al.,2006). SP4206 blocked IL-2 binding to IL-2R with 70 nM affinity.Importantly, it blocked IL-2 induced STAT phosphorylation in cellsexpressing IL-2R with an affinity of 3 uM.

Interestingly, this concept of designing small molecules that bind tocytokines to block the actions of these proteins, has in fact beenemployed to identify small molecule allosteric inhibitors of the NGFregulated RTKs. Niederhauser et al. (2000) identified a small molecule,Ro 08-2750 (see Fig. 1 for structure) that binds to the NGF dimers.Importantly, the NGF dimers bind to TrkA and p75NTR. Both receptorsform dimers in response to the NGF binding with TrkA receptoractivation promoting survival of neurons while the activation of p75NTR

causes apoptosis and cell death. Niederhauser et al. (2000) showed thatat low concentrations, Ro 08-2750 interacted with the NGF to block thebinding of NGF to p75NTR but not to TrkA. As a consequence, the NGFinduced apoptosis through its activation of p75NTR was abolished while

the ability of NGF to promote neuronal survival and neurite formationthrough the stimulation of TrkAwas not affected. The authors proposedthat Ro 08-2750 produced conformational changes in NGF presumablyto reduce the epitopes needed for binding to the p75NTR whilemaintaining the pharmacophore needed for binding to TrkA. As aconsequence, Ro 08-2750 can be viewed as an allosteric modulator thatmodifies the NGF to selectively activate an RTK without affecting othersignaling pathways, much like a designer drug.

Thus, one approach to allostericallymodulate RTKs is to devise smallmolecules that bind to the pharmacophore of the growth factor thatnormally regulates the RTK rather than devising small moleculestargeting the RTK itself. Furthermore, as shown in studies byMaliartchouk et al. (2007), small peptidomimetics based on thepharmacophore of the growth factors, NGF in this case, can be designedthat directly stimulate TrkA topromoteneuronal survival indicating thatit should be possible to discover small molecule agonists that directlybind to the extracellular domain of the TrkA to allosterically increaseRTK activity. Similarly, one might expect the same approach to be usedto design smallmolecule allosteric inhibitors that prevent the binding ofthe growth factor.

A second approach to develop allosteric inhibitors of RTKs focusedat interacellular sites is to block the interaction of the catalyticdomains of the RTK dimers to prevent autophosphorylation andactivation. This in fact was the approach used by Zhang et al. (2007) todevelop novel allosteric inhibitors of the EGF receptor. For thisreceptor, dimers form when EGF binds to the extracellular domain ofthe receptor. When this happens, the catalytic domains interact andthe C-terminal region of one monomer binds to the N-terminal regionof the other catalytic domain of the other to induce activity. Anendogenous protein, mitogen-induced gene 6 (MIG6), binds to theC-terminal region of the kinase domains preventing the interaction ofthe dimer catalytic domains and prevents activation. Importantly, themutant forms of the EGF receptor that are constitutively active stillrequire this asymmetric dimer formation for activity and the processis regulated by MIG6. Zhang et al. (2007) suggested that smallmolecules simulating the actions of MIG6 could provide a newgeneration of anti-cancer drugs highly selective for the EGF receptors.In fact, follow up studies have further defined the interaction of thecatalytic domains of the EGF receptors using both crystallography andmolecular simulation studies providing structural basis for developingselective small molecule inhibitors that could be effective in treatingEGF receptor related cancers (Jura et al., 2009a, 2009b).

In summary, if structural and mutagenesis data exists on anindividual RTK and/or its growth factor, it should be possible toemploy the Tethering technology (Arkin & Wells, 2004) as well asother structural approaches described above to design and developsmall molecule allosteric inhibitors against a number of RTKs. Thetethering approach is a unique technology that can be employed todevelop small molecule modulators of the protein–protein interac-tion. It has been employed to discover novel allosteric regulators ofthe cytokine receptors and enzymes such as the caspases (Hardy et al.,2004) and should be amenable for developing inhibitors of RTKsproviding a rational approach for drug discovery against this family ofkinases and also providing a new generation of drugs to treatdisorders involving over active growth factor and their receptors suchas in the case of proliferative disorders like cancer and inflammationas well as CNS diseases where certain growth factors and cytokinescause neurodegeneration and cell death.

3. Allosteric modulators of soluble protein kinases

3.1. Allosteric modulators of the tyrosinekinase Bcr–Abl overcome Gleevec resistance

While the development of allosteric modulators against RTKsholds great promise in the future for the development of novel drugs,

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extensive studies have also focused on developing allosteric inhibitorsand activators of soluble tyrosine and serine/threonine kinases. One ofthe more successful targets that have led to clinically useful drugs isthe –Bcr-Abl fusion protein, which has been a focus for novel tyrosinekinase drug discovery over the last few decades. The Bcr fusion to Ablkinase creates constitutive activity and causes chronic myelogenousleukemia (CML). Gleevec, a novel type 2 kinase inhibitor wasoriginally targeted to this mutant kinase (Zhang et al., 2009). It wasone of the first molecules found to bind near the ATP binding region ofthe inactive form of this or any kinase. Its binding prevented theopening of the activation loop needed for catalytic activity thusforcing the kinase to remain in an inactive state. In fact, Gleevecselectively binds to the inactivate state of this mutant enzyme andwas one of the first drugs approved using this unique mechanism ofaction.

While in treating CML and approved by FDA, some advanced–stagepatients relapse as a result of the emergence of clones expressingGleevec-resistant forms of Bcr–Abl (Gorre et al., 2001; Shah et al.,2004). Specifically, some mutations occur in the kinase to reduce thebinding of Gleevec. This can be a particular problem in anti-cancerdrug development since cells have an incredible ability to exertmechanisms to resist drug effects. It is as if cells can generate multipleforms of a particular kinase with subtle mutations that do not affectthe activity but seem predestined to overcome any chemical forms ofinhibition. When a drug like Gleevec inhibits Bcr–Abl, other clones towhich Gleevec can't affect take over the activity.

In a continuing battle to find inhibitors of Bcr–Abl, the crystalstructure analysis of Gleevec–Bcr–Abl binding was used as a basis, toidentify the newer Bcr–Abl inhibitors nilotinib (AMN107) anddasatinib (BMS-354825) which have been approved by FDA (Shahet al., 2004;Weisberg et al., 2005). These compounds interact with theATP-binding region of the Bcr–Abl with much higher affinity thanGleevec and were effective in inhibiting Bcr–Abl with mutations thatconfer Gleevec resistance.

However, even with these newer drugs, some patients becomeresistant to treatment because of the ‘gatekeeper’ T315I mutation,located in the center of the ATP-binding cleft (Bradeen et al., 2006;Zhang et al., 2010). This mutation, within the ATP binding sitesubstitutes a large isoleucine for the threonine needed for Gleevecbinding but does not affect ATP binding, so that Gleevec and thenewer drugs developed don't work at inhibiting this mutant kinase.

Using an entirely different approach to those employed indeveloping previous Bcr–Abl inhibitors, Adrian et al. (2006) andZhang et al. (2010) developed GNF-2 (see Fig. 1 for structure) andGNF-5, which are allosteric, non-ATP competitive inhibitors of Bcr–Abl. GNF-2 was identified in an unbiased cytotoxicity assays usingthe Bcr–Abl expressing cells. GNF-2 inhibited Bcr–Abl activity andreduced Stat5 tyrosine phosphorylation in cells but did not affect theBcr–Abl kinase activity in vitro nor did it affect the activity of anumber of other kinases in vitro. It did bind to the Bcr–Abl in vitro butdid not affect the binding of ATP or Gleevec to the kinase. GNF-2 bindsto the myristoyl pocket in Bcr–Abl and its binding to the kinase wasselectively blocked in the competition assays by myristoylatedpeptides. Furthermore, the mutations in the myristoylated pocketprevented GNF-2 from inhibiting the proliferation of cells expressingthe mutant kinase but did not affect the inhibitory ability of Gleevec.Subsequent crystal structure analysis and NMR spectroscopy haveshown that GNF-2 binds to the myristoylated pocket while Gleevecbound near the ATP binding domain. Importantly, GNF-2 allosterismdid not affect Gleevec binding but facilitated Gleevec's ability toinhibit Bcr–Abl. GNF-2 was found to produce conformational changesin the kinase to preserve the inactive form of the enzyme to reduceATP binding as assessed through the use of hydrogen-exchange massspectroscopy. Furthermore, GNF-5, an analog of GNF-2, with Gleevecproduced additive inhibition of the kinase and overcame resistanceconferred by the T315I mutation as well as other mutations. GNF-5

was also effective in a murinemodel of the Bcr–Abl induced leukemia.The use of these novel allosteric inhibitors not only provides anapproach to treat CML more effectively than presently available, butalso supports the utility of the allosteric modulators in overcomingresistance to more classical protein kinase drug inhibitors.

3.2. Allosteric inhibitors of the serine/threonine kinase, Akt

Akt consists of a family of three serine/threonine protein kinaseswhich have high sequence similarity, especially in their catalyticdomains. The enzymes have a critical role in proliferative diseasessuch as cancer and have been a major target of the pharmaceuticalindustry in its efforts to develop newer and more effective chemother-apeutic agents (Carnero et al., 2008; Tokunaga et al., 2008). The Aktkinases have an unusual N-terminal pleckstrin homology (PH) domainwhich is important for anchoring the enzymes to the cell membranewhere they come in contact with phosphoinositide dependent kinase 1(PDK-1)which phosphorylates the Akt kinases on the activation loop toproduce continued catalytic activity. When not bound to the plasmamembrane, the PH domain occludes the activation loops of the Aktkinase preventing PDK1 activation and maintaining the kinases in aclosed conformation. The high sequence similarity of theAkt isoforms inthe kinase domain suggested that targeting the PH domain may be aviable approach to develop subtype selective inhibitors of the Aktisoforms.

Studies by Barnett et al. (2005) and his colleagues focused onidentifying selective Akt kinase inhibitors. They employed an Aktactivity assay in screening a small molecule library and by luckidentified several compounds (Akt-I-1 and Akt-I-1,2) (see Fig. 1 forstructure) that inhibited Akt1 in an ATP and substrate non-competitive manner. Neither compound affected the activity of Akt3nor a large number of other protein kinases. Both inhibitors effectivelykill the tumor cell lines overexpressing Akt1 in vitro. They wereeffective alone and also synergized with other chemotherapeuticagents (Hirai et al., 2010). They were also effective in vivo in a tumorxenograft model (LNCaP prostate cancer xenografts) (Cherrin et al.,2010). Furthermore, one of the inhibitors (MK-2206) was recentlyshown to be safe in humans in Phase 1 studies after oral administration(Lindsley, 2010).

The selectivity of these compounds was remarkable given the highsimilarity of the Akt isoforms. Subsequent studies showed thecompounds bind to the PH domain maintaining the kinase in a closedformation preventing PDK1 phosphorylation of Akt1 and reducingAkt1 phosphorylation of the downstream substrates. The evidencethat these compounds acted as allosteric inhibitors of Akt1 and boundto the PH domain was supported by the FRET based studies.

Interestingly, these inhibitors may also be working by affecting thetranslocation of Akt. The studies by Calleja et al. (2010) showed thatthese inhibitors blocked the translocation of a GFP-Akt1-mRFP to theplasma membrane induced by PDGF, presumably by hindering theassociation of PH with anchoring sites in the membrane. Thistranslocation is necessary for localizing Akt1 with PDK1 to cause theactivation of Akt1. Interestingly, Kim et al. (2010) have also identifiedthe allosteric inhibitors of Akt1 that bind to the PH domain. The smallmolecule blocks the translocation of Akt to the cell membrane, toinhibit the activity and prevent the proliferation of tumor cells thatexhibit high activity. Itwas also effective in vivo in reducing the sizes ofthe ovarian and pancreatic tumors in mice that have high Akt activity.

They also found that their compound inhibited the activity ofmutant forms of Akt, in particular Akt E17Kmutation. This mutation isfound in the human breast, colorectal and ovarian cancers and is in thePH domain of the kinase. By enhancing the electrostatic interaction ofAkt1 with phosphoinositide in the plasma membrane, the mutationcauses “pathological” localization of the kinase to the plasmamembrane, to cause the cell transformation and leukemia in mice.Interestingly, Carpten et al. (2007) have shown this mutation to

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reduce the affinity of Akt1 for MK-2206 by over 4-fold suggesting thatthe amino acid substitution affects the conformation of the PH bindingpocket for this small molecule. In contrast, the Akt allosteric inhibitoridentified by Kim et al. (2010) was equally effective in inhibiting themutant and wild type Akt suggesting it may overcome potentialresistance that may form to MK-2206.

The results with the PH domain allosteric inhibitors and theirability to block the translocation of Akt to inhibit the activity suggestthat a potently important direction for the discovery of novel, highlyselective allosteric kinase inhibitors could focus on modulating theintracellular movement of the enzymes, rather than purely focusingon enzyme conformation, since the importance of the activity of manykinases is dependent on their subcellular distribution.

3.3. Allosteric inhibitors of other serine/threonine kinases

In addition to Akt, unique allosteric modulators have beendiscovered against a number of other serine/threonine proteinkinases as described previously (Eglen & Reisine, 2009, 2010). Forexample, much progress has been made in developing novelregulators of the Checkpoint kinase 1 (CHK1). This enzyme controlsthemovement from S to G2 phase in cell cycle and is important for theDNA damage control. As such, it is important in cancer treatment andsensitivity of malignant cells to chemotherapeutics. Thus, theallosteric inhibitors of this kinase have been identified by firstscreening chemical libraries in classical kinase phosphorylation assaysand then picking out “hits” based on their lack of dependence on ATP,that is they were not competitive with ATP (Converso et al., 2009).Recent studies by Vanderpool et al. (2009) have now described twonew allosteric CHK1 inhibitors which bind near the substrate bindingsite of the kinase but which do not compete directly with thesubstrate binding. They have been proposed to produce conforma-tional changes that subsequently hinder the substrate interaction inthe catalytic domain. These findings are particularly interesting sincerelatively few small molecule drugs have been identified that canaffect substrate binding either directly or indirectly and such inhibitorsmight be expected to confer a high degree of selectivity, which iscertainly needed when regulating a kinase controlling cell cycle.

p38 MAPK has been another serine/threonine kinase for whichextensive efforts have been made to discover novel and highlyselective allosteric inhibitors because of its important role in thedisease (see Eglen & Reisine, 2010). The allosteric inhibitors have beenidentified that block the translocation of this kinase to the nucleus incells preventing its access to target sites (Almholt et al., 2004; Trask etal., 2009) and by nature these inhibitors are not competitive with ATP.Furthermore, Diskin et al. (2008) have identified novel lipid allostericbinding sites on p38 kinase that can lead to the inhibition of activity.Furthermore, Simard et al. (2009) developed a novel assay to measuremovement of the activation loop of p38 which they used to identifynovel type 2 inhibitors of the kinase there were highly selective.

Similarly, major progress has been made in developing novelallosteric modulators of the phosphoinsositide-dependent proteinkinase-1 (PDK1), which can control the activation of a large number ofprotein kinase including the protein kinase A and C families bycatalyzing the phosphorylation of residues in the activation loop (Gao& Harris, 2006). PDK1 has been found to have a docking site that bindsthe substrates, the so called PDK1 interacting fragment (PIF) site. PIFsequences are found in many PDK1 substrates. In fact, the PIF bindingsite in PDK1 is a hydrophobic groove, uniquely located N-terminal tothe catalytic site and allows this kinase to sense other target kinases assubstrates. Importantly, the binding of substrate to this docking siteactivates PDK1 by inducing conformational changes in the catalyticdomain. Thus, the binding of a substrate kinase to PDK1 via thesubstrates PIF motif stimulates the PDK1 activity which thenphosphorylates the activation loops of the substrate kinase (Engelet al., 2006; Gao & Harris, 2006).

A polypeptide corresponding to the PIF motif was shown to bind toPDK1, by surface plasmon resonance and activate the kinase and X-raycrystallography has been employed to study the mode of interaction ofthe activating PIF-tide and PDK1 as a basis for small molecule drugdiscovery (Gao & Harris, 2006). Using this structural information, Engelet al. (2006) was able to devise a strategy to identify the allostericactivators of PDK1 that bound to the PIF docking site. This involved insilico compound screening and testing the ability of PDK1 tophosphorylate an artificial substrate consisting of segments of theactivation loop of PKB. Several small molecule compounds whereidentified that activated PDK1 and this activation was blocked by thePIF-tide suggesting that the small molecules are bound to the PIFdocking site.

Similarly, Stockman et al. (2009) employed library screening usingNMR analysis to identify the compounds that bound to the PDK1–PIFbinding domain. The compounds identified as binding were analyzedforwhether theywere ATP competitive or selectively blocked by the PIFpeptides and then subjected to functional assays involving the ADPdepletion assays. Using this approach, these authors also identified thepotential allosteric activators of PDK1.

To further identify the allosteric inhibitors of PDK1, Bobkova et al.(2010) a TR-FRET assay was employed using a fusion of the fragment ofthe activation loop substrate to screen small molecule libraries. Usingthe assay, small molecule alkaloidswere identified that bound to the PIFdomain in PDK1 and inhibited the kinase activity. In fact, these allostericinhibitors were highly selective, since they were unable to affect theactivity of over 96 other protein kinases. Since PDK1 is a master kinaseinvolved in regulating a larger number of other serine–threoninekinases and is involved in a number of proliferative diseases, suchhighlyselective allosteric inhibitors may be developed into unique and highlyvalue anti-cancer therapies.

3.4. Allosteric inhibitors that affect protein kinase translocation

The studies of other protein kinases have shown the importance oftranslocation in both normal activity and dysfunction in a disease. Theimportance of the translocation of the protein kinases with regard tofunctionwasmost clearly shown for protein kinaseC (PKC) (Budas et al.,2007). The PKC family consists of a number of different isoforms, manyof which have distinct functions in cells. The kinases interact with thereceptors for the activatedC-kinase (RACK)proteins. TheRACKs interactwith the allosteric sites in the PKCs to stabilize the kinases in an openactivated form and transport the enzymes to the subcellular compart-ments where they have access to their target substrates (Budas et al.,2007). The functional role of the RACKs and the translocation in PKCfunctionwas shown by the discovery of the allosteric peptide inhibitorsof PKC–RACK association that revert the PKCs to an inactive form andreduce the access of the kinases to their targets. The peptide inhibitorscorresponding to the unique contact sites of each PKC isoformwith theirRACKs were developed that could be used to investigate the selectivefunctions of each isoform and in some cases have been developed intotherapeutics (Liron et al., 2007). For example, a peptide inhibitor ofδPKC–RACK was discovered that was effective in reducing the infarctsize in the animal models of myocardial infarction and stroke. Thepeptide was found safe in human trials and is now undergoing laterstage efficacy studies for the treatment of cardiovascular disorders(Budas et al., 2007). Peptidomimetics targeting other PKC isoform–

RACK complexes have been shown in preclinical studies to be useful intreating cardiac hypertrophy, ischemia and some pain responses. Thus,the compounds, in particular small molecules targeting the allostericsites of the RACK–PKC interaction could have a number of potentiallyuseful therapeutic applications.

Similarly, translocation is a critical factor in the functions of cAMPdependent protein kinase (PKA) (Patel et al., 2010). The translocation ismediated by a family of 13 different A kinase anchoringproteins (AKAP)which both transport and anchor PKA to target substrates and the site of

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cAMP gradients in cells (Ruehr et al., 2004; Patel et al., 2010). AKAPsinteract with allosteric sites in PKA, much like RACKs bind to theallosteric regions of PKC. They interact with the regulatory subunits ofPKA and the peptides corresponding to the PKA binding region of AKAPexhibit low nM affinities for the regulatory regions. The binding hasbeen evaluated by NMR showing the dynamics unique for the protein–protein interaction (Kinderman et al., 2006; Sarma et al., 2010). Crystalstructure analysis has also been made on the AKAP binding to theregulatory subunits revealing that AKAP and peptide analogs interactwith a groove on the surface of the regulatory subunit of PKA involving astrong hydrophobic protein–protein interaction (Kinderman et al.,2006; Taylor et al., 2008).

A recent study by Patel et al. (2010) further established theimportance of AKAP in PKA function in studies in which a peptideinhibitor of the interaction blocked the PKA translocation in cardiacmyocytes, reduced the kinase phosphorylation of selected targets andaffected myocyte contractility, heart rate and ventricular pressure.Furthermore, recent studies have shown that SNPs are present insome of the PKA binding domains of AKAP, one of which increasesAKAP–PKA binding. This SNP has been shown to increase the risk forsudden cardiac death and ventricular arrhythmias. This suggests thatallosteric inhibitors targeting this particular AKAP–PKA might have atherapeutic benefit, supporting the role of the allosteric modulatorysites of transporters of kinases as potential targets for the novel drugdiscovery.

In fact, the translocation between cellular compartments appearsto be a common and important factor in the function of many proteinkinases, including the G protein linked receptor kinases (GRKs),p38 MAPK and others. In all cases, the translocation process involvesthe allosteric sites on the protein kinases to which the cellular proteinsinvolved in the transport of the kinases bind to both affect theconformation of the kinase and anchor the enzymes to sites near theirtarget substrates (see Trask et al., 2009; Eglen & Reisine, 2010).The assays that measure the protein kinase translocation could providedirected approaches for the discovery of the allosteric inhibitors ofa number of different protein kinases. For example, Almholt et al. (2004)developed cellular distribution assay for p38 MAPK using fluorescenttags and confocalmicroscopy tomeasure themovement of the kinase inan HTS format. Similar assays have been developed using a comple-mentation assay format and secondary assays can be employed todistinguish the direct inhibitors of the kinase catalytic activity fromthe allosteric inhibitors (see Eglen & Reisine, 2009, 2010).

3.5. Allosteric modulators of G protein linked receptor kinases (GRK)s

G protein linked receptor kinases (GRK) are a family of 7 serine/threonine kinases that are critical regulators of the G protein linkedreceptor (GPCR) function and as such are important in neurotrans-mission, and the action of hormones and growth factors (Krupnick &Benovic, 1998; Pitcher et al., 1998). These kinases recognize activated,agonist occupied GPCRs (Boguth et al., 2010) and catalyze thephosphorylation of the receptors to attract β-arrestins whichuncouple the receptors from G proteins, terminating the signalingthrough the G protein pathways. GPCRs then couple to other signalingpathways, in some cases to growth factor signaling, to alter theirfunctional profile and change the biological actions of the activatingtransmitter or hormone on the target cells (Lefkowitz & Shenoy,2005). Thus, GRKs restrict the activities of the GPCRs and areresponsible for generating alternative cellular regulation by thesecell surface receptors.

GRKs are soluble and contain catalytic domains similar to mostother kinases. However, they have an abundance of allostericregulatory sites that are involved in binding and recognizing targetGPCRs as well as the regions needed to anchor the kinase in theplasma membrane to allow for association with the receptors (Huanget al., 2009; Boguth et al., 2010). Furthermore, GRKs are recruited to

membrane bound GPCRs by βγ subunits. These subunits are releasedfrom the G protein following the agonist activation of the GPCR tointeract with multiple elements in signaling pathways includingenzymes, ionic channels and GRKs to turn off continued stimulation.The βγ subunit bind to additional allosteric sites in the GRKs to allowthe kinases to associate with the receptors and the binding alsoinduces allosterism to catalyze phosphorylation of the intracellularsites in the receptor (Pitcher et al., 1992; Tesmer et al., 2010).

Studies by Boguth et al. (2010) have identified some of thestructural elements of GRKs, in this case GRK6, by crystallography,that are responsible to the association with GPCRs and have identifiedthe physical nature of some of the allosteric sites in the kinase. Inparticular, GRKs have unique N-terminal regions that make directcontact with GPCRs and thus are responsible for receptor recognitionandmay also be involved in detecting agonist bound conformations ofthe receptor. GPCRs bind to allosteric sites on GRKs to induceconformational changes in the GRKs to promote activity. Adjacent tothe N-terminus is a highly basic, relatively flat region essential for theassociation of GRKs with phospholipids in the plasma membrane andas such are important for anchoring the kinase near the target GPCR.In addition to these regions, GRKs have unique C-terminal regions alsoinvolved in membrane targeting and possibly interaction with otherproteins, such as the βγ subunits and therefore contains allostericsites.

Both peptides and small molecules have been identified that affectthe GRK activity via the allosteric mechanisms. The regions of GRK2which bind βγ subunits have been identified and the peptidescorresponding to these regions block the activity of the G proteins andalso prevent the recruitment of GRK to the plasma membranefollowing receptor activation (Rockman et al., 2002; Hata & Koch,2003). Furthermore, small molecules that bind to βγ, including M119and gallein (Davis et al., 2005; Bonacci et al., 2006; Lehmann et al.,2008) block the association of the G proteins to GRK2 in the cell freesystems and in HL60 cells (Casey et al., 2010). Thus, M119 and galleinare the allosteric modulators of GRKs and act by binding to the regionsof G proteins that are needed to recruit the GRKs to the cell membraneto phosphorylate GPCRs.

These small molecules have been used to show the potentialtherapeutic importance of developing drugs targeting GRK (Caseyet al., 2010). β-adrenergic receptors and excessive βγ subunit activityhave been associated with pathophysiological mechanisms involvedin heart failure (Bristow et al., 1982; Ungerer et al., 1993; Koch et al.,1995; Iaccarino et al., 1999; Rockman et al., 2002; Hata et al., 2006;Matkovich et al., 2006; Raake et al., 2008; Dorn, 2009). Excessivestimulation of β-adrenergic receptors can result in the excessiverelease of βγ subunit and extensive recruitment of GRK2 to theplasma membrane to phosphorylate and desensitize and eventuallydownregulate the β-receptor. Over-expressing GRK2 in the cardiactissue can also lead to heart failure while the knockdown of the kinasecan be cardioprotective. Furthermore, the peptides that block βγsubunit interaction with GRKs have been found effective in improvingthe cardiac function in the models of heart failure.

Similarly, Casey et al. (2010) found that the small molecules M119and gallein, which block βγ subunit interaction with GRKs, blocked theβ-agonist induced recruitment of GRK2 to the cell membranes of thecardiomyocytes. They were also cardioprotective in several models ofthe heart failure. Specifically, continuous administration of isoprotere-nol will cause receptor desensitization and cardiac dysfunctionwhereascotreatment with M119 blocked the cardiac dysfunction and main-tained normal contractile activity. Furthermore, in a genetic model ofheart failure in mice with cardiac overexpression of calsequestrin,gallein administration prevented cardiac degeneration.

These studies suggest that small molecule drugs that block GRK2translocation to the cell membrane could be effective in treating cardiacdisorders. Importantly,GRKover-activityhas been linked to anumber ofdisorders and diseases including opiate tolerance development and

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addiction. Since GRKs are generally inactive and are only stimulatedwhen associatingwith GPCRs, then compounds targeting allosteric sitesonGRKs involved in coupling toGPCRs andG proteins could be effectivein a host of disorders by blocking the accessibility of the kinase to thetarget substrate without affecting the activity per se of the catalyticdomain. Furthermore, if GRKs show selectivity in which GPCRs theyassociate with, then the allosteric modulators could be designed toselectively affect the GRK regulation of different GPCRs allowing forfocused regulation of the actions of the specific neurotransmitters,hormones or growth factors.

There are assays that can be employed to discover novel inhibitorsof GRK translocation. Tagging the GRKs with fluorescent moleculesand tracking movement using confocal microscopy, much like whathas been done previously with β-arrestin (Ferguson & Caron, 2004)can be employed to study GRK recruitment to the cell membrane inresponse to the GPCR activation and a number of drug discoveryprograms in the pharmaceutical industry have develop HTS confocalscreening technologies for the drug discovery. Similarly, complemen-tation assays have been developed tomeasure protein translocation incells (Eglen, 2005; Fung et al., 2006; Zhao et al., 2008).

4. Assays to discover novelallosteric modulators of protein kinases

Numerous assays have been employed for the discovery of drugstargeting protein kinases, such as standard phosphorylation assays aswell as more recently developed ADP depletion or formation assayswhich have been described elsewhere (Eglen & Reisine, 2009, 2010).The readout of these assays, when combined with the structuralanalysis, has resulted in the discovery and development of a numberof useful and effective drugs. The mode of action of the compoundsidentified can generally be distinguished in secondary assays in whichthe effectiveness of the inhibitor is assessed with respect to ATPconcentration. Compound binding can then be further evaluatedusing surface plasmon resonance spectroscopy, mass spectroscopyand even crystallography to facilitate the rational design of drugs.While such assays have been employed to discover some allostericmodulators, newer technologies have now been developed to identifynovel allosteric modulators.

4.1. Receptor tyrosine kinase assays for allosteric modulators

Themajority of drugs targeting RTKs are either protein therapeutics,such as antibodies targeting the extracellular domains to block growthfactor interaction, or smallmolecules targeting theATPbindingdomainsof the catalytic subunit of the kinase. As indicated above, there are someexamples of small molecule allosteric activators of the receptor tyrosinekinaseswhichmimic the actions of the protein growth factors aswell asthe small molecule inhibitors. The small molecule allosteric inhibitorscould be particularly useful clinically because of their increasedselectivity compared to the ATP competitive drugs.

The assays to measure the RTK activity are available that eitherdetect receptor phosphorylation as a response to receptor activation oremploy downstream readouts of an activity, such as the phosphoryla-tion of the transcription factors. More recently, an RTK assay wasdeveloped that employedaβ-galactosidase complementation assay andthe assay is referred to as the PathHunter (Olson & Eglen, 2007; Eglen,2007). The assay employs two fragments of the enzymeβ-galactosidasethat when separated have no enzymatic activity but when in closeproximity recombine to have full enzyme activity which can result in ahighly amplified luminescent response. For the RTK assay, one of thefragments of the β-galactosidase, enzyme acceptor (EA), is associatedwith anSHdomainwhile the smaller fragment, Prolabel, is embedded inthe C-terminus of the receptor tyrosine kinase. When the kinase isstimulated by the growth factor, the catalytic domain is phosphorylatedattracting the SH-EA fragmentwhich complements with the Prolabel in

the receptor to form anactiveβ-galactosidase. The assay technology canalso be employed to measure the dimerization of the RTKs using anapproach developedbyWehrman et al. (2002, 2005, 2006, 2007). In thisformat, EA and Prolabel are incorporated into the C-terminal regions ofthe different subunits of the receptor tyrosine kinase. When dimeriza-tion occurs, the fragments can recombine to form activeβ-galactosidaseand luminescent response. This assay can then identify the allostericantagonists that block the dimer formation to inhibit receptor kinaseactivity.

Similarly, Leuchowius et al. (2010) andWeibrecht et al. (2010) havedeveloped a proximity ligation assay to measure the dimer formationand RTK activity. Unlike the previously described assay, this approachinvolves the use of antibodies, one selective for the RTK intracellulardomain and the other targeting the tyrosine phosphorylation sites. Theantibodies are conjugated to oligonucleotides and when the antibodiesare in close proximity, the oligonucleotides serve as templates forligating additional oligonucleotides into a cyclic DNA construct and theantibody attached oligonucleotides are extended by rolling circleamplification. Hybridizing fluorophore-labeled oligonucleotides pro-vided and amplified the reaction product revealing an intensefluorescent signal. Due to the intensity of the signal, the assay can beemployed in cells expressing low levels of receptor such as in theprimary cells to study the native receptor tyrosine kinases aswell as therecombinant receptors and the assayhas to be adapted toanHTS format.This provides an important advantage if there are cellular factorsaffecting the receptor dimer formation or activity that are not easilyincluded in the recombinant receptor assay formats and if the receptordensity and expression levels are a critical factor in the drug discovery,as they are in the GPCR drug discovery. Disadvantages are that theproximity ligation assay requires the use of antibodies against thereceptor, is not homogenous, and is more laborious to perform as aresult.

4.2. Assays for allosteric modulatorsof soluble kinases — subunit interactions

Many soluble protein kinases either consist of subunits or proteinmodulators that bind to the kinase to regulate catalytic activity. Thesemodulators can interact with the allosteric sites to affect theconformation of the kinase to either increase or diminish the activity.Consequently, the small molecules targeting those allosteric sites canaffect the activity either by blocking the interaction of the proteinmodulator or by inducing the conformational changes in the kinase toalter the catalytic activity. The assays to measure the small moleculeinteractionwith those sites can be employed to discover novel allostericmodulators of the kinases.

Cyclic AMP dependent protein kinase has been employed exten-sively to develop novel assays for the allosteric modulators. This kinaseconsists of regulator (R) and catalytic (C) subunits with R suppressingactivity of C. The cAMP binding to R causes dissociation of the complexand increasing activity. Thus, the small molecules targeting the cAMPbinding site on R as well as multiple other sites on R and even the Rbinding site in C not only can affect the catalytic activity but alsomodulate the R–C association.

A commonly employed protein–protein interaction assay to mea-sure the R–C association is the AlphaScreen/AlphaLISA assay. The assayconsists of acceptor and donor beads coated with either streptavidin oranti-GST antibodies. C is biotinylated andR is constructedwith aGST tagas described in Gesellchen et al. (2006). In this bead proximity assay, anintense luminescent response occurs when the R–C complex forms andthe response is lostwhen the cAMPor its analogs are added to cause thecomplex to dissociate. The assay is homogenous and is easily formattedfor the cell free based HTS. Importantly, the assay format could beemployed for most protein–protein interactions and therefore could beused to discover the allostericmodulators of a large number of differentprotein kinases. In fact, recent studies by Bobkova et al. (2010) used the

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AlphaScreen to identify the novel allosteric inhibitors of PDK1 usingultra high throughput screening.

Similarly, bioluminescent resonance energy transfer (BRET) assayformats can be used tomeasure R and C interaction and protein–proteininteractions in general. As described by Gesellchen et al. (2006), theassay can be set up by tagging R or C with either Renilla luciferase as abioluminescent donor and GFP as a the acceptor and the constructs canthen be expressed in cells by transfection. This provides a homogenouscell based assay to measure protein–protein interaction allowing thedetermination of both of the molecules that can affect haloenzymeformation and those that are cell permeable.

Both the AlphaScreen/LISA and BRET assays are robust and highlysensitive and have been used extensively for compound screening.However, they require themodification of the target proteins. These caninvolve relatively large additions such as either GST or GFP.While thesemodificationsdonot seem to affect themeasurementof the PKAsubunitinteraction, such modifications could affect other target proteinsespecially if the additions are larger than the target proteins themselves.

The technologies to measure the physical interaction of the kinasesubunits or regulators can be employed and that do not require themodification of the interacting proteins. These include simple spectro-photometric analysis and surface plasmon resonance. However, theseassays, which are cell free, are not easily employed for HTS like theAlphaScreen and BRET assay formats. Interestingly, Saldanha et al.(2006) developed an assay for PKA to identify the allosteric modulatorsbased on a fluorescence polarization (FP) format. The assay uses theendogenous inhibitor of protein kinase (PKI) as a probe formeasuring Rand C interaction. Both PKI and R compete for similar sites on C. Rexhibits a much higher affinity for C than PKI, thus when R–C arecomplexed, PKI does not bind whereas when C is free, PKI can bind.Thus, these investigators coupled a fragment of PKI with carboxyfluor-escien and developed a PPI assay that they adapted for HTS for theallosteric activators of PKA. The assay is homogenous, highly sensitiveand, unlike the AlphaScreen and BRET assays, does not require themodification of the target kinase to be implemented.

Conceivably the FP assay format can be employed to discover thenovel smallmolecule allosteric inhibitors and activators of other proteinkinases. In fact, an FP assay was used to identify the small moleculeinhibitors of JIP1 binding to the kinase JNK1 (Chen et al., 2009). JIP1 is aprotein activator of JNK1 and binds to the allosteric sites on the kinase.These authors labeled JIP1 with the fluorescent molecule TAMRA anddeveloped a binding assaywhich they screenedwith the smallmoleculelibraries for the allosteric inhibitors of this kinase. Similar types of assayscould be employed for the IKKB regulation byNEMOand the CDK familyas well as likely a number of other kinases.

5. Summary

Studies on the kinase structure and functionhave led to the advancesin knowledge on the molecular properties of this protein family andfacilitated the discovery of many effective drugs to treat cancer andother diseases. Newer technologies allow a broader scope of drugdevelopment against kinases, especially the development of theallosteric inhibitors. This class of drugs provides opportunities toidentify more selective drugs as well as those able to modulate thekinase activity inways additional to those previously developed. First, itis possible to activate the kinases with the allosteric modulators whichhave importance in treating disorders associated with a lack of activity,such as in a neurodegenerative disease. Secondly, it provides ways toblock the activation of some kinases without affecting basal activity,which is in itself, physiologically important. Thirdly, it is nowpossible tomodulate intracellular movement of kinases to specific locations toinhibit the activity of some functions but not others, in effect, making itfeasible to develop highly targeted kinase drugs. Finally, and potentiallymost important, is the potential development of the small molecule

allosteric agonists and antagonists of the growth factor receptorsopening up a new field of receptor targeted therapies.

Acknowledgments

The authors wish to thank Crist Filer for preparing the structuresshown in Fig. 1.

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