structure, function and inhibition of the phosphoinositide 3-kinase p110α enzyme

120 Biochemical Society Transactions (2014) Volume 42, part 1

Structure, function and inhibition of thephosphoinositide 3-kinase p110α enzymeJack U. Flanagan*†1 and Peter R. Shepherd*†‡*Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1042,

New Zealand

†Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag 92019, Auckland 1042, New Zealand

‡Department of Molecular Medicine and Pathology, The University of Auckland Medical School, Private Bag 92019, Auckland 1042, New Zealand

AbstractThe PI3K (phosphoinositide 3-kinase) p110α isoform is activated by oncogenic mutations in many cancers.This has stimulated intense interest in identifying inhibitors of the PI3K pathway as well as p110α-selectiveinhibitors, and understanding the mechanisms underlying activation by the oncogenic mutations. In thepresent article, we review recent progress in the structure and function of the p110α enzyme and twoof its most common oncogenic mutations, the development of isoform-selective inhibitors, and p110α

pharmacology.

IntroductionThe class I PI3Ks (phosphoinositide 3-kinases) are a familyof closely related enzymes that are among the first level ofcell signalling molecules regulated by tyrosine kinases, bysmall G-proteins Rac and Ras and by the βγ subunits ofheterotrimeric G-proteins [1]. They are encoded by fourgenes (PIK3CA, PIK3CB, PIK3CG and PIK3CD), eachof which codes for a protein of approximately 110 kDa(p110α, p110β, p110γ and p110δ respectively). The kinasedomain of these enzymes is closely related to class II andclass III PI3Ks and to the phosphoinositide 3-kinase-relatedkinases, including mTOR (mammalian target of rapamycin),DNA-PK (DNA-dependent protein kinase), ATM (ataxiatelangiectasia mutated) and ATR (ataxia telangiectasia- andRad3-related) [1].

Regulation of class I PI3Ks by tyrosine kinases occursbecause three of the enzymes (p110α, p110β and p110δ)are tightly associated with a regulatory subunit encodedby the PI3KR1, PI3KR2 and PI3KR3 genes. These containtwo SH2 (Src homology 2) domains that allow recruitmentto tyrosine-phosphorylated proteins and explains how thePI3K isoforms are activated during normal stimulation ofRTKs (receptor tyrosine kinases) (e.g. in insulin receptorsignalling [2]) and also how they can be continually activatedby oncogenic forms of RTKs [1,3]. Regulation via Gβγ

subunits is by direct interaction with the p110β and p110γ

catalytic subunits. All four isoforms contain a Ras-bindingdomain that allows interactions with a range of small GTPases

Key words: cancer, H1047R mutant, p110α, phosphoinositide 3-kinase (PI3K).

Abbreviations: ABD, adaptor-binding domain; INPP4b, inositol polyphosphate-4-phosphatase;

IRS1, insulin receptor substrate 1; iSH2, coiled-coil inter-SH2; mTOR, mammalian target of

rapamycin; mTORC, mTOR complex; nSH2, N-terminal SH2; PI3K, phosphoinositide 3-kinase;

PKB, protein kinase B; pS6, p70 S6 kinase; PTEN, phosphatase and tensin homologue deleted on

chromosome 10; RTK, receptor tyrosine kinase; SH2, Src homology 2.1To whom correspondence should be addressed (email [email protected]).

[4]. These mechanisms result in recruitment to the plasmamembrane where the substrate is located.

Class I PI3Ks phosphorylate the 3-position of the inositolring of the ubiquitous membrane phospholipid PtdIns(4,5)P2

producing PtdIns(3,4,5)P3 [5]. PtdIns(4,5)P2 is a relativelyabundant membrane phospholipid, but PtdIns(3,4,5)P3 isfound at much lower levels. PtdIns(3,4,5)P3 levels rise rapidlyafter stimulation of many RTKs and G-protein-coupledreceptors and fall rapidly when the stimulus is removed.In most cells, the decrease is due to lipid phosphatasesincluding INPP4b (inositol polyphosphate-4-phosphatase),SHIP (SH2-domain-containing inositol phosphatase) 1 and2, and PTEN (phosphatase and tensin homologue deletedon chromosome 10) [3,6,7]. Class I PI3Ks can also act asprotein kinases, although very little is known about thecellular role of this capability [8,9]. PtdIns(3,4,5)P3 recruitsa limited subset of PH (pleckstrin homology)-domain-containing proteins, and a consequence is transient co-localization of signalling proteins. The most widely studiedsuch event is co-localization of PDK1 (phosphoinositide-dependent kinase 1) with Akt/PKB (protein kinase B), whichresults in phosphorylation of Akt/PKB on Thr308 and theactivation of a wide range of downstream signalling eventsinvolved in regulating cell growth, cell division and cellmetabolism [3,7].

Hyperactivation of this pathway is observed in manycancers; it can arise from oncogenic mutations in growthfactor receptors and also explains, in part, the powerfuloncogenic effects of Ras mutations. Another activatingmechanism in tumours is loss of the phosphatases thatdephosphorylate the lipids. Both PTEN and INPP4b arecommonly bi-allelically mutated or deleted in tumours [7].Overexpression of various class Ia PI3K isoforms has alsobeen reported in many tumours [10]. Oncogenic mutationsin the p110α enzyme are also common in tumours [11],and an oncogenic p110β mutation was reported recently

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[12]. The mutations in PIK3CA cluster in two hotspots[11], with the most common mutations including the helicaldomain E545K and kinase domain H1047R substitutions.Both cause constitutive activation of the kinase, but resultin different kinetic properties [13]. They activate p110α

signalling through the PI3K–Akt/PKB–mTORC (mTORcomplex) pathway, and are oncogenic in vivo [14]. Mutationsare also found in the PI3KR1 gene encoding the p85α

regulatory subunit, and these also appear to act throughp110α [15]. Contributions of PI3Ks to tumour developmentdo not have to be intrinsic to the tumour cells themselves.Some of the responses of stromal cells that support tumourgrowth are dependent on different PI3K isoforms includingp110α [16] and p110δ [17]. These findings have led tointense interest in developing inhibitors that selectively blockindividual isoforms while retaining therapeutic efficacy in thehope of gaining a greater therapeutic window. In the presentarticle, we review the biology behind efforts to developselective inhibitors of p110α.

Enzyme structure and functionX-ray crystal structures of wild-type p110α [18] and itsH1047R oncogenic mutant have been determined [19], theformer with small-molecule inhibitors PIK108 [20] andBYL719 [21] bound in the ATP-binding site, the latterwith wortmannin bound [19]. These structures illustrate thethree-dimensional arrangement of the domains that make upthe p110 catalytic unit including the ABD (adaptor-bindingdomain), RBD (Ras-binding domain), C2 domain, helicaldomain and the kinase domain. They also show where theN-terminal SH2 (nSH2) and coiled-coil inter-SH2 (iSH2)domains of p85α bind to the p110α catalytic unit. The iSH2domain interacts with the ABD and C2 domains, whereasthe nSH2 domain interacts with the C2 and helical domains.The phosphotyrosine-binding site of the nSH2 domain formsan interface with the p110α helical domain [19] and inhibitskinase activity [22].

The p110α–p85α structures provide insight into thespatial arrangement of cancer-associated mutations within thetwo proteins, and in some cases their mechanistic effects.The E545K mutation is located at the interface betweenthe helical and nSH2 domains [19], whereas the H1047Rmutation is found in the C-terminal tail of the kinasedomain in a helical region described as the regulatoryarch [23,24]. Structural [19] and biochemical [25] dataindicate that the E545K substitution disrupts the helicaland nSH2 domain interaction, relieving the inhibitory effectand rendering the p110α enzyme unresponsive to furtheractivation by phosphotyrosine-containing peptides; it is,however, still responsive to activation by Ras. By contrast, theactivated state of the H1047R kinase domain mutation retainsresponsiveness to phosphotyrosine peptides [26], and is notactivated further by Ras [13]. This suggested that the differentmutations mimic activated states of the enzyme [13,27].

Detailed insight into the conformational changes duringactivation for the wild-type and oncogenic p110α–p85α

complexes upon phosphotyrosine peptide binding andmembrane binding was provided recently by deuteriumexchange experiments [28]. Burke et al. [28] proposed that thewild-type enzyme undergoes four distinct events in movingfrom its inactive cytosolic form to the active membrane-bound state. Although the order is unknown, these includebreaking the nSH2–helical domain interface, disruptingthe iSH2–C2 interface, movement of the ABD relative to thekinase domain and interaction of the kinase domain withlipid. Mutations activate the enzyme by either mimickingor enhancing different steps. The H1047R mutation affectsthe lipid-interaction surface within the C-lobe of the kinasedomain, and the E545K helical domain mutant affected thep85α nSH2 interface as well as the iSH2–C2 interaction andABD–kinase domain orientation.

The effect of these mutations on lipid binding wasinvestigated recently [20]. Hon et al. [20] showed thatprimary lipid-binding sites in the wild-type enzyme includehydrophobic residues in the C-terminal tail as well as basicresidues in the activation loop, with lipid binding involvingboth an electrostatic and a hydrophobic component. Astrong correlation between lipid kinase activity and lipidbinding was noted for both wild-type and oncogenicmutant enzymes. Releasing the p85α nSH2 domain byphosphotyrosine peptide binding or E545K mutationincreased membrane binding, with basic residues in theactivation loop contributing to the electrostatic component,whereas the C-terminal tail has a more complex contribution.Upon phosphotyrosine peptide binding, the H1047R mutanthad increased hydrophobic and electrostatic interactions [20].On the basis of a superimposition of existing p110α/p85α

protein structures, Hon et al. [20] also proposed thatrelease of the nSH2 domain constraint promotes a concertedconformational change that propagates from the helicaldomain and may effect changes in the conformation of theactivation loop and C-terminal region rendering them lipid-binding-competent.

Conformational differences between the wild-type andoncogenic mutant proteins also provide opportunity foraltered interactions with other proteins as reported recentlyfor the E545K mutant and the IRS1 (insulin receptor substrate1) protein [29]. Here, the oncogenic mutation facilitated ap110α–IRS1 interaction independent of p85 binding to IRS1phosphotyrosine amino acids and may contribute to theoncogenic effect of the E545K mutation.

Isoform-selective inhibitorsSince PI3K isoforms play such an important role in signallingpathways, it is not surprising that drugs targeting all isoformshave significant side effects [30,31]. This has led to effortsto develop isoform-selective inhibitors in the hope that thesewill have greater therapeutic index.

Few p110α-specific compounds have been reported todate, with the aminothiazole-based molecules developedby Novartis, including A66 [32] and the closely relatedNVP-BYL719 [21], being the most widely studied selective

C©The Authors Journal compilation C©2014 Biochemical Society


compounds [15,33,34]. The latter compound entered clinicaltrials in 2010 [21,34]. In the absence of protein structureinformation for these compounds bound to p110α, moleculardocking and analogue synthesis was used to characterizefeatures of A66 that control its isoform-specificity. Selectivityand potency for p110α and its H1047R and E545K oncogenicforms was associated with the inhibitors’ carboxamidegroup and a predicted hydrogen bond with the p110α-specific amino acid Gln859 [21,33]. Site-directed mutagenesisillustrated the role of Gln859 as the main ATP-binding siteresidue influencing A66 activity [35], and a crystal structure ofBYL719 bound to the p110α enzyme confirmed the predictedelectrostatic interaction [21]. Identifying interactions thatinfluence the p110α-selectivity of PIK75 [36] and relatedcompounds [37] has also been the subject of medicinalchemistry [38], molecular modelling [39–41] and biochemicalstudies [35,42] and is likely to involve the p110α-specificamino acid Ser773, although other residues may play a role[35]. Nacht et al. [43] reported the first p110α-specificirreversible inhibitor on the basis of the chemical scaffold ofGDC-0941 that covalently modifies p110α-specific Cys862.

In contrast with the electrostatic interaction underlyingthe p110α-selectivity of A66 and BYL719, p110δ-specificinhibitors including IC87114 and PIK-39 achieve isoform-specificity by steric effects. These ligands have a ‘propeller’shape in the p110δ ATP-binding site [44] and requireformation of an allosteric pocket termed the specificitypocket. This pocket is created by Met752 side chainrearrangement in the ATP-binding site [44], and is alsoformed in p110γ with PIK-39 bound [45] and in p110α

with PIK-108 bound [20] by rearrangement of analogousmethionine amino acids. Differences in flexibility of thisregion is the likely controller of selectivity as indicated byMD simulations [44] and site-directed mutagenesis studies[46]. Not all p110δ-specific inhibitors use the specificitypocket. The inhibitor AS15 was shown to make p110δ-specific interactions in a site adjacent to Met752 with its sidechain in a more conventional conformation [44].

p110α pharmacologyThe discovery of p110α-selective inhibitors improves thepharmacological tool set that can be used to investigatespecific PI3K isoform signalling in cell or animal systems.We used p110α-selective inhibitors A66 and PIK75 to probethe role of p110α in PI3K signalling through the PI3K–Akt/PKB–mTORC pathway in a panel of 12 cancer celllines of different tissue origin including low-passage-numbermelanoma cell lines [33]. These studies found that, uponinsulin stimulation after serum starvation, the cell lines couldbe classified as sensitive or resistant to drug treatment onthe basis of Akt/PKB phosphorylation, a proximal markerof pathway activation. Sensitive cells harboured the p110α

H1047R mutation, whereas resistant cell lines harboured theE545K mutation, or were PTEN-null, implicating mutation-dependent differences in p110α signalling from the insulingrowth factor receptor. Combinations of p110α-, p110β-

and p110δ-specific inhibitors were able to reduce Akt/PKBphosphorylation in PIK75-resistant cells, pointing to residualphosphorylation by isoforms other than the p110α, andfunctional redundancy reported previously for insulinsignalling [47]. Sensitivity may in part relate to the total levelsof H1047R oncogenic protein expressed and its levels relativeto other p110 isotypes. Drug sensitivity or resistance was re-tained by tumour xenografts in mice, and growth of tumoursfrom A66-sensitive cell lines was restricted by drug treatment,and corresponded to decreased activity of the PI3K–Akt/PKB–mTORC pathway, as indicated by decreased phos-phorylation of Akt/PKB and pS6 (p70 S6 kinase), markers ofproximal and distal pathway activation respectively [33].

Resistance to PI3K inhibitors can arise from activationof downstream components of the PI3K signalling pathwaywhich can partially compensate for the loss of PI3K activity.Elkabets et al. [34] found that across a panel of 20 breastcancer cell lines harbouring a range of p110α mutations,including the H1047R and E545K, some were sensitive andothers resistant to p110α-specific inhibition by BYL719 withrespect to proliferation. Resistance both in vitro and in vivowas related to persistent mTORC1 signalling indicated byphosphorylation of the distal PI3K pathway marker pS6,whereas phosphorylation of Akt/PKB, remained sensitiveto p110α blockade. Notably, resistance to p110α inhibitionby BYL719 and non-selective PI3K inhibition by GDC-0941 could be acquired in vitro by reactivation of mTORC1signalling and increased S6 phosphorylation after long-term drug exposure. In tumour biopsies from patients thatresponded to BYL719 treatment, strong suppression of S6phosphorylation was observed, whereas tumours that didnot respond showed only weak suppression. Moreover, inpatients with a BYL719-sensitive tumour that progressed,S6 phosphorylation was increased. Resistance could besuppressed in cell lines and xenografts by combining BYL719with the mTORC1 inhibitor RAD001 [34].

p110α inhibition in cancerMany compounds under clinical development are dual classI PI3K and mTOR inhibitors (reviewed recently in [10,48]),although the p110α-specific BYL719 was reported to showpromising clinical activity [34]. Recent analysis of clinicaltrials of mTORC1 and PI3K inhibitors has shown anassociation between the H1047R mutation and a positiveresponse, and is in line with our pre-clinical findings, althoughit was noted that specifically designed clinical studies areneeded to explore this observation [49].

It also seems likely that p110α inhibition will have arange of side effects. Animal studies have shown that A66induces impairment of in vivo insulin action indicating thatp110α is the most important form of PI3K in the pathwaysacutely regulating glucose metabolism [30,31]. Furthermore,detrimental effects on bone structure after 1 month of dosingmice with A66 suggested that osteoporosis might be a sideeffect of treatment with p110α inhibitors. These effects maylimit the therapeutic window for a standard p110α-selective


Signalling 2013: from Structure to Function 123

inhibitor, and taken together with the possibility of mutation-specific responses indicate that new strategies that targetcancer-specific p110α signalling or that increase relative drugconcentration selectively in the tumour might need to beemployed.

Future directions for drug discoverySince oncogenic mutations mimic or enhance differentactivation steps of the p110α enzyme, exploiting theconformational differences may lead to novel inhibitors withsome selectivity for the oncogenic mutant forms, and thesemight have a broader therapeutic window. Indeed, very recentreports indicate that this may be possible [29]. Also, the recentreport of a p110α-selective irreversible inhibitor indicatesthe potential to develop PI3K inhibitors that might achievebetter therapeutic responses by more persistent knockdownof tumour signalling.

Finally, another area of interest in selectively targetingoncogenic PI3K signalling might be in disrupting theinteractions between p110 catalytic subunits and the smallGTPases Rac or Ras, or Gβγ subunits, as emerging evidenceindicates that isoform-selective responses could be generated[4]. It was shown recently that p110β/Gβγ signalling could beblocked in cells using a peptide targeting the interaction [50].

In conclusion, the overall aim of the next wave ofdrug discovery in this area will be to explore these newpossibilities to determine whether such second-generationPI3K inhibitors might be more therapeutically advantageousthan the first-generation ones.

Acknowledgements

We thank Roger Williams and John Burke for comments on the paper

before submission.

Funding

This work was supported by the Health Research Council of New

Zealand [programme grant number 13/763] along with the Maurice

Wilkins Centre for Biodiscovery.

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Received 11 November 2013doi:10.1042/BST20130255


structure, function and inhibition of the phosphoinositide 3-kinase p110α enzyme

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