Discovery of Novel

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137ISSN 1756-891910.4155/FMC.09.5 2009 Future Science Ltd Future Med. Chem. (2009) 1(1), 137155 ISSN 1756-8919

Review

Phosphatidyl-inositol lipids can be bona dedivided into two classes based on their cellularunctions: they are either essential componentso mammalian plasma membrane, or essentialregulators o cellular processes. The dynamic oanabolic and catabolic processes governing theregulation o these entities has been linked to thepathological status o certain diseases [1]. Thisreview will emphasize the interplay between thephosphatidyl-inositol 4,5-bisphosphate (PIP2)and the phosphatidyl-inositol-3,4,5-trisphos-phate (PIP3), and its regulation mediated bysmall molecule therapeutic intervention in can-cer. The conversion o PIP2 into PIP3 is cata-lyzed by phosphatidylinositol-3-kinase (PI3K).The lipid kinase PI3K adds a phosphate grouponto the position 3 alcohol group o the inositolmoiety and, conversely, the tumor-suppressorlipid phosphatase and tensin homolog (PTEN)removes this phosphate group. Four dierentcatalytic paralogs (a, b, d and g) composingthe class I PI3Ks have been described so ar.PI3K is a hetero-dimer composed o a regula-tory (commonly called p85 or class Ia: p85a,

b or g, p50a and p55a; and p101 or class Ib)and a catalytic subunit (commonly known asp110: p110a, b or d or class Ia and p110g orclass Ib). class II and III PI3Ks contain highlyhomologous kinase domains, but these proteinspossess dierent substrate anities than class IPI3Ks [2].

The identication o the pleckstrin homol-ogy (PH) domain containing serine/threoninekinase Akt/protein kinase B (PKB), and itsimplication in cell survival, has been a majorstep toward the elucidation o the key mecha-

nism or the apparition and maintenance o

tumorigenicity. It is now well understood thatconstitutive activation o this kinase occursrequently in tumors and is associated witha generally low prognosis. Moreover, the past10 years have been critical in identiying andcharacterizing the genetic events that can leadto PI3K pathway activation. Hence, the geneproducts controlling the intracellular PIP3 lev-els have been ound to be either constitutivelyactive or missing[3]. For example, constitutivePI3K pathway could be the result o either theloss o the PTEN gene or the apparition oinactivating mutations in the PTENgene [4].Conversely, activating mutations have been dis-covered in the gene encoding the PI3Ka para-log (PIK3CA), with high prevalence in tumorrom various lineages [5]. These mutationshave been shown to be oncogenic in vitro andin vivo. Using a doxycycline-inducible system,Engelman and colleagues have urther demon-strated that the expression o an oncogenic allelein mice is sucient to drive and maintain lungadenocarcinoma ormation [6].

Among other downst ream eectors , the

PI3K/Akt pathway has been shown to exerttumorigenic potential by activating the mTORcomplex 1 (mTORC1; composed o mTOR the mammalian target o rapamycin com-plexed to raptor). The best example to illustratethis is with an allosteric mTORC1 inhibitingrapamycin derivative which is able to suppressAkt induced prostatic intraepithelial neoplesia(PIN) lesions in mice [7]. Recent evidence hasled to the identication o the second mTOR-containing complex, mTORC2 (composedo mTOR and rictor) as one o the direct Akt

upstream activators [8].

Discovery of novel anticancer therapeutics

targeting the PI3K/Akt/mTOR pathway

Background: Among promising targeted therapies for cancer treatment, phosphatidylinositol 3-kinase pathway

inhibitors have in the last 3 years continued to retain the attention of both academic institutions and pharmaceutical

companies. The large amount of published clinical and preclinical data has indeed conrmed the preponderant role

of this so-called survival pathway for tumor maintenance. Discussion: Global efforts have, therefore, been deployed

that have led to the genesis of a panoply of small molecule inhibitors. This review will focus on updating the reader

on the current medicinal chemistry efforts targeting this pathway. Conclusions: Recent discoveries important for

patient stratication, quantication of target modulation in humans and combination therapies will be presented

and discussed.

Sauveur-Michel Maira,

Pascal Furet & Frdric

Stauffer

NIBR GDC and Oncology,

Novartis Pharma AG, CH4002

Basel, Switzerland

Tel.: +41 616 968 749

Fax: +41 616 966 246

E-mail: frederic.stauffer@

novartis.com

Pi3K

An enzyme involved in theregulation of key, secondmessenger of signal transduction

mTOR

mTor is a critical cellularcomponent integrating inputfrom both nutrients and growthfactor signaling pathways, andultimately regulating proteintranslation and cell growth


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Future Med. Chem. (2009) 1(1)138 future science group

Review| Maira, Furet & Stauffer

These discoveries have unraveled the basic ele-ments to allow the development o new opportu-nities or therapeutic intervention. Thus, directlytargeting the enzymatic activities or activationo the various players in the PI3K/Akt/mTOR

pathway(Figure 1) is now considered as prob-ably one o the most promising opportunitiesor the development o new targeted therapies.The aim o this review is to provide the readerwith a state-o-the art overview o the drug-dis-covery activities using small molecules to targeteither the lipid kinase PI3K, the protein kinaseAkt/PKB, its upstream act ivator PDK1(3 -phosphoinositide-dependent protein kinase-1),mTOR and mTORC1, and to ocus on thosethat will possibly have an impact on the regis-tered oncology therapeutic arsenal within the

next hal decade.

Inhibitors of the PI3K/Akt/mTOR

pathway

Allosteric inhibitors of mTORC1In mammalian cells, the mTOR kinase comesin two varieties. It is either complexed with rap-

tor or rictor proteins to give the mTORC1 andmTORC2 complexes, respectively. In contrastto mTORC2, the mTORC1 complex also con-tains the FKBP12 protein (FK506-binding pro-tein 1A, 12kDa). FKBP12 has a high anityto rapamycin and derivatives. Hence, or thisreason, mTORC1 but not mTORC2 kinaseactivity is exquisitely sensitive to these agents.The role and unctions o mTORC1 have beenstudied extensively. This complex is mainlyinvolved in protein translational control, oper-ating as a growth actor and nutrient sensitive

apparatus or growth and prolieration regula-tion [9]. The elucidation o mTORC2 unctionsemerged more recently with the breakthroughin the understanding o Akt signaling, as it hasbeen shown that this complex is in act the long-awaited serine 473-Akt kinase [8]. Rapamycin(sirolimus, Wyeth, compound 1 [Figure 2]),a macrolide isolated rom Streptomyces hygro-scopicus is an allosteric inhibitor o mTORC1activity displaying antiungal [10] and immuno-suppressive eects [11]. The unavorable physico-chemical properties o rapamycin have triggeredchemical modications o the hydroxyl unc-

tions at position 28 and 40. A simple methyla-tion o these alcohol unctionalities o the par-ent compound at either position was tolerated inrespect o FKBP12 binding, but only the alkyla-tion at the later retained substantial inhibitoryeect on mTORC1 catalytic activity[12]. Threerapamycin derivatives modied on hydroxyl atposition 40 are, thereore, being developed andare currently undergoing Phase II/III clinical tri-als as anticancer agents: everolimus (RAD001,Novartis, compound 2 [Figure 2]) is substituted

with a hydroxyethyl group [201] allowing or oral

bioavailability and ormulation in tablet orm;Temsirolimus (CCI-779, Wyeth, compound 3)is acylated with 2,2-bis(hydroxymethyl)pro-pionyl [202] and is administered in the clinic byinusion; and more recently discovered, deoro-limus (AP23573, Merck/Ariad, compound 4)is a dimethylphosphinate derivative that can beormulated or intravenous administration orin enteric coated tablets or oral administration(Figure 2)[203].

The current status o the allosteric mTORinhibitors or which clinical data are available

has recently been extensively reviewed [13]. A

Growth factors

IRS1PI3KPDK1

mTOR

PIP3 PIP3 PIP2

T308S473

TSC2

TSC1

GTP

S6

PRAS40

Raptor

mTOR

4E-BP1

FKBP12

p70S6K

Effectors

of Akt

Rictor

Sin1

mTORC2

mTORC1

PTEN

Rheb

RTK

Future Med. Chem. Future Science Group (2009)

Figure 1. Phosphatidylinositol-3-kinase/Akt/mTOR pathway.Upon ligandbinding, RTK can recruit and activate PI3K. PIP2 is then converted into thesecondary messenger PIP3. The tumor-suppressor phosphatase PTEN antagonizesPI3K activity. PIP3 has a high afnity to PH domain-containing proteins such asAkt/PKB or PDK1. Once at the membrane, Akt is phosphorylated by PDK1 andmTORC2 on residue Thr 308 and Ser 473, respectively. Fully activated Akt hasnumerous downstream eectors involved in cell metabolism, growth,prolieration, survival and in angiogenesis, including two upstream regulators omTORC1: TSC2 and PRAS40. Among mTORC1 eectors, p70S6K promotespathway signal termination either via phosphorylation and subsequentdestabilization o IRS1 or IRS1 gene transcription. The PI3K super-amily members(PI3K, mTOR) and the AGC kinase amily members (Akt, PDK1, p70S6K) areschematically represented as rectangles or diamond-shaped boxes, respectively.PI3K: Phosphatidylinositol-3-kinase; PTEN: Phosphatase and tensin homolog.

SmallmOleculeinhibiTORS

Synthetic entities aiming atblocking specic functions of

specic targets, for a

therapeutic benet


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139future science group www.future-science.com

Discovery of novel anticancer therapeutics targeting the PI3K/Akt/mTOR pathway | Review

large number o clinical trials, both as as sin-gle agents or in combination have been initi-ated or these compounds [301]. The prodrugo rapamycin TAFA93 (Isotechnika, Canada)has also been clinically evaluated. Most nota-

bly, temsirolimus is the rst mTORC1 inhibitorto be registered or the treatment o advancedrenal cell carcinoma [14]. In the same indica-tion, everolimus has shown a avorable increasein progression-ree survival [15]. The mTORC2complex is rapamycin-insensitive but long-termtreatment could result in decreased Akt activity,by decreasing rictor association to mTOR [16].These ndings have raised interest in develop-ing mTOR catalytic inhibitors, that would havean impact on both mTORC1 and mTORC2complexes, hence targeting both downstream

mTORC1 and Akt signaling pathways. The rea-sons that pure mTORC1 blockade, as a singleintervention, seems not to be as ecient in otherindications (breast cancer, glioblastoma, neuro-endocrine tumors) is still a matter o debate.This is urther discussed later in the section othis review titles Feedback loops, cross-talks andcombination therapies.

Inhibitors of the PI3K super-familymembers PI3K & mTORThe rst generation o PI3K inhibitors includeswortmannin (compound 5 [Figure 2]), a natural

product produced byPenicillium wortmanni, [17]and the synthetic PI3K inhibitor LY294002(compound 6, Eli Lilly); the latter resulting romthe optimization o the broader spectrum kinaseinhibitor quercetin (compound 7 [Figure 2]).Most notably, the morpholine replacement oits catechol moiety[18] has been a breakthroughstarting point or the development o severalseries o inhibitors with dierent degrees oselectivity modulation within the PI3K super-amily [19], also including the drug discoverywork conducted at Kudos leading to PI3K-

related protein kinase (PIKK) DNA-dependentprotein kinase (DNA-PK) or ataxia telangiecta-sia mutated (ATM)-specic inhibitors, such asKU55399 [204], which all outside the scope othis review. The cocrystal structures o PI3Kgtogether with the ligand quercetin or LY294002revealed dierences in the binding mode. In thecase o LY294002, morpholine was playing therole o the hinge binder (Figure 3A) [20]. Bothcompounds have been key tools in the elucida-tion o PI3K-dependent events important ortumor initiation and maintenance. Wortmannin

has been shown to be an irreversible inhibitor

o PI3K, which orms a covalent bond with aconserved lysine o the active site [21] and dis-plays a single-digit nanomolar-apparent IC

50

value [22]. A prodrug strategy has been appliedto generate the pegylated thiocarbonate deriva-

tive o 17b-hydroxy-wortmannin PWT-458(compound 8 [Figure 2]) [23]. The reactiveurane embedded in the wortmannin struc-ture renders the molecule chemically unstabletoward nucleophilic attack. However, reactingwortmannin with diethylamine led to a com-pound with reduced, but still potent, activity(PI3K: IC

50= 80 nM) [24].

Follow-up medicinal chemistry activi-ties allowed the identiication o PX-866(Oncothyreon, compound 9 [Figure 2]) [25][205].The prodrug strategy has also been applied

with LY294002 by linking the molecule on themorpholine nitrogen with a vascular-targetedRGDS solubilizing tag (integrin [avb3/a5b1]binding component) [26]. SF-1126 (Semaore,compound 10 [Figure 2]) by itsel is not a PI3Kinhibitor. The molecule has been designed tobe acid stable and liberate the active component(LY294002) at neutral pH. The RGDS moietywas added to increase the solubility o LY294002and target the molecule to the tumor compart-ment. A dose o 20 mg/kg administered eithersubcutaneously or intravenously three-timesa week, produced tumor stasis against either

U87MG or PC3 tumor models in mice, respec-tively. This eect was correlated with a tran-sient glucose elevation. This, together with theact that SF-1126 showed reduced 18FDG-PETuptake upon treatment in orthotopic mam-mary MDA-MB-468 tumors, has proven thatthe molecule is indeed pharmacologically ableto modulate the PI3K pathwayin vivo. SF-1126is currently being assessed in a Phase I clinicaltrial by intravenous inusion over 1.5 h with atwice-weekly schedule over a 4-week cycle.

The elucidation o the crystal structure o

PI3Kg [27] has geared up the discovery o newchemical entities with inhibitor properties, tar-geting specic isoorms. This was mostly drivenor nononcology indications, such as autoim-mune, infammatory disorders or cardiovas-cular diseases. For instance, the morpholinoPI3Kb speciic inhibitor TGX155 (Cerylid,compound 11) [206] was obtained by replacingthe chromenone core o LY294002 by a quino-linone core. Although the compound retainssome activity against the PIKK (class IV PI3K)amily member DNA-PK, it entered a Phase I

clinical trial in 2003 or antithrombotic therapy


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Sirolimus

(Rapamycin)

Everolimus

Temsirolimus

Deforolimus

1 R = H

2 R =

3 R =

4 R = 5

Wortmannin

8

PWT-458

N

O

O

O O

O

O

O

O

O

O

OH

O

R

OH

OH

O

OH

OHP

O

O

OO

OO

O

O

O

O

OO

O

O

O

O

OO

SPEG

O

O

O

O

N

O

O

O

OH

OH

OH

OH

HO

OH

O

O

OO

O

N

6

LY294002

7

Quercetin

9

PX-866

N+

O

O

O

O

O

NH

O

NH

O

NH

NH

NH2

NH

O

O

NH

OH

O

OHO

OH

O

NH

O

N

O

F

N

N

O

N

OHN

10

SF-1126

11

TGX155

12

TGX221

N

NN

N

NH2

NH2

OH

HO

OH

N

S

NH

O

N

SO

O

N

N

N

S

HNHN

O

O

S

O

O

OF

14

TG100-115

15

AS-605240

16

AS-25242413

N N

N NN

N

N

OO

F

FNS

HN

O

O

N

N

N

N

NH

N

H

NH

O

N

N N

N

N

O

18

ZSTK474

17

GSK105615

19

D-106669

20

NVP-BEZ235

Figure 2. Inhibitors of the phosphatidylinositol-3-kinase/Akt/mTOR pathway (compounds 120) .


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www.future-science.com 141future science group


beore being discontinued [28]. Follow-up opti-mization eorts on this series led to the discov-ery TGX221 (compound 12 [Figure 2]) [207]that displays an alternative isosteric pyrido-pyrimidin-4-one core scaold and is a new-

generation potent PI3Kb-selective inhibitor(PI3Kb: IC

50= 5 nM, 20-times more selective

to PI3Kd and 3 log units to PI3Ka,g) [29].High-throughput screening technology

directed toward the discovery o PI3Kg inhibi-tors or anti-infammatory therapy has allowedthe identication o novel potent class I PI3Kinhibitors, such as compound 13 (Novartis,[Figure 2]), [30] bearing a 2-acetylamino-4-methylthiazole hinge binder (Figure 3B). Thisbasic scaold has been substantially exploitedby scientists, or instance at Boehringer, who

have urther evolved this scaold in a rigidiedtricyclic system [208209] or at Serono, who haveattached an alternative thiazole anity pocketmoiety and proposed an alternative oxazolereplacement or the hinge binding moiety[210].One o the early PI3K inhibitors to enter theclinic (currently in Phase II) was TG100-115(TargeGen, compound 14 [Figure 2]) [211] show-ing mainly inhibition o PI3Kg (IC

50= 83 nM,

three-times selective against PI3Kd and 1 logunit against PI3Ka and b). The molecule hasquite a short human hal-lie (~2 h) that couldpossibly be suitable to adequately address the

intended protective action in the indication oacute myocardial inarction [31]. AS-605240(Merck-Serono, compound 15 [Figure 2])[212] is an other PI3Kg-selective inhibitor(PI3Kg: IC

50= 8 nM, ~1 log unit selective to

PI3Ka, b and d) worth mentioning due to itsoutstanding PI3Kg binding ligand eciency oapproximately 0.62 kcalmol-1 per heavy atombased on PI3Kg IC

50 Kd [32]; this value com-

pares with approximately 0.37 or compound 14and reaches twice the value or compound 6(Figure 2). Including the thiazolidine-2,4-dione

moiety in urther inhibitors, such as AS-252424(Merck-Serono, compound 16 [Figure 2]),allowed or a more diverse and less classicalpotent hinge binder that could possibly displayalternative physicochemical properties or selec-tivity prole (PI3Kg: IC

50= 30 nM, 1.52 log

selectivity against PI3Ka,b,d). A unique set oavorable interactions with the thiazolidine-2,4-dione moiety, especially an ionic interaction withthe conserved lysine, occurs or this moleculein the anity pocket [33]. Such compoundshave been shown to have preclinical therapeu-

tic potential outside the oncology eld [34].

Combining the thiazolidine-2,4-dione moietywith a classical hinge binder 4-pyridinylquino-line (Figure 3C), has resulted in the discovery oGSK105615 (GlaxoSmithKline, compound 17[Figure 2]) [213] a very potent pan-class I PI3K

inhibitor (PI3Ka: Ki = 0.42 nM,


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The optimization o the privileged kinaseinhibitor scaold imidazoquinoline [41] has ledto the imidazoquinolin-2-one NVP-BEZ235(Novartis, compound 20 [Figure 2]) [216]. Thiscompound is a potent class I PI3K inhibitor

(PI3Ka: IC50 = 4 nM, no selectivity againstPI3Kg and d and


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with advanced solid tumors enriched by patientswith advanced breast cancer. A second inhibitorrom the same company (NVP-BGT226, struc-ture not disclosed) has entered Phase I clinicaltrials in 2008 or patients with advanced solid

tumors, including advanced breast cancer.PI-103 (compound 21 [Figure 4]) [217] has beenpresented as a dual PI3Ka/mTOR inhibitor[43]. The compound has demonstrated single-digit nanomolar potency with promiscuity orthe class Ia PI3K and inhibition within 1-logunit o potency, as well as or class Ib PI3Kgand PIKK DNA-PK. Less potent activity isreported or mTOR and class II PI3K C2b com-pared with class Ia PI3K [44]. PI-103 has beenmodied urther to give GDC-0941 (integratedin Genentech/Roche portolio, compound 22[Figure 4]

)[218],

with a classical replacement oboth the phenol with an indazole and the pyri-douropyrimidine core with a thienopyrimidinecore, and through the introduction o a weaklybasic solubilizing group. GDC-0941 has beendescribed as a potent dual PI3Ka/d inhibitor(PI3Ka/d: IC

50= 3 nM, 1 log unit selectivity

against PI3Kb/g) with an improved selectivityprole within the PI3K super-amily as com-pared with PI-103 [45]. GDC-0941 is an orallyavailable compound that has been shown toproduce stasis or even regression when admin-istered at a dose o 75 mg/kg twice daily to

mice bearing either PC3 PTEN-null prostateor MDA-MB361 ErbB2 amplied breast tumorxenograts, respectively. The antitumor eectwas correlated with the reduction o phos-phorylation levels o Akt, PRAS40, RPS6 andp70S6K, demonstrating ecient target modula-tion in tumors. Phase I clinical trials with thiscompound were initiated in patients with solidtumors in April 2008. An interim report romthe rst our cohorts o the dose-escalation study(daily 1560 mg) disclosed an already promis-ing relationship between pS473-Akt levels in

platelet-enriched ractions and drug concentra-tion in plasma. At these dose levels, the com-pound appears to be tolerated, with no grade3/4 drug-related adverse event reported with aschedule o 3 weeks on, 1 week o, good PK doseproportionality and a moderate-to-long hal-lie(828 h) [46].

Exelixis have taken an aggressive clinicaltrial approach with the simultaneous develop-ment o three molecules, each with a dierentinhibition prole within the PI3K/Akt/mTORpathway. XL-147 (structure not disclosed) has

been described as a pan-class I PI3K inhibitor

(PI3Ka: C50

= 39 nM, not selective againstPI3Kg and d, showing 1-log unit selectivityagainst PI3Kb, DNA-PK and mTOR) [47,48]. Inpatients with solid tumor, orally administereddaily as a gelatin capsule, with a schedule cycle

o 3 weeks on, 1 week o, the compound hasbeen escalated rom 30 to 900 mg (Phase I tri-als). The treatment is well tolerated, with grade 3rash as the most prominent adverse eect and, ingeneral, no eect on glucose levels and minimalinsulin increase. Stable disease (more than threecycles) was achieved or eight out o 23 patients.The mean terminal hal-lie o the compoundis 3.76.3 days with steady state achieved ater1520 days [49]. Further Phase I trials have beeninitiated with XL-147 to assess the combina-tion o XL-147 with paclitaxel, carboplatin and

erlotinib (Tarceva

) in patients with solid tumors.XL-765 (structure not disc losed) has beendescribed as a dual class I PI3K/mTOR inhibi-tor showing the same level o potency on PI3Kaas XL-147 (PI3Ka: IC

50= 39 nM, activity

within 1 log unit or class I PI3K) and show-ing activity against mTOR (IC

50= 157 nM)

and DNA-PK [50]. XL-765 also dierentiatesitsel rom XL-147 with its pharmacokineticproperties (median terminal hal-lie o 311 hand steady state reached by day 8). XL-765 wasadministered twice daily without a resting period(15120 mg) or once daily 100 mg. This dose is

currently being considered together with 50 mgtwice daily or urther studies. The highest dosetested (120 mg twice daily) showed clear signso adverse eects, including grade 34 increasein hepatic transaminases. Analyses o glucosehomeostasis markers revealed blood insulin lev-els increased in an exposure-dependent manner,but glucose levels were minimally impacted [51].As or XL-147, ollow-up Phase I clinical combi-nation studies have also been initiated in patientswith solid tumors together with erlotinib, and inpatient with malignant gliomas together with

temozolomide. The third and last compoundrom Exelixis, XL-148, is an Akt inhibitor, and isdescribed in the section titled Inhibitor o AGCkinase amily members Akt and PDK1.

Specic inhibitors of the mTOR kinaseOver the last 2 years, in a similar manner toPI3K, a new generation o mTORC1/2 inhibi-tors has emerged rom screening and drug-dis-covery eorts directed toward the kinase activesite o mTOR. These molecules are also enteringPhase I clinical trials as anticancer agents. This

mode o action allows a direct modulation o


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Review| Stauffer, Maira & Furet

both complexes mTORC1 and mTORC2. Anexample o such an inhibitor, originating romthe classical kinase inhibitor scaold 8-amino-imidazopyrazine [52], present in the IGF-1Rinhibitor OSI-906 (compound 20 [Figure 4]; in

Phase I clinical trial) and optimized or selec-tive mTOR inhibition [219,220], is OSI-027 (OsiPharmaceuticals, structure not disclosed). Thiscompound has been in Phase I clinical trialssince June 2008 in the same patient populationas reported or the PI3K inhibitor GSK105615.Another molecule rom the same company(OXA-01) has been described more recently as apotent mTORC1 (IC

50= 29 nM) and mTORC2

(IC50

= 7 nM) inhibitor. In the presence o thecompound, the phosphorylated 4E-BP1, RPS6and Akt levels were completely inhibited in

the breast cancer cell lines MDA-MB-231 andBT474. Moreover, active apoptosis inductionin a subset o tumor cell lines was also noted.However, these activities occur at much higherconcentrations compared with the mTORC1/2inhibition (23-log unit shit versus the cell-reeassay IC

50). In vivo, administration o OXA-01

at a dose o 75 mg/kg twice daily produced sta-sis o MDA-MB231 breast tumors grown sub-cutaneously in mice. A good correlation wasobserved between phosphorylated 4E-BP1 levelsand the drug concentration in plasma[53]. Usingthe isosteric and classical promiscuous protein

kinase inhibitor scaold 4-amino-pyrazolopy-rimidine and the structural knowledge aroundexisting inhibitors, dierent selectivity proleshave been generated depending on the substitu-tion pattern at positions 1 and 3; the prolesgoing rom broad-spectrum tyrosine kinase todual to specic PI3K superamily kinase inhibi-tors, such as PP242 (compound 24 [Figure 4]),a potent mTOR inhibitor (IC

50= 8 nM and

>12 log selectivity against PI3Ks and tyrosinekinases) [54]. KU0063794 (Astra-Zeneca, com-pound 25 [Figure 4]) [221] has been described as a

potent specic mTOR inhibitor (IC50 = 2.5 nMand >2 log unit selectivity against other PI3Ksuperamily members). Comparison o com-pound 25 and rapamycin on a panel o 70 tumorcell lines in vitro showed a clear broader spec-trum o antiprolierative eect by inhibiting thecatalytic activity o mTOR compared with theallosteric inhibition o mTORC1. KU0063794has shown almost stasis in a U87MG gliobla-soma tumor xenograt model in mice whenadministered daily at 75 mg/kg [55]. Celgenehave presented new mTOR inhibitors exhibiting

preclinical properties which distinguish them

rom rapamycin. These molecules have beenshown to inhibit the prolieration o both rapa-mycin-sensitive and rapamycin-insensitive celllines, to inhibit Akt phosphorylation more read-ily (1 h) compared with rapamycin (24 h), and to

inhibit the IGF-1 induced Akt phosphorylationmediated by mTORC1 inhibition [56].

Exelixis have disclosed new selective mTORinhibitors which are still at the preclinical stage,or instance, EXEL-4431 and EXEL-7518, thatinhibit mTORC1 with subnanomolar potencyand with 34 log units o selectivity againstclass I and III PI3Ks and DNA-PK[57].

Inhibitor of AGC kinase family membersAkt & PDK1Indeed, much less progress has been made

toward the development o kinase inhibitors tar-geting either Akt or PDK1. Because Akt plays acentral role in the PI3K/Akt/mTOR pathway, itrepresents a well-validated and attractive target.The complexity o the regulatory interactionsbetween the dierent domains composing thiskinase, together with the act that Akt inhibitionis oten linked to promiscuity within the AGCkinase amily (protein A, G and C kinases) havebeen limiting actors to the discovery and devel-opment o classical Akt ATP active site-directedinhibitors. One breakthrough has been the dis-covery o allosteric inhibitors by Merck, such as

compound 26 (Figure 4) [222], that even allowedthe generation o isoorm selectivity, despite thehigh degree o amino acid homology ound inthe three Akt proteins; Akt3 has approximately80% protein sequence identity with Akt1 andAkt2 and an ident ica l act ive site cle t [58].Compound 26 has been described as a low micro-molar Akt1 inhibitor hit (Akt1: IC

50= 3.4 M,

Akt2: IC50

= 23 M and Akt3: IC50

> 50 M)identied by high-throughput screening againstthe ull-length Akt paralogs [59], inactive againstPH domain-truncated Akt [60], and that has been

urther optimized to more potent compoundsby modication o the quinoxaline core andextension at the isopropylamine substituent [59].These PH domain-dependent, isozyme-specicinhibitors are the most specic Akt tool sparingAGC kinase amily members described so ar.They have already been used to assess the di-erent Akt para log contribution to the apoptoticresponse o tumor cells to a variety o chemo-therapies [61]. However, it is the new pan Aktinhibitor GSK690693 (GlaxoSmithKline, com-pound 27 [Figure 4]) [223] (Akt1: IC

50= 2 nM,

Akt2: IC50 = 13 nM and Akt3: IC50 = 13 nM)


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directed toward the ATP active site that hasrecently initiated its clinical development.GSK690693 is a highly unctionalized tetra-substituted imidazopyridine that displays adegree o selectivity against a broad panel o

kinases with 13 kinases substantially inhibited(IC

50< 100 nM), including AGC amily mem-

bers PKA, PrkX and PKC isozymes. The Aktinhibitory potential has also been establishedin cellular settings assessing the inhibition ophosphorylation o dierent Akt substrates(GSK3a/b, FKHR/FKHRL1, p70S6K andPRAS40) that could be achieved without con-comitant inhibition o upstream receptors or theMAPK pathway. Total nuclear translocation oFOXO3A-GFP (FOXO3A is a transcriptionactor or which the cellular localization is con-

trolled directly by Akt. The GFP moiety servesas a fuorescence marker to easily determine thelocalization o the exogenously expressed chi-meric FOXO3A-GFP protein in the presence orabsence o a given test item) in stably transectedU2OS could be achieved with 1 M o the inhib-itor. Sensitivity to the antiprolierative eects oGSK690693 are variable, with IC

50values less

than 1 M observed ve out o eight breasttumor cell lines. Cells sensitive to the compoundwere ound to express high levels o phosphory-lated S473-Akt, but not all cells presenting thiseature or genetic deects on the PTENgene were

sensitive to the compound. In vivo, GSK690693showed moderate statistically signicant ecacyon BT474, HCC-1954 (breast), LNCaP (pros-tate) and Sk-Ov-3 (ovarian) tumor xenogratmodels in mice when administered intraperito-neally at 30 mg/kg once daily. At two thirds othis dose the compound is already inducing anobservable increase in blood insulin and bloodglucose (maximal eect around 4 h) indicativeo a strong block o liver insulin signaling via theinsulin receptor and the PI3K/Akt pathway[62].The hyperglycemic eect o this compound is

being used as a pharmacodynamic marker inman. In turn, the overall glucose homeosta-sis may, as described in preclinical models, benormalized with the concomitant use o a lowcarbohydrate-content diet. This modality is alsoused or the current Phase I clinical study that isbeing conducted in patients with solid tumors,including lymphoma, or which the compoundis given by an intravenous inusion.

Early work has illustrated the diculty togenerate selectivity within the AGC kinase am-ily or Akt inhibitors; or instance PKA displays

only three dierent residues in its active site clet

compared with Akt, but is much easier to cocrys-tallize. Using PKA-mutated structure-basedoptimization, ()-balanol derivatives have beenoptimized rom dual Akt/PKA inhibitors to apotent Akt inhibitor (compound 28 [Figure 4],

IC50 = 20 nM), displaying 2 log units o selec-tivity against PKA[63]. The use o a PKAPKBchimera has acilitated structural elucidationo these inhibitors. Additionally, cocrystalliza-tion o PKA and phosphorylated Akt2 with thesubnanomolar Akt inhibitor A-443654 (Abbott,compound 29 [Figure 4],Akt1: IC

50= 0.16 nM,

>2 log selectivity against PKA), rom the inda-zolepyridine series [63], has allowed the valida-tion o key structural dierences. The dierentorientation o the indole moiety o the ligandA-443654 explains the selectivity observed or

this compound[64]

. This series has been urtheroptimized, with the replacement o the indolewith a phenyl, at the price o cardiovasculareects. Further substitution o the phenyl witha trifuoromethyl at position 3 and introductiono a nitrogen at position 6 o the indazole haveyielded molecules devoid o undesired cardiovas-cular eects [65]. A-443654 has been described asa cellular inducer o the S473-Akt phosphoryla-tion, independent o the downstream mTORC1inhibition [66]. This conormational or unc-tional eect on the phosphorylation turnover oS473-Akt is puzzling. The sequence containing

the serine 473 belongs to the C-terminal endhydrophobic moiety classically present in theAGC kinase amily members. This hydrophobicmotive is involved in regulating their unctionby interacting with PDK1, or with their ownso-called PIF pocket, located in the small lobeo the kinase domain [67]. An example o allo-steric agonistic modulation o PDK1 by inter-acting with the PIF pocket has been recentlydescribed [68]. It is now well established that theN-terminal PH domain exerts an inhibitoryactivity on the nonphosphorylated kinase that

is lost upon recruitment at the membrane byPIP3. The subsequent sequence o events andtheir interdependence, leading to the ull activa-tion o Akt by phosphorylation on threonine 308and serine 473, are less well understood.

A more comprehensive discussion o Akt inhi-bition has been made elsewhere, [69,70], includingdescription o the agents disrupting the PIP3/PHdomain interaction; design o such agents couldbe guided by the structural inormation avail-able on this interaction between phosphoinositoland the PH domain [71]. This is a valid strategy

or inactivation o Akt since it will prevent the


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membrane translocation o Akt and, possibly,may also disrupt the translocation o PDK1, butit is not without selectivity challenges. Periosine(Keryx Biopharmaceuticals, compound 30[Figure 4]) is one clinically relevant agent that is

in part mediating its eects by blocking Akt acti-vation. Periosine is a zwitterionic phospholipidthat is thought to interere with the PIP3/PHdomain interaction [72]. Several Phase I/II clini-cal trials with this compound are currently ongo-ing. Finally, XL-418 is a dual Akt/p70S6K inhibi-tor rom Exelixis that has entered Phase I clinicaltrial in April 2007 but has been suspended due tothe very low systemic compound levels observedin patients treated with this molecule.

As previously mentioned, PDK1 belongs tothe AGC kinase amily and is responsible or the

phosphorylation o a subset o kinases belong-ing to its own amily, including Akt (Thr-308,Akt1 numbering). Specically or the substrateAkt, PDK1 regulation occurs via its PH domainmediated translocation to the membrane andcolocalization with Akt upon building up o thePIP3 secondary messenger pool [73]. Despite theact that PDK1 is present as a unique enzyme,achieving specic active site inhibition has beenalmost as dicult as or the Akt kinase. The nat-ural product UCN-01 (compound 31 [Figure 4])isolated rom Streptomyces is a PDK1 inhibitorthat is currently undergoing clinical trials [74].

UCN-01 is closely related to staurosporine and,as such, shares some promiscuity, most notablywithin the AGC kinase amily. Another exam-ple o a compound described as a modulator oPDK1-mediated activation o Akt is celecoxib(Merck, compound 32 [Figure 4]), [224], which isa cyclooxygenase-2 (COX-2) inhibitor with well-documented clinical history, currently undergo-ing multiple Phase I/II clinical trials as singleagent, or in combination with conventionalchemotherapeutics or radiation. This compoundhas been described as a weak micromolar ATP

competitive inhibitor o PDK1. A medicinalchemistry eort to diverge the PDK1 activityrom the COX-2 activity has led to the discoveryo more potent molecules, such as OSU-03012(compound 33 [Figure 4]). This compound hasbeen shown to exert the desired proapototic eectagainst the tumor cell line PC3 with the samelevel o potency as enzymatic PDK1 inhibition(PDK1: IC

50= 5 M) [75] arguing or the possibility

o o-target eects. One possible candidate is thep21-activated kinase (PAK) [76]. NVP-BAG956,rom the imidazo-quinoline series, (compound 34

[Figure 4]) has been described as a selective dual

PI3K/PDK1 inhibitor (PI3Ka: IC50

= 56 nM,PDK1: IC

50= 245 nM). This is an unprec-

edented prole with inhibition within both thePI3K amily and the AGC kinase amily. Thecompound was ound to reduce threonine 308-

Akt levels in U87MG glioblastoma tumor cellswith an IC

50o 45 nM, as well as downstream

pharmacologic markers. This compound ismarginally ecacious in mice against prostatePC3M tumors when administered twice daily,orally, at a dose 75 mg/kg[41]. This compoundwas however successully used to enhance theinhibitory eects o the c-Abl and PKC/Flt-3inhibitors imatinib, nilotinib or PKC412,against BCRABL-expressing or FLT3 mutantcells, respectively[77].

Toward isoform-specic class I PI3Kinhibitors in oncology

Evaluating the epidemiology o PIK3CA muta-tions leads to the attractive possibility o PI3Ka-speciic inhibition to treat cancer patientsstratiied or PI3Ka-activating mutants. Inaddition, recently isolated natural product romthe sponge Aka coralliphaga liphagal (Wyeth,compound 35 [Figure 4]) has been claimed tobe a PI3Ka-specic inhibitor (IC

50= 100 nM,

1 log selectivity against PI3Kg)[78].Several che-motypes inhibiting the PI3K superamily havebeen described. Through an understanding

o the key structural eatures that are govern-ing their selectivity proles [34,79], the urtherelaboration o more selective inhibitors o thedierent PI3K isoorms remains a possibility.The recent resolution o the PI3Ka x-ray struc-ture will undoubtedly impact on the design omore-specic PI3Ka inhibitors [80]. The activat-ing mutation o PI3Ka is remote to the ATPactive site clet; thereore, it is not expected thata classical ATP active site-directed inhibitorwill generate specic inhibition o the activatedmutant compared with wild-type PI3Ka. For

instance, the most requent mutation o PI3Ka(H1047R) does not alter the active site geometrybut rather stabilizes an active conormation othe activation loop in a similar manner to thatseen in wild-type paralog PI3Kg[81].

The elucidation o the roles o the dierentclass I isoorms in normal nonpathological tis-sues is still being carried out in several institu-tions worldwide. These studies are generallyconducted in genetically manipulated mice.The rst sets o studies have encompassed thetotal abrogation o gene expression by knockout

o the various isoorms. Little inormation has


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Figure 4. Inhibitors of the phosphatidylinositol-3-kinase/Akt/mTOR pathway and related scaffolds (compounds 2136).

N N

N

OO

N

O H

N N

N

OS

N

H

N

NNSO

O

N

N

O

N

N

O

N

O

N

N

NH2

NH2

NH2

NH2

NH2

N

N

N

H

OH

OH

OH

OH

N

H

N

NN

NO

N

ON N

O

O

N

H

NH

OO

N

N

H

O

N

NH

N

N

ON

OP

O-

OO

N+

NH

OH

O

N

N

O

N

H

N

N

SO

O

F

FF

N

N

N

N

N

N

N F

FF

N

H

H2N

H2N

O

N

N

NHO

HO

HO

N

N

N

N

N

N

O

KU0063794 GSK690693

25 26 27 28

21

PI103

22

GDC-0941

23

OSI906

24

PPP242

33

OSU-03012

34

NVP-BAG956

35

Liphagal

36

IC87114

29 30 31 32

A-443654 Perifosine UCN-01 Celecoxib

HN


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been retrieved rom these studies or the PI3Kaand PI3Kb isoorms as the ablation was oundto be embryonic lethal [82,83]. The conditionalknockout o the PIK3CA and PIK3CBgenes or,even better, replacement o the endogenous gene

by a kinase-dead version was published recently.Not surprisingly, p110a has been ound to becritical or regulation o cell growth, cell signal-ing through growth actors and cell transorma-tion [84,85]. The surprise came with the ndingsthat p110b seems to also have a strong involve-ment in cell growth and metabolism and thatthis is in part due to its activation by G-proteincoupled receptors, a eature that was believed tobe the hallmark o p110g [86,87]. Indeed, unc-tional redundancies between these two isoormswere identied [88]. Even more striking is the act

that PTEN-null-induced prostate intra-epithelialneoplasia phenotype could be reverted by losso p110b expression [86]. Supporting this, theablation o p110b in the PTEN null cell linePC3 was ound to reduce its tumorigenic-ity[89]. To date, onlyd-specic inhibitors havebeen disclosed or cancer treatment, based onthe act that this isoorm is highly expressedin hematological malignancies. In July 2008,CAL-101 (Calistoga) has entered Phase I cl ini-cal tria l in patients with blood cancer. This mol-ecule is an orally bioavailable PI3Kd-selectiveinhibitor belonging to the series o IC-87114

(ICOS/Lilly, compound 36 [Figure 4]) and isthe rst class I PI3K isoorm specic to be testedor oncology use.

Biomarkers &

pharmacodynamic readouts

Which tumor types will respond to PI3K inhibi-tors and what readouts could be used to monitorPI3K inhibition? These are probably the recur-ring questions or all pharmaceutical companiesthat have or will have PI3K clinical developmentcandidates. Both questions require the use o

validated predictive and pharmacodynamic orresponse biomarkers, respectively.

Predictive biomarkersSince PI3K activation is oten linked to geneticabnormalities in specic genes (e.g., PIK3CA,PTEN, EGFRand KRAS), their genetic statuswill probably dictate the clinical outcome [90].The best examples to illustrate this is the use othe c-Abl/c-kit inhibitor Gleevec or the treat-ment o Philadelphia positive (BcrAbl translo-cated) chronic myeloid melanomas or mutated

c-kit containing GISTs. What is the evidence

that would allow a similar conclusion or thePI3K or PI3K pathway components? Using theMerck allosteric Akt 1/2 inhibitor, She and col-laborators were able to demonstrate, in a mono-layer prolieration assay, a higher sensitivity to

the agent in breast cancer cell lines bearing eitherErbB2 amplication, or PIK3CA mutation [91].Moreover, the cell lines harboring these geneticalterations seemed to be more prone to cell death,when exposed to the inhibitor. Similar obser-vations were made with the pure pan-class Iinhibitor GDC-0941 [92], or with the dual PI3K/mTOR inhibitors GSK105615 [35,36,93], NVP-BEZ235 [94] and SF-1126 [26]. More studies arerequired to ully understand the underlyingmechanisms responsible or both insensitivityand sensitivity toward these pharmacological

tools. The observed stratication might, orexample, be used to establish predictive molecu-lar signatures, that could be use to better delin-eate the target population. Such signatures couldbe established preclinically, in case o PTEN-nulltumors [95,96].

Hence, or Phase I and urther Phase II and IIItrials, it is important to have in place the genomictechnologies that will allow a rapid and preciseassessment o gene copy number (e.g., ErbB2 orHER1 in breast or lung cancer, respectively) andexon sequencing (e.g., or PIK3CA- or KRAS-activating mutations). The loss o certain key

tumor-suppressor genes, such as PTEN, couldbe detected by quantication o the correspond-ing mRNA. In this particular case, the presenceor absence o the protein should be conrmedby immunohistochemical analysis, as epigen-etic silencing or post-translational modica-tions might occur. To complete this task, it is,thereore, important that good-quality tumormaterial is collected prior therapy. Since tumorbiopsies are sometime ineasible or provide low-quality samples, alternative approaches exist.Tumor cells escaping rom the primary masses

could be detected in the blood system. Recenttechnologies have been deployed or the recoveryand enrichment o these circulating tumor cellsrom the blood o patients. Moreover, circulat-ing tumor cells could be used to detect geneticlesions, such as ErbB2 amplication [97]. It is alsopossible to collect cell-ree tumor DNA in variousfuids, including blood and urine, rom patients.Although it is probably premature to generalizethis or all clinical trials, this technology hasalready been successully employed to detectthe KRAS mutational status rom DNA isolated

rom patients with colorectal carcinoma[98].


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Invasive pharmacodynamic biomarkersThe identity o the pharmacodynamic bio-markers or the PI3K pathway, are reason-ably straightorward. Indeed, the phosphory-lated levels either o Akt, orkhead actors or

PRAS40, p70S6 K

, RPS6, or 4E-BP1 couldindicate the relevant inhibition o PI3K, PI3Kand Akt or PI3K, Akt and mTORC1, respec-tively. In preclinical models, these readoutshave been extensively used to ollow the phar-macokineticpharmacodynamic relationshipo the PI3K inhibitor GDC-0941 [99], the dualPI3K/mTOR inhibitors NVP-BEZ235 [42],GSK105615 [35,93,36] and, or the mTOR cata-lytic inhibitors, KU-0063794 [55,100]. Althoughinitially appearing to be a rather trivial exer-cise when using preclinical tumor models, the

detection and quantication o these eventsare more complex when using tumor materialrom biopsies. Indeed, preclinically one canuse regular western-blotting or ELISA assays,the only way that this can be done with humansamples is immunohistochemistry. From thisaspect, the PI3K inhibitors have inherited a lotrom the rapamycin and rapamycin derivativesclinical trials. This is best exemplied withinthe extensive pharmacodynamic studies thathave been carried out with the Novartis com-pound RAD001 (everolimus), in human solidtumors [101]. Although easible, the technology

is bound by the variations imposed by the pro-cedures rom the surgical resection to tissuexation, and by the heterogeneity inherent inthe tumor material. It is, thereore, o generalconcern that using tumor material might notbe suicient to dynamically monitor theseread outs. To palliate this, a variety o surro-gate tissues have been ound to be suitable orthese purposes. Among them, the use o pre-Bmononuclear cells isolated rom blood sam-pling[102], or the use o hair ollicles [103] havebeen recently ound to be valuable surrogate

tissues, and are currently being used in clini-cal trials and are already demonstrating goodoutputs as shown or the Exelixis compoundsXL-147 and XL-765 [104]. It is probably sae tospeculate that these will become the standardbiomarker assays or orthcoming Phase II clini-cal trials. Finally, the determination o bloodglucose and insulin levels is still considered,although caution should be taken when usingthese homeostasis components. One reason isthat, in contrast to the expectations that PI3Kinhibitors would increase both parameters, it

seems that only insulin levels will increase and

then only in a transient manner. This is in sharpcontrast to what is observed with Akt inhibitors,such as the Merck Akt allosteric inhibitors thatwould show both strong upregulated insu linand glucose blood levels [105]. The mechanistic

phenomenon that probably explains these di-erences still remains to be elucidated. The sec-ond reason would be that both levels are linkedto the general metabolic status o the patients,and would require some normalization, as donethrough a asting period.

Noninvasivepharmacodynamic biomarkersCompleting the biomarker arsenal are the non-invasive imaging technologies. Taking advan-tage o the implications o the PI3K pathway in

glucose uptake, and the avidity o tumor cellsor glucose metabolism, positron emission tomo-graphy (PET) using18F-deoxy-glucose (FDG) asa radioactive tracer might represent the best solu-tion or showing pathway inhibition. In mice,H1047R-PIK3CA lung-induced carcinoma caneasily be imaged by FDG-PET, and the signal intumor is readily shut o with the PI3K inhibi-tor NVP-BEZ235 [6]. As an extension, it is con-ceivable that this methodology could be used toselect patients with pathway addiction, revealedby FDG-PET imaging, but this remains to bevalidated retrospectively, in stratied Phase II

trials. Alternatively, the implication o PI3K inangiogenic events, downstream o VEGF hasrecently been strongly validated genetically,and pharmacologically[106,107]. Indeed, strongin vitro and in vivo anti-angiogenic propertieshave been demonstrated or the pure pan-class IPI3K inhibitors GDC-0941 [108], SF1126 [26] andZSTK474 [109], and or the dual PI3K/mTORinhibitor NVP-BEZ235 [110]. By extension, theseinhibitors are expected to inhibit tumor vascu-lature permeability, as demonstrated or NVP-BEZ235 [110], using a DCE-MRI approach with

the contrasting agent P792 (Vistarem). Thevalidation o this approach in Phase I clinicaltrials is currently ongoing.

Feedback loops, cross-talks

& combination therapies

Feedback loops & redundant pathways:when PI3K & Ras pathways talk togetherIn normal cells, signaling pathways are tightlyregulated, such that their activation is not tran-sient and terminated rapidly. Termination sig-nals are oten provided by the downstream eec-

tors themselves. Activation o the Ras pathway,


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or example, can be readily terminated whenERK kinases phosphorylate the integrator mol-ecule Grb2, promoting the dissociation o theRas GEF protein SOS [111]. The same principleapplies to the PI3K pathway [112]. It is now

well established that PI3K recruitment to someRTKs, such as the insulin receptor, is rapidlyabolished upon ligand binding, due to IRS phos-phorylation-induced degradation. This eect isprincipally mediated by the mTORC1 eectorp70S6K, and the consequence o mTORC1 block-ade being IRS stabilization, and subsequentPI3K pathway reactivation. As a matter o act,rapamycin derivatives have been shown to pro-mote Akt activation in vitro [113] and this wasalso conrmed in human tumors [101]. Moreover,it was recently demonstrated that PI3K reactiva-

tion by rapamycin could lead to Ras pathwayengagement and ERK activation [114]. Hence,pure mTORC1 inhibition might lead to limitedecacy. In principle, PI3K inhibitors should notinduce such pathway reactivation, as in bothcases this would require the lipid kinase activ-ity. Similarly, mTOR and dual mTOR/PI3Kinhibitors are not expected to behave like allo-steric mTORC1 inhibitors, as mTOR catalyticinhibition is also impacting mTORC2, which isrequired or ull activation o Akt [8].

Since pathways are not linear, but ratherbranched to others, the possibility that tumors

escape rom certain targeted therapy is notunlikely. For example, activation o the ribo-somal protein RPS6, one o the mTORC1downstream eectors through p70S6K, can alsobe achieved through the ERK substrate p90RSK.In a more general manner, tumors might haveactivated constitutively dierent pathways con-verging on similar important eectors such asRPS6 or 4E-BP1 [115]. The antiapoptotic pro-tein BAD has also been shown to be a commonRas and PI3K pathways target, important ortumor cell survival [116]. It is, thereore, impor-

tant to plan ahead or such a scenario, andidentiy best cotreatments that will synergizewith PI3K inhibitors. Inhibitors o the Ra andMEK kinases are certainly the best candidatesto test this hypothesis. Validation o this concepthas actually been provided using mice bearingKRAS induced lung adenocarcinoma. Althoughtreatment with either the PI3K/mTOR inhibitorNVP-BEZ235 or the Astra Zeneca/ARRY MEKinhibitor AZD6244 lead to only modest anti-tumor eects, complete tumor shrinkage wasobserved when both drugs were administered

simultaneously[6].

PI3K pathway & acquired resistance toupstream targeted agentsAcquired resistance during treatment is a recur-rent theme in cancer therapy. For small-molecu-lar-weight kinase inhibitors that inhibit through

binding into the catalytic pocket, the molecularmechanisms or resistance could be due to theselection and expansion o mutational event thatreduces or abolishes the anity o the inhibitor tothe target. This is the case or the EGFR inhibitorsgetinib (Iressa) and erlotinib that are inactiveagainst EGFR containing the threonine 790 tomethionine (T790M-EGFR) gatekeeper residueconversion. This mutation induces an increasedanity or ATP, hence reducing the competi-tive inhibition with the inhibitors [117]. However,this could also be attributed to a rewiring o the

signaling pathways through alternate routes.Abrogation o the cell-death-induced eects bygetinib in non-small-cell lung cancer (NSCLC)models could be readily achieved when an onco-genic orm o PI3K is expressed, underlining theimportance o the PI3K pathway[118].

The PI3K pathway is critically involved inNSCLCs, as per its recruitment and activation byErbB3, the third and only member o the EGFRamily that has no kinase activity. In addition,ErbB3 is oten co-amplied in NSCLC [119].More evidence indicating a central role o ErbB3in mediating resistance to tyrosine kinase inhibi-

tors, through recruitment o PI3K is rst, in thepresence o tyrosine kinase inhibitors, the intrin-sic equilibrium driving the activity o ErbB3 ischanged toward a more active state [120] , second,cMet amplication can substitute or EGFR-mediated ErbB3 activation [121] and, third, asimilar situation could occur involving IGF-1Ractivation, through reduced expression o thenegative secreted regulator IGFBP-3 [122]. ForErbB2-amplied breast cancers, activation othe PI3K pathway was genetically shown to bea major determinant o resistance to anti-ErbB2

therapy, such as herceptin [123]. As or NSCLCs,ErbB3 probably plays an important role in thisphenomenon [124]. PI3K inhibition might, there-ore, play an important role in circumventingtumor resistance upon EGFR therapies [125].Evidences already exist that this is the case, asdescribed or NVP-BEZ235 [126,127].

Conclusions & future perspective

The development o PI3K and/or mTOR inhibi-tors has matured to the stage where we havenow multiple candidates with dierent selec-

tivity proles addressing the inhibition o the


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PI3K/Akt/mTOR pathway in cancer patients.A new generation o molecules will undoubtedlybenet rom the lessons currently being learned inthe clinical trials and will enable a rened prole.New reports o potential PDK1 inhibitor develop-

ment candidates with appropriate selectivity pro-les will undoubtedly appear in the next coupleo years, and this also applies to improved PKBinhibitors, based on the drug-discovery eortsdedicated to those two targets reported in therecent patent literature. It will be very interest-ing to compare a selective PDK1 inhibitor witha selective PI3K inhibitor or a selective mTORcatalytic inhibitor in a clinical setup, since allthree enzymes are strongly connected to Aktactivation but are, at the same time, involved inother important processes and, consequently, may

display a very dierent tolerability prole. Withthe emerging understanding o the role o the di-erent PI3K isoorms, the role o the selective orsparing PI3K isoorm inhibitors should increase,

especially on stratied patient populations andor combination therapy. However, it is unclearat this stage as to whether redundant inhibitionwithin the PI3K/Akt/mTOR pathway will ulti-mately not be needed or optimal ecacy[128].

The upcoming 2 years will be crucial in revealingi the compounds currently trialed in clinics willlive up to their promises or cancer therapy andor tolerability.

Financial & competing interests disclosure

The authors are employees and shareholders o Novartis

Pharma AG. The authors have no other relevant aliations

or nancial involvement with any other organization or

entity with a nancial interest in or nancial confict with

the subject matter or materials discussed in the manuscript.

This includes employment, consultancies, honoraria, stock

ownership or options, expert testimony, grants or patentsreceived or pending, or royalties.

No writing assistance was utilized in the production o

this manuscript.

Executive summary

Phosphatidylinositol-3-kinase (PI3K) and the downstream eectors on the pathway are well-validated and drugable targets. Strong involvement rom the scientifc community has helped to better defne the roles and interconnections o each components on

the pathway in tumor cells.

Strong involvement rom the pharmaceutical sector (biotech and large companies) has helped to adequately resource innovativemedicinal chemistry or PI3K/Akt/mTOR pathway inhibition.

An array o PI3K, PI3K/mTOR, mTOR and Akt inhibitors are already available to the scientifc community. Some o theses inhibitors have entered Phase I clinical trials in 2007/2008, and the frst pharmacokinetic and pharmacodynamic data are

already available.

Preclinical evidence o PIK3CAs status as a predictive marker has been presented and confrmed by dierent groups. A need to better understand the eects on pathway re-activation upon treatment with a PI3K inhibitor has been recognized by

the community.

The need or efcient combination therapies is still a matter o intense investigation.

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