Review Article mTOR Kinase: A Possible Pharmacological ...downloads.hindawi.com/journals/bmri/2015/394257.pdf · mTOR Kinase: A Possible Pharmacological Target in the Management of

Review ArticlemTOR Kinase A Possible Pharmacological Target in theManagement of Chronic Pain

Lucia Lisi1 Paola Aceto2 Pierluigi Navarra1 and Cinzia Dello Russo1

1 Institute of Pharmacology Catholic University Medical School Largo F Vito 1 00168 Rome Italy2Department of Anesthesiology and Intensive Care A Gemelli Hospital Catholic University Medical School 00168 Rome Italy

Correspondence should be addressed to Cinzia Dello Russo cinziadellorussormunicattit

Received 16 May 2014 Accepted 12 September 2014

Academic Editor Livio Luongo

Copyright copy 2015 Lucia Lisi et alThis is an open access article distributed under theCreative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Chronic pain represents a major public health problem worldwide Current pharmacological treatments for chronic painsyndromes including neuropathic pain are only partially effective with significant pain relief achieved in 40ndash60 of patientsRecent studies suggest that the mammalian target of rapamycin (mTOR) kinase and downstream effectors may be implicated inthe development of chronic inflammatory neuropathic and cancer pain The expression and activity of mTOR have been detectedin peripheral and central regions involved in pain transmission mTOR immunoreactivity was found in primary sensory axons indorsal root ganglia (DRG) and in dorsal horn neurons This kinase is a master regulator of protein synthesis and it is criticallyinvolved in the regulation of several neuronal functions including the synaptic plasticity that is a major mechanism leading to thedevelopment of chronic pain Enhanced activation of this pathway is present in different experimental models of chronic painConsistently pharmacological inhibition of the kinase activity turned out to have significant antinociceptive effects in severalexperimental models of inflammatory and neuropathic pain We will review the main evidence from animal and human studiessupporting the hypothesis that mTOR may be a novel pharmacological target for the management of chronic pain

1 Introduction

Chronic pain represents a major public health problemworldwide affecting approximately 37 of the US popula-tion with an economic burden of up to US$ 635 billion peryear [1] In Europe the prevalence of chronic pain syndromesranges between 25 and 30 [2] Physiologically nociceptivepathways are activated in response to traumatic or noxiousstimuli Acute pain which is primarily due to nociceptionserves as an adaptive and protectivemechanism to detect loc-alize and limit tissue damage on the contrary chronic painwhich persists after a reasonable time for healing to occur(ranging between 1 and 6 months in most definitions) canbe regarded as a form of maladaptive response in whichpain is no longer protective or strictly linked to the initialstimulus After application of an intense and prolongedinjury ongoing excitation of primary nociceptive neuronsleads to neuronal changes both in the primary afferents(peripheral sensitization) and in the spinal dorsal horn neu-rons (central sensitization) contributing to the development

of chronic pain [3] In this condition pain arises in theabsence of noxious stimulus may be stimulated by normallyinnocuous stimuli (allodynia) is exaggerated and prolongedin response to noxious stimuli (primary hyperalgesia) andspreads beyond the site of injury (secondary hyperalgesia)[3] Chronic pain has a neuropathic origin in approximately20 of the patients [2] Neuropathic pain may arise from adirect damage of somatosensory nerves or nerves innervatingvisceral organs or from a disease affecting the somatosensorynervous system which implies an indirect injury resultingfromvarious causes includingmetabolic stress autoimmunedegenerative or chronic inflammatory conditions and idio-pathic origins [4]

Neuropathic pain is characterized by pain hypersensitiv-ity that is mediated by both peripheral and spinal neuronalsynaptic plasticity (leading toperipheral and central sensiti-zation resp) involving pre- and posttranslational changesin the expression and functions of receptors enzymes andvoltage-dependent ion channels in sensory neurons [3] Inaddition other biochemical events contribute to the hyper-activity of the somatosensory system including phenotypic

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 394257 13 pageshttpdxdoiorg1011552015394257

2 BioMed Research International

mTOR

mTORC1

darr Deptor

MLST8G120573L

uarr PRAS40

uarr Raptor

(a)

mTORC2

mTOR

mSin1

Protor

MLST8G120573L

darr Deptoruarr Rictor

(b)

Figure 1 Schematic representing the molecular partners of mTOR forming (a) mTOR complex 1 (mTORC1) and (b) mTOR complex 2(mTORC2) The down-arrows indicate the inhibitory proteins whereas the up-arrows indicate activator factors on mTOR function

neuronal switch (ie large myelinated A120573 fibers expressingneuropeptides directly involved in pain transmission such assubstance P and calcitonin gene-related peptide) sproutingof nerve endings (iemyelinatedA120573fibers establishing directcontacts with nociceptive projecting neurons in the lamina I-II of the spinal dorsal horn) loss of spinal inhibitory controland increased activity of descending excitatory pathways[3] Moreover synaptic plasticity within key cortical regionsinvolved in pain processing (ie the anterior cingulated cor-tex the insular cortex primary and secondary sensory cor-tices and the amygdala) has been also observed in relationto neuropathic pain [4] Finally activation of glial cells withrelease of pronociceptive mediators can directly modulateneuronal excitability and thus pain transmission contribut-ing to central sensitization and to the occurrence of neuro-pathic pain [5]

Multimodal pharmacological treatments for chronic painsyndromes including neuropathic pain are based on theuse of antiepileptics antidepressants local anesthetics opioidanalgesics or tramadol These treatments are only partiallyeffective with significant pain relief achieved in 40ndash60 ofpatients [4] A relatively recent modality of neuropathic paintherapy which represents the future challenge of upcomingresearches involves specific cellular targets implied in neu-ronal synaptic plasticity andor glial activation [6] Inter-estingly recent studies show that the mammalian target ofrapamycin (mTOR) kinase and downstream effectors may beimplicated in the development of chronic inflammatory neu-ropathic and cancer painThis kinase is a master regulator ofprotein synthesis and it is critically involved in the regulationof several neuronal functions including synaptic plasticityand memory formation in the central nervous system (CNS)[7] As mentioned above neuronal synaptic plasticity both atperipheral level and in the CNS is amajormechanism leadingto the development of chronic pain thus suggesting thatmTOR may be a novel pharmacological target for the man-agement of chronic pain In addition mTOR has been alsoreported to regulate astrocyte and microglial activity (as wehave recently reviewed [8]) thus suggesting an additionaltherapeutic target in the treatment of chronic pain syndromesthat involve increased glial activation The main evidence

from animal studies as well as clinical reports supporting thishypothesis is reviewed in the present paper

2 mTOR the lsquolsquoMechanisticrsquorsquoTarget of Rapamycin

The mTOR kinase now officially known as ldquomechanisticrdquoTOR is a conserved serinethreonine protein kinase belong-ing to the phosphoinositide 3-kinase (PI3K) family thatregulates multiple intracellular processes [9] In mammalsmTOR is encoded by a single gene [10] and interacts withseveral proteins to form two distinct complexes referred to asmTORC1 and mTORC2 These complexes display differentsensitivity to the inhibitory action of rapamycin whichmainly suppresses mTORC1-dependent activities in acutetreatments [11] Notably the two complexes promote the acti-vation of different signalling pathways and recognize distinctupstream regulators as well as downstream targets whosespecificity is determined by the specificity of the interactingproteins Both complexes include the inhibitory proteinDEPTOR and the adaptor protein mLST8G120573L (mammalianLST8G-protein 120573-subunit like protein) [12] However therole of mLST8G120573L in the regulation of mTORC1 functionremains unclear at present since its chronic loss does notaffect mTORC1 activity in vivo [13] As shown in Figure 1mTORC1 specifically contains the regulatory-associated pro-tein of mTOR (raptor) and the inhibitory protein PRAS40(proline-richAKT substrate of 40KDa) [14] Raptor regulatesmTORC1 assembly and serves as a scaffold for the recruit-ment of specific substrates such as the eukaryotic initiationfactor-4E-binding protein 1 (4E-BP1) [15 16] Similarly otherproteins reside uniquely within complex 2 that is therapamycin-insensitive companion ofmTOR (rictor) the pro-tein observedwith rictor (PROTOR) and the stress-activatedprotein kinase-interacting protein 1 (mSIN1) (Figure 1) [17]Much like raptor rictor is necessary for mTORC2 assemblytogether with mSIN1 for mTORC2 catalytic activity and itmay also be involved in the selective recruitment of specificsubstrates [14]

BioMed Research International 3

Growth factors (ie insulininsulin-like growth factors)

4E-BP1S6K1

eiF-4BeiF-4E

mRNA translation eEF2 kinase

Protein elongation

Cell growth and proliferationAutophagy

mRNA translation

S6Ribosomal biogenesis

Lysosomalbiogenesis

PKBAKT

Energy metabolism

PI3K

IRS1

Cytokines

Rheb

ERK12

Ras

Hypoxialow cell ATP

AMPK

Aminoacid content(ARG-LEU)

RAG AB

mTORC1

TSC1 TSC2

Lysosomes

(TNF120572)

I120581BK

Figure 2 Schematic representing the main intracellular targets as well as the main cellular processes regulated by mTORC1

The mTOR complex 1 is activated in response to differentintracellular and extracellular cues that is growth factorscytokines energy status oxygen and amino acids to controlmultiple functions related to cell growth andmetabolism [12]As shown in Figure 2 several upstream regulators ofmTORC1 activity converge on theheterodimer consisting oftuberous sclerosis 1 (TSC1 also known as hamartin) andTSC2 (also known as tuberin) In this complex TSC1 stabi-lizes TSC2 by preventing its degradation while TSC2 acts asaGTPase-activating protein (GAP) for the small GTPase pro-tein Rheb (Ras homolog enriched in brain) [18] Rheb in itsGTP-bound state binds to and activates mTORC1 whereasthe TSC12 complex normally inhibits mTORC1 activity byfavoring the GDP-bound inactive state of Rheb As recentlyreviewed by Laplante and Sabatini [12] growth factors (suchas insulin and insulin-like growth factors) increase mTORC1activity by promoting the phosphorylation and degradationof the TSC12 complex This occurs via ligand-dependentactivation of receptor tyrosine kinases (RTKs) like theinsulin receptors followed by activation of the PI3K and RaspathwaysThe effector kinases of these pathways namely theprotein kinase B (PKBAKT) and the extracellular-signal-regulated kinases (ERK) 1 and 2 induce TSC12 phosphoryla-tion (Figure 2) In addition AKT can directly phosphorylatethe inhibitory protein PRAS40 promoting its dissociationfrom raptor and further contributing to mTORC1 activation

Proinflammatory cytokines like TNF120572 increase mTORC1activity via I120581B kinase- (IKK-) dependent inactivation of theTSC12 complex whereas in response to hypoxia or lowenergy status the adenosinemonophosphate activated kinase(AMPK) blocks mTORC1 activity by increasing TSC2function and directly inhibiting raptor (Figure 2) Finallyincreased intracellular levels of aminoacids particularly argi-nine and leucine promote mTORC1 activation by inducingits binding to a distinct family of GTPases the Rag GTPasestogether with its translocation to the lysosomal surface [1920] It has been hypothesized that translocation of mTORC1to the lysosomes allows GTP-bound Rheb to interact withmTORC1 promoting its activation only when aminoacids areavailable Additional details on the regulation ofmTORC1 canbe retrieved in the above mentioned review article [12]

Protein synthesis is the best-characterized intracellularprocess regulated by mTORC1 whose activation generallyincreases the cellular capacity of protein generation [14] Thetwo main downstream targets of mTORC1 4E-BP1 and theACG-family protein S6 kinase 1 (S6K1) are key componentsof the protein translation machinery Phosphorylation of 4E-BP1 causes its dissociation from the eukaryotic translationinitiation factor- (eIF-) 4E (Figure 2) This allows eIF-4E toassociate with eIF-4G leading to the formation of eIF-4Fwhich facilitates the loading of ribosomes onto the mRNABy this molecular mechanism mTORC1 can control the


translation of specific mRNAs including the so-called 51015840-TOP mRNAs that mostly encode for components of thetranslational machinery In addition phosphorylation andactivation of S6K1 promote protein translation by phosphory-lation of several substrates including eIF-4B the eukaryoticelongation factor 2 (eEF2) kinase and the ribosomal S6protein (Figure 2) Local protein synthesis within sensoryneurons contributes to their nociceptive functions bothunder physiological conditions and during chronic pain Asdescribed in detail in Section 4 by controlling protein trans-lation mTORC1 can regulate the activity of sensory neuronsin periphery as well as in the CNS The activity of S6K1 isalso important in the control of RTK activation In fact S6K1(activated by mTORC1) promotes also the phosphorylationand inactivation of IRS1 the insulin receptor substrate 1 Thelatter is a docking protein that in its tyrosine-phosphorylatedform couples the insulin receptor to its downstream effectors[21]This is part of a retroinhibitory feedbackmechanism thatreduceRTKactivation thus the activity ofAKT andERK [12]The latter is also a kinase critically involved in the regulationof pain processing (see Section 4) The mTOR complex1 is also involved in the regulation of several metabolicpathways regulating the expression of genes encoding dif-ferent steps of glycolysis and the pentose phosphate pathwayas well as critical enzymes in the de novo biosynthesisof lipids [22] Finally mTORC1 can favor cell growth bynegatively regulating macroautophagy (autophagy) the cen-tral degradative process in cells and lysosome biogene-sis [12 14] As discussed in detail in Section 4 the acti-vation of autophagy in Schwann cells can limit the extent ofaxonal degeneration after nerve injury and promote regener-ation and myelination thus favoring analgesic effects

In contrast tomTORC1mechanisms leading tomTORC2activation are less characterized It seems that mTORC2activation is directly promoted by PI3K via phosphorylationof specific mTORC2 interactors including rictor (Figure 3)[17] Thus mTORC2 appears to be also responsive to growthfactors but insensitive to nutrients mTORC2 regulates theactivity of several proteins belonging to the ACG familyincluding AKT the serum- and glucocorticoid-induced pro-tein kinase (SGK1) and protein kinase C- (PKC-) 120572 AKT is akey regulator of cell survival and proliferation withmTORC2promoting its phosphorylation at Ser

473in the hydrophobic

motif and maximal activation [23 24] In this way mTORappears to be both a downstream effector of AKT (iemTORC1) and an important upstream regulator of the kinaseactivity (ie mTORC2) (Figure 4) Moreover it has beenshown that an intricate crosstalk exists between the twocomplexes (Figure 4) since S6K can negatively control theactivation of mTORC2 [24] whereas TSC12 positively con-tributes to mTORC2 activation [25] The mTOR complex 2also promotes the activity of SGK1 another important kinasein the control of cell proliferation [26] Finally activation ofPKC120572 by mTORC2 along with other effectors (like paxillinand Rho GTPases) regulates the dynamic of actin cytoskele-ton [27 28] Direct regulation of AKT activity together witha role in the control of actin dynamics suggests a possibleinvolvement of mTORC2 in the control of neuronal functionas discussed in Section 4

Cell growth proliferation

AKT

Growth factors

Cytoskeleton

Energy metabolism

SGK1

PI3K

mTORC2

PKC120572Rho

Figure 3 Schematic representing the main intracellular targets aswell as the main cellular processes regulated by mTORC2

mTORC1 mTORC2S6K

Rheb

AKT

TSC1 TSC2

Figure 4 Schematic representing the main mTORC1-mTORC2crosstalks

3 mTOR Inhibitors

The mTOR kinase now officially known as ldquomechanisticrdquoTOR was initially identified as ldquomammalian target ofrapamycinrdquo because the kinase is the main target of an anti-fungal compound derived from Streptomyces hygroscopicusrapamycin [29] This drug discovered in soil samples col-lected from Easter Island (Rapa Nui from where the name)was originally found to have antifungal proprieties butrapidly its immunosuppressive activity became its moreimportant property Actually rapamycin is widely used inpreventing clinical allograft rejection and in treating someautoimmune diseases [30] In the 1980s rapamycin was alsofound to have anticancer activity although the exact mecha-nism of action remained unknown until many years later

Rapamycin (or sirolimus)mainly inhibitsmTORC1 activ-ity by forming a trimolecular complex with mTOR and theimmunophilin FKBP12 (FK506-binding protein of 12 kDaalso known as PPIase FKBP1A) The drug associates withFKBP12 and the resulting complex interacts with the FRB(FKBP12-rapamycin binding) domain located in the carboxylterminus of mTOR the interaction disrupts the associationwith raptor and thus uncouples mTORC1 from its substrates


inducing a block of mTORC1 signaling [31 32] Howevernot all the functions mediated by mTORC1 are sensitive torapamycin the inhibition of cap-dependent translation andthe induction of autophagy are in part resistant to rapamycin[33] Originally the effects of rapamycin were thought tobe only related to the inhibition of mTORC1 but studiesof Sabatinirsquos group have shown that rapamycin given athigher concentrations and in chronic treatments also inter-feres with mTORC2 regulatory functions [11] In particularhigh intracellular levels of rapamycin inhibit the bindingand subsequent assembly of mTORC2-specific componentsmSIN1 and rictor [11]

Rapamycin readily crosses the blood brain barrier (BBB)thus exerting direct effects within the CNS [34] However inorder to ameliorate the pharmacokinetic profile of rapamy-cin novel drugs have been developedThis first generation ofmTOR inhibitors displays the same binding sites for mTORand FKBP12 and is thus so-called rapalogs (ie rapamycinand its analogs) Rapalogs includes CCI-779 (temsirolimus)RAD-001 (everolimus) and AP23573 (ridaforolimus ordeforolimus) (Table 1) Among these mTOR inhibitors CCI-779 is a prodrug of rapamycin which delays tumor pro-liferation [35] and it is actually used for the treatment of renalcell carcinoma whereas RAD-001 a 40-O-(2-hydroxyethyl)derivative of rapamycin is currently used as an immuno-suppressant to prevent rejection of organ transplants andlike CCI-779 for the treatment of renal cell cancer andsubependymal giant cell astrocytoma [36]

However the use of rapalogs unmasked the feedback loopbetween mTORC1 and AKT in certain type of cells ThemTORC1 inhibition induced by these drugs fails to repressthe negative feedback loop that results in phosphorylationand activation of AKT and it is unable to block the mTORC2positive feedback to AKT [37] The elevation of AKT activitycan promote a longer survival in some cell types and mayalso be associated to pain hypersensitivity (as described inSection 4)These limitations have led to the development of asecond generation of mTOR inhibitors the ATP-competitivemTOR inhibitors which block both mTORC1 and mTORC2activity [38] Unlike rapamycin which is a specific allostericinhibitor of mTORC1 these ATP-competitive inhibitors tar-get the catalytic site of the enzyme thus promoting a broadermore potent and sustained inhibition of mTOR and prevent-ing the activation of PI3KAKT caused by the derepression ofnegative feedbacks [39] This is due to the effective inhibitionof rapamycin-insensitive mTORC2 activity in addition tomTORC1 inhibition and also to a more comprehensive andsustained mTORC1 inhibition as demonstrated by sustainedreduction of 4E-BP1 phosphorylation [38] Actually manycompounds with different chemical structures show thesefunctions (see Table 1) and some of them are being tested inclinical trials

Finally the close interaction of mTOR with the PI3Kpathway has also led to the development of mTORPI3Kdual inhibitors [40] Compared with drugs that inhibit eithermTORC1 or PI3K these drugs have the benefit of inhibitingboth mTORC1 and mTORC2 and all the catalytic isoforms ofPI3K Interestingly because of the high sequence homology

between mTOR and PI3K some compounds (like wortman-nin) originally identified as PI3K inhibitors were later shownto inhibit mTOR as well [41] The activity of these smallmolecules differs from rapalog activity for a more specificblock of both mTORC1-dependent phosphorylation of S6K1and mTORC2-dependent phosphorylation of AKT at theSer473

residue Dual mTORPI3K inhibitors include NVP-BEZ235 BGT226 SF1126 and PKI-587 (Table 1) and manyof them are being tested in early-stage of preclinical trials

A detailed list of mTOR inhibitor drugs is providedin Table 1 together with specific information on theirmolecular properties (including in vitro mTOR IC50 andcellular potency towards mTORC1 and mTORC2) DualmTORC1mTORC2 inhibitors have been developed by coun-terscreening their inhibitory activity against the most closelyrelated kinases class I and class III PI3K lipid kinases and thePI3K-related kinase (PIKK) family members [42] whereasdual PI3KmTORC inhibitors were optimized to inhibit classI PI3Ks [43]Thus the in vitro inhibitory potency against classI PI3Ks has been also included in Table 1 As far as the ATP-competitive mTOR inhibitors (first generation and secondgeneration) are concerned we calculated the ratio betweenPI3K and mTOR IC50 and marked those drugs with a ratiolt500 since inhibition of PI3K by these drugs may becomerelevant in cellular or in vivo systems This informationshould provide the reader with a better understanding of thebiological effects of these novel drugs

4 mTOR in the Control of Chronic Pain

41 Histology The expression and activity of mTOR havebeen extensively detected in peripheral and in central regionsinvolved in pain transmission both under physiological con-ditions and in several experimental models of inflammatoryand neuropathic pain In the adult rat and mouse cutaneoustissue mTOR immunoreactivity was found in a subset ofprimary sensory axons and in nonneuronal cells surroundingthe peripheral axons in the dermis [44 45] Using specificmarkers that distinguish between C- and A-fibers it has beenshown that mTOR positive axons are mainly myelinated A-fibers and only less than 5 are peptidergic fibers coexpress-ing CGRP Interestingly these fibers were stained also for theactive form of mTOR evaluated by measurement of mTORphosphorylation at Ser

2448 This phosphorylation primarily

reflects a feedback signal from the mTORC1 downstreamtarget S6K1 (also known as p70S6 kinase p70S6K) and it istherefore considered a reliable marker of mTORC1 activationwithin the cells [46] Moreover myelinated sensory fibers inthe rat skin also express phosphorylated downstream targetsof mTORC1 including 4E-BP1 S6K and the S6 ribosomalprotein [44] suggesting that mTORC1 may regulate localprotein synthesis within these axons thus contributing totheir nociceptive functions under physiological conditionsThese fibers particularly the large A-beta (A120573) fibers arenormally involved in the conduction of nonnociceptiveinputs such as light touchmovement or vibration [47] How-ever amplification of their signals by sensitized dorsal horn


Table 1 mTOR inhibitor drugs

Classes DrugsmTOR

(in vitro kinaseIC50)

mTORC1(cellular potency

EC50)

mTORC2(cellular

potency EC50)

Class I PI3K(in vitro kinase

IC50)References

Rapamycin174120583M

(2 nM1 in presenceof FKBP12)

04ndash35 nM2 [82ndash84]

RapalogsRAD001 04ndash35 nM2 [82]CCI-779 176 120583M lt20 nM 10ndash20120583M [84]AP23573 02 nM [85]

ATP-competitivemTOR inhibitors(first generation)

KU-0063794 25 nM1 660 nM3 240 nM3 gt53ndashgt30 120583M [86ndash88]PP2424 8 nM 300ndash400 nM 010ndash22 120583M [89 90]PP304 80 nM 068ndash58120583M [89]Torin 14 43 nM 2ndash10 nM 2ndash10 nM 017ndashgt10120583M [90 91]

WEY-6004 9 nM1 300 nM 1 120583M 196ndash845 120583M [92]WYE-3544 5 nM1 300 nM 1 120583M 189ndash737 120583M [92]CC214-1 2 nM 40 nM 18 nM 138 120583M [93]OSI-0274 4 nM 042ndashgt30 120583M [94]X-3874 23 nM1 012ndashgt03 120583M [95]

ATP-competitivemTOR inhibitors(second generation)

AZ8055 013 nM1 27 nM3 24 nM3 32ndash189 120583M [42 88]AZ2014 28 nM1 200 nM3 80 nM3 38ndashgt30 120583M [88]

INK128MLN01284 1 nM lt10 nM lt10 nM 022ndash529 120583M [96]WYE-125132 019 nM1 20 nM 200 nM 118ndashgt10120583M [97]CC214-2 106 nM 386 nM 315 nM gt30 120583M [93]

ATP-competitivemTORPI3K dualinhibitors

Wortmannin 02 120583M1 01 nM [83 98]LY294002SF11015 15120583M1 05ndash16 120583M [83 99ndash101]

PI-1035 In vitro kinaseIC50 20 nM

In vitro kinaseIC50 83 nM 2ndash15 nM [40 100 101]

Torin 2 281 nM 025 nM 10 nM 468ndash175 nM [102 103]GSK2126458 ND Low nM 018ndash041 nM 004 nM [104]NVP-BEZ2355 207 nM lt250 nM 8nM 4ndash75 nM [43 101]NVP-BGT2266 4ndash63 nM [105]

SF1126 (RDGSconjugated SF1101)

Not significantinhibitory activityuntil hydrolyzed to

SF1101

[106]

PKI587 14 nM1 lt30 nM lt10 nM 06ndash8 nM [107]In vitromTOR kinase IC50 was evaluated using either the immunoprecipitated or the recombinant full length enzyme Cellular potency for the two differentmTOR complexes was calculated after short term incubation ranging between 30min and 2 h of different cell lines with mTOR inhibitors and subsequentanalysis of the phosphorylation status of specific mTORC1 (S6K or S6) or mTORC2 (AKT at Ser

473) substrates In vitro PI3K and PIKK IC50 were measured

using specific biochemical assays1A truncated mTOR enzyme was used in the in vitro kinase assay2Cellular potency was evaluated by inhibition of cell proliferation using vascular smooth muscle cells stimulated by fetal calf serum3Cellular potency was evaluated using a high throughput immunocytochemical assay carried out in the MDA-MB-468 cell line4Ratio between PI3K and mTOR IC50 is lt5005The reader should also consider IC50 values reported by Hayakawa et al 2007 [99] and Kong et al 2009 [101]6NVP-226 is considered a dual mTORPI3K inhibitor However in vitro preclinical data on the mTOR inhibitory activity for this compound were not foundthrough Medline Search

neurons is thought to account for secondary hyperalgesia andallodynia clinical features of chronic pain (as summarized inSection 1) Consistently active mTORC1 and its downstreamphosphorylated targets (4E-BP1 and the S6 ribosomal pro-tein) have been also detected in the adult rat dorsal rootsmostly in myelinated axons [48] Local or intrathecal admin-istration of rapamycin significantly reduced phosphorylation

of downstream targets of mTORC1 both in the peripheralfibers [44] and in the central spinal cord neurons [48]Similarly to these data phospho-mTOR was also found to beexpressed by a subset of myelinated fibers in the adult mousedorsal roots [45] whereas in rat dorsal root ganglia (DRG)positive immunoreactivity for mTOR and S6K1 was detectedmainly in the cell body of small nociceptors coexpressing


substance P or IB4 positive [49] These data suggest thatmTOR and its downstream targets are mostly transported tomyelinated peripheral and central fibers in the medium andlarge (but not small) DRG neurons [44 48] In addition atthe DRG level a predominant expression of 4E-BP1 has beendetected in GFAP positive satellite cells [49] Satellite cells arespecialized glial cells that surround the cell body of sensoryneurons both in DRG and in trigeminal ganglia and likecentral glia are involved in the development of chronic pain[50] Studies from our laboratory have shown that satelliteglial cells contribute to neuronal sensitization of trigeminalneurons in vitro [51] and that they can express functionalCGRP receptors which increase the stimulatory effects ofcytokines [52] Finally Xu et al [49] have documented theexpression ofmTOR andmTORC1 downstream targets in thedorsal horn of rat spinal cordHowever these authors failedto detect the phosphorylated proteins both at the DRG andspinal cord level On the other hand Geranton et al [48]using a tyramide signal amplification- (TSA-) enhancedimmunofluorescent staining demonstrated that phospho-mTOR phospho-S6 ribosomal protein and phospho-4E-BP1are strongly expressed in lamina IIII projection neurons ofthe dorsal horn that is those neurons critically involved inthe development and maintenance of chronic pain [53]

Consistently with a possible involvement of mTOR inchronic pain processes it has been shown that peripheralinflammation increases mTOR activation in the spinal corddorsal horn In particular intraplantar injection of capsaicinsignificantly increased the phosphorylation level of the S6ribosomal protein after 2 h in the dorsal horn neurons aneffect abolished by intrathecal administration of rapamycin[48] Intraplantar injection of carrageenan increased themagnitude of S6 phosphorylation in the ipsilateral spinalcord dorsal horn but not in the contralateral one 4 h aftertreatment In these animals phospho-S6 together with Rhebthe positive regulator of mTOR activity was mainly detectedin neurons but not in spinal glia (astrocytes and microglia)Moreover basal S6 activity was also detected in larger motorneurons in the ventral horn but it was not modified bycarrageenan peripheral administration [54] Finally periph-eral inflammation due to intraplantar injection of completeFreundrsquos adjuvant significantly increased mTOR activationin DRG neurons with a minor increase observed in spinalcord dorsal horn [55] Enhanced activation of the mTORpathway was also found in different experimental models ofneuropathic pain In adult male rat undergoing L5-L6 spinalnerve ligation (SNL) increased phospho-mTOR staining wasdetected in sensory neurons mainly in myelinated fibers ofinjured nerves [56] However Asante et al [57] in the samemodel measured reduced levels of CGRP and S6K1 in thesuperficial dorsal horn neurons just at the ligated L5-L6nerves but not in the uninjured L4 spinal cord IncreasedmTOR activation was instead found in a mouse model ofchronic crush injury (CCI) at the spinal cord level and itwas significantly reduced by intrathecal administration ofrapamycin In particular rapamycin reduced phospho-mTOR expression at 7 and 14 days after surgery together withsignificant reduction of the phosphorylation level of S6K1 and4E-BP1 at 7 (but not 14) days after surgery [58] In contrast no

differences in the level of mTOR activation were detected inthe spinal cord dorsal horn 7 days after surgery in a differentmodel of neuropathic pain induced by spared nerve injury(SNI) of the sciatic nerve [48] All together these data suggestthat mTORC1 activity is significantly elevated in dorsal hornneurons in different models of chronic pain (even tough notin all models) thus suggesting a possible involvement inmediating central neuronal plasticity and thus pain hypersen-sitivity In support of this hypothesis a recent paper by Xuet al [59] has demonstrated increased mTORC1 activation inthe spinal cord dorsal horn (at the lumbar level but not atcervical and thoracic level) in a rat model of morphineinduced tolerance and hyperalgesia thus suggesting that thereduced analgesic effects of morphine observed in long-termtreatments may be due to the upregulation of mTORC1activity within the spinal cord It is therefore possible thatinhibition of mTORC1 activity may have beneficial effects inchronic pain syndromes

42 Electrophysiology In addition to these data there isalso consistent electrophysiological evidence which pointout mTOR signaling pathways as important modulators ofchronic pain [44 57 60] The electrophysiological analysisconducted by Jimenez-Dıaz et al [44] revealed an increasedmechanical threshold of subsets of myelinated fibers the A-fiber nociceptors after intraplantar injection of rapamycinfollowing capsaicin induced injury in rats The authorsproposed that ongoing local protein synthesis is essential forthe complete response of this subset of nociceptors and werepioneers in supposing the possibility that a similar process isoperating at the sites of termination of sensory afferentswithin the dorsal horn [44] The role of mTOR in the neuroninjury-induced hyperexcitability was demonstrated later in astudy conducted by Asante et al in which rapamycin (50120583Lof 250 nM) directly applied on the spinal cord at L4-L5level was found to produce inhibitory effects on nociceptiveC-fiber activity in lamina V wide dynamic range (WDR)neurons in response to mechanically and thermally stimulusin rats after removal of lumbar vertebral segments L1ndashL3 ofthe spinal cord [60] For the thermally induced response asignificant inhibition was only found at 35∘C with a trendtowards reduction at higher temperatures Also formalin-induced hyperexcitability was attenuated by spinally admin-istered rapamycin with significant effects only in the secondphase of formalin test which is thought to be due tocentral sensitization of dorsal horn neurons as a result of theinitial attenuation of input from nociceptive C-fiber afferentsoccurring during the first phase The same authors demon-strated similar effects of anisomycin a general inhibitor ofprotein translation on the formalin test confirming thatthese effects were indeed due to inhibition of mRNA trans-lation [60] In a second paper Asante et al demonstratedthe spinal effects of the rapamycin analogue CCI-779 onneuronal responses by using in vivo single-unit extracellularrecordings from spinal cord neurons of rats following L5-L6SNL [57] The authors found that spinal administration of250 120583M CCI-779 significantly attenuated specific neuronalresponses to mechanical stimuli from SNL rats compared


to predrug responses and sham rats whereas no effect wasestablished on thermally evoked responses In particularCCI-779 inhibitedC-fiber-mediated transmission ontoWDRneurons A further significant inhibitory effect was seenon WDR neuronal postdischarge and on ldquowind-uprdquo phe-nomenon a potentiated response mediated by nociceptiveC-fibers activity and a measure of central hypersensitivitymechanisms The limitation of this study is represented bythe fact that no differences in electrophysiological responseswere found between sham and neuropathic rats before druginjection The authors stated that the increased excitabilityof L4 WDR neurons in neuropathic rats could have maskedthe adjacent L5 ligation according to a previous study inwhich periphery connected spinal neurons were shown toexpand their receptive fields after the samenerve ligation [61]The observation that CCI-779 was quite ineffective on neu-ronal responses in the absence of nerve injury [57] allows toconfirm that mTOR signaling at the spinal level is an integralelement of nociception and that its role in central sensitiza-tion likely contributes to the persistence of pain

43 Behavioral Studies The antinociceptive effects of rapam-ycin and its analogous CCI-779 (thus mTOR inhibition)have been documented in several experimental models ofinflammatory and neuropathic pain Using the formalintest it has been shown that rapamycin (administered bothintrathecally or locally in the paw) significantly reduced thesecond phase of behavioral pain in mice [62] Similarly inadult rats intrathecal administration of rapamycin produceda significant inhibition of formalin-induced second phaseflinches [63] The formalin test is a well-characterized behav-ioral model of chemically induced pain consisting of twoconsecutive pain behavior phases of which the second onehas been suggested to involve central sensitization of the noci-ceptive system [64] thus suggesting an involvement of spinalmTOR in inflammation induced hyperalgesia On the otherhand rapamycin injected directly through the skin at L5-L6 spinal cord level 30 minutes before the formalin test wasshown to significantly reduce both pain behavioral phasesof the formalin test in adult rats indicating an involvementof mTOR in peripheral sensitization as well [60] A possiblerole of mTOR kinase (and thus local protein translation)in mediating both peripheral and central neuronal sensiti-zation is also suggested by other studies based on differentmodels of inflammatory pain Inflammatory pain can beinduced by intradermal injection of other nociceptive andalgogenic substances including capsaicin carrageenan andthe complete Freundrsquos adjuvant (CFA) [64] For example theinjection of capsaicin produces both peripheral sensitizationof C- and A120575-nociceptors and central sensitization of dorsalhorn neurons [48] The hallmark of peripheral sensitizationis represented by increased thermal sensitivity most likelysupported by ERK activation in the cell body of nociceptorsfollowed by synthesis of TRPV1 receptors and their transportto the axon terminals in the inflamed cutaneous tissuewhereas increased mechanical sensitivity is mainly due tocentral sensitization of spinal cord neurons [65] In thisexperimental model (in adult rats) rapamycin administered

either centrally or locally significantly reduced mechanicalhyperalgesia without affecting thermal hypersensitivity inadult rats [44 48] In a similar manner local and centralinjection of CCI-779 reduced mechanical hyperalgesia butnot thermal hypersensitivity developing in response to intra-dermal injection of carrageenan in adult mice [45] Themechanical hypersensitivity that occurs in the undamagedarea surrounding the site of injury in these models isknown to be mostly transmitted by A-fibers and amplified bysensitized dorsal horn neurons

In this regard electromyographic studies have shown thatboth local and intrathecal injection of rapamycin significantlyincreased threshold temperatures for paw withdrawal evokedby fast heat ramps (activating A-fiber nociceptors) comparedto control injections of vehicle thus suggesting a direct effecton this subset of myelinated nociceptors known to be impor-tant for the increased mechanical sensitivity that followsinjury [44 48] However intrathecal injections of rapamycininhibited the activation of downstream targets of mTOR indorsal horn and dorsal roots thus suggesting a modulatoryeffect on both primary afferents and central neurons [48]Consistently with these observations centrally administeredrapamycin was shown to reduce mechanical allodynia in sev-eral models of inflammatory pain [54 55 59] However thesestudies reported variable effects on thermal sensitivity withinhibitory effects observed after intraplantar injections ofcarrageenan [54 63] and minor albeit significant reductionsof thermal hyperalgesia induced by intraplantar injection ofCFA [55] Considering that the expression of mTOR andother components of the translational machinery has notbeen detected inC-nociceptors these results further suggest adirect role of spinal mTOR in the modulation of central painprocessing Interestingly the PI3K inhibitor wortmanninwas more effective at reducing pain hypersensitivity inresponse to carrageenan thus suggesting the involvement ofother pathways (including ERK) in the development ofcentral sensitization [63] Moreover intradermal injection ofcarrageenan increased the phosphorylation level of AKT atSer473

in the dorsal horn of spinal cord thus suggesting a con-comitant activation ofmTORC2 in parallel with developmentof hyperalgesia in response to peripheral inflammation [63]Therefore wortmannin by inhibiting PI3K can simultane-ously affect both mTOR complexes and other signalingpathways (including ERK as described in Sections 2 and 3)potentially important in mediating peripheral and centralsensitization thus resulting more effective than rapamycin inchronic pain

mTOR inhibitors also display beneficial effects in severalmodels of neuropathic pain which are also characterizedby the development of secondary hyperalgesia In summaryintraplantar or intrathecal administration of rapamycin sig-nificantly reducedmechanical allodynia developing after SNIin rats [44 48] This model consists in a tightly ligation fol-lowed by distal sectioning of the common peroneal and tibialbranches of the sciatic nerve with preservation of the suralnerve It is characterized by increased mechanical sensitivityobserved 6 days after surgery in the spared sural territory thatis the lateral part of the hind paw [44 48] Similarly inmice intraplantar or intraperitoneal injection of CCI-779


significantly reduced mechanical allodynia observed in thelateral part of the hind paw three days after surgery [45]Interestingly in this study the authors also evaluated theeffect of repeated administration of CCI-779 (4 injectionsevery 24 hours) observing a persistent reduction in mechan-ical allodynia which was gradually lost 48 hours after the lastadministration [45] Moreover in agreement with a possiblerole of mTORC2 in mediating central neuronal plasticity thedual mTOR inhibitor Torin 1 appeared to be more effectivethan rapamycin in this experimental model either afterone injection or after repeated administrations [45] Interest-ingly daily administration of metformin (which also inhibitsmTOR by promoting AMPK activation (see Section 2))reduced mechanical allodynia in a mouse model of SNI [56]In addition to these data mTOR inhibitors were found toexert beneficial effects in other models of neuropathic painFor example CCI-779 or metformin significantly reducedmechanical allodynia in a model of persistent pain caused bySNL [56 57] and rapamycin reduced mechanical hypersen-sitivity in mouse models of CCI [58 66] In agreement withthese observations Cui et al have shown using adult femaleSprague Dawley which chronic (14 days) intrathecal admin-istration of rapamycin reduced both the mechanical allody-nia and the thermal hypersensitivity induced by CCI [67]However variable effects of rapamycin on thermal sensitivityhave been reported in this experimental model as well [58]suggesting a main role of mTOR in the regulation of centralsensitization Notably peripheral nerve injury can induce adifferent spectrum of glial (both microglia and astrocytes)activation in the dorsal horn of the spinal cord These cellscan release inflammatory and pronociceptive mediators thussignificantly contributing to neuronal sensitization and tothe establishment of chronic pain syndromes [5] Recentexperimental evidence from our group and others suggest adirect role of mTOR in the regulation of glial inflammatoryresponses and a potential beneficial role of rapamycin inneuroinflammatory based diseases [8] including chronicpain syndromes In fact rapamycin (centrally administered)reduced CCI induced astrogliosis [67] but did not mod-ify microglial activation when injected peripherally in theaffected hind paw [66] These data suggest that rapamycinmay have multiple cellular and molecular targets that cancontribute to the therapeutic effects observed in vivo In thisregard Marinelli et al have demonstrated the critical role ofautophagy particularly in Schwann cells (SCs) in reducingneuronal damage clearing myelin debris and facilitatingneuronal regeneration after injury [66] In these animalsrapamycin significantly increased the autophagic flux in SCstheir proliferation and improved myelination in injurednerves [66] Consistently rapamycin improved myelinationin explant cultures from neuropathic mice by activatingautophagic mechanisms [68] However mice lacking mTORin SCs display hypomyelinated sciatic nerves [69] furtherunderlying the relevance of the mTOR pathway in theregulation of myelination in both peripheral and centralnervous system [70 71]

In this field recent evidence from our group suggests thatmTOR inhibitors can reduce signs of neuropathic pain in achronic model of a demyelinating disease the experimental

autoimmune encephalomyelitis (EAE) We have shown thatchronic administration of rapamycin was able to increase thesensitivity threshold for mechanical allodynia which is usu-ally reduced at the clinical onset of disease [72] In this studywe observed that rapamycin ameliorates clinical andhistolog-ical signs of EAE when administered to already ill animals atthe peak of disease (therapeutic approach) Interestingly thehistological study of the brains at the end of the experimentrevealed a significant improvement in the myelination of thecorpus callosum in the rapamycin treated animals whichmay be the consequence of reduced neuroinflammationas well as a direct effect of rapamycin [72] In additionrapamycin was also found beneficial in a mouse modelof cancer metastatic pain In fact repeated treatment withrapamycin reduced both thermal and mechanical hyper-sensitivity developing in response to intratibial injection ofprostate cancer cells In this model of chronic cancer painrapamycin was also effective when administered in a ther-apeutic manner that is once daily 5 days after injection ofcancer cells within the tibia [73] Rapamycinwas also effectivein a similar model of metastatic cancer pain induced byintratibial inoculation of breast cancer cells [74] Finallyrapamycin reduced hyperalgesia associated with chronicadministration of morphine in rats [59] thus further sup-porting a potential clinical use of mTOR inhibitors in themanagement of chronic pain syndromes

44 Preclinical and Clinical Evidence of a Pronociceptive Rolefor mTOR Inhibitors Despite the promising results from thepreclinical studies reviewed above the role of rapamycinand rapalogs in the clinical treatment of chronic pain isundermined by the clinical evidence that chronic treatmentof patients with these mTORC1 inhibitors is associated withincreases in the incidence of pain [75 76] including the possi-ble development of complex regional pain syndrome (CRPS)[77] In addition the anticancer agents RAD001 or AP23573are associated with a number of unique toxicities with oneof the most significant being the so-called painful mTORinhibitor-associated stomatitis (mIAS) [78] However mech-anistic data are lacking concerningwhether and how rapalogsare linked to the development of pain in patients chronicallytreated with these drugs hence more and appropriate clinicalstudies are necessary to clarify this important issue In thisregard the only preclinical study carried out in C57bl6 micethat show possible negative effects of long-term mTOR inhi-bition with respect to pain hypersensitivity is a recent paperfrom Melemedjian et al [79] These authors demonstratedthat chronic rapamycin treatment (intraperitoneally injectedfor 9 days) induced mechanical allodynia in sham-operatedanimals while reducing mechanical hypersensitivity in SNLanimals in agreement with data reviewed in Section 43Similarly rapamycin partially reversed mechanical allodyniain mice with SNI while producing mechanical allodyniain sham animals Chronic administration of rapamycinappeared to increase ERK andAKT phosphorylation in shamanimal sciatic nerves thus suggesting that the abrogation ofthe negative feedback loops on these other pathways due tothe incomplete blocking of mTORC1 (see Sections 2 and 3)


may cause activation of other signaling pathways responsiblefor pain development [79] For example increased ERKactivity can induce sensory neuron sensitization mechanicalhypersensitivity and spontaneous pain Interestingly theclinically available antidiabetic drugmetformin which is alsoa AMPK activator thus mTOR inhibitor (see above) preventsrapamycin-induced ERK activation and the developmentof mechanical hypersensitivity and spontaneous pain [79]These data suggest that a more complete inhibition of themTOR pathway can overcome these side effects of rapamycinand its analogs In this regard in a retrospective studyconducted on diabetic patients the use of metformin hasbeen found significantly associated with reduction of painsymptoms in patients affected by lumbar radiculopathy avery frequent form of chronic pain syndrome [80 81]

5 Conclusions

Data presented in this review paper strongly suggest thatmTOR has a critical role in several mechanism of painprocessing including a role in the development of chronicpain However clinical evidence suggests that chronic useof first generation mTOR inhibitors may be associated withdevelopment of pain hypersensitivity thus underlying theinvolvement of other signaling pathways including PI3Kdownstream effectors (ERK AKT and more interestinglymTORC2) A more comprehensive understanding of thesesignaling pathways may lead to improved treatments for themanagement of chronic pain A vast array of novel inhibitorswith a broader range of activity is becoming available forclinical testing These have been mostly developed for cancertreatment but may also be employed in the management ofchronic pain

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

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[2] S P Cohen and J Mao ldquoNeuropathic pain mechanisms andtheir clinical implicationsrdquo British Medical Journal vol 348Article ID f7656 2014

[3] A Latremoliere and C J Woolf ldquoCentral sensitization a gener-ator of pain hypersensitivity by central neural plasticityrdquo Journalof Pain vol 10 no 9 pp 895ndash926 2009

[4] B Xu G Descalzi H-R Ye M Zhuo and Y-W WangldquoTranslational investigation and treatment of neuropathic painrdquoMolecular Pain vol 8 article 15 2012

[5] P M Grace M R Hutchinson S F Maier and L R WatkinsldquoPathological pain and the neuroimmune interfacerdquo NatureReviews Immunology vol 14 no 4 pp 217ndash231 2014

[6] C Voscopoulos and M Lema ldquoWhen does acute pain becomechronicrdquo British Journal of Anaesthesia vol 105 supplement 1pp i69ndashi85 2010

[7] C A Hoeffer and E Klann ldquomTOR signaling at the crossroadsof plasticity memory and diseaserdquo Trends in Neurosciences vol33 no 2 pp 67ndash75 2010

[8] C D Russo L Lisi D L Feinstein and P Navarra ldquomTORkinase a key player in the regulation of glial functions relevancefor the therapy of multiple sclerosisrdquoGlia vol 61 no 3 pp 301ndash311 2013

[9] S Wullschleger R Loewith and M N Hall ldquoTOR signaling ingrowth andmetabolismrdquoCell vol 124 no 3 pp 471ndash484 2006

[10] N Hay and N Sonenberg ldquoUpstream and downstream ofmTORrdquo Genes and Development vol 18 no 16 pp 1926ndash19452004

[11] D D Sarbassov S M Ali S Sengupta et al ldquoProlonged rapa-mycin treatment inhibits mTORC2 assembly and AktPKBrdquoMolecular Cell vol 22 no 2 pp 159ndash168 2006

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[15] K Hara Y Maruki X Long et al ldquoRaptor a binding partner oftarget of rapamycin (TOR) mediates TOR actionrdquoCell vol 110no 2 pp 177ndash189 2002

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[20] Y Sancak L Bar-Peled R Zoncu A L Markhard S Nada andD M Sabatini ldquoRagulator-rag complex targets mTORC1 to thelysosomal surface and is necessary for its activation by aminoacidsrdquo Cell vol 141 no 2 pp 290ndash303 2010

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[38] D Benjamin M Colombi C Moroni and M N Hall ldquoRapa-mycin passes the torch a new generation of mTOR inhibitorsrdquoNature ReviewsDrugDiscovery vol 10 no 11 pp 868ndash880 2011

[39] S A Wander B T Hennessy and J M Slingerland ldquoNext-generation mTOR inhibitors in clinical oncology how pathwaycomplexity informs therapeutic strategyrdquo Journal of ClinicalInvestigation vol 121 no 4 pp 1231ndash1241 2011

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[55] L Liang B Tao L Fan M Yaster Y Zhang and Y-X TaoldquomTOR and its downstream pathway are activated in the dorsalroot ganglion and spinal cord after peripheral inflammationbut not after nerve injuryrdquo Brain Research vol 1513 pp 17ndash252013

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with neuropathy in the ratrdquo Journal of Pain vol 11 no 12 pp1356ndash1367 2010

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[64] D Le Bars M Gozariu and S W Cadden ldquoAnimal models ofnociceptionrdquo Pharmacological Reviews vol 53 no 4 pp 597ndash652 2001

[65] I Obara S M Geranton and S P Hunt ldquoAxonal proteinsynthesis a potential target for pain reliefrdquo Current Opinion inPharmacology vol 12 no 1 pp 42ndash48 2012

[66] S Marinelli F Nazio A Tinari et al ldquoSchwann cell autophagycounteracts the onset and chronification of neuropathic painrdquoPain vol 155 no 1 pp 93ndash107 2014

[67] J Cui W He B Yi et al ldquomTOR pathway is involved in ADP-evoked astrocyte activation and ATP release in the spinal dorsalhorn in a rat neuropathic pain modelrdquo Neuroscience vol 275pp 395ndash403 2014

[68] S Rangaraju J D Verrier I Madorsky J Nicks W A DunnJr and L Notterpek ldquoRapamycin activates autophagy andimproves myelination in explant cultures from neuropathicmicerdquo The Journal of Neuroscience vol 30 no 34 pp 11388ndash11397 2010

[69] D L Sherman M Krols L-M NWu et al ldquoArrest of myelina-tion and reduced axon growthwhen Schwann cells lackmTORrdquoJournal of Neuroscience vol 32 no 5 pp 1817ndash1825 2012

[70] C Norrmen and U Suter ldquoAktmTOR signalling in myelina-tionrdquo Biochemical Society Transactions vol 41 no 4 pp 944ndash950 2013

[71] T L Wood K K Bercury S E Cifelli et al ldquomTOR a linkfrom the extracellular milieu to transcriptional regulation ofoligodendrocyte developmentrdquo ASN Neuro vol 5 no 1 ArticleID e00108 2013

[72] L Lisi P Navarra R Cirocchi et al ldquoRapamycin reduces clin-ical signs and neuropathic pain in a chronic model of exper-imental autoimmune encephalomyelitisrdquo Journal of Neuroim-munology vol 243 no 1-2 pp 43ndash51 2012

[73] M-H Shih S-C Kao W Wang M Yaster and Y-X TaoldquoSpinal cord NMDA receptor-mediated activation of mam-malian target of rapamycin is required for the development andmaintenance of bone cancer-induced pain hypersensitivities inratsrdquo Journal of Pain vol 13 no 4 pp 338ndash349 2012

[74] D M Abdelaziz L S Stone and S V Komarova ldquoOsteolysisand pain due to experimental bone metastases are improved bytreatment with rapamycinrdquo Breast Cancer Research and Treat-ment vol 143 no 2 pp 227ndash237 2014

[75] K Budde ldquoHow to use mTOR inhibitors The search goes onrdquoAmerican Journal of Transplantation vol 11 no 8 pp 1551ndash15522011

[76] F X McCormack Y Inoue J Moss et al ldquoEfficacy and safetyof sirolimus in lymphangioleiomyomatosisrdquo The New EnglandJournal of Medicine vol 364 no 17 pp 1595ndash1606 2011

[77] CMassard K FizaziMGross-Goupil andB Escudier ldquoReflexsympathetic dystrophy in patients with metastatic renal cellcarcinoma treated with Everolimusrdquo Investigational New Drugsvol 28 no 6 pp 879ndash881 2010

[78] M A De Oliveira F M E Martins Q Wang et al ldquoClinicalpresentation and management of mTOR inhibitor-associatedstomatitisrdquo Oral Oncology vol 47 no 10 pp 998ndash1003 2011

[79] O K Melemedjian A Khoutorsky R E Sorge et al ldquoMTORC1inhibition induces pain via IRS-1-dependent feedback activa-tion of ERKrdquo Pain vol 154 no 7 pp 1080ndash1091 2013

[80] A Taylor A H Westveld M Szkudlinska et al ldquoThe use ofmetformin is associated with decreased lumbar radiculopathypainrdquo Journal of Pain Research vol 6 pp 755ndash763 2013

[81] A Taylor A H Westveld M Szkudlinska et al ldquoThe use ofmetformin is associated with decreased lumbar radiculopathypain [Erratum]rdquo Journal of Pain Research vol 2014 no 7 pp89ndash90 2014

[82] W Schuler R Sedrani S Cottens et al ldquoSDZ RAD a newrapamycin derivative pharmacological properties in vitro andin vivordquo Transplantation vol 64 no 1 pp 36ndash42 1997

[83] L Toral-Barza W-G Zhang C Lamison J LaRocque JGibbons and K Yu ldquoCharacterization of the cloned full-lengthand a truncated human target of rapamycin activity specificityand enzyme inhibition as studied by a high capacity assayrdquoBiochemical and Biophysical Research Communications vol 332no 1 pp 304ndash310 2005

[84] B Shor W-G Zhang L Toral-Barza et al ldquoA new pharmaco-logic action of CCI-779 involves FKBP12-independent inhibi-tion of mTOR kinase activity and profound repression of globalprotein synthesisrdquo Cancer Research vol 68 no 8 pp 2934ndash2943 2008

[85] V M Rivera R M Squillace D Miller et al ldquoRidaforolimus(AP23573 MK-8669) a potent mtor inhibitor has broadantitumor activity and can be optimally administered usingintermittent dosing regimensrdquo Molecular Cancer Therapeuticsvol 10 no 6 pp 1059ndash1071 2011

[86] J M Garcıa-Martınez J Moran R G Clarke et al ldquoKu-0063794 is a specific inhibitor of the mammalian target ofrapamycin (mTOR)rdquoBiochemical Journal vol 421 no 1 pp 29ndash42 2009

[87] K Malagu H Duggan K Menear et al ldquoThe discoveryand optimisation of pyrido[23-d]pyrimidine-24-diamines aspotent and selective inhibitors ofmTOR kinaserdquoBioorganic andMedicinal Chemistry Letters vol 19 no 20 pp 5950ndash5953 2009

[88] K G Pike KMalaguM G Hummersone et al ldquoOptimizationof potent and selective dual mTORC1 and mTORC2 inhibitors


the discovery of AZD8055 and AZD2014rdquo Bioorganic amp Medic-inal Chemistry Letters vol 23 no 5 pp 1212ndash1216 2013

[89] M E Feldman B Apsel A Uotila et al ldquoActive-site inhibitorsof mTOR target rapamycin-resistant outputs of mTORC1 andmTORC2rdquo PLoS Biology vol 7 no 2 article e38 2009

[90] Q Liu S Kirubakaran W Hur et al ldquoKinome-wide selectivityprofiling of ATP-competitive mammalian target of rapamycin(mTOR) inhibitors and characterization of their binding kinet-icsrdquo The Journal of Biological Chemistry vol 287 no 13 pp9742ndash9752 2012

[91] C C Thoreen S A Kang J W Chang et al ldquoAn ATP-competitive mammalian target of rapamycin inhibitor revealsrapamycin-resistant functions of mTORC1rdquo The Journal ofBiological Chemistry vol 284 no 12 pp 8023ndash8032 2009

[92] K Yu L Toral-Barza C Shi et al ldquoBiochemical cellular and invivo activity of novel ATP-competitive and selective inhibitorsof the mammalian target of rapamycinrdquo Cancer Research vol69 no 15 pp 6232ndash6240 2009

[93] D S Mortensen J Sapienza B G S Lee et al ldquoUse of coremodification in the discovery of CC214-2 an orally availableselective inhibitor of mTOR kinaserdquo Bioorganic and MedicinalChemistry Letters vol 23 no 6 pp 1588ndash1591 2013

[94] S V Bhagwat P C Gokhale A P Crew et al ldquoPreclinicalcharacterization of OSI-027 a potent and selective inhibitor ofmTORC1 and mTORC2 distinct from rapamycinrdquo MolecularCancer Therapeutics vol 10 no 8 pp 1394ndash1406 2011

[95] S-M Chen J-L Liu X Wang C Liang J Ding and L-HMeng ldquoInhibition of tumor cell growth proliferation andmigration by X-387 a novel active-site inhibitor of mTORrdquoBiochemical Pharmacology vol 83 no 9 pp 1183ndash1194 2012

[96] A C Hsieh Y Liu M P Edlind et al ldquoThe translational land-scape of mTOR signalling steers cancer initiation and metasta-sisrdquo Nature vol 485 no 7396 pp 55ndash61 2012

[97] K Yu C Shi L Toral-Barza et al ldquoBeyond rapalog therapypreclinical pharmacology and antitumor activity of WYE-125132 an ATP-competitive and specific inhibitor of mTORC1andmTORC2rdquoCancer Research vol 70 no 2 pp 621ndash631 2010

[98] A Arcaro and M P Wymann ldquoWortmannin is a potent phos-phatidylinositol 3-kinase inhibitor the role of phosphatidyli-nositol 345-trisphosphate in neutrophil responsesrdquo Biochem-ical Journal vol 296 no 2 pp 297ndash301 1993

[99] M Hayakawa H Kaizawa K-I Kawaguchi et al ldquoSynthesisand biological evaluation of imidazo[12-a]pyridine derivativesas novel PI3 kinase p110120572 inhibitorsrdquo Bioorganic and MedicinalChemistry vol 15 no 1 pp 403ndash412 2007

[100] F I Raynaud S Eccles P A Clarke et al ldquoPharmacologic char-acterization of a potent inhibitor of class I phosphatidylinositide3-kinasesrdquo Cancer Research vol 67 no 12 pp 5840ndash5850 2007

[101] D Kong S-I Yaguchi and T Yamori ldquoEffect of ZSTK474a novel phosphatidylinositol 3-kinase inhibitor on DNA-dependent protein kinaserdquo Biological and Pharmaceutical Bul-letin vol 32 no 2 pp 297ndash300 2009

[102] Q Liu J Wang S A Kang et al ldquoDiscovery of 9-(6-amino-pyridin-3-yl)-1-(3-(trifluoromethyl)phenyl)benzo[h][16]naph-thyridin-2(1 H)-one (torin2) as a potent selective and orallyavailable mammalian target of rapamycin (mTOR) inhibitorfor treatment of cancerrdquo Journal of Medicinal Chemistry vol54 no 5 pp 1473ndash1480 2011

[103] Q Liu C Xu S Kirubakaran et al ldquoCharacterization of Torin2an ATP-competitive inhibitor of mTOR ATM and ATRrdquoCancer Research vol 73 no 8 pp 2574ndash2586 2013

[104] S D Knight N D Adams J L Burgess et al ldquoDiscovery ofGSK2126458 a highly potent inhibitor of PI3K and the mam-malian target of rapamycinrdquo ACS Medicinal Chemistry Lettersvol 1 no 1 pp 39ndash43 2010

[105] B Markman J Tabernero I Krop et al ldquoPhase I safety phar-macokinetic and pharmacodynamic study of the oral phos-phatidylinositol-3-kinase and mTOR inhibitor BGT226 inpatients with advanced solid tumorsrdquo Annals of Oncology vol23 no 9 pp 2399ndash2408 2012

[106] J R Garlich P De N Dey et al ldquoA vascular targeted pan phos-phoinositide 3-kinase inhibitor prodrug SF1126 with antitu-mor and antiangiogenic activityrdquoCancer Research vol 68 no 1pp 206ndash215 2008

[107] A M Venkatesan C M Dehnhardt E D Delos Santos et alldquoBis(morpholino-l35-triazine) derivatives potent adenosine5-triphosphate competitive phosphatidylinositol-3-kinasemammalian target of rapamycin inhibitors discovery of com-pound 26 (PKI-587) a highly efficacious dual inhibitorrdquo Journalof Medicinal Chemistry vol 53 no 6 pp 2636ndash2645 2010

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


BioMed Research International


Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


Neural Plasticity


Parkinsonrsquos Disease


Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Psychiatry Journal


Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases


Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


mTOR

mTORC1

darr Deptor

MLST8G120573L

uarr PRAS40

uarr Raptor

(a)

mTORC2

mTOR

mSin1

Protor

MLST8G120573L

darr Deptoruarr Rictor

(b)

Figure 1 Schematic representing the molecular partners of mTOR forming (a) mTOR complex 1 (mTORC1) and (b) mTOR complex 2(mTORC2) The down-arrows indicate the inhibitory proteins whereas the up-arrows indicate activator factors on mTOR function

neuronal switch (ie large myelinated A120573 fibers expressingneuropeptides directly involved in pain transmission such assubstance P and calcitonin gene-related peptide) sproutingof nerve endings (iemyelinatedA120573fibers establishing directcontacts with nociceptive projecting neurons in the lamina I-II of the spinal dorsal horn) loss of spinal inhibitory controland increased activity of descending excitatory pathways[3] Moreover synaptic plasticity within key cortical regionsinvolved in pain processing (ie the anterior cingulated cor-tex the insular cortex primary and secondary sensory cor-tices and the amygdala) has been also observed in relationto neuropathic pain [4] Finally activation of glial cells withrelease of pronociceptive mediators can directly modulateneuronal excitability and thus pain transmission contribut-ing to central sensitization and to the occurrence of neuro-pathic pain [5]

Multimodal pharmacological treatments for chronic painsyndromes including neuropathic pain are based on theuse of antiepileptics antidepressants local anesthetics opioidanalgesics or tramadol These treatments are only partiallyeffective with significant pain relief achieved in 40ndash60 ofpatients [4] A relatively recent modality of neuropathic paintherapy which represents the future challenge of upcomingresearches involves specific cellular targets implied in neu-ronal synaptic plasticity andor glial activation [6] Inter-estingly recent studies show that the mammalian target ofrapamycin (mTOR) kinase and downstream effectors may beimplicated in the development of chronic inflammatory neu-ropathic and cancer painThis kinase is a master regulator ofprotein synthesis and it is critically involved in the regulationof several neuronal functions including synaptic plasticityand memory formation in the central nervous system (CNS)[7] As mentioned above neuronal synaptic plasticity both atperipheral level and in the CNS is amajormechanism leadingto the development of chronic pain thus suggesting thatmTOR may be a novel pharmacological target for the man-agement of chronic pain In addition mTOR has been alsoreported to regulate astrocyte and microglial activity (as wehave recently reviewed [8]) thus suggesting an additionaltherapeutic target in the treatment of chronic pain syndromesthat involve increased glial activation The main evidence

from animal studies as well as clinical reports supporting thishypothesis is reviewed in the present paper

2 mTOR the lsquolsquoMechanisticrsquorsquoTarget of Rapamycin

The mTOR kinase now officially known as ldquomechanisticrdquoTOR is a conserved serinethreonine protein kinase belong-ing to the phosphoinositide 3-kinase (PI3K) family thatregulates multiple intracellular processes [9] In mammalsmTOR is encoded by a single gene [10] and interacts withseveral proteins to form two distinct complexes referred to asmTORC1 and mTORC2 These complexes display differentsensitivity to the inhibitory action of rapamycin whichmainly suppresses mTORC1-dependent activities in acutetreatments [11] Notably the two complexes promote the acti-vation of different signalling pathways and recognize distinctupstream regulators as well as downstream targets whosespecificity is determined by the specificity of the interactingproteins Both complexes include the inhibitory proteinDEPTOR and the adaptor protein mLST8G120573L (mammalianLST8G-protein 120573-subunit like protein) [12] However therole of mLST8G120573L in the regulation of mTORC1 functionremains unclear at present since its chronic loss does notaffect mTORC1 activity in vivo [13] As shown in Figure 1mTORC1 specifically contains the regulatory-associated pro-tein of mTOR (raptor) and the inhibitory protein PRAS40(proline-richAKT substrate of 40KDa) [14] Raptor regulatesmTORC1 assembly and serves as a scaffold for the recruit-ment of specific substrates such as the eukaryotic initiationfactor-4E-binding protein 1 (4E-BP1) [15 16] Similarly otherproteins reside uniquely within complex 2 that is therapamycin-insensitive companion ofmTOR (rictor) the pro-tein observedwith rictor (PROTOR) and the stress-activatedprotein kinase-interacting protein 1 (mSIN1) (Figure 1) [17]Much like raptor rictor is necessary for mTORC2 assemblytogether with mSIN1 for mTORC2 catalytic activity and itmay also be involved in the selective recruitment of specificsubstrates [14]



4E-BP1S6K1

eiF-4BeiF-4E


Protein elongation


mRNA translation


Lysosomalbiogenesis

PKBAKT

Energy metabolism

PI3K

IRS1

Cytokines

Rheb

ERK12

Ras

Hypoxialow cell ATP

AMPK


RAG AB

mTORC1

TSC1 TSC2

Lysosomes

(TNF120572)

I120581BK











AKT

Growth factors

Cytoskeleton

Energy metabolism

SGK1

PI3K

mTORC2

PKC120572Rho


mTORC1 mTORC2S6K

Rheb

AKT

TSC1 TSC2


3 mTOR Inhibitors

















Classes DrugsmTOR



EC50)

mTORC2(cellular

potency EC50)


IC50)References

Rapamycin174120583M

















SF1101

[106]

























5 Conclusions




References






























































































































Neural Plasticity










Psychiatry Journal









Journal of




4E-BP1S6K1

eiF-4BeiF-4E


Protein elongation


mRNA translation


Lysosomalbiogenesis

PKBAKT

Energy metabolism

PI3K

IRS1

Cytokines

Rheb

ERK12

Ras

Hypoxialow cell ATP

AMPK


RAG AB

mTORC1

TSC1 TSC2

Lysosomes

(TNF120572)

I120581BK











AKT

Growth factors

Cytoskeleton

Energy metabolism

SGK1

PI3K

mTORC2

PKC120572Rho


mTORC1 mTORC2S6K

Rheb

AKT

TSC1 TSC2


3 mTOR Inhibitors

















Classes DrugsmTOR



EC50)

mTORC2(cellular

potency EC50)


IC50)References

Rapamycin174120583M

















SF1101

[106]

























5 Conclusions




References






























































































































Neural Plasticity










Psychiatry Journal









Journal of








AKT

Growth factors

Cytoskeleton

Energy metabolism

SGK1

PI3K

mTORC2

PKC120572Rho


mTORC1 mTORC2S6K

Rheb

AKT

TSC1 TSC2


3 mTOR Inhibitors

















Classes DrugsmTOR



EC50)

mTORC2(cellular

potency EC50)


IC50)References

Rapamycin174120583M

















SF1101

[106]

























5 Conclusions




References






























































































































Neural Plasticity










Psychiatry Journal









Journal of
















Classes DrugsmTOR



EC50)

mTORC2(cellular

potency EC50)


IC50)References

Rapamycin174120583M

















SF1101

[106]

























5 Conclusions




References






























































































































Neural Plasticity










Psychiatry Journal









Journal of




Classes DrugsmTOR



EC50)

mTORC2(cellular

potency EC50)


IC50)References

Rapamycin174120583M

















SF1101

[106]

























5 Conclusions




References






























































































































Neural Plasticity










Psychiatry Journal









Journal of





















5 Conclusions




References






























































































































Neural Plasticity










Psychiatry Journal









Journal of
















5 Conclusions




References






























































































































Neural Plasticity










Psychiatry Journal









Journal of









5 Conclusions




References






























































































































Neural Plasticity










Psychiatry Journal









Journal of




5 Conclusions




References






























































































































Neural Plasticity










Psychiatry Journal









Journal of






































































































Neural Plasticity










Psychiatry Journal









Journal of




































































Neural Plasticity










Psychiatry Journal









Journal of



































Neural Plasticity










Psychiatry Journal









Journal of














Neural Plasticity










Psychiatry Journal









Journal of


Documents

Review Article mTOR Kinase: A Possible Pharmacological ...downloads.hindawi.com/journals/bmri/2015/394257.pdf · mTOR Kinase: A Possible Pharmacological Target in the Management of