cance s
A icle
Di e en ial E ec s o IGF-1R Small Molecule
Ty osine Kinase Inhibi o s BMS-754807 and OSI-906
on Human Cance Cell Lines
Ma ía Fuen es-Baile 1,†, Ma ía P. Ven e o 2,†, JoséA. Encina 3,* , Pila Ga cía-Mo ales 3,
Ma ía Po eda-Del ell 3, Elizabe h Pé ez-Valenciano 3, Víc o M. Ba be á1,4,
Ja ie Gallego-Plazas 5,Ál a o Rod íguez-Lescu e 5, JoséMa ín-Nie o 6and
Miguel Saceda 1,3,*
1Unidad de In es igación, Fundación pa a el Fomen o de la In es igación Sani a ia y Biomédica de la
Comunidad Valenciana (FISABIO), Hospi al Gene al Uni e si a io de Elche, 03203 Elche (Alican e), Spain;
[email p o ec ed] (M.F.-B.); [email p o ec ed] (V.M.B.)
2Unidad de In es igación, Ins i u o de In es igación Sani a ia y Biomédica de Alican e (ISABIAL),
Hospi al Gene al Uni e si a io de Alican e, 03005 Alican e, Spain; [email p o ec ed]
3
Ins i u o de Biolog
í
a Molecula y Celula (IBMC) and Ins i u o de In es igaci
ó
n, Desa ollo e Inno aci
ó
n en
Bio ecnología Sani a ia de Elche (IDiBE), Uni e sidad Miguel He nández, 03202 Elche (Alican e), Spain;
[email p o ec ed] (P.G.-M.); mpo eda@in e eady.com (M.P.-D.); [email p o ec ed] (E.P.-V.)
4Unidad de Gené ica Molecula , Hospi al Gene al Uni e si a io de Elche, 03203 Elche (Alican e), Spain
5Se icio de Oncología, Hospi al Gene al Uni e si a io de Elche, 03203 Elche (Alican e), Spain;
[email p o ec ed] (J.G.-P.); [email p o ec ed] (Á.R.-L.)
6Depa amen o de Fisiología, Gené ica y Mic obiología, Facul ad de Ciencias, Uni e sidad de Alican e,
03080 Alican e, Spain; [email p o ec ed]
*Co espondence: [email p o ec ed] (J.A.E.); [email p o ec ed] (M.S.); Tel.: +34-966658432 (M.S.)
†These au ho s con ibu ed equally o his wo k.
Recei ed: 24 No embe 2020; Accep ed: 9 Decembe 2020; Published: 11 Decembe 2020
Simple Summa y:
We ha e es ed he e ec s o IGF-1R y osine kinase inhibi o s BMS-754807 (BMS)
and OSI-906 (OSI) on human colon, panc ea ic ca cinoma cell, and glioblas oma cell lines and p ima y
cul u es. Al hough OSI and BMS a e able o inhibi IGF-1R ac i i y a low doses, he di e en ial
e ec on cell p oli e a ion and cell-cycle phase dis ibu ion shown by bo h compounds p obes ha
many e ec s obse ed a e media ed by BMS o - a ge in e ac ions. Using MAPKs ELISAs and
phospho-RTK a ay analysis, we ha e iden i ied se e al BMS egula ed pu a i e kinases able o
media e BMS o - a ge e ec s. In e es ingly, molecula docking assays sugges ha BMS could
a ec hese kinases no only by blocking hei ATP-binding domain, bu also by means o allos e ic
in e ac ions. Since BMS has an impo an an ineoplas ic e ec on hese poo p ognosis ypes o cance ,
hese compounds could be aken in conside a ion o ea men independen ly o IGF-1R s a us.
Abs ac :
We ha e de e mined he e ec s o he IGF-1R y osine kinase inhibi o s BMS-754807 (BMS)
and OSI-906 (OSI) on cell p oli e a ion and cell-cycle phase dis ibu ion in human colon, panc ea ic
ca cinoma, and glioblas oma cell lines and p ima y cul u es. IGF-1R signaling was blocked by
BMS and OSI a equi alen doses, al hough bo h inhibi o s exhibi ed di e en ial an ip oli e a i e
e ec s. In all panc ea ic ca cinoma cell lines es ed, BMS exe ed a s ong an ip oli e a i e e ec ,
whe eas OSI had a minimal e ec . Simila esul s we e ob ained on glioblas oma p ima y cul u es,
whe e HGUE-GB-15, -16 and -17 displayed esis ance o OSI e ec s, whe eas hey we e inhibi ed in
hei p oli e a ion by BMS. Di e en ial e ec s o BMS and OSI we e also obse ed in colon ca cinoma
cell lines. Bo h inhibi o s also showed di e en e ec s on cell cycle phase dis ibu ion, BMS induced
G
2
/M a es ollowed by cell dea h, while OSI induced G
1
a es wi h no cell dea h. Bo h inhibi o s
also showed di e en e ec s on o he p o ein kinases ac i i ies. Taken oge he , ou esul s a e
indica i e ha BMS mainly ac s h ough o - a ge e ec s exe ed on o he p o ein kinases. Gi en ha
Cance s 2020,12, 3717; doi:10.3390/cance s12123717 www.mdpi.com/jou nal/cance s
Cance s 2020,12, 3717 2 o 18
BMS exhibi s a po en an ip oli e a i e e ec , we belie e ha his compound could be use ul o he
ea men o di e en ypes o umo s independen ly o hei IGF-1R ac i a ion s a us.
Keywo ds:
IGF-1R inhibi o ; ATP-binding domain; o - a ge inhibi ion; molecula docking;
panc ea ic ca cinoma; colon ca cinoma; glioblas oma; y osine kinase
1. In oduc ion
IGF-1R (UniP o KB code P08069) is a y osine kinase ecep o loca ed in he plasma memb ane,
which is in ol ed in he p ocesses o cell g ow h, de elopmen , and di e en ia ion. In addi ion,
i exhibi s a e y s ong an iapop o ic ac i i y [
1
]. The IGF1R gene is ansla ed in o a single polypep ide
p ecu so ha is clea ed o yield an
α
subuni , which con ains he ligand-in e ac ing domain, and a
β
subuni , which con ains he ansmemb ane and y osine kinase domains [
2
]. These wo subuni s
emain linked by disul ide bonds, he s uc u e o he ecep o being a he e o e ame wi h a
βααβ
con o ma ion [
3
]. The ecep o can also o m hyb id he e o e ame s wi h he
α
and
β
chains o
he insulin ecep o (IR) [
2
]. I s ligands a e insulin-like g ow h ac o s 1 and 2 (IGF-1 and IGF-2)
and insulin. IGFR-1R binds IGF-1 wi h high a ini y, and IGF-2 and insulin wi h lowe a ini y [
4
].
Ac i a ion o IGF-1R upon ligand binding leads o he au ophospho yla ion o i s y osine kinase
domain, wi h ensuing ac i a ion o he Ras-Ra -MAPK and PI3K-AKT/PKB signaling pa hways,
which is c ucial o IGF-1R o exe i s mi ogenic and an iapop o ic ac i i ies [3].
Nume ous s udies ha e shown ha IGF-1R is o e exp essed in p ima y umo s and cance -de i ed
cells. This inc ease in IGF-1R le els e lec s a e e sion o mo e p imi i e, less di e en ia ed and
oncogenic s a es ha a e cha ac e ized by high concen a ions o IGF-1R mRNA and IGF-binding
si es [
4
]. In his con ex , i has been shown ha ac i e IGF-1R can be ound o e exp essed in all
sub ypes o b eas cance , and ha he p esence o high le els o phospho yla ed IGF-1R is associa ed
wi h a lowe pa ien su i al [
5
]. O e exp ession o IGF-1R has also been associa ed wi h a highe
umo g ade, inhibi ion o apop osis, inc eased p oli e a ion a e, and angiogenesis in pa ien s wi h
panc ea ic duc al adenoca cinoma [
6
], and wi h a lowe su i al in pa ien s wi h colo ec al cance [
7
].
Mo eo e , he IGF-1R signaling pa hway is highly ac i e in di e en ypes o human umo s, as is he
case o me as a ic melanoma [
8
], and is known o play a c i ical ole in he ans o ma ion, g ow h and
su i al o glioblas oma mul i o me (GBM) cells [9,10].
Cu en ly, s a egies a e being de eloped in o de o exploi IGF-1R as a he apeu ic a ge [
3
].
Inhibi o so IGFo IGF-1Ra ebeing es edinclinical ials ha belong o h eemainclasses: monoclonal
an ibodies agains IGF-1R, monoclonal an ibodies agains IGF-1R ligands (IGF-1 and IGF-2), and IGF-1R
y osine kinase inhibi o s [
11
]. Agen s ha a ge IGF-1R include monoclonal an ibodies such as
cixu umumab (IMC-A12), dalo uzumab (MK-0646) and oba umumab (Sch717454), and he small
molecules ac ing as y osine kinase inhibi o s dubbed BMS-754807 (BMS-754807), linsi inib (OSI-906),
XL228 and AXL1717 [
12
]. Among hese, BMS and OSI-906 (OSI) a e aken o ally and cons i u e he
mos speci ic, ATP-compe i i e inhibi o s, whe eas o he s also inhibi ecep o y osine kinases beyond
he IGF-1R and IR amily [
11
]. BMS-754807 is a po en and e e sible inhibi o o bo h IGF-1R and IR,
wi h a hal -maximal inhibi o y concen a ion (IC50) o 1.8 nM and 1.7 nM, espec i ely, in cell- ee
assays [
13
]. On he o he hand, i is less po en on Me , Au o a A/B, T kA/B and Ron, and shows
li le ac i i y on Fl 3, Lck, MK2, PKA, PKC and o he p o ein kinases [
14
]. BMS-754807 e ec i ely
inhibi s he g ow h o a wide ange o human umo ypes
in i o
, including mesenchymal (Ewing
sa coma, habdomyosa coma, neu oblas oma, and liposa coma), epi helial (b eas , lung, panc eas,
colon, gas ic), and hema opoie ic (mul iple myeloma and leukemia) umo cell lines. I has been
shown ha his compound causes apop osis in a human habdomyosa coma cell line, associa ed
wi h an inc eased clea age o poly ADP- ibose polyme ase (PARP) and caspase-3 exp ession [
13
].
Rega ding OSI-906, his compound is a selec i e inhibi o o IGF-1R, wi h an IC50 o 35 nM in cell- ee
Cance s 2020,12, 3717 3 o 18
assays, and is modes ly po en agains he IR, wi h an IC50 o 75 nM. I is also known o ha e no ac i i y
owa ds Abl, ALK, BTK, EGFR, FGFR1/2, PKA and o he p o ein kinases [
15
,
16
]. OSI-906 inhibi s
he p oli e a ion o hepa ocellula ca cinoma (HCC) cell lines by a leas 40%. HCC cells sensi i e
o OSI-906 show highe le els o phospho yla ion o IGF-1R and IR han esis an cells, sugges ing
ha sensi i i y o OSI-906 is associa ed wi h he inhibi ion o bo h o hese ecep o s [
17
]. Mo eo e ,
OSI-906-induced apop osis and inhibi ion o cell p oli e a ion appea o be di ec ly linked o he
inhibi ion o AKT in se e al umo cell lines, including lung, panc eas and colo ec al cell lines [
18
].
In his con ex , OSI-906- ea ed colo ec al cance xenog a s show a dec ease in umo g ow h and
inc eased apop osis
in i o
and
in i o
[
19
]. In his sys em, OSI-906 has been ound o amelio a e cell
p oli e a ion by al e ing he cell cycle in he G0/G1phase.
In his wo k, we ha e add essed he e ec s o BMS-754807 and OSI-906 on cell p oli e a ion and
cell-cycle phase dis ibu ion in se e al human colon, and panc ea ic ca cinoma, and glioblas oma cell
lines and p ima y cul u es de i ed om glioblas oma pa ien s. Ou esul s show ha BMS-754807
mainly ac s h ough o - a ge e ec s exe ed on o he p o ein kinases independen ly o IGF-1R
inhibi ion. Gi en ha BMS-754807 exhibi s a po en an ip oli e a i e e ec s on glioblas oma, colon and
panc ea ic ca cinoma cellula models analyzed in his wo k, we belie e ha his compound could be
use ul o he ea men o di e en ypes o umo s independen ly o hei IGF-1R ac i a ion s a us.
2. Resul s
2.1. BMS-754807 and OSI-906 E ec on IGF-1R Phospho yla ion
Gi en ha bo h compounds, BMS and OSI, ha e been de eloped as IGF-1R and IR inhibi o s, we
decided o s udy whe he equi alen doses o hese d ugs we e able o inhibi IGF-1R phospho yla ion
o a simila ex en . Wi h his pu pose, we es ed he e ec s o a 10
µ
M dose o BMS o OSI on IGF-1R
phospho yla ion in he human panc ea ic cell lines IMIM-PC-2 and RWP-1. As shown in Figu e 1A,
bo h inhibi o s we e able o block almos comple ely IGF-1R phospho yla ion induced by 10% FBS,
as de e mined by using a comme cial human phospho-RTK a ay. In addi ion, IGF-1R phospho yla ion
was analyzed by wes e n blo ing using an ibodies agains phospho-IGF-1R (Ty -857) o IGF-1R. OSI
and BMS we e used a 500 nM and 10
µ
M (Figu e S1). Bo h compounds we e able o inhibi IGF-1R
phospho yla ion a low doses.
Cance s 2020, 12, x 3 o 19
IGF-1R, wi h an IC50 o 35 nM in cell- ee assays, and is modes ly po en agains he IR, wi h an IC50
o 75 nM. I is also known o ha e no ac i i y owa ds Abl, ALK, BTK, EGFR, FGFR1/2, PKA and
o he p o ein kinases [15,16]. OSI-906 inhibi s he p oli e a ion o hepa ocellula ca cinoma (HCC)
cell lines by a leas 40%. HCC cells sensi i e o OSI-906 show highe le els o phospho yla ion o
IGF-1R and IR han esis an cells, sugges ing ha sensi i i y o OSI-906 is associa ed wi h he
inhibi ion o bo h o hese ecep o s [17]. Mo eo e , OSI-906-induced apop osis and inhibi ion o cell
p oli e a ion appea o be di ec ly linked o he inhibi ion o AKT in se e al umo cell lines, including
lung, panc eas and colo ec al cell lines [18]. In his con ex , OSI-906- ea ed colo ec al cance
xenog a s show a dec ease in umo g ow h and inc eased apop osis in i o and in i o [19]. In his
sys em, OSI-906 has been ound o amelio a e cell p oli e a ion by al e ing he cell cycle in he G0/G1
phase.
In his wo k, we ha e add essed he e ec s o BMS-754807 and OSI-906 on cell p oli e a ion and
cell-cycle phase dis ibu ion in se e al human colon, and panc ea ic ca cinoma, and glioblas oma cell
lines and p ima y cul u es de i ed om glioblas oma pa ien s. Ou esul s show ha BMS-754807
mainly ac s h ough o - a ge e ec s exe ed on o he p o ein kinases independen ly o IGF-1R
inhibi ion. Gi en ha BMS-754807 exhibi s a po en an ip oli e a i e e ec s on glioblas oma, colon and
panc ea ic ca cinoma cellula models analyzed in his wo k, we belie e ha his compound could be
use ul o he ea men o di e en ypes o umo s independen ly o hei IGF-1R ac i a ion s a us.
2. Resul s
2.1. BMS-754807 and OSI-906 E ec on IGF-1R Phospho yla ion
Gi en ha bo h compounds, BMS and OSI, ha e been de eloped as IGF-1R and IR inhibi o s,
we decided o s udy whe he equi alen doses o hese d ugs we e able o inhibi IGF-1R
phospho yla ion o a simila ex en . Wi h his pu pose, we es ed he e ec s o a 10 µM dose o BMS
o OSI on IGF-1R phospho yla ion in he human panc ea ic cell lines IMIM-PC-2 and RWP-1. As
shown in Figu e 1A, bo h inhibi o s we e able o block almos comple ely IGF-1R phospho yla ion
induced by 10% FBS, as de e mined by using a comme cial human phospho-RTK a ay. In addi ion,
IGF-1R phospho yla ion was analyzed by wes e n blo ing using an ibodies agains phospho-IGF-1R
(Ty -857) o IGF-1R. OSI and BMS we e used a 500 nM and 10 µM (Figu e S1). Bo h compounds
we e able o inhibi IGF-1R phospho yla ion a low doses.
Nex , we assessed by molecula docking assays he in e ac ion be ween bo h inhibi o s, BMS
and OSI, on he IGF-1R p o ein s uc u e, as shown in Figu e 1B. Bo h compounds we e p edic ed o
bind p e e en ially o he ATP-binding si e o his ecep o wi h equi alen a ini ies. Mo eo e , he
calcula ed Gibbs ee ene gy changes (∆G) o bo h compounds we e qui e simila , o −9.75 and −9.45
kcal/mol o BMS and OSI, espec i ely.
Figu e 1. (A) Rep esen a i e image o a human phospho-RTK a ay (R&D Sys ems) analysis
pe o med on RWP-1 cells. The h ee panels below show a magni ica ion o he IGF-1R do s ob ained
om RWP-1 cells g own in he p esence o 10% FBS, con ol (un ea ed) and ea ed o 3 h wi h 10
µM OSI-906 o BMS-754807, C(+) show he posi i e con ol spo s used o he no maliza ion o
Figu e 1.
(
A
) Rep esen a i e image o a human phospho-RTK a ay (R&D Sys ems) analysis pe o med
on RWP-1 cells. The h ee panels below show a magni ica ion o he IGF-1R do s ob ained om RWP-1
cells g own in he p esence o 10% FBS, con ol (un ea ed) and ea ed o 3 h wi h 10 µM OSI-906 o
BMS-754807, C(+) show he posi i e con ol spo s used o he no maliza ion o luo escence in ensi y
be ween di e en il e s. (
B
) Docking analysis o he in e ac ion be ween he wo inhibi o s, BMS-754807
and OSI-906, on he IGF-1R s uc u e. Pu ples ellipses ep esen al e na i e heo e ical binding si es
o IGF-1R and ed ellipse ep esen he ATP-binding domain, which is also he highes a ini y si e in
bo h cases.
Cance s 2020,12, 3717 4 o 18
Nex , we assessed by molecula docking assays he in e ac ion be ween bo h inhibi o s, BMS and
OSI, on he IGF-1R p o ein s uc u e, as shown in Figu e 1B. Bo h compounds we e p edic ed o
bind p e e en ially o he ATP-binding si e o his ecep o wi h equi alen a ini ies. Mo eo e ,
he calcula ed Gibbs ee ene gy changes (
∆
G) o bo h compounds we e qui e simila , o
−
9.75 and
−9.45 kcal/mol o BMS and OSI, espec i ely.
2.2. BMS-754807 and OSI-906 E ec s on Cell Viabili y
In o de o analyze he e ec o bo h IGF-1R inhibi o s on cell lines de i ed om di e en ypes o
human umo s, we ca ied ou MTT cell-p oli e a ion assays in he p esence o BMS o OSI. The esul s
shown in Figu e 2illus a e ha he dec ease in he pe cen age o iable cells a e ea men wi h
10
µ
M BMS o OSI in di e en glioblas oma, colon and panc ea ic ca cinoma cell lines was qui e
di e en o he wo inhibi o s, wi h he esul ha BMS had a s onge inhibi o y e ec on cell g ow h
in almos all cell lines es ed as compa ed wi h OSI. Indeed, se e al cell lines we e esis an o OSI
bu we e inhibi ed by BMS, which was especially e iden o he h ee glioblas oma p ima y cul u es
and he IMIM-PC-2 panc ea ic ca cinoma cell line. In gene al, 10
µ
M OSI inhibi ed cell g ow h by
10–40%, whe eas he same concen a ion o BMS caused a 40–80% inhibi ion, depending on he cell line.
We also pe o med MTT assays using di e en concen a ions o bo h inhibi o s anging om 0.1 o
10
µ
M on all he cellula models s udied. The esul s ob ained in dose– esponse expe imen s ca ied
ou in ou cell lines a e shown in Figu e 3and in wo mo e cell lines, SW480 and RWP-1 in Figu e S2.
These esul s demons a e ha he di e en ial e ec s o he wo compounds on cell p oli e a ion we e
mani es a all concen a ions es ed.
Cance s 2020, 12, x 4 o 19
luo escence in ensi y be ween di e en il e s. (B) Docking analysis o he in e ac ion be ween he
wo inhibi o s, BMS-754807 and OSI-906, on he IGF-1R s uc u e. Pu ples ellipses ep esen
al e na i e heo e ical binding si es o IGF-1R and ed ellipse ep esen he ATP-binding domain,
which is also he highes a ini y si e in bo h cases.
2.2. BMS-754807 and OSI-906 E ec s on Cell Viabili y
In o de o analyze he e ec o bo h IGF-1R inhibi o s on cell lines de i ed om di e en ypes
o human umo s, we ca ied ou MTT cell-p oli e a ion assays in he p esence o BMS o OSI. The
esul s shown in Figu e 2 illus a e ha he dec ease in he pe cen age o iable cells a e ea men
wi h 10 µM BMS o OSI in di e en glioblas oma, colon and panc ea ic ca cinoma cell lines was qui e
di e en o he wo inhibi o s, wi h he esul ha BMS had a s onge inhibi o y e ec on cell
g ow h in almos all cell lines es ed as compa ed wi h OSI. Indeed, se e al cell lines we e esis an
o OSI bu we e inhibi ed by BMS, which was especially e iden o he h ee glioblas oma p ima y
cul u es and he IMIM-PC-2 panc ea ic ca cinoma cell line. In gene al, 10 µM OSI inhibi ed cell
g ow h by 10–40%, whe eas he same concen a ion o BMS caused a 40–80% inhibi ion, depending
on he cell line. We also pe o med MTT assays using di e en concen a ions o bo h inhibi o s
anging om 0.1 o 10 µM on all he cellula models s udied. The esul s ob ained in dose– esponse
expe imen s ca ied ou in ou cell lines a e shown in Figu e 3 and in wo mo e cell lines, SW480 and
RWP-1 in Figu e S2. These esul s demons a e ha he di e en ial e ec s o he wo compounds on
cell p oli e a ion we e mani es a all concen a ions es ed.
Figu e 2. BMS-754807 and OSI-906 e ec on cell iabili y in glioblas oma, colon and panc ea ic cance
cell lines. The indica ed cell lines we e ea ed wi h 10 µM BMS-754807 o OSI-906 o 72 h, and cell
p oli e a ion was e alua ed by MTT assays. Da a ep esen he mean ± SEM (n ≥ 6) o iable cell
pe cen age in he p esence o 10 µM BMS o OSI compounds, as compa ed o un ea ed cells aken as
100%. *, p < 0.05; **, p < 0.01.
Figu e 2.
BMS-754807 and OSI-906 e ec on cell iabili y in glioblas oma, colon and panc ea ic cance
cell lines. The indica ed cell lines we e ea ed wi h 10
µ
M BMS-754807 o OSI-906 o 72 h, and cell
p oli e a ion was e alua ed by MTT assays. Da a ep esen he mean
±
SEM (n
≥
6) o iable cell
pe cen age in he p esence o 10 µM BMS o OSI compounds, as compa ed o un ea ed cells aken as
100%. *, p<0.05; **, p<0.01.
Cance s 2020,12, 3717 5 o 18
Cance s 2020, 12, x 5 o 19
Figu e 3. Dose– esponse e ec o BMS-754807 and OSI-906 on cell p oli e a ion in di e en umo cell
lines. The indica ed cell lines we e ea ed wi h 0.01–10 µM BMS-754807 o OSI-906 o 72 h and cell
p oli e a ion was e alua ed by he MTT assay. (A) IMIM-PC-2 panc ea ic ca cinoma cell line; (B) T98
glioblas oma cell line; (C,D) HGUE-GB-17 and HGUE-GB-15 glioblas oma p ima y cul u es,
espec i ely. The solid line o each plo has been calcula ed by i ing he h ee pa ame e s o a sigmoid
equa ion (dose– esponse cu e) on he da a ha ep esen he decimal loga i hm o he inhibi o
concen a ion e sus he esponse ob ained. G aphPad P ism 5 so wa e (G aphPad So wa e Inc.,
San Diego, CA, USA) has been used. Da a ep esen he mean ±SEM (n ≥ 6) o iable cells pe cen age
wi h espec o un ea ed con ols, aken as 100%.
2.3. BMS-754807 and OSI-906 E ec s on Cell Cycle Phase Dis ibu ion
To de e mine he e ec o bo h inhibi o s, BMS and OSI, on he dis ibu ion o cells among he
di e en phases o he cell cycle, hey we e ea ed o no wi h 10 µM BMS o OSI o 24 h, and hen
hei DNA was labeled wi h p opidium iodide. Figu e 4A shows he esul s ob ained by low
cy ome y in he ou panc ea ic ca cinoma cell lines analyzed, e lec ing ha he wo inhibi o s
exe ed di e en ial e ec s on cell cycle phase dis ibu ion. While OSI p oduced no e ec o blockade
in he G1 phase, BMS elici ed an a es in he G2+M phases o he cell cycle. BMS also causes an inc ease
(albei small) in he ac ion o cells in sub-G1 phase (Figu e 4B), which was indica i e o cell dea h.
Simila esul s we e ob ained in colon and glioblas oma cell models. The sub-G1 phase a e BMS
ea men shown in Figu e 4B is s a is ically signi ican al hough small; howe e , i has o be aken
in o accoun ha he maximum e ec o OSI and BMS is shown in Figu e 3, which p esen s he MTT
es da a ca ied ou a e 72 h o ea men . A e 24 h o BMS ea men , a pe cen age o cells in he
sub-G1 phase is obse ed, bu also a much highe ac ion is blocked in he G2 + M phase, which a e
al eady ma ked o die. I we ollow he e ec on he cell cycle a 48 and 72 h, cells blocked in G2 + M
a e ansloca ed o he Sub-G1 phase (Figu e 4C).
Figu e 3.
Dose– esponse e ec o BMS-754807 and OSI-906 on cell p oli e a ion in di e en umo
cell lines. The indica ed cell lines we e ea ed wi h 0.01–10
µ
M BMS-754807 o OSI-906 o 72 h and
cell p oli e a ion was e alua ed by he MTT assay. (
A
) IMIM-PC-2 panc ea ic ca cinoma cell line;
(
B
) T98 glioblas oma cell line; (
C
,
D
) HGUE-GB-17 and HGUE-GB-15 glioblas oma p ima y cul u es,
espec i ely. The solid line o each plo has been calcula ed by i ing he h ee pa ame e s o a sigmoid
equa ion (dose– esponse cu e) on he da a ha ep esen he decimal loga i hm o he inhibi o
concen a ion e sus he esponse ob ained. G aphPad P ism 5 so wa e (G aphPad So wa e Inc.,
San Diego, CA, USA) has been used. Da a ep esen he mean
±
SEM (n
≥
6) o iable cells pe cen age
wi h espec o un ea ed con ols, aken as 100%.
2.3. BMS-754807 and OSI-906 E ec s on Cell Cycle Phase Dis ibu ion
To de e mine he e ec o bo h inhibi o s, BMS and OSI, on he dis ibu ion o cells among he
di e en phases o he cell cycle, hey we e ea ed o no wi h 10
µ
M BMS o OSI o 24 h, and hen hei
DNA was labeled wi h p opidium iodide. Figu e 4A shows he esul s ob ained by low cy ome y in
he ou panc ea ic ca cinoma cell lines analyzed, e lec ing ha he wo inhibi o s exe ed di e en ial
e ec s on cell cycle phase dis ibu ion. While OSI p oduced no e ec o blockade in he G
1
phase,
BMS elici ed an a es in he G
2
+M phases o he cell cycle. BMS also causes an inc ease (albei small)
in he ac ion o cells in sub-G1phase (Figu e 4B), which was indica i e o cell dea h. Simila esul s
we e ob ained in colon and glioblas oma cell models. The sub-G
1
phase a e BMS ea men shown
in Figu e 4B is s a is ically signi ican al hough small; howe e , i has o be aken in o accoun ha
he maximum e ec o OSI and BMS is shown in Figu e 3, which p esen s he MTT es da a ca ied
ou a e 72 h o ea men . A e 24 h o BMS ea men , a pe cen age o cells in he sub-G
1
phase is
obse ed, bu also a much highe ac ion is blocked in he G2 +M phase, which a e al eady ma ked o
die. I we ollow he e ec on he cell cycle a 48 and 72 h, cells blocked in G2 +M a e ansloca ed o
he Sub-G1 phase (Figu e 4C).
Cance s 2020,12, 3717 6 o 18
Cance s 2020, 12, x 6 o 19
Figu e 4. (A) E ec o BMS-754807 and OSI-906 on cell cycle phase dis ibu ion in panc ea ic ca cinoma
cell lines. RWP-1, IMIM-PC-1, IMIM-PC-2 and HS766T cell lines we e ea ed wi h 10 µM BMS-754807
o OSI-906 o 24 h and cell cycle phase dis ibu ion was analyzed by low cy ome y. Da a ep esen he
mean ± SEM (n ≥ 3) o he pe cen age o cells in each phase o he cell cycle. * p < 0.05, ** p < 0.01. (B) Cell
dea h induc ion by BMS-754807 and OSI-906 in panc ea ic ca cinoma cell lines. RWP-1, IMIM-PC-1,
IMIM-PC-2 and HS766T we e ea ed wi h 10 BMS-754807 µM o OSI-906 o 24 h. Da a ep esen he
mean ± SEM (n ≥ 3) o he pe cen age o dead cells (sub-G1) on he cell cycle analysis ep esen ed in A.
** p < 0.01. (C) E ec o 10 µM BMS-754807 o 24, 48 and 72 h in he panc ea ic ca cinoma cell line RWP-
1. Da a ep esen he inc ease in he pe cen age o cell dea h and he pa allel dec ease in cells in he G2
+ M phase o he cell cycle. Da a ep esen he mean ± SEM (n ≥ 3).
We ha e p e iously s udied he e ec o ano he inhibi o o IGF-1R, pic opodophyllin (PPP),
on glioblas oma cellula models, and de e mined ha he molecula mechanism o cell dea h induced
by his compound was no a caspase-dependen apop osis [20]. Acco dingly, we decided o es a
pan-caspase inhibi o in o de o de e mine whe he BMS-induced cell dea h occu ed o no by
means o caspase-dependen apop osis. Figu e 5 shows he e ec o he gene al caspase inhibi o , Z-
VAD-FMK, on BMS-754807-induced cell dea h in IMIM-PC-1 and IMIM-PC-2 panc ea ic ca cinoma
cell lines. Ou esul s e lec ed ha his pan-caspase inhibi o almos comple ely abolished cell dea h
induced by BMS, sugges ing ha his phenomenon ook place h ough a ypical caspase-dependen
apop o ic mechanism. This is an in e es ing inding since we ha e es ed h ee pu a i e inhibi o s o
IGF-1R in his and in ou p e ious a icle, and we ha e ound ha wo o hem (namely PPP and
BMS) induced cell dea h and blocked cell cycle in he G2 + M phase, al hough hei mechanisms o
cell dea h induc ion we e di e en (one is caspase dependen , while he o he one is caspase
independen ) [20].
Figu e 4.
(
A
) E ec o BMS-754807 and OSI-906 on cell cycle phase dis ibu ion in panc ea ic ca cinoma
cell lines. RWP-1, IMIM-PC-1, IMIM-PC-2 and HS766T cell lines we e ea ed wi h 10
µ
M BMS-754807
o OSI-906 o 24 h and cell cycle phase dis ibu ion was analyzed by low cy ome y. Da a ep esen he
mean
±
SEM (n
≥
3) o he pe cen age o cells in each phase o he cell cycle. * p<0.05, ** p<0.01. (
B
) Cell
dea h induc ion by BMS-754807 and OSI-906 in panc ea ic ca cinoma cell lines. RWP-1, IMIM-PC-1,
IMIM-PC-2 and HS766T we e ea ed wi h 10 BMS-754807
µ
M o OSI-906 o 24 h. Da a ep esen he
mean
±
SEM (n
≥
3) o he pe cen age o dead cells (sub-G1) on he cell cycle analysis ep esen ed in A.
** p<0.01. (
C
) E ec o 10
µ
M BMS-754807 o 24, 48 and 72 h in he panc ea ic ca cinoma cell line
RWP-1. Da a ep esen he inc ease in he pe cen age o cell dea h and he pa allel dec ease in cells in
he G2 +M phase o he cell cycle. Da a ep esen he mean ±SEM (n≥3).
We ha e p e iously s udied he e ec o ano he inhibi o o IGF-1R, pic opodophyllin (PPP),
on glioblas oma cellula models, and de e mined ha he molecula mechanism o cell dea h induced
by his compound was no a caspase-dependen apop osis [
20
]. Acco dingly, we decided o es
a pan-caspase inhibi o in o de o de e mine whe he BMS-induced cell dea h occu ed o no by
means o caspase-dependen apop osis. Figu e 5shows he e ec o he gene al caspase inhibi o ,
Z-VAD-FMK, on BMS-754807-induced cell dea h in IMIM-PC-1 and IMIM-PC-2 panc ea ic ca cinoma
cell lines. Ou esul s e lec ed ha his pan-caspase inhibi o almos comple ely abolished cell dea h
induced by BMS, sugges ing ha his phenomenon ook place h ough a ypical caspase-dependen
apop o ic mechanism. This is an in e es ing inding since we ha e es ed h ee pu a i e inhibi o s o
IGF-1R in his and in ou p e ious a icle, and we ha e ound ha wo o hem (namely PPP and BMS)
induced cell dea h and blocked cell cycle in he G2 +M phase, al hough hei mechanisms o cell dea h
induc ion we e di e en (one is caspase dependen , while he o he one is caspase independen ) [20].
Cance s 2020,12, 3717 7 o 18
Cance s 2020, 12, x 7 o 19
Figu e 5. E ec o a pan-caspase inhibi o on cell dea h induced by BMS-754807. IMIM-PC-1 and
IMIM-PC-2 cell lines we e ea ed wi h 10 µM BMS-754807 (BMS) in he p esence o absence o 25 µM
Z-VAD-FMK (ICn) o 24 h, and he numbe o cells in he sub-G1 phase o he cell cycle was
de e mined by low cy ome y. Da a ep esen he mean ± SEM (n ≥ 3), aking he numbe o BMS-
ea ed, ICn-un ea ed cells in sub-G1 phase as 100%. **, p < 0.01.
2.4. BMS-754807 and OSI-906 E ec s on he Ac i i y o In acellula P o ein Kinases
Gi en ha bo h compounds, BMS and OSI, a e inhibi o s o IGF-1R y osine kinase ac i i y, we
se o assess whe he hei obse ed di e en ial e ec s on cance cell lines we e a ibu able o hei
possible abili y o inhibi o - a ge p o ein kinases. Wi h his pu pose, we s a ed by analyzing hei
pu a i e e ec on he ac i i y o di e en MAP kinases. We pe o med hese expe imen s in he
panc ea ic ca cinoma cell line RWP-1, since bo h compounds inhibi ed hei p oli e a ion and a ec ed
hei cell cycle phase dis ibu ion (Figu e S2 and Figu e 4A,B). An ELISA es was used o de e mine
he phospho yla ion (and hence ac i a ion) s a us o a se o MAP kinases, namely, ERK 1 and 2, JNK
1, 2 and 3, and p38α, a e 1 and 6 h o ea men wi h 10 µM BMS o OSI. Since we we e looking o
pu a i e al e na i e a ge s o BMS, independen ly o IGF-1R, o maximize he di e en ial a ge s o
OSI and BMS we use a high dose o hese compounds, 10 µM. I is ob ious ha when we ea cells
in cul u e, we could add e y low doses, bu his would no be he eal si ua ion conce ning he doses
o hese d ugs ecei ed by he pa ien s ha , ob iously, a e no going o ecei e nM doses, so we need
o s udy he e ec s o eal doses, in o de o mimic he si ua ion in he umo cells inside he pa ien s.
The e a e se e al a icles ha s udy he concen a ion o linsi inib (OSI) in plasma and blood o
human pa ien s. The concen a ions o linsi inib in he plasma ange om 1789 ng/mL (app ox. 4.5
µM) o alues highe han 4500 ng/mL (app ox. 10.6 µM), which means ha ou da a wi h 10 µM jus
o maximize he di e ence be ween OSI and BMS o allow us iden i y BMS o a ge s is in he ange
o he dose ecei ed by he pa ien [21,22], no o men ion he doses used in mouse xenog a models,
whe e OSI and BMS a e used in doses as high as 40 mg/kg, ha is, a dose equi alen o se e al
hund ed µM. Ou esul s shown in Figu e 6A e lec ed ha bo h compounds nega i ely a ec ed
ERK1/2 phospho yla ion, al hough BMS exe ed a s onge inhibi o y e ec (by 80%) han OSI (by
30%). On he o he hand, bo h compounds elici ed an inc ease in he phospho yla ion o JNK1-3
(Figu e 6B), again BMS being mo e e ec i e (wi h a 100% inc ease o e con ol phospho yla ion)
han OSI (40% inc ease). The g ea es di e ence be ween he wo compounds was obse ed on he
ac i a ion o p38α (Figu e 6C), whe e OSI induced a highe han 100% inc ease in i s phospho yla ion
o e con ol le els, whe eas BMS esul ed in he inhibi ion o p38α phospho yla ion by 80%.
In an a emp o iden i y possible di e en ial a ge s o BMS and OSI, we used a human
phospho-kinase a ay. The mos signi ican esul s o hese expe imen s a e shown in Figu e 6D,
which alida ed he di e en ial e ec o bo h compounds on p38α, bu addi ionally allowed us o
Figu e 5.
E ec o a pan-caspase inhibi o on cell dea h induced by BMS-754807. IMIM-PC-1 and
IMIM-PC-2 cell lines we e ea ed wi h 10
µ
M BMS-754807 (BMS) in he p esence o absence o 25
µ
M
Z-VAD-FMK (ICn) o 24 h, and he numbe o cells in he sub-G
1
phase o he cell cycle was de e mined
by low cy ome y. Da a ep esen he mean
±
SEM (n
≥
3), aking he numbe o BMS- ea ed,
ICn-un ea ed cells in sub-G1phase as 100%. **, p<0.01.
2.4. BMS-754807 and OSI-906 E ec s on he Ac i i y o In acellula P o ein Kinases
Gi en ha bo h compounds, BMS and OSI, a e inhibi o s o IGF-1R y osine kinase ac i i y,
we se o assess whe he hei obse ed di e en ial e ec s on cance cell lines we e a ibu able o
hei possible abili y o inhibi o - a ge p o ein kinases. Wi h his pu pose, we s a ed by analyzing
hei pu a i e e ec on he ac i i y o di e en MAP kinases. We pe o med hese expe imen s in he
panc ea ic ca cinoma cell line RWP-1, since bo h compounds inhibi ed hei p oli e a ion and a ec ed
hei cell cycle phase dis ibu ion (Figu e S2 and Figu e 4A,B). An ELISA es was used o de e mine he
phospho yla ion (and hence ac i a ion) s a us o a se o MAP kinases, namely, ERK 1 and 2, JNK 1, 2
and 3, and p38
α
, a e 1 and 6 h o ea men wi h 10
µ
M BMS o OSI. Since we we e looking o pu a i e
al e na i e a ge s o BMS, independen ly o IGF-1R, o maximize he di e en ial a ge s o OSI and
BMS we use a high dose o hese compounds, 10
µ
M. I is ob ious ha when we ea cells in cul u e,
we could add e y low doses, bu his would no be he eal si ua ion conce ning he doses o hese
d ugs ecei ed by he pa ien s ha , ob iously, a e no going o ecei e nM doses, so we need o s udy
he e ec s o eal doses, in o de o mimic he si ua ion in he umo cells inside he pa ien s. The e a e
se e al a icles ha s udy he concen a ion o linsi inib (OSI) in plasma and blood o human pa ien s.
The concen a ions o linsi inib in he plasma ange om 1789 ng/mL (app ox. 4.5
µ
M) o alues
highe han 4500 ng/mL (app ox. 10.6
µ
M), which means ha ou da a wi h 10
µ
M jus o maximize
he di e ence be ween OSI and BMS o allow us iden i y BMS o a ge s is in he ange o he dose
ecei ed by he pa ien [
21
,
22
], no o men ion he doses used in mouse xenog a models, whe e OSI
and BMS a e used in doses as high as 40 mg/kg, ha is, a dose equi alen o se e al hund ed
µ
M. Ou
esul s shown in Figu e 6A e lec ed ha bo h compounds nega i ely a ec ed ERK1/2 phospho yla ion,
al hough BMS exe ed a s onge inhibi o y e ec (by 80%) han OSI (by 30%). On he o he hand, bo h
compounds elici ed an inc ease in he phospho yla ion o JNK1-3 (Figu e 6B), again BMS being mo e
e ec i e (wi h a 100% inc ease o e con ol phospho yla ion) han OSI (40% inc ease). The g ea es
di e ence be ween he wo compounds was obse ed on he ac i a ion o p38
α
(Figu e 6C), whe e OSI
induced a highe han 100% inc ease in i s phospho yla ion o e con ol le els, whe eas BMS esul ed
in he inhibi ion o p38αphospho yla ion by 80%.
Cance s 2020,12, 3717 8 o 18
Cance s 2020, 12, x 8 o 19
iden i y a numbe o p o ein kinases, such as GSK-3, AMPK, AKT, SRC, CHK2, among o he s, whose
phospho yla ion was inhibi ed by BMS, bu no by OSI. These da a poin ed ou ha many o he
di e en ial e ec s obse ed be ween OSI and BMS on ou es ed cell lines we e due o inhibi ion
p omo ed by BMS, bu no by OSI, o wo main in acellula signaling pa hways, namely,
PI3K/AKT/mTOR and TP53. In addi ion, Wes e n blo analysis o BMS and OSI e ec s on ERK ½ and
AKT in RWP-1 cells was pe o med, showing he same esul s.
Figu e 6. E ec o BMS-754807 and OSI-906 on he ac i a ion o o - a ge p o ein kinases. The RWP-
1 panc ea ic ca cinoma cell line was ea ed o 1 o 6 h wi h 10 µM BMS-754807 o OSI-906. ERK ½
(A), JNK 1, 2, 3 (B) and p38α (C) we e de e mined by using an Ins an One ELISA ki om eBioscience.
(D). RWP-1 cells we e ea ed wi h 10 µM BMS-754807 o OSI-906, o le un ea ed o 6 h, and hen
subjec ed o analysis on a human phospho -RTK a ay. The g aph shows he e ec o bo h inhibi o s
on he phospho yla ion s a us o he di e en p o ein kinases included in he a ay. Da a ep esen
he mean ± SEM (n ≥ 3) o phospho yla ion le els, aking hose o un ea ed cells as 100%. *, p < 0.05;
**, p < 0.01.
2.5. BMS-754807 and OSI-906 Po en ial In e ac ion wi h P o ein Kinases
Figu e 7 shows he ee ene gy a ia ion (ΔG, kcal/mol) calcula ed using Au oDock/ ina o he
bes docking sco es o BMS-754807 and OSI-906 in e ac ion wi h he ATP-binding si e in he ca aly ic
domain o se e al p o ein kinases iden i ied in he phospho -kinase a ay assay. The calcula ed KD
(KD = expΔG/RT) o compounds wi h a ΔG ≤ −10.5 kcal/mol was in he nanomola o subnanomola
ange [23,24]. As no iceable om alues shown in Figu e 7, only BMS-754807 displayed a ΔG below
ha alue o i s binding o PTK6, HCK (panel A) and FYN (panel B). The ΔG alues we e be ween
0.5 and 1.5 kcal/mol g ea e o OSI-906 han o BMS-754807, which would imply a highe a ini y o
he la e o he ATP-binding si e o p o ein kinases PTK6, SRC, p38α, mTOR, HCK, GSK-3β (Figu e
7A) and IGF-1R (Figu e 7B). These da a we e in ag eemen wi h ou expe imen al obse a ions om
phospho -RTK a ay analysis, showing ha whe eas BMS-754807 clea ly inhibi ed hese enzymes,
OSI-906 did no , o did i less e ec i ely (Figu e 6D). Howe e , o some o he p o ein kinases shown
in Figu e 7B, namely, AMPK, CHK2, AKT1 and AKT2, he ΔG alues o OSI-906 binding we e lowe
han o BMS-754807, implying ha he a ini y o he la e o hei ATP-binding si e would be
highe . In his las case, he molecula docking da a we e in disag eemen wi h he expe imen al da a
om phospho -kinase a ays. Bo h d ugs ha e been designed agains he ATP-binding si e and
should hus beha e as compe i i e inhibi o s. Howe e , OSI and BMS could bind wi h high a ini y
Figu e 6.
E ec o BMS-754807 and OSI-906 on he ac i a ion o o - a ge p o ein kinases. The RWP-1
panc ea ic ca cinoma cell line was ea ed o 1 o 6 h wi h 10
µ
M BMS-754807 o OSI-906. ERK
1
2
(
A
),
JNK 1, 2, 3 (
B
) and p38
α
(
C
) we e de e mined by using an Ins an One ELISA ki om eBioscience.
(
D
). RWP-1 cells we e ea ed wi h 10
µ
M BMS-754807 o OSI-906, o le un ea ed o 6 h, and hen
subjec ed o analysis on a human phospho -RTK a ay. The g aph shows he e ec o bo h inhibi o s on
he phospho yla ion s a us o he di e en p o ein kinases included in he a ay. Da a ep esen he
mean
±
SEM (n
≥
3) o phospho yla ion le els, aking hose o un ea ed cells as 100%. *, p<0.05; **,
p<0.01.
In an a emp o iden i y possible di e en ial a ge s o BMS and OSI, we used a human
phospho-kinase a ay. The mos signi ican esul s o hese expe imen s a e shown in Figu e 6D,
which alida ed he di e en ial e ec o bo h compounds on p38
α
, bu addi ionally allowed us
o iden i y a numbe o p o ein kinases, such as GSK-3, AMPK, AKT, SRC, CHK2, among o he s,
whose phospho yla ion was inhibi ed by BMS, bu no by OSI. These da a poin ed ou ha many
o he di e en ial e ec s obse ed be ween OSI and BMS on ou es ed cell lines we e due o
inhibi ion p omo ed by BMS, bu no by OSI, o wo main in acellula signaling pa hways, namely,
PI3K/AKT/mTOR and TP53. In addi ion, Wes e n blo analysis o BMS and OSI e ec s on ERK 1/2 and
AKT in RWP-1 cells was pe o med, showing he same esul s.
2.5. BMS-754807 and OSI-906 Po en ial In e ac ion wi h P o ein Kinases
Figu e 7shows he ee ene gy a ia ion (
∆
G, kcal/mol) calcula ed using Au oDock/ ina o he
bes docking sco es o BMS-754807 and OSI-906 in e ac ion wi h he ATP-binding si e in he ca aly ic
domain o se e al p o ein kinases iden i ied in he phospho -kinase a ay assay. The calcula ed K
D
(K
D
=exp
∆G/RT
) o compounds wi h a
∆
G
≤ −
10.5 kcal/mol was in he nanomola o subnanomola
ange [
23
,
24
]. As no iceable om alues shown in Figu e 7, only BMS-754807 displayed a
∆
G below
ha alue o i s binding o PTK6, HCK (panel A) and FYN (panel B). The
∆
G alues we e be ween 0.5
and 1.5 kcal/mol g ea e o OSI-906 han o BMS-754807, which would imply a highe a ini y o he
la e o he ATP-binding si e o p o ein kinases PTK6, SRC, p38
α
, mTOR, HCK, GSK-3
β
(Figu e 7A)
and IGF-1R (Figu e 7B). These da a we e in ag eemen wi h ou expe imen al obse a ions om
phospho -RTK a ay analysis, showing ha whe eas BMS-754807 clea ly inhibi ed hese enzymes,
OSI-906 did no , o did i less e ec i ely (Figu e 6D). Howe e , o some o he p o ein kinases shown
Cance s 2020,12, 3717 9 o 18
in Figu e 7B, namely, AMPK, CHK2, AKT1 and AKT2, he
∆
G alues o OSI-906 binding we e lowe
han o BMS-754807, implying ha he a ini y o he la e o hei ATP-binding si e would be highe .
In his las case, he molecula docking da a we e in disag eemen wi h he expe imen al da a om
phospho -kinase a ays. Bo h d ugs ha e been designed agains he ATP-binding si e and should
hus beha e as compe i i e inhibi o s. Howe e , OSI and BMS could bind wi h high a ini y o o he
a eas di e en om he ATP-binding si e, and he eby exe a ole as allos e ic modula o s o by
p e en ing in e ac ions wi h o he p o eins ac ing in ups eam o downs eam signaling cascades in
which he s udied p o ein kinases also pa icipa e. In o de o add ess his ques ion, we ca ied ou
500 uns o molecula docking assays o BMS-754807 and OSI-906 in e ac ions wi h he ull ca aly ic
domains o all he 12 p o ein kinases indica ed in Figu e 7, wi h he esul s depic ed in Figu e 8.
Fo each p o ein kinase, we ound a di e en numbe o clus e s o in e ac ion si es, anging om
one o i e, which di e ed by <5 Å in hei oo mean squa e de ia ion alues o BMS-754807 and
OSI-906 compounds. A i s unexpec ed obse a ion was ha he molecula docking assays did no
show any ho spo (clus e ) o BMS-754807 o OSI-906 in e ac ion wi h he ATP-binding si es o CHK2
(Figu e 8G) o p38
α
(Figu e 8T), espec i ely. In gene al, hese docking assays e ealed a g ea e
numbe o ho spo s o BMS-754807 han o OSI-906, which could explain he mo e e ec i e inhibi o y
e ec displayed on cells by BMS-754807 in ou expe imen al da a. Also no iceable was he p esence o
clus e s o binding o his inhibi o in he a ea o in e sec ion be ween he N- and C- e minal domains
o AKT2 (Figu e 8C), AMPK (Figu e 8E), mTOR (Figu e 8Q), p38
α
(Figu e 8S) and SRC (Figu e 8W).
I is emp ing o specula e ha BMS-754807 binding o his a ea would p e en he necessa y opening
and closing mo emen a ound he cle in he ca aly ic domain necessa y o ATP binding.
Cance s 2020, 12, x 9 o 19
o o he a eas di e en om he ATP-binding si e, and he eby exe a ole as allos e ic modula o s
o by p e en ing in e ac ions wi h o he p o eins ac ing in ups eam o downs eam signaling
cascades in which he s udied p o ein kinases also pa icipa e. In o de o add ess his ques ion, we
ca ied ou 500 uns o molecula docking assays o BMS-754807 and OSI-906 in e ac ions wi h he
ull ca aly ic domains o all he 12 p o ein kinases indica ed in Figu e 7, wi h he esul s depic ed in
Figu e 8. Fo each p o ein kinase, we ound a di e en numbe o clus e s o in e ac ion si es, anging
om one o i e, which di e ed by <5 Å in hei oo mean squa e de ia ion alues o BMS-754807
and OSI-906 compounds. A i s unexpec ed obse a ion was ha he molecula docking assays did
no show any ho spo (clus e ) o BMS-754807 o OSI-906 in e ac ion wi h he ATP-binding si es o
CHK2 (Figu e 8G) o p38α (Figu e 8T), espec i ely. In gene al, hese docking assays e ealed a
g ea e numbe o ho spo s o BMS-754807 han o OSI-906, which could explain he mo e e ec i e
inhibi o y e ec displayed on cells by BMS-754807 in ou expe imen al da a. Also no iceable was he
p esence o clus e s o binding o his inhibi o in he a ea o in e sec ion be ween he N- and C-
e minal domains o AKT2 (Figu e 8C), AMPK (Figu e 8E), mTOR (Figu e 8Q), p38α (Figu e 8S) and
SRC (Figu e 8W). I is emp ing o specula e ha BMS-754807 binding o his a ea would p e en he
necessa y opening and closing mo emen a ound he cle in he ca aly ic domain necessa y o ATP
binding.
Figu e 7. Compa ison o Gibbs ee ene gy a ia ion (ΔG, kcal/mol) o BMS-754807 and OSI-906
inhibi o s based on molecula docking agains he ATP-binding si e o se e al p o ein kinases (see
Table S1 o hei UniP o KB accession numbe s). The da a ha e been dis ibu ed in wo panels (A,B)
o acili a e compa ison. Panel A shows he esul s o molecula docking analyses o PTK6, SRC,
p38α, mTOR, HCK and GSK-3β p o ein kinases. Panel B shows he esul s o FYN, AMPK, IGF-1R,
CHK2, AKT1 and AKT2.p o ein kinases.
Figu e 7.
Compa ison o Gibbs ee ene gy a ia ion (
∆
G, kcal/mol) o BMS-754807 and OSI-906
inhibi o s based on molecula docking agains he ATP-binding si e o se e al p o ein kinases
(see Table S1 o hei UniP o KB accession numbe s). The da a ha e been dis ibu ed in wo
panels (
A
,
B
) o acili a e compa ison. Panel
A
shows he esul s o molecula docking analyses o
PTK6, SRC, p38
α
, mTOR, HCK and GSK-3
β
p o ein kinases. Panel
B
shows he esul s o FYN, AMPK,
IGF-1R, CHK2, AKT1 and AKT2.p o ein kinases.
Cance s 2020,12, 3717 16 o 18
Fo mal Analysis o da a, p epa ing da a o publica ion, w i ing he o iginal D a and Funding. All au ho s ha e
ead and ag eed o he published e sion o he manusc ip .
Funding:
This esea ch was unded by a G an om Ins i u o de Salud Ca los III G an PI012/02025 co-suppo ed
by FEDER unds and PRECIPITA c owd unding pla o m om Fundaci
ó
n Española pa a la Ciencia y la Tecnolog
í
a
(Fecy ) o M. Saceda and AMACMED (Asociaci
ó
n de muje es a ec adas po c
á
nce de mama de Elche y Coma ca)
and Monica Mo aleda dona ion o M. Saceda. The Spanish Minis y o Economy and Compe i i eness (MINECO,
P ojec RTI2018-096724-B-C21) and he Gene ali a Valenciana (PROMETEO/2016/006) suppo ed he wo k in he
Encina labo a o y.
Acknowledgmen s:
The au ho s a e g a e ul o he wo e iewe s o his pape and o ou labo a o y membe s
o help ul commen s.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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