Combined pharmacophore based small molecule design for direct inhibition of
the OLIG2 transcription factor complex
Rajesh Mukthavaram, Igor Tsigelny, Valentina Kouznetsova, Ying Chao, Sandra Pastorino, Jiang Pengfei, Sandeep Pingle, Wolf Wrasidlo, Milan Makale, Santosh Kesari
Moores Cancer Center, University of California, San Diego, CA.
OBJECTIVES
EXPERIMENTAL RESULTS
CONCLUSIONS
•Transcription factors (TFs) are a major
class of signaling proteins and are key to
many diseases
olig2/actin
40
•Drug design has mostly failed
30
•Transformed stem-like cells (CSCs) drive
the common and highly lethal brain tumor,
glioblastoma
20
0
• Computational modeling of the specific
OLIG2–E47 dimerization interface
KR
SK72
•Validation of the combined pharmacophore
approach was achieved by the identification
and screening of compounds that
suppressed human GBM in vitro and
suppressed OLIG2 target genes
Cancer stem like cells, nestin expression
E12
HTF4
E47
MyoD3
MYF5
LYL1
TAL2
MYOG
MYF6
NeuroD1
OLIG2
ATOH1
PTF1
HAND2
HAND1
TCF21
ASCL1
ASCL3
HES5
ID3
RRVANNARERLRVRDINEAFKELGRMCQLHL---NSEKPQTKLLILHQAVSVILNLEQQV
RRMANNARERLRVRDINEAFKELGRMCQLHL---KSEKPQTKLLILHQAVAVILSLEQQV
RRMANNARERVRVRDINEAFRELGRMCQMHL---KSDKAQTKLLILQQAVQVILGLEQQV
RRKAATMRERRRLSKVNEAFETLKRSTSSNP---NQRLP--KVEILRNAIRYIEGLQALL
RRKAATMRERRRLKKVNQAFETLKRCTTTNP---NQRLP--KVEILRNAIRYIESLQELL
RRVFTNSRERWRQQNVNGAFAELRKLLPTHP--PDRKLS--KNEVLRLAMKYIGFLVRLL
RKIFTNTRERWRQQNVNSAFAKLRKLIPTHP--PDKKLS--KNETLRLAMRYINFLVKVL
RRRAATLREKRRLKKVNEAFEALKRSTLLNP---NQRLP--KVEILRSAIQYIERLQALL
RRKAATLRERRRLKKINEAFEALKRRTVANP---NQRLP--KVEILRSAISYIERLQDLL
RRMKANARERNRMHGLNAALDNLRKVVPCYS--KTQKLS--KIETLRLAKNYIWALSEIL
LRLKINSRERKRMHDLNIAMDGLREVMPYAHGPSVRKLS--KIATLLLARNYILMLTNSL
RRLAANARERRRMHGLNHAFDQLRNVIPSFN--NDKKLS--KYETLQMAQIYINALSETP
LRQAANVRERRRMQSINDAFEGLRSHIPTLP--YEKRLS--KVDTLRLAIGYINFLSELV
CAHAGARGGARRTQSINSAFAELRECIPNVP--ADTKLS--KIKTLRLATSYIAYLMDLL
RKGSGPKKERRRTESINSAFAELRECIPNVP--ADTKLS--KIKTLRLATSYIAYLMDVL
QRNAANARERARMRVLSKAFSRLKTTLPWVP--PDTKLS--KLDTLRLASSYIAHLRQIL
AVARRNERERNRVKLVNLGFATLREHVPNGA--ANKKMS--KVETLRSAVEYIRALQQLL
FTRKRNERERQRVKCVNEGYAQLRHHLPEEY--LEKRLS--KVETLRAAIKYINYLQSLL
RRDRINSSIEQLKLLLEQEFARHQ------P---NSKLE--KADILEMAVSYLKHSKGER
GKGPAAEEPLSLLDDMNHCYSRLRELVPGVP--RGTQLS--QVEILQVVLAEPAPGPPDG
57
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49
56
i
ii
iii
Current Studies
•Comprehensive investigation and validation
of OLIG2 selective binding, using additional
biochemical and x-ray crystallographic
methods
Homology modeling and definition of the OLIG2 pharmacophore
Sequence alignment of transcription factors binding to E2A
• Definition of multiple pharmacophore
hypotheses forms the basis of our strategy
% Cell Viability
10
•Searches of conformational databases for
compounds predicted to bind all
pharmacophores, thus maximizing affinity
and specificity
•Biochemical and cell-based screening and
validation of identified compounds
BT74 GBM4 GBM8
120
100
80
60
40
20
0
Control
Inject
■ GBM4
▲ GBM8
▼ U87
♦ NHA
0.5 uM
-1.0
0.0
1.0
2.0
Venn diagram for four sets of
compounds resulted from
Four pharmacophorehypotheses based search
GBM8
U87
1.066
1.536
7.519
4
2
Veh
NHA
2.5 uM
IC50
Inactive
6
0
Inhibitor conc. log[µM]
GBM4
active
8
18.50
In-vitro anti-GBM potency of representative compound
0.5
2.5
5
Inhibitor Conc (µM)
Normalized OMG fold
change
APPROACH
1 astro BT70
Normalized P21 fold
change
Solution: Targeting the TF, OLIG2 using a
novel computational approach based on
related, multiple pharmacophores
• NCI database searches yielded structures
potentially able to bind all pharmacophores
10
•Glioblastoma CSCs express high levels of
OLIG2, a TF essential for their viability
• OLIG2 dimerizes with E47 for functional
activation, and inhibits P21 gene expression,
a tumor suppressor
• The combined pharmacophore approach
defines a parental pharmacophore and
multiple daughter pharmacophores
(subpharmacophores)
3
active
Inactive
2
•Inhibitors will be further assessed with
in vivo GBM models
1
0
Veh 0.5 2.5
5
Inhibitor Conc (µM)
OLIG2 inhibitor effects on expression levels of P21 and OMG
Acknowledgments
We would like to express our gratitude and
sincere appreciation for the American Brain
Tumor
Foundation and Francis X. Colden, III
In tribute to Francis X. Colden, III
for their generous grant that made this work
possible.
In tribute to Francis X. Colden, III