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

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Transcription factors (TFs) are a major class of signaling proteins and are key to many diseases

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Drug design has mostly failed

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Transformed stem-like cells (CSCs) drive the common and highly lethal brain tumor, glioblastoma

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Glioblastoma CSCs express high levels of OLIG2, a TF essential for their viability

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OLIG2 dimerizes with E47 for functional activation, and inhibits P21 gene expression, a tumor suppressor Solution: Targeting the TF, OLIG2 using a novel computational approach based on related, multiple pharmacophores

APPROACH

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Computational modeling of the specific OLIG2 –E47 dimerization interface

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Definition of multiple pharmacophore hypotheses forms the basis of our strategy

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Searches of conformational databases for compounds predicted to bind all pharmacophores, thus maximizing affinity and specificity

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Biochemical and cell-based screening and validation of identified compounds olig2/actin 40 30 20 10 0 1 astro BT70 BT74 GBM4 GBM8 KR SK72

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The combined pharmacophore approach defines a parental pharmacophore and multiple daughter pharmacophores (subpharmacophores)

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NCI database searches yielded structures potentially able to bind all pharmacophores

Cancer stem like cells, nestin expression E12 RR VA NN A R E R L R V R D I N E AF K E L G R M CQ L H L -- NS E K P QT K LLIL HQ AV S VIL N L E QQ V HTF4 RR MA NN A R E R L R V R D I N E AF K E L G R M CQ L H L -- K S E K P QT K LLIL HQ AVAVIL S L E QQ V E47 RR MA NN A R E R V R V R D I N E AF R E L G R M CQ M H L -- K S D K A QT K LLIL QQ AV Q VIL G L E QQ V MyoD3 RRK AA T M R E RRR L S K V N E AF E T L KR STSSN P -- NQ R LP - K V E IL R N AI R Y I E G L Q ALL MYF5 RRK AA T M R E RRR L KK V NQ AF E T L KR CTTTN P -- NQ R LP - K V E IL R N AI R Y I E S L Q E LL LYL1 RR VF TNS R E R W R QQN V NG AFA E L RK LLP TH P - P D RK L S - K N E VL R LAM K Y I G FLV R LL TAL2 RK IF TNT R E R W R QQN V NS AFA K L RK LIP TH P - P D KK L S - K N E T L R LAM R Y I N FLV K VL MYOG RRR AA T L R E KRR L KK V N E AF E AL KR ST LL N P -- NQ R LP - K V E IL R S AI QY I E R L Q ALL MYF6 RRK AA T L R E RRR L KK I N E AF E AL KRR T VA N P -- NQ R LP - K V E IL R S AI SY I E R L Q D LL NeuroD1 RR M K A N A R E R N R M HG L N AAL D N L RK VVP CYS - K TQ K L S - K I E T L R LA K NY IWAL S E IL OLIG2 L R L K I NS R E RKR M H D L N IAM D G L R E VMP Y A HG P S V RK L S - K IA T LLLA R NY ILML TNS L ATOH1 RR LAA N A R E RRR M HG L NH AF D Q L R N VIP S F N - N D KK L S - K Y E T L Q MA Q I Y I N AL S E T P PTF1 L R Q AA N V R E RRR M QS I N D AF E G L R SH IP T LP - Y E KR L S - K V D T L R LAI GY I N FL S E LV HAND2 C A H A G A R GG A RR TQS I NS AFA E L R E C IP N VP - A D T K L S - K I K T L R LA TSY IA Y LM D LL HAND1 RK GSG P KK E RRR T E S I NS AFA E L R E C IP N VP - A D T K L S - K I K T L R LA TSY IA Y LM D VL TCF21 Q R N AA N A R E R A R M R VL S K AF S R L K TT LPWVP - P D T K L S - K L D T L R LA SSY IA H L R Q IL ASCL1 AVA RR N E R E R N R V K LV N L G FA T L R E H VP NG A - A N KK M S - K V E T L R S AV E Y I R AL QQ LL ASCL3 F T RKR N E R E R Q R V K C V N E GY A Q L R HH LP EE Y - L E KR L S - K V E T L R AAI K Y I NY L QS LL HES5 RR D R I NSS I E Q L K LLL E Q E FA R HQ ----- P -- NS K L E - K A D IL E MAV SY L K HS K G E R ID3 G K G PAA EE PL S LL DD M NHCYS R L R E LVP G VP - R GTQ L S - Q V E IL Q VVLA E PAP G PP D G 56 56 56 56 49 56 57 57 57 55 55 56 56 55 55 56 58 56 56 56 Sequence alignment of transcription factors binding to E2A

i ii iii

Homology modeling and definition of the OLIG2 pharmacophore 120 100 80 60 40 20 ■ GBM4 ▲ GBM8 ▼ U87 ♦ NHA 0 -1.0

0.0

1.0

Inhibitor conc. log[µM] 2.0

Control 0.5 uM

GBM4 GBM8 U87 NHA Venn diagram for four sets of compounds resulted from Four pharmacophore hypotheses based search IC50 1.066

1.536

7.519

18.50

2.5 uM

In-vitro anti-GBM potency of representative compound •

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

Current Studies

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Comprehensive investigation and validation of OLIG2 selective binding, using additional biochemical and x-ray crystallographic methods 10

Inject

8 6

active Inactive

3 2

active Inactive

4 1 2 0 0 Veh 0.5

2.5

Inhibitor Conc (µM) 5 Veh 0.5

2.5

5 Inhibitor Conc (µM)

OLIG2 inhibitor effects on expression levels of P21 and OMG •

Inhibitors will be further assessed with in vivo GBM models

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