Combined pharmacophore based small molecule design for direct inhibition of the OLIG2 transcription factor complex

1. Combined pharmacophore based small molecule design for direct inhibition of the OLIG2 transcription factor complex E12 RRVANNARERLRVRDINEAFKELGRMCQLHL---NSEKPQTKLLILHQAVSVILNLEQQV 57 HTF4 RRMANNARERLRVRDINEAFKELGRMCQLHL---KSEKPQTKLLILHQAVAVILSLEQQV 57 E47 RRMANNARERVRVRDINEAFRELGRMCQMHL---KSDKAQTKLLILQQAVQVILGLEQQV 57 MyoD3 RRKAATMRERRRLSKVNEAFETLKRSTSSNP---NQRLP--KVEILRNAIRYIEGLQALL 55 MYF5 RRKAATMRERRRLKKVNQAFETLKRCTTTNP---NQRLP--KVEILRNAIRYIESLQELL 55 LYL1 RRVFTNSRERWRQQNVNGAFAELRKLLPTHP--PDRKLS--KNEVLRLAMKYIGFLVRLL 56 TAL2 RKIFTNTRERWRQQNVNSAFAKLRKLIPTHP--PDKKLS--KNETLRLAMRYINFLVKVL 56 MYOG RRRAATLREKRRLKKVNEAFEALKRSTLLNP---NQRLP--KVEILRSAIQYIERLQALL 55 MYF6 RRKAATLRERRRLKKINEAFEALKRRTVANP---NQRLP--KVEILRSAISYIERLQDLL 55 NeuroD1 RRMKANARERNRMHGLNAALDNLRKVVPCYS--KTQKLS--KIETLRLAKNYIWALSEIL 56 OLIG2 LRLKINSRERKRMHDLNIAMDGLREVMPYAHGPSVRKLS--KIATLLLARNYILMLTNSL 58 ATOH1 RRLAANARERRRMHGLNHAFDQLRNVIPSFN--NDKKLS--KYETLQMAQIYINALSETP 56 PTF1 LRQAANVRERRRMQSINDAFEGLRSHIPTLP--YEKRLS--KVDTLRLAIGYINFLSELV 56 HAND2 CAHAGARGGARRTQSINSAFAELRECIPNVP--ADTKLS--KIKTLRLATSYIAYLMDLL 56 HAND1 RKGSGPKKERRRTESINSAFAELRECIPNVP--ADTKLS--KIKTLRLATSYIAYLMDVL 56 TCF21 QRNAANARERARMRVLSKAFSRLKTTLPWVP--PDTKLS--KLDTLRLASSYIAHLRQIL 56 ASCL1 AVARRNERERNRVKLVNLGFATLREHVPNGA--ANKKMS--KVETLRSAVEYIRALQQLL 56 ASCL3 FTRKRNERERQRVKCVNEGYAQLRHHLPEEY--LEKRLS--KVETLRAAIKYINYLQSLL 56 HES5 RRDRINSSIEQLKLLLEQEFARHQ------P---NSKLE--KADILEMAVSYLKHSKGER 49 ID3 GKGPAAEEPLSLLDDMNHCYSRLRELVPGVP--RGTQLS--QVEILQVVLAEPAPGPPDG 56 i ii iii Rajesh Mukthavaram, Igor Tsigelny, Valentina Kouznetsova, Ying Chao, Sandra Pastorino, Jiang Pengfei, Sandeep Pingle, Wolf Wrasidlo, Milan Makale, Santosh KesariMoores Cancer Center, University of California, San Diego, CA. 120 100 ■ GBM4 ▲ GBM8 ▼ U87 ♦ NHA 80 % Cell Viability OBJECTIVES EXPERIMENTAL RESULTS CONCLUSIONS 60 40 20 • Transcription factors (TFs) are a major class of signaling proteins and are key to many diseases • Drug design has mostly failed • Transformed stem-like cells (CSCs) drive the common and highly lethal brain tumor, glioblastoma • 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 • Solution: Targeting the TF, OLIG2 using a novel computational approach based on related, multiple pharmacophores • The combined pharmacophore approach defines a parental pharmacophore and multiple daughter pharmacophores (subpharmacophores) • NCI database searches yielded structures potentially able to bind all pharmacophores • 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 0 -1.0 0.0 1.0 2.0 Inhibitor conc. log[µM] Cancer stem like cells, nestin expression APPROACH Current Studies • Computational modeling of the specific OLIG2–E47 dimerization interface • Definition of multiple pharmacophore hypotheses forms the basis of our strategy • 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 • Comprehensive investigation and validation • of OLIG2 selective binding, using additional • biochemical and x-ray crystallographic • methods • Inhibitors will be further assessed with • in vivo GBM models Homology modeling and definition of the OLIG2 pharmacophore Sequence alignment of transcription factors binding to E2A Control 0.5 uM 2.5 uM Inject In tribute to Francis X. Colden, III Acknowledgments We would like to express our gratitude and sincere appreciation for the American Brain Tumor Foundation and Francis X. Colden, III for their generous grant that made this work possible. OLIG2 inhibitor effects on expression levels of P21 and OMG Venn diagram for four sets of compounds resulted from Four pharmacophore-hypotheses based search In-vitro anti-GBM potency of representative compound In tribute to Francis X. Colden, III