UNIVERSITA’ DEGLI STUDI DI PALERMO FACOLTA’ DI FARMACIA DIPARTIMENTO FARMACOCHIMICO TOSSICOLOGICO E BIOLOGICO Design, synthesis and biological evaluation of new inhibitors of carcinogenic processes Dott. Ilenia Abbate

TUMOR PROGRESSION Cancer cells are less sensitive to the therapy and repopulate the tumor, facilitating tumor progression Migratory growth factor growth factor- induced mitogenesis STAT3 c-MYC PROTO-ONCOGENE PI 3 kinase Akt survival Ras ERK MERK Cyclin/cdks promotes G1/S phase

SIGNAL-TRANSDUCTION PATHWAYS CORRELATED TO TUMOR PROGRESSION Without fuctional HSP90, these proteins undergo proteasome-mediated degradation, leading to cell cycle arrest and apoptosis.

…and Signaling Pathways HSP90 chaperone stabilizes oncoproteins Inhibiting HSP90 degrades oncoproteins, stopping tumor growth

Multiple HSP90 client proteins mediate acquisition and maintenance of the six properties necessary for transformation of a normal cell into a cancer cell: ability to evade apoptosis ability to be self-sufficient for growth ability to invade surrounding tissue and to metastasize to distant sites ability to undergo limitless replication ability to promote neoangiogenesis ability not to respond to antigrowth signals.

HSP90: molecular chaperone Folding Biological fuction Stabilization/Degradation Activation CLIENTS PROTEINS Protein kinases (Her-2/ErbB2 e Akt) Cdk4/ciclin D complex c-Raf-1 HIF-1α p53 Steroid hormone receptors

HSP90 stress condition: heat, irradiation, ROS (reactive oxygen radicals), toxins, lack of nutrients, hypoxia, bacterial and viral infections. -C-terminal domain : 10kDa, omodimerization and co-chaperones recruitment super-chaperone complex; -middle region: 35kDa, binding site for client proteins and ATPase activity; -N-terminal domain: 25kDa, binding site for ATP/ADP and inhibitors, such as geldanamycin and radicicol.

INHIBITORS HSP90

HYDROPHOBIC INTERACTION: BINDING PROTEIN… geldanamycin HYDROPHOBIC INTERACTION: Leu48, Lys58, Met98, Thr109, Val136, Phe138, Val150, Val186 H-BOND ACCEPTORS/DONORS: Asp51, Asp54, Lys58, Asp93, Gly97, Lys112, Phe139,Thr184

PHARMACOPHORE APPROACH HSP90 INHIBITORS ZINC DATABASE 5,627,809 compounds PURINES 12 PYRAZOLES 28 SULFONAMIDES 9 Lipinsky’s Rule PU1 PU2 PI1 PI2 SU1 SU2 Molecular Docking HTVS Glide level MolecolareDocking SP Glide level HYPO1 HYPO2 MOLECULAR DOCKING/ PHARMACOPHORE APPROACH MATCHING HITS 112 COMPOUNDS

112 DERIVATIVES

dihydropyrazole 9 and 2-amino-3-cyanopyridine 10 SYNTHESIS dihydropyrazole 9 and 2-amino-3-cyanopyridine 10 a: R1=Cl, R2=R3=H b: R1=R2=H, R3=NMe2 c: R1=R3=H, R2=F d: R1=OCH3, R2=R3=H e: R1=H, R2=R3=OCH3 f: R1=R2=H, R3=OCH3 g: R1=R2=H, R3=OH h: R1=OH, R2=OCH3, R3=H i: R1=R3=H, R2=OCH3 j: R1=R2=H, R3=NEt2 k: R1=Br, R2=R3=H

pyridinothieno/benzothieno-triazolO-pyrimidines R=CN, Ph X= CH, N R’ A B C D E F G H I J K L M N O P Q R S T U V

NATIONAL CANCER INSTITUTE, Bethesda DEVELOPMENTAL THERAPEUTIC PROGRAM BIOLOGICAL SCREENING NATIONAL CANCER INSTITUTE, Bethesda DEVELOPMENTAL THERAPEUTIC PROGRAM

NATIONAL CANCER INSTITUTE DEVELOPMENTAL THERAPEUTICS PROGRAM MEAN GRAPH AND DOSE RESPONSE CURVES 16 side chain O LEUKEMIA RENAL CANCER CNS CANCER SN-12C:pGI50=7.96 SF-539:pGI50=7.69 K-562:pGI50=7.64

One DOSE RESPONSE CURVES MEAN GRAPH One DOSE RESPONSE CURVES 60 cell lines single dose of 10 uM

HSP90 CLIENT PROTEIN c-Kit EGFR / C-met AR / p-Akt HER2 / ER b-Raf (V800E) TUMOR GIST NSCLC PROSTATE (PTEN-/-) BREAST (HER2 +) MELANOMA

…THANKS FOR YOUR ATTENTION DIPARTIMENTO FARMACOCHIMICO, TOSSICOLOGICO E BIOLOGICO Prof. Anna Maria Almerico Prof. Antonino Lauria Prof. Gaetano Dattolo Prof. Maria A. Livrea Prof. Luisa Tesoriere Dott. Carla Gentile …THANKS FOR YOUR ATTENTION