Indenoisoquinolines

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Indenoisoquinolines Non-Camptothecin Top1
inhibitors
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Three Topoisomerase Types in human cells
Pommier et al. ACS Chem Rev 2009 http//discover.n
ci.nih.gov/pommier/pommier.htm
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Reversible transesterifications, differential
geometry and polarity (5 vs. 3 tyrosyl
linkage) of human topoisomerases
Pommier et al. ACS Chem Rev 2009 http//discover.n
ci.nih.gov/pommier/pommier.htm
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Relaxation of DNA by Topoisomerase I (top1)
Rotation Supercoil removal

Top1
Top1 is essential for transcription and
replication (repair?)
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Camptothecin and its derivatives
Camptothecin is an alkaloid from Camptotheca
acuminata Decne, a rapidly growing tree from
China. Discovered by Monroe Wall and Mansukh Wani
who also discovered taxol.
Hycamtin
GSK
Camptosar Irinotecan
Pfizer
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Camptothecins as one of Natures Paradigms for
Interfacial Inhibitors
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Why New Top1 Inhibitors?

• Because camptothecins are effective anticancer
  drugs.
• Hence, Top1 is a validated target for cancer
  treatment.
• Because agent with a common target have different
  pharmacology, toxicology and exhibit different
  anticancer activity (for instance top2 poisons or
  tubulin inhibitors colchicine lt-gt vinblastine).
• Because camptothecins have limitations
• Bone marrow and intestinal toxicity (adults).
• Drug efflux substrates (ABCG2).
• Chemically unstable E-ring opening.

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Pharmacological limitations of camptothecins
1. Unstable at physiological pH
Serum Albumin Binding
2. Camptothecins bind reversibly to the top1
cleavage complexes. Hence cleavage complexes
reverse rapidly after drug removal gt prolonged
infusions
9

• E-ring derivatives are
• Potent Top1 inhibitors
• gt E-ring opening not necessary for Top1
  trapping
• 2. Not ABCG2 substrates
• Overcome one limitation of camptothecin
• 3. Stable in blood
• gt Overcome another major limitation of
  camptothecins for clinical use

• LiaoBatesPommier Mol Pharmcol 2008
• TakagiBatesPommier Mol Cancer Ther 2007

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Non Camptothecin Top1 Inhibitors in Development
Indolocarbazole
Indenoisoquinolines
Dibenzonaphthyridones
IND filed Clinical trial upcoming
Phase I-II clinical
Preclinical Development
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Crystal structure of novel Top1 inhibitors and
the Top1-DNA complexes
Christophe Marchand, Smitha Antony, Gary Laco,
LMP Mark Cushman, Purdue University Lance
Stewart, Bart Stacker, deCode BioStructures Matthe
w Redinbo, University NC
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Indenoisoquinoline
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Co-crystal structure of Top1 inhibitors Trapping
of Top1-DNA complex interface by drug stacking
(intercalation) between the base pairs flanking
the Top1 cleavage site
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The drugs are bound to Top1 by a network of
H-bonds
Indenoisoquinolines
Camptothecins
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(No Transcript)
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Interfacial Inhibitors Camptothecins and other
natural products

• Bind to a stereospecific site at the interface of
  molecular complexes undergoing conformation
  changes during a reaction
• Protein-DNA (e.g. Top1-CPT)
• Protein-protein (e.g. ARF-GTP-EF-brefeldin A)

• Binding to this hotspot traps the complex
  The inhibition generally is reversible ltgt kinetic

• Natural products Top1 and 2 inhibitors, taxol,
  colchicine, a-amanitin, cyclosporine, rapamycin,
  ribosome-targeted antibiotics

• Implications for drug discovery
• Uncompetitive Inhibitors
• Assays based on stabilization rather than
  inhibition
• of macromolecular complexes.

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The Indenoisoquinolines From bedside to bench to
bedside
Mark Cushman Chemistry Purdue University
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Indenoisoquinolines Non-camptothecin Top1
inhibitors developed by NCI
COMPARE ANALYSIS - DTP DATABASES AND
TOOLS (Kohlhagen, G Mol Pharmacol 1998)
Antony, S. Cancer Res 2003
R
Antony Cancer Res 2007
Joint patent Purdue University Mark
Cushman - National Cancer Institute Yves Pommier
The Indenoisoquinolines Overcome the Chemical
instability (E-ring opening) of Camptothecins
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Indenoisoquinoline
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Molecular pharmacology for the indenoisoquinolines
selected for clinical trials NSC 725776 and NSC
724998
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Indenoisoquinolines are potent Top1
inhibitors They induce Top1 cleavage complexes
are sites that are different from those of
camptothecin
Substrate
Cleavage sites
Antony Cushman, Pommier, Cancer Res 2007
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Indenoisoquinolines induce Top1cc in cells, which
persist after drug removal
Before removal
R30 min
R60 min
1


NSC 725776
0.02
Indenoisoquinolines
1
NSC 724998


0.02
1
Fraction of total DNA retained on filter
CPT
0.02
1
Camptothecins
Topotecan
0.02
1
SN-38 (Active metabolite of irinotecan

0.02
Antony Cushman, Pommier Cancer Res 2007
0
4
8
12
16
0
4
8
12
16
0
4
8
12
16
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The Indenoisoquinolines Overcome Resistance to
Camptothecins in Cell Lines Overexpressing ABCG2
and MDR-1 Transporters
RR1 Relative resistance values obtained by
dividing the IC50 values of the ABCG2
overexpressing cell line (HEK-293-482R-5) by the
IC50 value of the respective parental cell
line. RR2 Relative resistance values obtained by
dividing the IC50 values of the
MDR-1/P-glycoprotein overexpressing cell line
(HEK-293-MDR-19) by the IC50 value of the
respective parental cell line. Values obtained
were from two separate experiments.
Antony Cushman, Pommier, Cancer Res 2007
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Antitumor activity of indenoisoquinolines in
animal models
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The indenoisoquinoline NSC 724998 selected for
clinical trial is more active and less toxic than
Topotecan (NSC 609699)
ndenoisoquinoline
Melanoma A375 Xenografts
Tumor weight
Topotecan
Indenoisoquinoline
(indenoisoquinoline)
(topotecan)
Body weight
Indenoisoquinoline
Topotecan
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Development of a Novel Pharmacodynamic Biomarker
for Clinical Development Activation of Histone
?-H2AX In response to Indenoisoquinolines
Histone H2AX and its phosphorylated form ?-H2AX
were discovered In the LMP-CCR by William
Bonner Collaboration William Bonner and James
Doroshow to transfer the assay from cell culture
to animal and human tissues
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Induction of ?-H2AX by Topoisomerase I cleavage
complexes (Top1cc) by camptothecin (CPT) has been
established in prior studies
Western blotting
Immunofluorescence confocal microscopy

• Furuta,Aladjem, Bonner, Pommier, JBC, 2003
• RaoHicksonPommier, MCB, 2005
• TakemuraPommier, JBC, 2006
• AntonyPommier, Cancer Res, 2007
• Seiler, Conti, Pommier, MCB, 2007

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?H2AX Response in Mice Bearing Human Tumor
Xenografts

• Profiling of ?H2AX response to topotecan
  treatment at various doses under Phase 0
  conditions (assay validation)
• Establishing time- and dose-dependency of ?H2AX
  response to indenoisoquinoline treatment
  (upcoming clinical trials)

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?-H2AX response to the indenoisoquinoline in A375
Xenografts
Vehicle
Single-Dose NSC 724998 (8.33 mg/kg)
200X enlargement
200X enlargement
Samples from 4 mice (40X)
Samples from 4 mice (40X)
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Bone Marrow Studies (SAIC-LHTP) and caveats of
mouse models

• Dose interpretation of PD and efficacy studies in
  mouse models
• Standard dose ranges of mouse MTD, 1/3 MTD, and
  1/10 MTD will include the predicted human marrow
  MTD for all compounds
• A dose of 1/20 MTD, a likely nontoxic dose in
  humans, is relevant to a Phase 0 setting

1Erickson-Miller CL, et al. Cancer Chemother
Pharmacol. 199739467.
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Indenoisoquinolines NSC 706744, 725776 and NSC
724998

• are potent and specific inhibitors of Top1 both
  in vitro (biochemical assays) and in cells
• chemically stable whereas camptothecins
  ring-open
• induce Top1 cleavage complexes at different
  genomic sites than camptothecins
• induce Top1 cleavage complexes that persist
  after drug removal
• (greater persistence than camptothecin).
• overcome ABCG2- and MDR-1-mediated drug
  resistance
• exert antitumor activity in mice models
• less toxic than topotecan and their bone marrow
  toxicity in mice better correlated with human
  toxicity than for topotecan
• Histone ?-H2AX is a sensitive biomarker

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• CONCLUSIONS
• Indenoisoquinolines NSC 706744, 725776 and NSC
  724998
• Discovered using NCI-DTP tools and databases
  (NCI-60 COMPARE analysis)
• Studied under dual funding mechanism (Intra and
  Extramural)
• Optimized using Top1-DNA crystal data, systematic
  SAR (recombinant Top1 camptothecin-resistant
  cell lines NCI-60 cell lines screening)
• Close collaboration between chemists,
  pharmacologists and structural biologists.

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Acknowledgements

• Mark Cushman (Purdue)
• William Bonner (LMP-CCR)
• Smitha Antony (LMP-CCR)
• George Vande Woude (DBS?)
• Susan Bates (COB-CCR)
• Robert Wiltrout (CCR)
• Patricia Steeg (LMP-CCR)
• Lyuba Varticovski (LHC-CCR)
• James Doroshow (DCTD-LMP-NCI)
• Joseph Tomaszewski (DCTD-NCI)
• Jerry Collins (DTP-DCTD-NCI)
• Barbara Mroczkowski (DCTD)
• Ralph Parchment (SAIC-FCRDC)
• Bob Kinders (SAIC-FCRDC)

• Kenneth Paull? (DTP)
• Melinda Hollingshead (DTP)
• Jiuping Ji (SAIC-FCRDC)
• Joe Covey (DTP)
• Liz Glaze (DTP)
• Prabhakar Risbood (DTP)
• Jim Cradock (DTP)
• Rao Vishnuvajjala (DTP)
• Sima Hayavi (DTP)
• Tiziano DiPaolo (DCTP)
• Vali Sevastita (DTP)
• Gina Uhlenbrauck (DTP)
• Shivaani Kummar (COB-CCR)
• Giuseppe Giaconne (COB-CCR)