1. Novel Synthetic Makaluvamine
Against Lung Cancer
Sushanta Sarkar
Department of Pharmaceutical Sciences
Texas Tech University of Health Science Center
Amarillo, Texas.
December 2, 2015.

2. Outline
• Introduction
• Anticancer drug from natural source
• Anticancer drug from marine source
• Makaluvamines
• Hypothesis
• Preliminary data
• Summery
• Future study
• Acknowledgement

3. Anticancer drug from natural source
• Nature is the vital source of anticancer drug.
• Demonstrates low-toxicity, low-side effect, high efficacy.
• Exerts their anticancer activity by modulating cell cycle , cell
progression, and apoptosis
• Paclitaxel and camptothecin, the two plant-derived natural
products were estimated to account for nearly one-third of
the global anticancer market

4. Latest statistics of anticancer drugs
• FDA approved anticancer small molecule in the last 35 years: 175
Among them 131(74.8%) were other than synthetic and 85(48.5%)
were derived from natural sources.
• FDA approved 7 anticancer drugs in 2010 and 5 drugs among them
were derived from natural sources.
Newman et al. J Nat prod. 2012

5. Anticancer drug from marine sources
• Toxins, alkaloids, and peptides are
very common marine secondary
metabolites.
• Pyrroloquinolone alkaloid inhibits
topoisomerase-I and
topoisomerase-II Image by: National Cancer Institute, Australia
• Didemnin B was the first marine depsipeptide in phase-I clinical
trial in 1988 and withdrawn because of excessive side effects.
• Dozens of alkaloids are currently in different phases of human
trial including PM1004, hemiasterlin, elisidepsin, plitidepsin,
tasidotin, and soblidotin etc.
• Cytarabine was approved by FDA in 1993.
Mayer et al. Trends Pharmacol sci.2010

6. Makaluvamines
• Makaluvamine A was first isolated in
1993.
• Pyrroloimmunoquinolone alkaloid from
marine sponges of genera Zyzzya,
Histodermella, and Spenospongia.
• Secondary metabolites of marine flora
and fauna.
• Fused chemical rings show biological
activities including anti-cancer, anti-
fungal, anti-viral, and anti-microbial.
Ireland CM et al. J Am Chem Soc. 1993
Image by: National Cancer Institute, Australia
• 16 makaluvamine analogues were isolated from sponges as well
as from plasmodial cells of myxomycete.
• Anti-topoisomerase activity is similar to or someway better than
other topoisomerase inhibitors like etoposide, m-AMSA, and
doxorubicine.

7. Makaluvamines
• Makaluvamine A and F
show high potency in xre-
6 cells in terms of TOPO-II
inhibition.1
• Makaluvamine A and C
inhibit tumor growth in
solid tumor model by DNA
intercalation.2
• Makaluvamine A and H
stabilize TOPO-II DNA
cleavage complex.3
1. Copp et al. anticancer drug disc. 1993.
2. Kelly et al. J Nat Prod. 2002.
3. Dijoux et al. Bioorg Med Chem. 2005.

8. Synthetic makaluvamines
Prototype Structure Modification Cytotoxic activity Mechanism
Addition of
lexitropsin-synthetic
DNA ligand
Significant cytoxicity
demonstrated against KB
(cervical cancer), HCT-
116 (colorectal cancer),
L1210 (lymphocytic
leukemia), MCF-7(breast
cancer) and CHO cell
lines.
Inhibition of
topoisomerases
Pyrrolothiazo
Very poor solubility
precludes biological
evaluation.
Not applicable
7-substitution on the
iminoquinone ring
with nitrogen
containing groups
Significant anti-
proliferative activity
against the leukemia cell
line L1210 with
submicromolar IC50
values.
Inhibition of
topoisomerases
Nag et al. Mol Cell Pharmacol. 2012

9. Synthetic makaluvamines
Methyl substitution at
pyrrolo nitrogen, 7-
substitution with
indole groups or 4-
hydroxy phenethyl
IC50 less than 11 µM
against NCI-H460 human
non-small cell lung
carcinoma cell line.
Highest potency shown by
compound with
thiomorpholine group
Topoisomerase II
inhibition
Substitution at
position 7 with alkyl
and phenyl
substituents.
Significant activity
demonstrated in 13
different cancer cell lines
including lung, breast,
prostate, colon cancer.
Inhibition of
topoisomerases
Modulation of cell
cycle proteins;
Inhibition of MDM2;
Apoptosis.
Nag et al. Mol Cell Pharmacol. 2012

10. Synthetic makaluvamines
• 7-(4-fluorobenzylamino)-1,3,4,8-tetrahydropyrrolo(4,3,2-de)quinolin-8(1H)-
one(FBA-TPQ)
• 7-(phenethylamino)-1,3,4,8-tetrahydropyrrolo(4,3,2-de)quinolin-8(1H)-one(PEA-
TPQ)
• 7-(3,4-methylenedioxyphenethylamino)-1,3,4,8-tetrahydropyrrolo(4,3,2-
de)quinolin-8(1H)-one(MPA-TPQ)
• 7-(3,4-dimethoxyphenethylamino)-1,3,4,8-tetrahydropyrrolo(4,3,2-de)quinolin-
8(1H)-one (DPA-TPQ)
Nag et al. Mol Cell Pharmacol. 2012

11. Synthetic makaluvamines: TCBA-TPQ
N-tosyl-7-(4-chlorobenzylamino)-1,
3, 4, 8-tetrahydroprrolo (4, 3, 2-
de)quinolin-8(1H)-one (TCBA-TPQ)
Most potent among all the Benzyl
analogs with Tosyl group
Nag et al. Mol Cell Pharmacol. 2012
1. Inhibition of Topoisomerase
2. Growth inhibition of cancer cells
3. Induction of apoptosis
4. Induction of cell cycle arrest in S-phase
5. Reactivation of p53
6. Down regulation of MDM2

12. Possible mechanism of action
Nag et al. Mol Cell Pharmacol. 2012

13. MDM2
• Murine double minute gene 2 (mdm2) was identified along with mdm1, and
mdm3 overexpressed by 50-fold in mouse BALB/c cell line.
• Locates in acentromeric extrachromosomal nuclear bodies.
• Human counterpart, hdm2 contains 12 axons and alternate splicing results in
different MDM2 isoform
• Contains 490 amino acids
MDM2 structure and binding sites of different interacting proteins
Nag et al. J Biomed Res. 2013

14. MDM2
• MDM2 has both p53 dependent and independent pathway to causes tumor
progression
• MDM2 gets overexpressed by p53 itself, and causes p53 degradation.
• Binds in transactivation domain of p53 and causes
ubiquitination and proteosomal degradation
• Prevents interaction with p53 and other transcriptional co-
activators
• Recruits transcriptional co-repressor
MDM2-p53 regulatory pathway
Nag et al. J Biomed Res. 2013

15. MDM2-p53 interaction
• Ribosomal proteins form a complex with p53 and MDM2 to inhibit
MDM2-mediated p53 ubiquitination and stabilization of p53.
• ARF and PML sequester the MDM2 in the nucleolus, inhibiting
MDM2 from binding and degrading p53.
• CK1 phosphorylates p53, localizes to the PML nuclear bodies.
• RYBP interacts with MDM2 to decrease MDM2-mediated p53
ubiquitination.
• HIPK2, tumor suppressor (Ts) protein phosphorylates MDM2,
promoting its proteasomal degradation.
Zhang et al. J Biol Chem. 2009, Nag et al. J Biomed Res. 2013

16. MDM2-p53 interaction
• MDMX forms heteroligomers with MDM2 and induces p53 degradation.
• RNF2 promotes p53 degradation.
Nag et al. J Biomed Res. 2013

17. MDM2-p53 interaction inhibitors
General strategies to inhibit the MDM2-p53 interaction
Nag et al. J Biomed Res. 2013

18. Problems with current MDM2 inhibitors
• Require wild-type p53 in the cancer cell
• Low activity in cells with mutant p53
• Poor “Drug-like” properties
• May even contribute to resistance in cell lines harboring
mutant p53
• Current compounds have not shown impressive activity in
clinical trials
Nag et al., Curr Med Chem, 2014

19. Hypothesis
Synthetic makaluvamine analogue TCBA-TPQ
inhibits lung cancer by down regulation of
MDM2

20. Lung cancer
• Causes 1.2 millions death worldwide
• Two major type:
Small cell lung cancer (SMCL)
Non-small cell lung cancer (NSCLC)
Adenocarcinoma
Large cell carcinoma
Squamous cell carcinoma
• 13 stages of NSCLC
• EGFR, ALK, RAS mutations are very common.1
• 210,828 people in the United States were diagnosed with lung cancer, including 111,395
men and 99,433 women.
• 157,423 people in the United States died from lung cancer, including 86,689 men and
70,734 women.2
1. https://nccd.cdc.gov/uscs/toptencancers.aspx
2. Cox A. D. et. al. Can bio & Ther. (2002)

21. Lung cancer treatment
• Surgery
• Radiation therapy
• Chemotherapy (Carboplatin, Topotecan, Erlotinib, Docetaxel, Irinotecan,
Doxorubicine, Cisplatin)
• Targeted therapy
• Lung cancer is usually treated with a combination of therapies
https://nccd.cdc.gov/uscs/toptencancers.aspx

22. Lung cancer treatment
• Tyrosin kinase inhibitors. Eg: Ceritinib, afatinib, Cirozotinib
etc.
• Immunotherapy by Cytotoxic T-lymphocyte associated
antigen-4 and Programmed cell death receptor protein-1
antagonist. Eg: Necitumumab
• Combination chemotherapy with small molecule inhibitor
MDM2. Eg: Nutlin-3, cisplatin and doxorubicine.

23. Growth Inhibitory Activity of Makaluvamine Analogs in Human Lung Cancer Cells. Cells were Exposed to Various Concentrations of the
Compounds for 72 hours followed by MTT Assay.
Preliminary data
Nadkarni et al. Med Chem. 2009

24. Preliminary data
Cells were exposed to various concentrations of the compounds
for 72 hours followed by MTT assay.
Nadkarni et al. Med Chem. 2009
Growth inhibitory activity of makaluvamine analogs Ia and Ic in lung
cancer and normal cells.

25. Preliminary data
Induction of cell cycle arrest by TCBA-TPQ lung cancer cells
The cells were exposed to various concentrations of TCBA-TPQ for 24 hours
followed by determination of cell cycle analysis.
A549
H1299
Nadkarni et al. Med Chem. 2009

26. Preliminary data
Induction of apoptosis by TCBA-TPQ lung cancer cells
The cells were exposed to various concentrations of TCBA-TPQ for 24 hours
followed by assessment of apoptosis.
A549
H1299
Nadkarni et al. Med Chem. 2009

27. Preliminary data
Cells were exposed to various concentrations of TCBA-TPQ for 24 hours, and the target
proteins were detected by immunoblotting with specific antibodies.
Nadkarni et al. Med Chem. 2009
Effects of TCBA-TPQ on the expression of various apoptosis-related proteins in human
lung cancer cells.

28. Pharmacokinetic parameter in rat
YU Jun-Xian et al. Chin J Nat Med. 2015
The plasma concentration-time curve and
pharmacokinetic parameter of TCBA-TPQ in
rat after IV administration of 5mg/kg

29. Summary
• Inhibition of cell growth of lung cancer cells
• Induction of apoptosis in a dose dependent manner
• Induction of cell cycle arrest
• Inhibition of expression of MDM2 in a dose
dependent manner
• Inhibition of MDM2 in a p53-independent manner

30. Future study
1. Determine in vivo efficacy of TCBA-TPQ in lung cancer xenograft
and orthotopic models
2. Elucidate the mechanisms of action for the anticancer activity of
TCBA-TPQ:
• MDM2 inhibition
• Other potential targets
3. Evaluate in vitro and in vivo pharmacological properties of TCBA-
TPQ:
• Plasma stability, protein binding, metabolism by S9 enzyme
• Plasma pharmacokinetics, tumor uptake, and tissue distribution
in CD1 mice and Nude mice bearing xenograft/orthotopic
tumors

31. Acknowledgements
Dr. Ruiwen Zhang
Dr. Wei Wang
Dr. Jiangjiang Qin
Dr. Subhasree Nag
Dr. Sukesh Voruganti
Ivan Marsic

32. Thank you