seminar slides final-2015

Novel Synthetic Makaluvamine Against Lung Cancer

Sushanta SarkarDepartment of Pharmaceutical Sciences

Texas Tech University of Health Science CenterAmarillo, Texas.

December 2, 2015.

Outline• Introduction

• Anticancer drug from natural source• Anticancer drug from marine source• Makaluvamines

• Hypothesis• Preliminary data• Summery• Future study• Acknowledgement

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

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

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• Didemnin B was the first marine depsipeptide in phase-I clinical trial in 1988 and withdrawn because of excessive side effects.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

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.

Makaluvamines

N

N

O R1

R2

R3HN

N

N

O R1

R2

NH

HO N

N

O R1

R2

NH

HO

HN

N

O MeH2N

HN

N

O HNH

S

HO

Br

R1 R2 R3

1, Makaluvamine A Me H H2, Makaluvamine C H Me H3, Makaluvamine H Me Me H4, Makaluvamine I H H H

5, Makaluvamine B 6, Makaluvamine F

R1 R2

9, Makaluvamine D H H 10, Makaluvamine J H Me11, Makaluvamine K Me H12, Makaluvamine P Me Me

R1 R2

13, Makaluvamine E Me H 14, Makaluvamine G Me Me15, Makaluvamine L H Me16, Makaluvamine M H H

HN

N

O HH2N

7, Makaluvamine N

BrHN

N

O HO

8, Makaluvamine O

Br

12

2a3

4

5

8b6

7 8a

• 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.

Synthetic makaluvamines

Prototype Structure Modification Cytotoxic activity Mechanism

NHOOC

CH2OCH3

HN

ON

HN

CH2OCH3

ONCH2OCH3

HN

ONH O

N

NH

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

N

N

O H

N

S

Pyrrolothiazo Very poor solubility precludes biological evaluation.

Not applicable

HN

N

O HRHN

CF3COO

MeN

MeH N

HorR =

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

Synthetic makaluvamines

N

N

O CH3

HNR

N

N

O CH3

HN

S

R =

NH

CH2CH2

or OHCH2CH2

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

HN

N

O RR2HN

CF3COO

R1 = H, Ts

R2 = CH3, CH2CH3, CH2

CH2CH2 CH2CH2 OH

CH2CH2 OH

Br

Br

CH2CH2 NH

a b c

d e

f g

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.


Synthetic makaluvaminesN

HN

HN

O

CF3CHOOH

R

FBA-TPQ

N

HN

HN

O

CF3COOHH

PEA-TPQ

N

HN

HN

O

CF3COOHH

MPA-TPQ

O

O

N

HN

HN

O

CF3COOHH

DPA-TPQ

H3CO

H3CO

TCBA-TPQ

N

N

H2CHN

O

SO

O

Cl

CF3COOHH

H3C

R = FBA-TPQ R = H

• 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)


Synthetic makaluvamines: TCBA-TPQ

N-tosyl-7-(4-chlorobenzylamino)-1, 3, 4, 8-tetrahydroprrolo (4, 3, 2-de)quinolin-8(1H)-one (TCBA-TPQ)

N

N

H2CHN

O

SO

O

Cl

CF3CHOOH

H3C

Most potent among all the Benzyl analogs with Tosyl group


1. Inhibition of Topoisomerase2. Growth inhibition of cancer cells3. Induction of apoptosis4. Induction of cell cycle arrest in S-phase5. Reactivation of p536. Down regulation of MDM2

Possible mechanism of action


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

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


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

MDM2-p53 interaction

• MDMX forms heteroligomers with MDM2 and induces p53 degradation.• RNF2 promotes p53 degradation.


MDM2-p53 interaction inhibitors

General strategies to inhibit the MDM2-p53 interactionNag et al. J Biomed Res. 2013

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

Hypothesis

Synthetic makaluvamine analogue TCBA-TPQ

inhibits lung cancer by down regulation of

MDM2

Lung cancer

• Causes 1.2 millions death worldwide• Two major type:

Small cell lung cancer (SMCL)Non-small cell lung cancer (NSCLC)AdenocarcinomaLarge cell carcinomaSquamous 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.aspx2. Cox A. D. et. al. Can bio & Ther. (2002)

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

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.

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

Preliminary data

Cells were exposed to various concentrations of the compounds for 72 hours followed by MTT assay.


Growth inhibitory activity of makaluvamine analogs Ia and Ic in lung cancer and normal cells.

Preliminary dataInduction 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


Preliminary dataInduction 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


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.


Effects of TCBA-TPQ on the expression of various apoptosis-related proteins in human lung cancer cells.

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

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

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

Acknowledgements

Dr. Ruiwen Zhang

Dr. Wei Wang

Dr. Jiangjiang Qin

Dr. Subhasree Nag

Dr. Sukesh Voruganti

Ivan Marsic

Thank you

seminar slides final-2015

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