Transcript
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• Didemnin B was the first marine depsipeptide in phase-I clinical trial in 1988 and withdrawn because of excessive side effects.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.
Nag et al. Mol Cell Pharmacol. 2012
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)
Nag et al. Mol Cell Pharmacol. 2012
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
Nag et al. Mol Cell Pharmacol. 2012
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
Nag et al. Mol Cell Pharmacol. 2012
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
Nag et al. J Biomed Res. 2013
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.
Nag et al. J Biomed Res. 2013
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.
Nadkarni et al. Med Chem. 2009
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
Nadkarni et al. Med Chem. 2009
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
Nadkarni et al. Med Chem. 2009
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.
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
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