1. the upper digestive tract is growing rapidly throughout

1.      Project Title: Synthesis and bio-evaluation of derivatives of
paniculatin, a plant derived limonoid in targeting autophagy in gastric cancer
in North East population of India

2.     
Project Description (1000
characters):

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Paniculatin is a
limonoid natural product isolated from the fruits
of a locally available plant in NE India. Recently, our group has reported the
antiproliferative property of the natural product which inhibits the growth of breast
and lung cancer cells targeting autophagy. Occurrence of gastrointestinal
cancer is predominant in NE states. Present proposal aims to identify molecular
signatures of autophagy in gastric cancer patient samples from NE region and
induce the autophagy process using paniculatin. We also like to identify more
potent synthetic derivatives of the natural product via detailed structure
activity relationship (SAR) study powered by in silico modification and docking study. Efficacy of compound(s)
will be evaluated and changes in molecular targets will be further studied at intracellular
signaling level. Finally, antitumor effect and in vivo tissue toxicity of lead compounds will be evaluated in
tumor bearing mice to establish plausible autophagy targeted therapy.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

PART III: TECHNICAL
DETAILS OF PROJECT

Introduction

 

Origin
of proposal:

The incidence of gastrointestinal cancer
in the upper digestive tract is growing rapidly throughout the world. In India
the situation is becoming more alarming because of the shortage of good
healthcare and early detection system. The diseases are more prevalent in
certain ethnic populations. Compared to other parts of India, the occurrence of
oesophagus and gastric cancer are more common in North-Eastern states.1,2
The multiethnic population residing in varied biodiversities and
social-economic conditions, environmental exposures, tobacco use, smoking,
alcohol consumption, dietary habits and susceptibility might play pivotal role
and may possibly explain high prevalence of these diseases.2  On the other hand, availability of effective
and affordable medicine against these cancers is still limited. Limonoid
natural products are well known for their activity against different types of
cancer cells.3 Paniculatin is a limonoid isolated from the fruits of
a plant Chisocheton paniculatus
Hiern, commonly observed in the North Eastern part of India.4 The
plant, locally known as Bandor-dima/gotadhara, is well known in various
traditional medical practices but their mode of action are still unknown. Our
recent publication shows that this natural product can inhibit growth of lung
and breast cancer cells in vitro 5
and molecular mechanism is attributed to targeting of autophagy. There are
conflicting results on role of autophagy in progression of cancer either
promoting tumor growth and cell survival or contributing to tumor suppression
and promote cell death. Both of these effects employ complex regulatory
networks, mediated by p53, PI3K/Akt/mTOR, Ras and microRNA. This project
originates to combine the necessity of new drug development for the prevention
of widespread occurrence of stomach cancer in the NE-region with the effective
anticancer activity of a natural product isolated from the same region that
target autophagy modulation.  

 

Rationale of the study supported by
cited literature:

Chemoprevention of cancer with
natural limonoids have become a topic of discussion in recent years.3
Natural limonoids are found to be effective against various types of cancers
like breast, lung, oral, colon, pancreas etc. (Figure 1).6-9
Paniculatin is a limonoid isolated from the fruits of Chisocheton paniculatus Hiern, commonly observed in the North
Eastern part of India.4 Study from our lab showed that paniculatin
has significant activity against breast cancer cell lines compared to their
normal counterparts.5 There are report of similar compounds from
other sources which are effective against breast, lung, and oral cancers
(Figure 1).10 But, so far, there is no report of any systematic
study of the mode of action and structure activity relationship (SAR) of those
molecules. 

In last few decades, more emphasis
has been given on synthesizing various heterocycle fused terpenoid and steroids
and many of the synthetic compounds are found to show significant anticancer
activity.11 However, study on synthetic limonoids are limited. In
this context, synthesis of libraries of heterocycle substituted and heterocycle
fused paniculatin will help to identify new drug like molecules.

 

 

 

 

Hypothesis:

It may be hypothesized that
synthetic modification of paniculatin, a natural limonoid, will lead to
development of new skeleton with improved efficacy targeting any of the
oesophageal and stomach cancers that are prevalent in North-Eastern region.
Additionally, our in vitro studies
may help to decipher novel molecular mechanisms to address mode of action of
these compounds for targeted therapy.

 

Key Questions:

The key questions are –

i)                   
What are the molecular
signatures of autophagy like expression of miRNA, LC3, Becklin-1, PC62 etc in
gastric cancer biopsy samples obtained from NE patients?

ii)                 
Whether paniculatin
shows anticancer property in gastric cancer cells targeting autophagy and how
to chemically synthesize its derivatives that can target autophagy in gastric
cancer with higher efficacy?

iii)               
What are intracellular
signaling mechanism behind anticancer activity of the natural product and its
derivatives targeting autophagy?

iv)               
Whether the lead
compound(s) will be effective in physiological in tumor bearing experimental
animal?

 

Through this proposal, we aim to
study the anticancer activity of paniculatin and its synthetic derivatives. Furthermore, we are
interested to investigate the intracellular pathway through which these
molecules will work against cancer cells. Our target is to develop new lead
active compound against stomach cancers specific
to NE of India. Finally we like to investigate an antitumor treatment modality
in rodent model.

 

Current status of research and
development in the subject:

North-Eastern region of India has
prevalence of certain cancers like oral, esophageal and stomach cancers.
Reports from various studies and cancer registry have indicated dietary habits,
drinking of alcohol, smoking, tobacco chewing and other environmental
carcinogens as lead factors.2 The specific epidemiological and
molecular signatures of these cancer types in North East India has been
intensely studied and profiled.12 As mentioned earlier, limonoid
natural products are effective against various types of cancers like breast,
lung, colon, pancreas etc.3,5-10 But, till date, there is no report on
their efficacy in gastric cancers and 
thorough study about their structural modification and structure
activity relationship (SAR) is grossly missing. Kataky et al.13
reported synthesis of a few paniculatin derivatives but none of them has been
tested against cancer cells. Our work, for the first time, will evaluate
anticancer activity of different synthetic derivatives of this natural compound
in different gastric cancers and propose a possible preclinical therapeutic
modality in animal models.

 

The
Relevance of Proposed Study

Cancer is becoming a devastating
disease of all at present now. Around the globe it is becoming the most
important public burden and it is continuously increasing. In India, North-East
is one of the worst affected regions. The prevalence of cancer in this region
is comparable only to the Australian population, which suffers from skin cancer
due to long exposure to the sun. Among the entire different types of cancers,
esophagus, oral, and gastric cancers are highly prevalent in North Eastern
states. Therefore, the development and evaluation of potential
anticancer compounds by understanding the molecular mechanism of their action
is an urgent necessity. Chisocheton
paniculatus Hiern, locally known as
Bandor-dima/Gotadhora, is a widely available plant in NE-region. The plant and
its different parts are well known in various traditional medical practices.
Recently, reports from our lab showed the effectiveness of paniculatin,
isolated from the fruits of the plant, against breast cancer. Due to the wide
spread occurrence of oral/gastrointestinal cancers in this region and
possibility of identifying an effective anticancer compound from the same
place, the project is highly relevant for the benefit of NE-region.

 

The
outcome of proposed study:

Our
multifaceted study will lead to identification of novel heterocycle fused
limonoid skeleton which will be potential drug candidate against any of the gastric,
esophageal and oral cancers, highly prevalent in North East states of India.
Our in vitro results would screen
synthesized molecules and decipher molecular mechanism of action in terms of
intracellular signaling at cancer cells. Animal studies will generate
physiologically relevant information on histocompatibility and antitumor effectiveness
of these compounds. Collectively, these informations would be instrumental in
proposing specific drugs for any or all of these cancers with targeted
therapeutic efficacy. Furthermore, hands-on training will generate highly
skilled human resource to contribute in biomedical research and development in
North-East context. Development of different new processes and assay techniques
used in study will be utilized for transfer of technology and final products
can be patented for drug candidate. The knowledge generated in the study will
be published in peer-reviewed journals and may be translated into clinical
trials benefiting NE India as well as National and global cancer scenario.

 

 The Preliminary work done so far:

 We
have established the process for the isolation of paniculatin from the fruit
extract of Chisocheton paniculatus and
shown its antiproliferative role in breast cancers (reported in publication ref
5).The molecular mechanism is attributed to increased autophagy that eventually
kills cancer cells but leaves normal cells.

 

Scope of the
Application indicating anticipated project and processes

The project has scope to identify effective active
molecules against oral and gastrointestinal cancers which can be used for
further drug development. Also identification of anticancer mechanism targeting
specific molecular profiles of these types of cancers might lead to development
of new targeted therapy.

 

Objective of the
proposal:

 

NEIST/Hospital
 part: 

 

1.     
Identification
of molecular signatures of autophagy in North East gastric patients.

2.     
Isolation
and purification of paniculatin from Chisocheton
paniculatus.

3.  In silico
modification of different functional groups of the natural product for
structure activity relationship (SAR) study targeting autophagy in gastric
cancer. Design and screening of new paniculatin derivatives by molecular
docking.

4.  Design and
chemical synthesis of different heterocycle substituted paniculatin derivatives
and  heterocycle fused paniculatin.

 

 

Amity University part:

1.     
Study
of in vitro cytotoxicity and
anticancer activity of paniculatin and its synthetic derivatives against
gastric cancer cell lines and cell isolated from NE patient samples to screen
effective lead compounds.

2.     
Investigation
of intracellular molecular targets and anticancer mechanism of the lead
compounds at cell signaling level.

3.     
In Vivo study of
toxicity-histocompatibility of the lead compounds and assessment of antitumor
activity in tumor bearing mouse.

Work plan:

NEIST /Hospital part:

1.     
Objective 1: Identification
of molecular signatures of autophagy in North East gastric patients.

In clinical samples, autophagy will
be evaluated by examining autophagy markers by immunohistochemistry analysis and
autophagy genes by quantitative reverse-transcription polymerase chain reaction
and western blot analysis. Markers
of autophagy (LC3-II, Beclin-1, p62, ATG5, ATG7, ATG12) and the upstream mTOR
signaling pathway (Raptor, ULK1, pS6K) will be studied from gastric biopsy from
patients compared to normal counterparts.

Sample
size will be obtained by formula and sample exclusion/inclusion criteria will
be done by following clinical guidelines

 

Objective
2. Isolation and purification of paniculatin from Chisocheton paniculatus

The natural product will be isolated from the fruits
of Chisocheton
paniculatus. Dried and finely powdered fruits
will be extracted with petroleum ether. Chromatographic separation of other
impurities will provide the natural product.

Objective
3: Modification of different functional groups of the natural product for
structure activity relationship (SAR) study

Paniculatin is a tetracyclic
terpenoid with a furan ring connected to C-17 position. There are five
different functional groups which can be modified for the SAR study. Direct or
aminative reduction of C-3 carbonyl will provide different derivatives of
compound 2. Michael addition of
different nucleophile at C-1 position of conjugated double bond will lead to
analogs of compound 3. The C-16 CH2
is an activated position which can be easily substituted by halogination or
direct oxidation. Further nucleophilic substitution or addition will lead to
synthesis of different structures like compound 4. C-14 double bond can also be modified by epoxidation followed by
nucleophilic attack. Due to steric constrain it is expected that the
substitution will only go to C-15 to give derivatives of compound 5. It is also possible to modify both
the cis-OH by deacetylation followed by ketalization which will provide
different analogs of compound 6 (Scheme
1). These reactions will lead to number of different derivatives with modified
functional groups. Activity guided screening of the compounds will lead to
detailed structure activity relationship (SAR).

In silico molecular docking studies
will be undertaken with targets of autophagy in gastric cancer with
conceptualised derivatives for better efficacy. Studies of the docking activity
of this library of compounds will lead to the understanding of the relation
between biological activity and structural features and help to screen lead
molecules.

 

Scheme
1: Synthesis of nucleophile substituted paniculatin for SAR study

 

Objective
4: Design and synthesis of different heterocycle substituted paniculatin & heterocycle
fused paniculatin

 

There are few heterocycles which are called
privileged molecules. It is well known that substitution of those privileged
skeletons increase the biological activity of compounds. A number of different
analogs can be synthesized by the addition of different heterocyclic compounds
at different positions of paninculatin (Scheme 2). Thus conjugate addition of
nitrogen containing heterocycles like imidazole, quinoxaline, quinazoline etc.
at C-1 will lead to synthesis of analogs of 7-9. A similar
nucleophilic attack at epoxide of C-14-15 double bond will lead to derivatives
of compound 10-12. Further functionalization at C-3 carbonyl and
C-16 position is also possible via oxime formation. All of these will lead to a
library of paniculatin derivatives with different privileged heterocyclic
motifs attached to it.

 

Scheme 2: Synthesis of
heterocycle substituted paniculatin

 

 

A number of different derivatives
of the natural product can be synthesized by preparing heterocycle fused skeleton
via functionalization at C-16 and C-3 (Scheme 3). It is possible to prepare
oxime at both the position which will lead to different oxazole derivatives
e.g. 16 and 20 through dipolar cycloaddition type of reaction. Also it is
possible to prepare haloaldehyde from those carbonyls which will lead to a
series of pyrimidine, pyrazolopyrimidine etc. fused skeletons e.g. 14, 15, 18, 19 etc. Cyclization via imine formation
will lead to pyridine derivatives like 13
and 17. All these reactions will
lead to synthesis of a number of different derivatives of paniculatin which
will be targeted via activity guided assay to find out lead skeleton.

 

Scheme 3: Synthesis of
heterocycle fused paniculatins

 

 

Amity University part:

 

1.     
Study
of in vitro cytotoxicity and
anticancer activity of paniculatin and its synthetic derivatives against
gastric cancer cell lines and cell isolated from NE patient samples to screen
effective lead compounds.

Stomach, esophageal cancer cell
lines like H314, SCC25, AGS, HS746T, FLO-1, OE-33, etc. along with normal cells will be
cultured and maintained. The lead compounds will
be screened against those cell lines for in
vitro cytotoxicity using MTT assay. The IC50 and
dose-response curve will be generated to screen for most effective drug
choices. Change in cell cycle and apoptosis will
be assessed in best effective drug choice in most responsive cancer cell lines.
Further
change of oncogenic phenotypes of these cells will be evaluated through colony
forming ability, anchorage independent growth by soft agar assay and
migration/invasion by Boyden chamber assay.finally Activation of the transcription
factor HIF-1? assessed by Western blot and by quantifying mRNA expression
levels of VEGF, a target gene of HIF-1?. Autophagy was monitored by Western
blot for the conversion of LC3-I to LC3-II and by confocal microscopy

 

Objective
2: Investigation of intracellular molecular targets and anticancer mechanism of
the lead compounds at cell signaling

 

The molecular mechanism of
functioning of the compounds will be investigated by assessing survival and
apoptotic signaling of NF-kB, TGF-b, ERK1/2, PI-3/AKT etc. The intracellular
targets of the compound will be assessed through up/down regulation of key molecules
like P53, PTEN, KLF2, Caspase-3, 8, 9, BCL, BAX etc. (depending upon the cancer
cell types).

 

Objective
3: In Vivo study of
toxicity-histocompatibility of the compounds in vital organs and assessment of
antitumor activity in tumor bearing mouse

 

Normal
mice will be injected i.p, i.v and orally in different acute and chronic doses
and histopathological section staining of their vital organs like heart, liver,
kidney, brain etc. will be undertaken to assess toxicity. Furthermore, function
of these organs will be evaluated by measuring levels of serum glutamic
oxaloacetic transaminase (SGOT), serum glutamic-pyruvic transaminase (SGPT),
cretinine, acetylcholinesterase etc. Finally, preferred dose modality will be
used in tumor bearing mice and regression of tumor will be assessed compared to
vehicle treated control.