Structural Modification of Carboxyl Esterase with Lithium to

Enhance the Specific Activity for Drug Metabolism in Brain

Tumor Cell Lines

Mini Project Report

Submitted to

Visvesvaraya Technological University, Belgaum

In partial fulfillment for the award of

BACHELOR OF ENGINEERING
in
Biotechnology
By
ABHISHEK S N - 1MS17BT001
CHETHAN V G -1MS17BT007
GOWTHAM N -1MS17BT012
OMKAR M - 1MS17BT028

Internal guide
Dr Prabha M
Department of Biotechnology
RIT, Bangalore
 RAMAIAH INSTITUTE OF TECHOLOGY
(Autonomous Institute, Affiliated to VTU)
M.S.R Nagar, M.S.R.I.T Post, Banglore – 560054
DEPARTMENT OF BIOTECHNOLOGY

CERTIFICATE
It is certified that the project work entitled “Structural Modification of Carboxyl Esterase with
Lithium to Enhance the Specific Activity for Drug Metabolism in Brain Tumor Cell Lines.” is
a Bonafede work carried out by Mr. Abhishek S N, Mr. Chethan V Ghorpade, Mr. Gowtham
Nallampalli, and Mr. Omkar Mallinath bearing USNs 1MS17BT001, 1MS17BT007,
1MS17BT012, and 1MS17BT028, in partial fulfilment for the award of Bachelor of Engineering/
Bachelor of Technology in Biotechnology of Vishweshwaraya Technological University, Belgaum,
during the year 2020. It is certified that all corrections indicated for internal assessment have been
incorporated in the report and deposited in the departmental library. The project report has been
approved as it satisfies the academic requirements in respect of project work prescribed for the said
degree.

Name and signature of the Guide

Signature of HOD

Name and signature of Internal Examiner

 ACKNOWLEDGEMENT

This project was successful largely due to the effort of a number of wonderful people who have
always given their valuable advice or lent a helping hand. We sincerely appreciate the inspiration,
support and guidance of all those people who have been instrumental in making this project a
success. We would like to express our gratitude to, Dr. N.V.R. Naidu, principal, M.S. Ramaiah
Institute of technology, Bangalore, for his inspiration, constant support in development of project and
for providing all the necessary facilities.

First and foremast, we take this opportunity to express my profound gratitude and deep regards to
our guide Dr. Prabha M, for her exemplary guidance, monitoring and constant encouragement
throughout the course of this project work.

We would like to mention sincere thanks to Dr. Bindu S, Head of Department, Department of
Biotechnology, for her constant encouragement and guidance.

We also would like to thank our project committee member of Biotechnology Department, RIT, Dr.
Abhijith sir who devoted their time and knowledge in implementation of our project.

We would further like to thank our friends who have helped us in our lab work.

Nevertheless, we thank almighty and extended gratitude to our parents for their co-operation and
support.
DECLARATION

I here by declare that this project report is based on my original work except for
citations and quotations which have been duly acknowledged. I also declare that it has
not been previously and concurrently submitted by any other student/person or at any
other institutions or for any other purpose.

Signature: _____________________
Name: Abhishek S N (1MS17BT001)

Signature: _____________________
Name: Chethan V Ghorpade (1MS17BT007)

Signature: ______________________
Name: Gowtham Nallampalli (1MS17BT012)

Signature: ______________________
Name: Omkar Mallinath (1MS17BT028)

Date: May 2020

Contents

1. Abstract………………………………….06

2. Introduction and Objectives…………….07

3. Literature Review……………………….8-22

4. Materials and Methods………………….23-31

5. Results and Discussion………………….32-42

6. Summary and conclusion……………….43

7. Scope for future work…………………..44

8. References………………………………45-47

9. List of figures……………………………18

10. List of graphs……………………………01

11. List of tables……………………………..01

 Abstract

The project involves in the Structural Modification of Carboxyl Esterase with Lithium to Enhance
the Specific Activity for Drug Metabolism in Brain Tumor Cell Lines. The binding sites of the
carboxyl esterase enzyme were predicted using the database. The substrate (here alpha-naphthyl
acetate) binds to the catalytic domain of the CES and the enzyme catalyzes the hydrolysis reaction.
CES also has a regulatory domain to which a regulatory compound binds and regulates the activity of
the enzyme. The catalytic domain consists of 3 major residues namely Glu-354, His-468 and Ser-221
forming a catalytic triad to which a carboxylic ester bind. The small rigid active site pocket is
adjacent to the oxyanion hole formed by Gly 123-124 and is lined by several hydrophobic residues.
CES are major determinants of the pharmacokinetic behavior of most prodrugs, and the activity can
be influenced by direct interactions with a variety of compounds, either directly or by enzyme
regulation.

The previous study done by selecting the brain and liver to assay carboxyl esterase and acid
phosphatase enzymes for monitoring and enhancing enzyme activity using lithium chloride as the
modulator showed elevated activity for both the enzymes compared to their controls. Lithium is used
as a modulator in this project because it is one of the lightest elemental metal and is normally found
in low concentrations. Lithium salts are highly water soluble, Narrow therapeutic window between
toxicity and effectiveness. No psychotropic effects in normal individuals, it has potent mood
stabilizing properties in patients with bipolar disorders and recurrent depression. Common side
effects include metallic taste, polyuria, polydipsia and weight gain. Uncommon side effects include
hypothyroidism. The interaction between the substrate and the enzyme (CES) in the presence of
lithium showed elevated activity suppressing tumor inducing proteins. The activity of the enzyme
was increased several folds and found effective against the tumor proteins which is useful in the
treatment of brain tumor. Lithium is a promising modulator for carboxyl esterase activity that helps
by hydrolytic function in the modulation of neuronal function. This study was aimed to investigate
the modulatory effect of lithium chloride on protein activity.
INTRODUCTION

Hydrolytic enzymes, also referred as hydrolases belong to the CLASS III of Enzyme Classification
(EC 3) that catalyzes the hydrolysis of esters which gives rise to acid and alcohol. During hydrolysis,
a water molecule is added to the polymer and it cleaves the covalent bond between the two monomer
units. Hydrolytic enzymes are involved in the breakdown or hydrolysis of proteins, esters,
carbohydrates, peptides, glycosides and fat molecules into simpler units. For example, a nuclease is a
hydrolase that cleaves nucleic acids.

Hydrolases can be further classified into several subclasses, based upon the bonds they act upon like
ester bond, sugars, ethers, peptide bonds, acid anhydrides, etc. The Hydrolases those present in
lysosomes comprises of phosphatases, carboxylesterases, β glucurinodases and β galactosidases,
ribonucleases and protease. Earlier studies have shown that in developing human brain, lysosomal
hydrolases indicate brain growth in alternate phases of structural plasticity and trophic acquisition.
Hydrolases, believed to play an important role in neurodegenerative disorders like Alzheimer’s,
lysosomal disorders, brain tumors and also inherited metabolic disorders.

In esterase family, mainly the carboxylesterases (EC 3.1.1.1) are members of the serine hydrolase
superfamily and they can efficiently hydrolyze a variety of ester, amide, and carbamate containing
xenobiotics to their respective free acids. They are the class of esterases that are ubiquitously present
from bacteria to man. They are common in mammalian liver, microsomes. These enzymes efficiently
catalyze the hydrolysis of variety of esters and amide containing chemicals as well as drugs to the
respective free acids. They are involved in detoxification or metabolic activation of various drugs,
environment toxicants and carcinogens. They act as an effective biological barrier to limit the
distribution of substrate specificity, depending on species and location, they are present virtually
throughout the body, including intestines, blood, brain, skin and tumor.

Carboxyl esterase was reported in endothelial cells and blood brain barrier of human brain. There are
several different isozymes of carboxyl esterases based on sequence homology and similarity of
characteristics, the isozymes have recently been classified into five subfamilies, CES1, CES2, CES3,
CES4 and CES5.
A surprising number of drugs contain metals. This theme relies on the study of the design and
mechanism of action of metal-containing pharmaceuticals and compounds that interact with
endogenous metal ions in enzyme active sites. The diverse field includes the platinum and ruthenium
anti cancer drugs, gold drug chaperons and lithium. Lithium is the lightest elemental metal and is
normally found in low concentrations in human tissue. Lithium salts are highly water soluble and
have been used medically for many years.

Lithium levels are generally monitored because of the narrow therapeutic window between toxicity
and effectiveness. While lithium has no psychotropic effects in normal individuals, it has potent
mood stabilizing properties in patients with bipolar disorders and recurrent depression. Common side
effects include metallic taste, polyuria, polydipsia and weight gain. Uncommon side effects include
hypothyroidism.

This study is to know the effect of lithium on brain tumor cells of human. From this study, results
have shown the positive effect of lithium on brain cells which showed increased level of carboxyl
esterase for lithium treated cell lines.
REVIEW OF
LITERATURE
2. REVIEW OF LITERATURE

2.1 Protein

Proteins are large biomolecules or macromolecules consisting of one or more long chains of amino
acid residues. Proteins perform a vast array of functions within living organisms, including
catalyzing metabolic reactions, replication responding and transporting molecules from one location
to another. Proteins differ from one another primarily in their sequence of amino acids, which is
dictates by the nucleotide sequence of their genes, and which usually results in protein folding into a
specific three-dimensional structure. A protein contains at least one long polypeptide. Shortly after or
even during synthesis the residues in a protein are chemically modified by posttranslational
modification which alters the physical and chemical properties. Most proteins consist of linear
polymers built from series of up to 20 different L-alpha-amino acids.
All acids possess common structural features including an alpha carbon to which an amino group, a
carboxyl group and a variable side chain are bonded. Proteins can be found in all cells of the body
especially in the muscles. Protein is a vital part of the brain growth during development. Neurons are
mostly fat and may be fueled by glucose, but they use proteins to communicate with one another and
control what happens in the body. The enzymes, neurotransmitters, hormones help carry signals and
accomplish the tasks. For example- tyrosine prompts the brain to manufacture and not epinephrine
and dopamine.
The microtubule associated protein tau has been isolated and purified from both white and grey
matter of the brain. Tau proteins stabilize microtubules. Diseases of the nervous system such as
Alzheimer’s and Parkinson’s disease are associated with tau proteins have become defective and no
longer stabilize microtubules properly. With modulators it is known that the proteins are modified
and the difference in protein activity was found in various mechanisms. Thus modulators are treated
to find out how the proteins are modified and to bring out the difference in protein activity in various
cellular function.
2.2 Hydrolytic Enzymes

Hydrolases are classified as EC 3 in the EC number classification of enzymes. Hydrolytic enzymes

split different group of biomolecules such as esters, peptides and glycosides. Hydrolytic enzymes
break down protein, lipids, nucleic acids, carbohydrate and fat molecules into their simplest units.
Lysosomal hydrolase’s comprising phosphates, carboxyl esterase’s, glucoronidases and beta-
galactosidase, ribonuclease and acid proteins are believed to play an important role in brain tumor.
there are few studies of the lysosomal hydrolase’s in developing human brain indicate brain growth
occurs in alternate phases of structural plasticity and trophic acquisition. However, there are not
many studies that have elucidated the role of these enzymes in the normal human brain. In the
absence of precise quantitative and topographic distribution of the hydrolytic enzymes in normal
human brain, interpretation of results obtained in pathological conditions can either be fallacious or
incomplete. Even amongst the few studies reported in literature, several studies have highlighted the
importance of studying hydrolytic enzymes in a variety of pathological conditions such as brain
tumors and neurodegenerative diseases such as Alzheimer’s disease and inherited metabolic
disorders. (Prabha.M.et al.2015).

Hydrolytic enzymes in various diseases

AD is a progressive neurodegenerative disorder characterized by cognition and memory impairment.
AD brains are characterized by two pathological hall marks in the cerebral cortex and hippocampus:
senile plaques (Sps), consisting of deposits of beta-amyloid peptide (Abeta) and neurofibrillary
tangles (NFTs), composed of an abnormally phosphorylated form of the cytoskeleton-associated
protein Tau. The pathological accumulation of Abeta and hyperphosphorylation of Tau may develop
concomitantly within synaptic terminals and then induce loss of synapses, which is considered to be
closely correlated with the cognitive decline in AD. In case of Alzheimer’s disease both enzymes are
present in the brain, which is detected in neurofibrillary tangles and neurotic plaques in cholinergic
neurons. Cholinergic neurons are widely distributed throughout the mammalian central nervous
system that exist as both projection neurons and inter neurons. There is evidence that central
cholinergic systems may be important neural substrates for attention, an important component of the
larger process termed cognition. A better understanding of the normal functions of central
cholinergic systems will be a key step in exploring age related memory loss (peter et al., 2000).In
order to understand this concept earlier research studies showed that the causes and pathology of
Alzheimer’s disease has been identified a number of biochemical changes that occur within the cells
and throughout the brain, which contribute to improper cell functioning and cell death.

2.3 Carboxyl Esterase

They are the class of esterases that are ubiquitously present from bacteria to man. They are common
in mammalian liver, microsomes. These enzymes efficiently catalyze the hydrolysis of variety of
esters and amide containing chemicals as well as drugs to the respective free acids. They are
involved in detoxification or metabolic activation of various drugs, environment toxicants and
carcinogens.
Carboxyl esterase (EC 3.1.1.1) is one of the hydrolytic enzymes which play an important role in the
maintenance of the metabolic advantages and essential for neuronal function.

Fig.1 EC tree of Carboxyl Esterase

Carboxyl esterase (CE) is a drug metabolizing enzyme hydrolyzes molecules containing functional
groups such as a carboxylic acid ester, amide, and thioester mainly localized in microsomes (Satoh
and Hosokawa 1998). In brain, Carboxyl esterase was reported in endothelial cells and blood brain
barrier of human brain (Zhang et al. 2002). Biochemical studies have proved that carboxyl esterases
are efficient in hydrolyzing small substrates, but show vast differences with increasingly larger
substrates. For example, the human liver hCE1 was 100 to 1000-fold less efficient in metabolizing
CPT-11 than rabbit liver rCE, even though the two enzymes have over 80% amino acid identity
(Wadkins et al. 2001). Therefore, carboxylesterases play a critical role in the activation of various
antiviral, anticancer, and antibiotic prodrugs (Potter et al. 2006).

Reaction and mechanism

Its chemical reaction is of the form:

Carboxylic ester + H2O --  an alcohol + a carboxylate.
 alp
ha-naphthyl acetate alpha-naphthyl acetic acid
Fig.2 Mechanism of carboxyl esterase. The substrate alpha-naphthol acetate is cleaved by a type of non-
specific carboxylic ester hydrolase called esterase.

Fig.3 graphical view of the EC number of Carboxyl Esterase and is various substrates

Role of carboxyl esterase in drug activation

The carboxyl esterase’s constitute a heterogenous group of isoenzymes that can catalyze the
hydrolysis of a wide range of esters, amides and thioesters. Therefore, they play an important role in
the metabolism of drugs and lipids. Carboxyl esterase’s such as the esters of naphthol and
nitrophenols, are commonly used to measure the esterase activity. Many participate in phase 1
metabolism of xenobiotics such as toxins or drugs; the resulting carboxylates are then conjugated by
other enzymes to increase solubility and eventually excreted.
Carboxyl esterase activity can be influenced by interactions of a variety of compounds either directly
or at the level of enzyme regulation. Since a significant number of drugs are metabolized by
carboxylesterase, altering the activity of this enzyme class has important clinical implications.
Carboxyl esterase activity can be influenced by interactions of a variety of compounds including
some member of drugs containing metals which acts as modulators involved in the activation of
enzymes. Lithium is one of such modulators for enzyme and protein, which is an antidepressant and
mood stabilizer (Trevor Young et al.2004) either directly or at the level of enzyme regulation.

Fig.4 subcellular location of CES

It is known that drug activation is due to the activity of some hydrolytic enzymes for drug metabolism. It has
been proved that lithium enhances the activity of carboxyl esterase in brain cells (Prabha m et al , 2015)

2.4 Brain Tumor

A brain tumor is a collection of abnormal cells in the brain. Brain tumors can be either malignant or
benign. However, there is a limited amount of space in the skull. Therefore, any brain tumor even
one that is benign can interfere with the functions of your brain and body. Brain tumors can destroy
brain cells, increase inflammation, and elevate the pressure in the brain.
A primary brain tumor starts in your brain. When cancer cells from other parts of your body cause a
tumor in your brain, it is called a "secondary" or "metastatic brain tumor." Secondary brain tumors
are three times more common than primary brain tumors. All secondary brain tumors are malignant.
WHO classification of brain tumor
In the past, classification of tumors was dependent on histology. But now the use of "integrated"
diagnostic methods which include genotypic and phenotypic parameters for CNS classification of
tumors, adds a level of objectivity, that was missing in traditional diagnostic methods.
A compelling example of this refinement relates to the diagnosis of oligoastrocytoma a diagnostic
category that has always been difficult to define and that suffered from high interobserver
discordance, with some centers diagnosing these lesions frequently and others diagnosing them only
rarely. Using both genotype (i.e., IDH mutation and 1p/19q codeletion status) and phenotype to
diagnose these tumors results in nearly all of them being compatible with either an astrocytoma or
oligodendroglioma, with only rare reports of molecularly “true” oligoastrocytomas consisting of
histologically and genetically distinct astrocytic and oligodendroglia tumor populations . As a result,
both the more common astrocytoma and oligodendroglioma subtypes become more homogeneously
defined. In the 2016 CNS WHO, therefore, the prior diagnosis of oligoastrocytoma and anaplastic
oligoastrocytoma are now designated as NOS categories, since these diagnoses should be rendered
only in the absence of diagnostic molecular testing or in the very rare instance of a dual genotype
oligoastrocytoma.

Grading of selected CNS tumors according to the 2016 CNS WHO:

Fig.5 WHO grading of selected CNS tumors.

Grading of brain tumors

The grade of a tumor refers to the way the cells look under a microscope:
 Grade I: The tissue is benign. The cells look nearly like normal brain cells, and they grow
slowly.
 Grade II: The tissue is malignant. The cells look less like normal cells than do the cells in a
Grade I tumor.
 Grade III: The malignant tissue has cells that look very different from normal cells. The
abnormal cells are actively growing (anaplastic).
 Grade IV: The malignant tissue has cells that look most abnormal and tend to grow quickly.

Fig.6 A simplified algorithm for classification of the diffuse gliomas based on histological and genetic features (see text
and 2016 CNS WHO for details). A caveat to this diagram is that the diagnostic “flow” does not necessarily always
proceed from histology first to molecular genetic features next, since molecular signatures can sometimes outweigh
histological characteristics in achieving an “integrated” diagnosis. A similar algorithm can be followed for anaplastic
level diffuse gliomas; * Characteristic but not required for diagnosis. (David N. Louis et al, 2016)
Meningioma

The WHO classification divides meningiomas into three grades:

Grade I: Benign Meningioma

Grade II: Atypical Meningioma
Grade III: Malignant (Anaplastic) Meningioma
Most meningiomas are non-malignant, but cause disabilities and are life threatening, unlike most
non-malignant tumors. Some of them grow slowly, others rapidly and sometimes in sudden spurts.
The grow from the meninges. [Brian P. Walcott at al, 2013].
Meningioma comprises about one fourth of all primary tumors of the central nervous system(CNS).
It is the most common primary intracranial neoplasm and the most diversified in histologic patterns
among all primary tumors of the CNS. Meningiomas, as defined by the World Health Organization
(WHO), are meningothelial (arachnoid) cell neoplasms, typically attached to the inner surface of the
dura mater, and these tumors fall into WHO grades I, II, and III. (Perry A et al, 2007).

Molecular Genetics

Monosomy 22 is a unique cytogenetic alteration. (Zang KD et al, 2001) Mutation and/or deletion of
the NF2 gene on chromosome 22q12 is present in most NF2-associated meningiomas and about
50%-80% of sporadic meningiomas. The NF2 gene codes for merlin(schwannomin), and its normal
function is poorly understood. This gene is critical in constituting the early tumorigenic event in the
2-hit model of tumor-suppressor inactivation proposed by Knudson. (Knudson AG Jr, 1971) In
general, solitary meningiomas are clonal tumors as per X-chromosome inactivation studies. (Jacoby
LB et al, 1990) In one study, most meningiomas shared the same single mutation, and recurrent
tumors had the same clonality as the primary tumor.(Von Deimling A et al, 1999) .In multiple
meningiomas, it has been shown that most of these tumors share the same NF2 gene mutation
(Stangl AP et al)Stangl AP, Wellenreuther R, Lenartz D, Kraus JA, Menon AG, Schramm J, et al.
Clonality of multiple meningiomas. J Neurosurg. 1997 May and inactivation of the same copy of X-
chromosome. Larson JJ, Tew JM Jr, Simon M, Menon AG. Evidence for clonal spread in the
development of multiple meningiomas. J Neurosurg. 1995 Oct. Dural spread and peritumoral
implants may well be the mechanism in cases with multiple meningiomas. Borovich B, Doron Y.
Recurrence of intracranial meningiomas: the role played by regional multicentricity. J Neurosurg.
1986 Jan. Genetic events in the tumorigenesis and malignant progression of meningiomas. Various
genetic alterations have been identified in the malignant progression of meningiomas and are
summarized below (as adapted from Meningiomas in WHO Classification of Tumors of the Central
Nervous System.

Treatment
If there are no symptoms, then the doctor will monitor the tumors using MRI scans. The standard
procedure for treatment is surgery and chemotherapy are not normally advised as they are still
undergoing clinical trials. There are less invasive methods available, such as endoscopic removal of
meningioma through the nose, keyhole microsurgical removal through eyebrow incision, endoport
removal.
Gliomas
Low-grade gliomas do not spread outside the brain, but instead grow into the normal brain tissue,
creating symptoms as the tumor grows locally. This can disrupt connections between normal brain
cells and can also create pressure on the nearby brain.
There are three types of low-grade glioma:
 Low grade astrocytoma, which affects the type of glial cells called astrocytes.
 Low grade oligodendroglioma, which affects oligodendrocytes.
 Mixed glioma, which includes astrocytes and oligodendrocytes.

Oligodendroglioma
Treatment
If the oligodendroglioma is easily accessible, then surgery is performed, to remove the tumor.
Biopsies are typically performed to confirm diagnosis and determine the grade of tumor. Recurrent
low-grade tumor can be treated with surgery, radiation therapy and chemotherapy. Grade (II)
oligodendroglioma: Follow-up MRI scans after the removal of tumor tissue. If some traces of tumor
remain, also known as residual tissue, then the patient may undergo radiation after surgery. The
preferred timing for radiation to be conducted is under clinical trials.
Molecular genetics
The most common structural deformity is found in the chromosome arm 1p and 19q.
The striking feature of this glial tumor is the high frequency of co-deletion. Allelic losses of the
chromosomes are either separate or combined. In this one study, it was observed that, for 1p there
was a loss in 35 out of 42 (83%) cases and for 19q there was a loss in 28 of 39 (around 72%) and in
for both combined it was 27 out of 39 (69%). (Barbashina V et al, 2005).

Glioblastoma multiforme

Molecular Genetics
Phillips described three subclasses of high-grade gliomas (termed proneural, mesenchymal, and
proliferative) associated with different outcomes; specifically, prolonged survival of the proneural
subclass. Similar classification of GBMs was also detected in a larger cohort of mixed gliomas. In
2010, unsupervised clustering of gene expression data from adult GBM samples from the TCGA
identified four different molecular subtypes: Proneural, neural, classical, and
mesenchymal. Proneural GBMs were subdivided into G-CIMP-positive and G-CIMP-negative GBM
subsets on the basis of characteristic DNA methylation patterns that strongly correspond with IDH1
mutation status. (Brennan CW et al, 2013) Another later study, which compared DNA methylation
patterns across both pediatric and adult patients with GBM, found a similar clustering in tumors from
adult patients and further identified three more distinct clusters that predominantly consisted of
children and adolescents. Recently, Liu et al. profiled the genetic features of multifocal GBM and
found that M-GBMs had no IDH1, ATRX, or PDGFRA mutations, significantly associated with the
mesenchymal subtype. They also identified the CYB5R2 gene to be hypomethylated and
overexpressed in M-GBMs. (Noushmehr H et al, 2010).
The recent reports published on the Nature Genetics and NEJM were comprehensively analyzed by
whole-exome sequencing and/or targeted deep sequencing as well as array comparative genomic
hybridization. In the Nature Genetics article, grade II and III gliomas were divided into and
exhausted by the genetically well-defined type I–III subtypes. Type III tumors represented the IDH
wild-type grade II and III tumors in the current cohort, showing an OS rate more similar to that of
GBM. Similarly, the report from TCGA research network independently identified similar groups,
using unsupervised clustering analyses of DNA mutation, RNA expression, DNA copy number, and
DNA methylation data. The integration of genome-wide data from multiple platforms delineated
three molecular classes of lower-grade gliomas (grade II/III gliomas) that were more concordant with
IDH, 1p/19q, and TP53 status than with histologic class. This multi-platform approach yielded three
groups similar to those initially described by Jiao's model. The large majority of lower-grade gliomas
without an IDH mutation had genomic aberrations and clinical behavior strikingly similar to those
found in primary GBM (Eckel-PassowJE.et.al,2015).
The report from Mayo Clinic and UCSF defined apriorism groups that were based on the presence or
absence of TERT promoter mutations, IDH mutations, and 1p/19q codeletion and found consistent
associations between the molecular groups and age at diagnosis, survival, patterns of acquired
alterations, and germline variants across the three data sets.( Herringer U et al, 2015). The group
with only TERT mutations has a high prevalence of loss of chromosome 4 and acquired PIK3CA or
PIK3R1 mutations. Gliomas with only TERT mutations are primarily grade IV gliomas. These tests
(for IDH mutations, 1p/19q codeletion, and TERT promoter alterations) can be used to define five
principal groups of gliomas with characteristic distributions of age at diagnosis, clinical behavior,
acquired genetic alterations, and associated germline variants.

Treatment
It is very difficult to treat glioblastoma due to several complicating factors:
 The tumor cells are very resistant to conventional therapies.
 The brain is susceptible to damage due to conventional therapy.
 The brain has a very limited capacity to repair itself.
 Many drugs cannot cross the blood–brain barrier to act on the tumor.
 Treatment of primary brain tumors and brain metastases consists of both symptomatic and
 palliative therapies.

Symptomatic therapy
Supportive treatment focuses on relieving symptoms and improving the patient’s neurologic
function. The primary supportive agents are anticonvulsants and corticosteroids. Historically, around
90% of patients with glioblastoma underwent anticonvulsant treatment, although it has been
estimated that only approximately 40% of patients required this treatment. Recently, it has been
recommended that neurosurgeons not administer anticonvulsants prophylactically, and should wait
until a seizure occurs before prescribing this medication. Those receiving phenytoin concurrent with
radiation may have serious skin reactions such as erythema multiforme and Stevens–Johnson
syndrome. Corticosteroids, usually dexamethasone given 4 to 8 mg every 4 to 6 h, can reduce
peritumoral edema (through rearrangement of the blood–brain barrier), diminishing mass effect and
lowering intracranial pressure, with a decrease in headache or drowsiness.

Palliative therapy
Palliative treatment usually is conducted to improve quality of life and to achieve a longer survival
time. It includes surgery, radiation therapy, and chemotherapy. A maximally feasible resection with
maximal tumor-free margins is usually performed along with external beam radiation and
chemotherapy. Gross total resection of tumor is associated with a better prognosis.

Surgery
Surgery is the first stage of treatment of glioblastoma. An average GBM tumor contains 10 11 cells,
which is on average reduced to 10 9 cells after surgery (a reduction of 99%). Benefits of surgery
include resection for a pathological diagnosis, alleviation of symptoms related to mass effect, and
potentially removing disease before secondary resistance to radiotherapy and chemotherapy occurs.
The greater the extent of tumor removal, the better. Removal of 98% or more of the tumor has been
associated with a significantly longer healthier time than if less than 98% of the tumor is removed in
retrospective analyses. The chances of near-complete initial removal of the tumor may be increased
if the surgery is guided by a fluorescent dye known as 5-aminolevulinic acid. GBM cells are widely
infiltrative through the brain at diagnosis, and so despite a total resection of all obvious tumor, most
people with GBM later develop recurrent tumors either near the original site or t more distant
locations within the brain. Other modalities, typically radiation and chemotherapy, are used after
surgery in an effort to suppress and slow recurrent disease.

Radiotherapy
Subsequent to surgery, radiotherapy becomes the mainstay of treatment for people with
glioblastoma. It is typically performed along with giving temozolomide (TMZ). A pivotal clinical
trial carried out in the early 1970s showed that among 303 GBM patients randomized to radiation or
no radiation therapy, those who received radiation had a median survival more than double those
who did not. Subsequent clinical research has attempted to build on the backbone of surgery
followed by radiation. On average, radiotherapy after surgery can reduce the tumor size to 10 7 cells.
Whole-brain radiotherapy does not improve when compared to the more precise and targeted three-
dimensional conformal radiotherapy. A total radiation dose of 60–65 Gy has been found to be
optimal for treatment. GBM tumors are well known to contain zones of tissue exhibiting hypoxia
which are highly resistant to radiotherapy. Various approaches to chemotherapy radiosensitizers have
been pursued with limited success as of 2016. As of 2010 newer research approaches included
preclinical and clinical investigations into the use of an oxygen diffusion-enhancing compound such
as trans sodium crocetinate (TSC) as radiosensitizers, and as of 2015 a clinical trial was underway.
Boron neutron capture therapy has been tested as an alternative treatment for glioblastoma
multiforme but is not in common use. (D. Williams Parsons et al, 2008).

Chemotherapy

Chemotherapy uses anti-cancer (cytotoxic) drugs to destroy brain tumour cells. The drugs circulate
throughout your body in the bloodstream. It can be difficult to treat brain tumours with some
chemotherapy drugs because the brain is protected by the blood brain barrier. This is a natural filter
between the blood and the brain which protects the brain from harmful substances. Most studies
show no benefit from the addition of chemotherapy. However, a large clinical trial of 575
participants randomized to standard radiation versus radiation plus temozolomide chemotherapy
showed that the group receiving temozolomide survived a median of 14.6 months as opposed to 12.1
months for the group receiving radiation alone. This treatment regime is now standard for most cases
of glioblastoma where the person is not enrolled in a clinical trial. Temozolomide seems to work by
sensitizing the tumor cells to radiation. High doses of temozolomide in high-grade gliomas yield low
toxicity, but the results are comparable to the standard doses. Antiangiogenic therapy with
medications such as bevacizumab control symptoms but do not affect overall survival.

2.5 Modulator

An enzyme modulator is a type of any kind of substrate/drug/protein which modulates enzymes.

They include enzyme inhibitors and enzyme inducers. For example: lithium, cytokine as modulator.
Lithium is the lightest elemental metal and in human tissue it is found in low concentrations. Earlier
researchers have highlighted its role in enhancement of hippocampus neurogenesis (Chen et al. 2000)
and it is a mood stabilizer (Trevor Young et al. 2004).
Lithium as a Modulator

Lithium is the lightest elemental metal and is normally found in low concentrations in human tissue.
Lithium salts are highly water soluble and have been used medically for many years. Lithium levels
are generally monitored because of the narrow therapeutic window between toxicity and
effectiveness. While lithium has no psychotropic effects in normal individuals, it has potent mood
stabilizing properties in patients with bipolar disorders and recurrent depression. Common side
effects include metallic taste, polyuria, polydipsia and weight gain. Uncommon side effects include
hypothyroidism.
The mechanism by which lithium might help to regulate mood include the alteration of gene
expression.
LiCl salt is a typical ionic compound, although the small size of the Li+ ion gives rise to properties
not seen for the other alkali metal chlorides, such as extraordinary solubility in polar solvents and
lithium chloride data have showed that cardiac function, hemodynamics and heart rate are not
directly affected by LiCl at concentrations of 0.1 to 10 mM. only at 100Mm LiCl, a concentration
beyond that shows toxicity effects. Two other forms of low dose lithium supplementation, lithium
aspartate and lithium orotate may protect the brain and encourage the growth of gray matter in the
cerebral cortex and prevent and allow the progression of Alzheimer’s disease, senile dementia and
Parkinson’s disease. In the 1930’s and 40s, lithium chloride was sold in stores as a salt substitute.
But (as frequently happens) some people used it way too much and suffered toxic overdoses, so it
fell out of common use. Fortunately, lithium toxicity is entirely preventable, and it’s also easily
treatable.
Objectives

 To study the kinetics and the modulatory effect of lithium chloride on

protein activity.

 To investigate the interaction between the enzyme and substrate in the

presence of lithium.
 MATERIALS
AND METHODS
3. Materials and Methods:

3.1 Obtaining the structure of enzyme

The Protein Data Bank (PDB) archive is the single worldwide repository of information
about the 3D structures of large biological molecules, including proteins and nucleic acids. These are
the molecules of life that are found in all organisms including bacteria, yeast, plants, flies, other
animals, and humans. Understanding the shape of a molecule deduce a structure's role in human
health and disease, and in drug development. The structures in the archive range from tiny proteins
and bits of DNA to complex molecular machines like the ribosome.

Fig.7 protein data bank official server page.

3.2 PhyMOL visualization of the 3-D structure.

PyMOL is an open-source tool available from (www.pymol.org) to visualize molecules. It is
available for platforms like Windows, Linux and MacOS. PyMOL is capable of producing high-
quality images and animations from 3D structures. The program also has advanced functions for
structure manipulation and analysing their chemical properties. It also supports scripts and plugins.
PyMOL has been developed using Python language.
 Fig.8 phyMOL visualization of 3-Dimensional view of
Carboxyl Esterase

3.3 Prediction of binding sites

AlloFinder
The binding site of the enzyme is a place where the substrate binds and interacts with the enzyme. It
is also called as active site which is a 3-dimensional cleft formed by the assembly of various amino
acid residues coming from various sequence of the protein. The binding site has binding region and a
catalytic region, the binding region is responsible for the binding of the substrate and the catalytic
region involves in the catalysis of the enzyme reaction to obtain the product and free enzyme.
There are various servers for the prediction of these binding sites and one of those sever is
AlloFinder, it helps in allosteric modulator and allosterome analysis.
It requires an input of the enzyme to be studied in the form of pdb format, and it gives us access to
various ligand databases such as ZINC, ChemBL, Endogenous ligand etc. and it also has an option
for the user defined ligand entry.

Fig.9 AlloFinder web server for allosterome analysis.

Schrodinger

Transform drug discovery and materials research with advanced molecular modeling. This tool helps
us to study the structures of various enzymes, and it also gives access to modify the structure of
enzymes for detailed studies.
The ability to scale our calculations of key drug properties to ultra-large idea sets of over a billion
molecules to enable more rapid and successful identification of high-quality drug candidate
molecules via integration of next-generation machine-learning methods with our physics-based
techniques, as well as large-scale utilization of internal and cloud computing resources. The ability to
assess key properties of drug-like molecules using physics-based calculations with accuracy
comparable to that of experimental laboratory assays, to facilitate optimization of drug properties,
including drug potency, selectivity, and bioavailability.
The ability to generate, access, and analyze the data derived from complex calculations integrated
with assay data through a powerful and user-friendly graphical interface. Prediction with a high
degree of accuracy materials properties, such as glass transition temperature, surface tension, heat
capacity, etc., and to generate new de novo materials solutions with desired properties within the
specified chemical space. It also gives access to sophisticated scientific modeling, powerful
analytics, plots, and 3D visualization tool. The web-based, collaborative interface also enables
intuitive sifting of corporate data with customized extraction, transform and load (ETL).

Fig.10 Picture showing the surface view of Carboxyl Esterase in the Schrodinger tool.
Maestro
Maestro is the interface for all Schrödinger software. Significantly enhanced usability built on
Maestro’s long-standing impressive visualization and analysis tools makes Maestro a versatile
modeling environment for all researchers
Completely reimagined interface:
By working closely with our users, our UX designed the Maestro interface to anticipate user actions,
streamline common tasks, and organize data in an intuitive fashion.

Model generation:
Maestro supports many common file formats for structural input. In addition, Maestro provides an
intuitive, full-featured building tool for constructing molecular models of any type.

Flexible visualization:
Maestro provides many viewing options to accommodate the varied needs of different applications.
From biomolecular systems to complex materials, Maestro brings clarity to a wide range of modelled
systems.

3D realism:
Maestro's superior rendering and stereographic capabilities allow researchers to view complex
molecular systems as three-dimensional objects with unrivalled realism.

Quantitative structural analysis:

Maestro includes versatile measurement tools that give the user the ability to precisely quantify a
molecule's structural features. Superimposition tools make possible detailed comparisons between
structures.

Customization scripts:
Maestro offers the ability to customize and automate tasks as well as manage workflow via scripting.
Rather than a proprietary language, Maestro scripts are written in the industry-standard Python
language.

Molecular properties:
Computed properties such as vibrational modes, molecular orbitals, or electron density are easily
visualized in Maestro. The unique Sitemap feature locates areas within a protein that correspond to
hydrophobic or hydrophilic regions.
Data management and analysis:
Maestro employs a data system that automatically archives structure-related properties. A built-in
plotting facility helps elucidate structure-property relationships.

Publication and presentation:

Maestro outputs high-resolution, presentation-quality images that can be easily incorporated into
documents for publication or for sharing data with colleagues.

Cross-platform support:
Maestro runs natively on Linux, Windows, and Mac

3.4 SWISSDOCK

This website provides an access to a web service to predict the molecular interactions that may occur
between a target protein and a small molecule. S3DB, a database of manually curated target and
ligand structures, inspired by the Ligand-Protein Database. SwissDock is based on the docking
software EADock DSS, whose algorithm consists of the following steps:

1. Many binding modes are generated either in a box (local docking) or in the vicinity of all
target cavities (blind docking).

2. Simultaneously, their CHARMM energies are estimated on a grid.

3. The binding modes with the most favourable energies are evaluated with FACTS, and
clustered.

4. The most favourable clusters can be visualized online and downloaded on computer.

Fig.11 SwissDock web server for bioinformatics

3.5 RCSB PDB

The Protein Data Bank (PDB) is a database for the three-dimensional structural data of large

biological molecules, such as proteins and nucleic acids. The data, typically obtained by X-ray
crystallography, NMR spectroscopy, or, increasingly, cryo-electron microscopy, and submitted
by biologists and biochemists from around the world, are freely accessible on the Internet via the
websites of its member organisations. The PDB is overseen by an organization called the Worldwide
Protein Data Bank, wwPDB.

The PDB is a key in areas of structural biology, such as structural genomics. Most major scientific
journals, and some funding agencies, now require scientists to submit their structure data to the PDB.
Many other databases use protein structures deposited in the PDB.

Fig.12 RCSB PDB showing the sequence and 3-D structure of Human Carboxyl Esterase
ProBis
ProBiS is a computer software which allows prediction of binding sites and their corresponding
ligands for a given protein structure. Initially ProBiS was developed as a ProBiS algorithm by Janez
Konc and Dušanka Janežič in 2010[1] and is now available as ProBiS server, ProBiS CHARMMing
server, ProBiS algorithm and ProBiS plugin. The name ProBiS originates from the purpose of the
software itself, that is to predict for a given Protein structure Binding Sites and their corresponding
ligands.
Detect structurally similar binding sites
This tool takes as an input a query protein or a binding site. The ProBiS algorithm structurally
compares the query independently of sequence or fold with a database of non-redundant protein
structures. The output of this tool is a 3D query protein colored by degrees of structural conservation
from blue (unconserved) to red (structurally conserved) in Jmol viewer and a table of similar
proteins.
Pairwise local structural alignment
This tool takes as an input two proteins or binding sites. The ProBiS algorithm compares structures
based on geometry as well as physicochemical properties and returns their local structural alignment.
ProBiS web server RESTful Web Services
The ProBiS web server features RESTful (Representational State Transfer) web services to make the
binding site similarities and local pairwise alignments for any PDB protein structure easily accessible
from any script.
ProBiS-Database access
ProBiS-Database can be accessed directly from ProBiS (CHARMMing) server, ProBiS-Database
widget, which can be included in any web page to provide access to the ProBiS-Database, or
RESTful Web Services, which make ProBiS-Database easily accessible from any script

There are two ProBiS web pages out there: ProBiS at http://probis.cmm.ki.si maintained at the
National Institute of Chemistry, Slovenia, and ProBiS-CHARMMing
at https://probis.nih.gov situated at the National Institutes of Health, USA. ProBiS-CHARMMing has
additional functions only available from inside NIH: it enables energy minimization of predicted
protein-ligand complexes and their interaction energy calculation
Fig.13 ProBis showing the binding sites for different ligands
 RESULTS
AND
DISCUSSIONS
4. Results and discussion
4.1 Enzyme parameters

Atomic composition:
Carbon C 2843
Hydrogen H 4423
Nitrogen N 733
Oxygen O 811
Sulphur S 21

Number of amino acids: 567

Molecular weight: 62521.08
Theoretical pI: 6.15
Formula: C2843H4423N733O811S21
Total number of atoms: 8831
Extinction coefficients:
Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 87110
Abs 0.1% (=1 g/l) 1.393, assuming all pairs of Cys residues form cystines
Ext. coefficient 86860
Abs 0.1% (=1 g/l) 1.389, assuming all Cys residues are reduced
Estimated half-life:
The N-terminal of the sequence considered is M (Met).
The estimated half-life is: 30 hours (mammalian reticulocytes, in vitro).
>20 hours (yeast, in vivo).
>10 hours (Escherichia coli, in vivo).
Instability index:
The instability index (II) is computed to be 37.73
This classifies the protein as stable.
Aliphatic index: 88.41
Grand average of hydropathicity (GRAVY): -0.109
4.2 Interaction of lithium with enzyme

Carboxyl Esterase was interacted with lithium using in-silico techniques resulting in the covalent
binding which influences the decrease in the Km value of the enzyme. The lithium ion was bound to
the CES at regulatory domain of the enzyme. The allosteric region was determined using the tool
AlloFinder, which enables us to analyze the binding properties of the lithium with CES. Various
amino acid residues present at the site of binding lithium was predicted; the following amino acid
residues were predicted by AlloFinder tool:
SER 1075 A GLY 2325 B
GLY 1061 A HIS 2284 B
TYR 1118 A THR 2278 B
PRO 1085 A THR 2278 B
PHE 1076 A ASP 2324 B
ASP 1115 A VAL 2281 B
ASP 1115 A HIS 2184 B
PRO 1058 A HIS 2184 B
ASP 1115 A ALA 2280 B
TRP 1074 A GLU 2183 B
PHE 1076 A LEU 2329 B
LEU 1060 A HIS 2284 B
SER 1075 A MET 2326 B
LYS 1078 A ASP 2182 B
PHE 1076 A ILE 2323 B
GLU 1292 A LYS 2275 B
PRO 1073 A GLU 2183 B
SER 1113 A VAL 2281 B
GLU 1291 A LYS 2275 B
SER 1113 A THR 2277 B
SER 1075 A ARG 2186 B
SER 1075 A ASP 2324 B
TRP 1074 A ARG 2186 B
PRO 1054 A GLU 2183 B
PRO 1073 A ARG 2186 B
PHE 1076 A GLY 2325 B
PRO 1085 A THR 2277 B
PRO 1062 A HIS 2284 B
SER 1113 A THR 2278 B
PRO 1058 A ALA 2280 B
LEU 1060 A ALA 2280 B
LYS 1078 A HIS 2184 B
LEU 1112 A THR 2277 B
GLU 1072 A GLU 2183 B
ASP 1115 A THR 2278 B
TYR 1118 A HIS 2184 B
LYS 1078 A GLU 2183 B
LEU 1060 A VAL 2281 B
GLU 1114 A VAL 2281 B
GLU 1114 A HIS 2284 B
LYS 1111 A THR 2277 B
ASP 1115 A SER 2279 B
PHE 1076 A LEU 2328 B

Km Value:
Km is the concentration of substrates when the reaction reaches half of Vmax. A small Km indicates
high affinity since it means the reaction can reach half of Vmax in a small number of substrate
concentration. This small Km will approach Vmax more quickly than high Km value.
Michaelis-Menten Kinetics

In biochemistry, Michaelis–Menten kinetics is one of the best-known models of enzyme kinetics. It

is named after German biochemist Leonor Michaelis and Canadian physician Maud Menten. The
model takes the form of an equation describing the rate of enzymatic reactions, by relating reaction
rate V(rate of formation of product [P] to [S]) to , the concentration of a substrate S. Its formula is
given by,

This equation is called the Michaelis–Menten equation. Here, Vmax represents the maximum rate

achieved by the system, happening at saturating substrate concentration. The value of the Michaelis
constant Km is numerically equal to the substrate concentration at which the reaction rate is half of
Vmax. Biochemical reactions involving a single substrate are often assumed to follow Michaelis–
Menten kinetics, without regard to the model's underlying assumptions.

Graph.1 Michaelis–Menten saturation curve for an enzyme reaction showing the relation between the
substrate concentration and reaction rate.

Table.1 Km values of Carboxyl Esterase against different substrates

UniprotKB

UniProt is a freely accessible database of protein sequence and functional information, many entries
being derived from genome sequencing projects. It contains a large amount of information about the
biological function of proteins derived from the research literature. It is maintained by the UniProt
consortium, which consists of several European bioinformatics organizations and a foundation
from Washington, DC, United States.

UniProtKB

UniProt Knowledgebase (UniProtKB) is a protein database partially curated by experts, consisting of

two sections: UniProtKB/Swiss-Prot (containing reviewed, manually annotated entries) and
UniProtKB/TrEMBL (containing unreviewed, automatically annotated entries). As of
19 March 2014, release "2014_03" of UniProtKB/Swiss-Prot contains 542,782 sequence entries
(comprising 193,019,802 amino acids abstracted from 226,896 references) and release "2014_03" of
UniProtKB/TrEMBL contains 54,247,468 sequence entries (comprising 17,207,833,179 amino
acids).
UniProtKB/Swiss-Prot

UniProtKB/Swiss-Prot is a manually annotated, non-redundant protein sequence database. It

combines information extracted from scientific literature and biocurator-evaluated computational
analysis. The aim of UniProtKB/Swiss-Prot is to provide all known relevant information about a
particular protein. Annotation is regularly reviewed to keep up with current scientific findings. The
manual annotation of an entry involves detailed analysis of the protein sequence and of the scientific
literature.

Sequences from the same gene and the same species are merged into the same database entry.
Differences between sequences are identified, and their cause documented A range of sequence
analysis tools is used in the annotation of UniProtKB/Swiss-Prot entries

Relevant publications are identified by searching databases such as PubMed. The full text of each
paper is read, and information is extracted and added to the entry. Annotation arising from the
scientific literature includes, but is not limited to:

 Protein and gene names

 Function
 Enzyme-specific information such as catalytic activity, cofactors and catalytic residues
 Subcellular location
 Protein-protein interactions
 Pattern of expression
 Locations and roles of significant domains and sites
 Ion-, substrate- and cofactor-binding sites
 Protein variant forms produced by natural genetic variation, RNA editing, alternative
splicing, proteolytic processing, and post-translational modification

Annotated entries undergo quality assurance before inclusion into UniProtKB/Swiss-Prot. When new
data becomes available, entries are updated.

UniProtKB/TrEMBL

UniProtKB/TrEMBL contains high-quality computationally analysed records, which are enriched

with automatic annotation. It was introduced in response to increased dataflow resulting from
genome projects, as the time- and labour-consuming manual annotation process of
UniProtKB/Swiss-Prot could not be broadened to include all available protein sequences. The
translations of annotated coding sequences in the EMBL-Bank/GenBank/DDBJ nucleotide sequence
database are automatically processed and entered in UniProtKB/TrEMBL. UniProtKB/TrEMBL also
contains sequences from PDB, and from gene prediction, including Ensembl, RefSeq and CCDS.

UniParc

UniProt Archive (UniParc) is a comprehensive and non-redundant database, which contains all the
protein sequences from the main, publicly available protein sequence databases. Proteins may exist
in several different source databases, and in multiple copies in the same database. In order to avoid
redundancy, UniParc stores each unique sequence only once. Identical sequences are merged,
regardless of whether they are from the same or different species. Each sequence is given a stable
and unique identifier (UPI), making it possible to identify the same protein from different source
databases. UniParc contains only protein sequences, with no annotation. Database cross-references in
UniParc entries allow further information about the protein to be retrieved from the source databases.
When sequences in the source databases change, these changes are tracked by UniParc and history of
all changes is archived.

Fig. 14 uniprot web database showing the carboxyl esterase 3-d structure obtained through x-ray method
Fig.15 Computer-assisted docking of cocaine or heroin into the active sites of hCE1 (CES1) or hCE
(CES2). (A) An overlay of the ribbon representations of the hCE1 (CES1) (taupe) and hCE (CES2)
(orange/brown) structures used for the docking studies. As can be seen one loop of hCE1 (CES1)
(highlighted in pink) is displaced (marked by the white arrow) and projects towards the active site
catalytic amino acids (Ser, His, Glu). (B) An overlay of the catalytic amino acids in hCE1 (CES1)
(taupe) and hCE (CES2) (orange/brown) with the distances between the relevant atoms indicated. (C,
D) – Docking of cocaine into the active sites of hCE (CES2) and hCE1 (CES1), respectively. The
active site serine is depicted and the distance from the Og atom to the carbonyl carbon atoms in the
drug are displayed. (E, F) – Docking of heroin into the active sites of hCE (CES2) and hCE1 (CES1),
respectively. The active site serine is depicted and the distance from the Og atom to the carbonyl
carbon atoms in the drug are displayed. In all panels, the nitrogen and oxygen atoms displayed in
blue and red, respectively, and distances are marked in Å

Fig.16 3-d structure of CES showing the ligand binding sites

Amino acid composition

Ala (A) 46 8.1%
Arg (R) 16 2.8%
Asn (N) 20 3.5%
Asp (D) 23 4.1%
Cys (C) 5 0.9%
Gln (Q) 22 3.9%
Glu (E) 37 6.5%
Gly (G) 45 7.9%
His (H) 13 2.3%
Ile (I) 24 4.2%
Leu (L) 63 11.1%
Lys (K) 38 6.7%
Met (M) 16 2.8%
Phe (F) 27 4.8%
Pro (P) 39 6.9%
Ser (S) 35 6.2%
Thr (T) 32 5.6%
Trp (W) 12 2.1%
Tyr (Y) 14 2.5%
Val (V) 40 7.1%
Pyl (O) 0 0.0%
Sec (U) 0 0.0%

Total number of negatively charged residues (Asp + Glu): 60

Total number of positively charged residues (Arg + Lys): 54
Experimental Validation

Fig.17 A proposed mechanism for the formation of the tetrahedral intermadiate. (A) Hydrolysis of
substrate start with an attack by the oxygen atom of the hydroxy group of Ser 203 on the carbonyl
carbon atom of the substrate. (B) The carbon–oxygen bond of this carbonyl group becomes a single
bond, and the oxygen atom acquires a net negative charge. The four atoms now bonded to the
carbonyl carbon are arranged as in a tetrahedron. The formation of this transient tetrahedral
intermediate from a substrate is made possible by hydrogen bonds between the negative charged
oxygen anion (called an oxyanion) and main-chain NH group. This site is called oxyanion hole.

5 Summary and Conclusion

Fig.18 PhyMOL visualization of the ligand interaction with the CES.

 The project involves in the Structural Modification of Carboxyl Esterase with Lithium to
Enhance the Specific Activity for Drug Metabolism in Brain Tumor Cell Lines.
 The binding sites of the carboxyl esterase enzyme were predicted using the database. The
substrate (here alpha-naphthyl acetate) binds to the catalytic domain of the CES and the
enzyme catalyzes the hydrolysis reaction.
 CES also has a regulatory domain to which a regulatory compound binds and regulates the
activity of the enzyme.
 The catalytic domain consists of 3 major residues namely Glu-354, His-468 and Ser-221
forming a catalytic triad to which a carboxylic ester bind.
 The small rigid active site pocket is adjacent to the oxyanion hole formed by Gly 123-124
and is lined by several hydrophobic residues.
 CES are major determinants of the pharmacokinetic behavior of most prodrugs, and the
activity can be influenced by direct interactions with a variety of compounds, either directly
or by enzyme regulation.
  The previous study done by selecting the brain and liver to assay carboxyl esterase and acid
phosphatase enzymes for monitoring and enhancing enzyme activity using lithium chloride as
the modulator showed elevated activity for both the enzymes compared to their controls.

 Lithium is used as a modulator in this project because it is one of the lightest elemental metal
and is normally found in low concentrations.

 Lithium salts are highly water soluble, Narrow therapeutic window between toxicity and
effectiveness. No psychotropic effects in normal individuals, it has potent mood stabilizing
properties in patients with bipolar disorders and recurrent depression.

 Common side effects include metallic taste, polyuria, polydipsia and weight gain. Uncommon
side effects include hypothyroidism.

 The interaction between the substrate and the enzyme (CES) in the presence of lithium
showed elevated activity suppressing tumor inducing proteins.

 The activity of the enzyme was increased several folds and found effective against the tumor
proteins which is useful in the treatment of brain tumor.

 Lithium is a promising modulator for carboxyl esterase activity that helps by hydrolytic
function in the modulation of neuronal function.

Scope for future work:

 In future to identify the proteins mass spectrometry with higher resolution, SDS page, Native
page, 2D electrophoresis can be used.
 The proteins cam also be identified by using western blot analysis.
 In-vivo and In-vitro analysis of enzyme.
 Modification and clinical trials on brain tumor patients.
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