“ 2D QSAR STUDIES ON HUMAN GLUTAMYL CYCLASE

INHIBITORS AS ANTI-ALZHEIMER’S AGENTS ”

B.Sc. Dissertation

Submitted to St. Francis College for Women

in partial fulfillment of the requirements for the award of the

Degree of Bachelor of Science

By

SANA NAAZ

Roll number: 121319061028

Under the guidance of

Dr. V. Radhika

Department of Chemistry
St. Francis College for Women
Begumpet, Hyderabad-500 016
(Autonomous and Affiliated to Osmania University)
March 2022
 CERTIFICATE

This is to certify that this bonafide project work titled “ 2D QSAR STUDIES ON HUMAN

GLUTAMYL CYCLASE INHIBITORS AS ANTI-ALZHEIMER'S AGENTS ”, has

been carried out by SANA NAAZ, bearing Roll No:121319061028 towards partial

fulfillment of the requirements for the award of Degree of Bachelor’s of science from St.

Francis College for Women, Begumpet in the academic year 2021-2022.

Supervisor Head of the Department

External Examiner Controller of Examinations

1
 DECLARATION

The current study “ 2D QSAR STUDIES ON HUMAN GLUTAMYL CYCLASE

INHIBITORS AS ANTI-ALZHEIMER’S AGENTS ” has been carried out under the

supervision of Dr. V. Radhika, St. Francis College for Women. I hereby declare that the present

study that has been carried out by me, Sana Naaz, during April 2022 is original and no part of

it has been carried out prior to this date.

Date: 09/05/2022

Signature of Candidate:

2
 ACKNOWLEDGEMENT

I take the golden opportunity and immense pleasure to express my gratitude for the help

rendered to complete my dissertation. I would like to extend my sincere thanks towards

St. Francis College for Women, Hyderabad and Sr. Sandra Horta, Principal, St. Francis

College for Women, Dr. D Sumalatha, Head of Department of Chemistry, St. Francis

College for Women. Also, I am thankful to Dr. V. Radhika and Ms.Shravya, St. Francis

College for Women for their valuable guidance, scholarly inputs, and consistent

encouragement throughout the project work. I would like to express my deepest gratitude

to my family and friends. This dissertation would not have been possible without their

warm love, continued patience, and endless support. The guidance and support received

from each and everyone was vital for the success of the project.

3
 CONTENTS
Chapter Title Page No
no.
1 INTRODUCTION 9
1.1 ALZHEIMER’S DISEASE 10
1.2 CAUSES 11
1.3 TYPES OF ALZHEIMER’S DISEASE 13
1.4 PATHOPHYSIOLOGY 14
1.5 SYMPTOMS 15
1.6 EPIDEMIOLOGY 17
1.7 DIAGNOSIS 17
1.8 TREATMENT 19
1.9 COMPUTER AIDED DRUG DESIGN (CADD) 20
1.10 HISTORY OF CADD 20
1.11 LEAD IDENTIFICATION 21
1.12 LEAD OPTIMIZATION 21
1.13 PHARMAKOKINETICS DRUG INTERCTIONS 22
1.14 PHARMACODYNAMIC INTERACTIONS 23
1.15 TECHNIQUES INVOLVED IN CADD 24
1.16 LIGAND BASED DRUG DESIGN (LBDD) 24
1.17 QSAR(QUANTITATIVE STRUCTURE ACTIVITY 24
RELATIONS
2 REVIEW OF LITERATURE 26
3 MATERIALS AND METHODS 31
3.1 CHEMSKETCH 32

4
3.2 DATASET COLLECTION 32
3.3 SMILES 32
3.4 DESCRIPTOR CALCULATION USING pkCSM 33
3.5 2D QSAR MODEL DEVELOPEMENT 33
3.6 2D QSAR DEVELOPEMENT USING MLR 33
3.7 CALCULATION OF PREDICTED ACTIVITY 34
3.8 CALCULATION OF SD 34
3.9 CALCULATION OF PRESS 34
3.10 CALCULATION OF R2 34
4 RESULTS AND DISCUSSION 35
4.1 2D QSAR 36
4.2 CORRELATION ANALYSIS 37
4.3 RESIDUAL VALUE 38
4.4 COEFFICIENT ANALYSIS 39
4.5 CORRELATION MATRIX 40
4.6 SCATTER GRAPHS/CHARTS 42
5 CONCLUSION 50
6 REFERENCES 52

5
 LIST OF TABLES

TABLE TITLE PAGE

NO. NO.
1 HUMAN GLUTAMYL CYCLASE(QC) INHIBITORS 43
-53 MOLECULES

6
 LIST OF FIGURES

FIGURE TITLE PAGE

NO. NO.
1 Neurons in healthy brain vs Alzheimer’s disease with 10
amyloid plaques
2 Hypothesis for the pathophysiology of Alzheimer's 15
disease
3 Schematic outline for structure-based lead identification 21
and optimization
4 Flowchart of pharmacokinetic and pharmacodynamic 23
interactions

5 QSAR equation using MLR method 36

6 Correlation Analysis Graph 37
7 Analysis of Variance and fitting parameters 38
8 Residual table of the set 39
9 Coefficient Analysis of predictors 40
10 Correlation matrix of chosen descriptors 40
11 Full analysis of molecules 41
12 Actual activity vs predicted activity of the test set 42

7
 ABSTRACT
Alzheimer's disease (AD) is one of the most common neurodegenerative diseases and is
considered to be the main cause of cognitive impairment in elderly people. Glutaminyl cyclase
(QC) is involved in the formation of toxic amyloid plaques producing N-terminal pyroglutamic
acid of β-amyloid peptide (pGluAβ).In the cause of Alzheimer's disease, a library of glutamyl
cyclase (QC) Inhibitors was designed based on the proposed binding mode of the preferred
substrate, Aβ3E42. An in vitro structure-activity relationship study identified several excellent
QC inhibitors demonstrating 5- to 40-fold increases in potency compared to a known QC
inhibitor. Several compounds of this series displayed promising activity compared with a lead
sulindac analog. Structure-activity relationship studies like 2D QSAR, ADME studies were
performed to design a chemical entity. The training set had a good correlation coefficient (R2
=0.932). The set also exhibited good predictive power confirmed by the high value of the
cross-validated correlation coefficient (Q2 = 0.731). The outcomes of this work provide useful
insights into the development of new QC inhibitors as a potential treatment option for AD.

8
 CHAPTER 1

INTRODUCTION

9
1.1. ALZHEIMER’S DISEASE

Alzheimer's disease is a neurological ailment that causes the brain to shrink (atrophy) and
the death of brain cells. Alzheimer's disease is the most frequent form of dementia, which
is defined as a progressive loss of cognitive, behavioral, and social abilities that impairs a
person's capacity to operate independently. [1]

Alzheimer's disease is a progressive brain disease that wreaks havoc on memory and
thinking skills, as well as the capacity to carry out even the most basic tasks. Symptoms of
late-onset type occur in the mid-60s in the majority of patients with the condition.
Early-onset Alzheimer's disease is extremely rare and occurs between the ages of 30 and
60. The most prevalent cause of dementia in elderly people is Alzheimer's diseas e.

Figure 1. Neurons in healthy brain vs Alzheimer’s disease with amyloid plaques

Dr. Alois Alzheimers is the name of the disease. Dr. Alzheimer discovered abnormalities in
the brain tissue of a woman who died of an uncommon mental condition in 1906. Memory
loss, linguistic difficulties, and erratic conduct were some of her symptoms. He examined
her brain after she died and discovered several aberrant clumps (now known as amyloid

10
plaques) and tangled bundles of fibers (now called neurofibrillary, or tau, tangles). These
plaques and tangles in the brain are still regarded to be some of Alzheimer's disease's most
prominent symptoms. The loss of connections between nerve cells (neurons) in the brain is
another hallmark. Neurons communicate between different sections of the brain as well as
between the brain and muscles and organs throughout the body. Alzheimer's disease is
thought to be caused by a variety of additional complex brain alterations.

The entorhinal cortex and hippocampus, which are crucial in memory, are the first areas of
the brain to be damaged. Later on, it affects parts of the cerebral cortex involved in
language, logic, and social interaction. Many other parts of the brain are eventually
affected. [2]

1.2. CAUSES

The aberrant build-up of proteins in and around brain cells is assumed to be the origin of
Alzheimer's disease. Amyloid is one of the proteins involved, and deposits of it create
plaques around brain cells. The other protein is tau, which forms tangles within brain cells
as deposits. Although the actual cause of this process is unknown, scientists have
discovered that it begins several years before symptoms manifest.

Chemical messengers (known as neurotransmitters) essential in delivering messages, or

signals, between brain cells diminish when brain cells become impacted. In the brains of
persons with Alzheimer's disease, levels of one neurotransmitter, acetylcholine, are
extremely low. Different parts of the brain diminish throughout time.

Although it's still unknown what triggers Alzheimer's disease, several factors are known to
increase your risk of developing the condition.

➢ Age

Age is the single most significant factor. The likelihood of developing Alzheimer's disease
doubles every 5 years after you reach 65. But it's not just older people who are at risk of

11
developing Alzheimer's disease. Around 1 in 20 people with the condition are under 65.
This is called early- or young-onset Alzheimer's disease and it can affect people from
around the age of 40.

➢ Family history

The genes you inherit from your parents can contribute to your risk of developing
Alzheimer's disease, although the actual increase in risk is small. But in a few families,
Alzheimer's disease is caused by the inheritance of a single gene and the risks of the
condition being passed on are much higher.

➢ Down's syndrome

People with Down's syndrome are at a higher risk of developing Alzheimer's disease. This
is because the genetic changes that cause Down's syndrome can also cause amyloid plaques
to build up in the brain over time, which can lead to Alzheimer's disease in some people.

➢ Head injuries

People who have had a severe head injury may be at higher risk of developing Alzheimer's
disease, but much research is still needed in this area.

➢ Cardiovascular disease

Research shows that several lifestyle factors and conditions associated with cardiovascular
disease can increase the risk of Alzheimer's disease. These include

● obesity
● diabetes
● high blood pressure

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 ● high cholesterol
➢ Other risk factors

In addition, the latest research suggests that other factors are also important, although this
does not mean these factors are directly responsible for causing dementia. These include:

● hearing loss
● untreated depression (though depression can also be one of the symptoms of
Alzheimer's disease)
● loneliness or social isolation
● a sedentary lifestyle[3]

1.3 .TYPES OF ALZHEIMER’S DISEASE

● Early-onset Alzheimer's- This is a kind that affects people under the age of 65.
When they're diagnosed with the disease, they're usually in their 40s or 50s.
Early-onset Alzheimer's affects up to 5% of all Alzheimer's patients. It is more
common in people with Down syndrome. Early-onset Alzheimer's differs from other
varieties of the illness in a few respects, according to researchers. People who have
it are more likely to develop the brain abnormalities associated with Alzheimer's
disease. The early-onset type appears to be connected to an abnormality in
chromosome 14, a section of a person's DNA. Myoclonus, a type of muscular
twitching and spasm, is also more common in people with early-onset Alzheimer's.
● Late-onset Alzheimer's- This is the most common type of dementia, which affects
persons aged 65 and up. It could run in families or not. Researchers have yet to
discover a specific gene that causes it. Nobody knows why some people receive it
while others do not.
● Familial Alzheimer's disease (FAD) is a type of Alzheimer's disease that has been
linked to genes by doctors. At least two generations of individuals of affected
families have had the condition. FAD accounts for fewer than 1% of all Alzheimer's

13
 cases. FAD is seen in the majority of persons with early-onset Alzheimer's
disease.[4]

1.4. PATHOPHYSIOLOGY

The two pathologic hallmarks of Alzheimer's disease are

● Extracellular beta-amyloid deposits (in senile plaques)

● Intracellular neurofibrillary tangles (paired helical filaments)

The beta-amyloid deposition and neurofibrillary tangles lead to loss of synapses and
neurons, which results in gross atrophy of the affected areas of the brain, typically starting
at the mesial temporal lobe.[5]

The mechanism by which beta-amyloid peptide and neurofibrillary tangles cause such
damage is incompletely understood. There are several theories.

The amyloid hypothesis posits that progressive accumulation of beta-amyloid in the brain
triggers a complex cascade of events ending in neuronal cell death, loss of neuronal
synapses, and progressive neurotransmitter deficits; all of these effects contribute to the
clinical symptoms of dementia.

A sustained immune response and inflammation have been observed in the brain of
patients with Alzheimer's disease. Some experts have proposed that inflammation is the
third core pathologic feature of Alzheimer's disease.

Prion mechanisms have been identified in Alzheimer's disease. In prion diseases, a normal
cell-surface brain protein called prion protein becomes misfolded into a pathogenic form
termed a prion. The prion then causes other prion proteins to misfold similarly, resulting in
a marked increase in the abnormal proteins, which leads to brain damage. In Alzheimer's
disease, it is thought that the beta-amyloid in cerebral amyloid deposits and tau in
neurofibrillary tangles have prion-like, self-replicating properties.[6]

14
Figure 2. Hypothesis for the pathophysiology of Alzheimer's disease

1.5. SYMPTOMS

Generally, the symptoms of Alzheimer's disease are divided into 3 main stages.

1.Early symptoms

In the early stages, the main symptom of Alzheimer's disease is memory lapses. For
example, someone with early Alzheimer's disease may:

● forget about recent conversations or events

● misplace items
● forget the names of places and objects
● have trouble thinking of the right word
● ask questions repetitively
● show poor judgment or find it harder to make decisions
● become less flexible and more hesitant to try new things

15
There are often signs of mood changes, such as increased anxiety or agitation, or periods of
confusion.

2.Middle-stage symptoms

As Alzheimer's disease develops, memory problems will get worse. Someone with the
condition may find it increasingly difficult to remember the names of people they know
and may struggle to recognize their family and friends.

Other symptoms may also develop, such as:

● increasing confusion and disorientation – for example, getting lost, or wandering

and not knowing what time of day it is
● obsessive, repetitive, or impulsive behavior
● delusions (believing things that are untrue) or feeling paranoid and suspicious about
carers or family members
● problems with speech or language (aphasia)
● disturbed sleep
● changes in mood, such as frequent mood swings, depression, and feeling
increasingly anxious, frustrated, or agitated
● difficulty performing spatial tasks, such as judging distances
● seeing or hearing things that other people do not (hallucinations)

Some people also have some symptoms of vascular dementia. By this stage, someone with
Alzheimer's disease usually needs support to help them with everyday living. For example,
they may need help eating, washing, getting dressed, and using the toilet.

3.Later symptoms

In the later stages of Alzheimer's disease, the symptoms become increasingly severe and
can be distressing for the person with the condition, as well as their carers, friends, and
family.

16
Hallucinations and delusions may come and go over the course of the illness but can get
worse as the condition progresses. Sometimes people with Alzheimer's disease can be
violent, demanding, and suspicious of those around them.

A number of other symptoms may also develop as Alzheimer's disease progresses, such as:

● difficulty eating and swallowing (dysphagia)

● difficulty changing position or moving around without assistance
● weight loss – sometimes severe
● unintentional passing of urine (urinary incontinence) or stools (bowel incontinence)
● gradual loss of speech
● significant problems with short- and long-term memory

In the severe stages of Alzheimer's disease, people may need full-time care and assistance
with eating, moving, and personal care.[7]

1.6. EPIDEMIOLOGY

The number of dementia patients worldwide is estimated to reach 152 million by

mid-century, with the highest growth forecast in low- and middle-income nations.
According to Alzheimer's disease facts and figures for 2020, the number of AD patients (65
years and older) in America could rise dramatically from 5.8 million to 13.8 million by
2050. Over the last few decades, community-dwelling studies in Japan and China have
revealed a clearly elevated prevalence of Alzheimer's disease. Women's age-specific global
prevalence was 1.17 times higher than men's, and women's age-standardized mortality rate
was also higher than men's, implying that women's domination was not just due to their
longer longevity.[8]

17
1.7 DIAGNOSIS

No single test can confirm Alzheimer’s disease. To diagnose Alzheimer's disease many

tests are conducted. Tests may include:

1.Physical exam and medical history: We assess your overall health with a physical exam

and discuss your medical history. It’s a good idea to bring a family member with you. This

person can help remember details, especially if you’re having memory trouble.

Your medical history includes:

● Symptoms

● Previous and current illnesses or conditions

● Medications you’re taking

● Family medical history, including relatives with Alzheimer’s or other dementias

2.Mood assessment: We ask questions to check for signs of depression, anxiety, or other

disorders. These conditions can affect memory. They can also cause symptoms similar to

those of Alzheimer’s.

3.Brain imaging: We may order scans to check for or rule out other conditions such as a

brain tumor, stroke, or head injury. At OHSU, our brain imaging technologies include:

● Computerised tomography: CT scans use special X-ray equipment to produce

cross-sectional images of the brain.

18
 ● Magnetic resonance imaging: MRI scans use radio waves and a powerful magnet

to create detailed images. They help rule out other conditions or check for brain

shrinkage.

● Positron emission tomography: PET scans involve an injection of a small amount

of a radioactive substance and a special camera. A PET scan shows blood flow and

other activity in the brain. In some cases, we can use advanced PET technology to

see the buildup of a brain protein that may be an early sign of Alzheimer’s.

4.Blood tests: We can often rule out other causes by measuring how your thyroid gland is

working and whether you lack certain vitamins.

5.Spinal tap (lumbar puncture): We may collect a small sample of the brain and spinal

cord fluid. This test helps us rule out infections that can cause similar symptoms.

6.Genetic testing: Researchers have found some inherited DNA changes related to

Alzheimer’s disease. Routine testing is not recommended. If you or a loved one has

early-onset Alzheimer’s or a strong family history of it, a genetic counselor can help you

understand options. [9]

1.8.TREATMENT

There’s no cure for Alzheimer’s disease. But there are medicines that seem to slow down

its progress, especially in the early stages. Others can help with mood changes and other

behavior problems.

● Aducanumab-avwa (Aduhelm)
● Donepezil (Aricept)

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 ● Galantamine(Razadyne, formerly known as Reminyl)
● Andrivastigmine (Exelon)
● Memantine (Namenda)
● Memantine-Donepezil (Namzaric)
● Tacrine (Cognex)[10]

1.9. COMPUTER-AIDED DRUG DESIGN (CADD)

CADD has now become an indispensable tool in the long process of drug discovery and
development. It also provides options for understanding chemical systems in different
ways, yielding information that is not easy to obtain in laboratory analysis, with
considerably less cost and effort than experiments. Initially, CADD had a rocky perception
in the field of drug development, and perhaps some over-hyping of its promises, as is
present in the initial stages of almost any new technology or development. Today, one can
say that the discipline of computational medicinal chemistry has begun to mature and is a
routinely used component of the modern drug discovery process. Mastering different kinds
of CADD approaches and their software and utilizing all computational resources that are
valuable for drug design are certainly essential for becoming a successful computational
medicinal chemist in today’s world. In addition, having skills in one or more programming
languages, such as Python or JAVA will help smooth routine drug-design work. SBVs and
LBVs are also very likely to become routine in drug-discovery projects if they are not
considered to have already done so. The use of more accurate methods like MD and QM
continues to grow. In conclusion, CADD is beneficial for pharmaceutical development in
the areas of prediction of 3D structures, design of compounds, prediction of druggability, in
silico ADMET prediction however, it must be realised that computational predictions need
to be integrated with experimental approaches for successful drug discovery and
development.[11]

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1.10. HISTORY OF CADD

During the early 19th century, medicinal compounds were extracted from plants i.e
cocaine, opium, and morphine. As time progressed towards the late 19th century, synthetic
compounds were preferred and used for cosmetics and medical use. John Langley in 1905,
proposed the theory of “ Respective substances”. The first rational development of
synthetic drugs was carried out by Paul Ehrlich and Sacachiro Hata who produced
arsphenamine(Salvarsan) in 1910. Drug discovery is the step-by-step process by which
new candidate drugs are discovered.

1.11 LEAD IDENTIFICATION

Once the target has been identified the next step is to find the “Lead compound” which
shows the desired pharmaceutical activity. (ss) The lead compound is a chemical substance
that exhibits the desired pharmacological and biological activity. Lead compounds are used
as starting materials or precursors of drug designing. Possible sources of lead compounds
include - Natural products - Chemical libraries - Computational medicinal chemistry.[12]

1.12. LEAD OPTIMIZATION

It is assumed that inhibitory potency increases with increasing molecule-inhibitor binding.
So, on the computational side, the key for lead optimization is an accurate prediction of
biomolecule-ligand binding affinities. Once the lead is identified, the desirable traits of the
lead are optimized. (ss) The most promising hit series, once they are identified, advance
into the lead optimization stage of drug discovery. The goal of this stage is to optimize both
the biological activity and properties of the lead series by in vitro and in vivo assays.
[13]Lead optimization is majorly for experimental testing and confirmation of the
compound based on ADMET which is in vitro and in vivo tools.

21
 Figure 3. schematic outline for structure-based lead identification and optimization

1.13. PHARMACOKINETICS DRUG INTERACTION

Pharmacokinetics describes the relationship between the drug and the receptor and is
concerned with “ what the body does to the drug”. A pharmacokinetic drug interaction
occurs when one drug alters the concentration of another and alters “How much” and
“How long” it is present at the site of action.[14]
❖ Drug absorption interactions.
For a compound to reach the tissues, it must be absorbed into the bloodstream via mucous
membranes. How the drug is absorbed by our body is very important and there are various
factors that contribute towards poor absorption of a molecule. If the drug is poorly soluble
then the absorption will be ineffective. If the drug is unstable at acidic pH (stomach). But to
overcome this problem, different routes of administration are used to ensure maximum
absorption of the drug by our body. [15]
For example, insulin is destroyed under acidic conditions of the gastric juices hence, it is
administered via the subcutaneous route.
❖ Drug distribution interactions.
After the drug is absorbed into the bloodstream, it must make its way to the target site. For
effective drug distribution, the bloodstream plays a major role. Blood transports the drug
from one part of the body to another. [16]
The distribution rate can be affected by various factors like

22
• The ability of the drug to cross the blood-brain barrier
• Molecular size of the molecule
• Polarity of the drug molecule
• Highly polar drugs cannot readily pass through the lipid membrane and are not given
orally.[17]
❖ Drug metabolism interactions.
After the drug is absorbed and distributed, it undergoes a series of metabolic changes in the
form of chemical transformation. Some drugs are administered in their inactive form and
after they get absorbed, they transform into active agents and exert a therapeutic effect.
[18]

❖ Drug elimination or excretion interactions.

Drugs and their metabolites are excreted from the body mainly by the action of our kidneys
in the form of urine. They are also excreted by the biliary and fecal excretion. If the
compounds are not excreted then they get accumulated and adversely affect the normal
metabolism of the body.[19]

1.14.PHARMACODYNAMIC INTERACTIONS
Pharmacodynamic interaction occurs when two drugs given together act at the same or
similar receptor site and result in an overall increase or decrease of the effect. It is
concerned with “what drugs do to the body”. Pharmacodynamic interactions can be of
three different types:
• Additive interactions: occurs when two drugs of the same class, act on the same receptor
with the same mechanism[20]
• Synergistic interactions: occurs when two drugs are administered to produce the same
effect but act on different receptors and via different mechanisms.
• Antagonistic interactions: occurs when the drugs administered act against each other
resulting in an overall decrease in the effect.[21]

23
 Figure 4. Flowchart of pharmacokinetic and pharmacodynamic interactions

1.15. TECHNIQUES INVOLVED IN CADD

CADD is classified based on whether the 3D structure of the molecule is known or not.
● Ligand Based Drug Designing (LBDD)- it again includes 2D QSAR, 3D QSAR.[22]
● Structure-Based Drug Designing (SBDD)- it again includes Docking, Virtual
screening, Similarity search.

1.16.LIGAND-BASED DRUG DESIGN(LBDD)

In other words, it is also called indirect drug design and it is based on the molecules which
can bind to the biological target active site. This technique is used to develop novel
compounds which interact with the biologically active target molecule.[23] This model is
used to define the minimum necessary structural characteristics a molecule must have in
order to bind to the target. Thus, a model which has a biological target can be built
depending upon the knowledge of what binds to it. This model is then used to design new
molecules that can interact or bind to the target.
It is further classified into 2D & 3D QSAR.

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1.17.QSAR (Quantitative Structure-Activity Relations)
QSAR is an approach that is concerned with the identification and quantification of
physicochemical properties of a drug to see its effect on a drug's biological activity. A
quantitative structure-activity relation or QSAR is a mathematical equation of measurable
molecular properties of small molecules. [24]
The main steps of QSAR are
• Selecting datasets and extracting structural o empirical descriptors
• variable selection
• construction of model
• Validation evaluation

2D QSAR
2D QSAR is a very useful technique for explaining the relationships between chemical
structures and experimental observations.[25] 2D QSAR techniques are very often used
during the process of optimization of a chemical series towards a candidate for clinical
trials. The main elements of this method include:
• Numerical descriptors are used to translate the chemical structure into mathematical
variables.
• Quality of the data observed
• Statistical methods used for deriving the relationship between observations and
descriptors.[26]

OBJECTIVES
• The objective of Computer-aided drug designing is to find a chemical compound that can
fit a specific cavity on a protein target both geometrically and also chemically.
• To be able to understand how to relate chemical structure to biological activity.
• To discover, design, and also optimize new effective and safe drugs. [27]
• To predict drug activity and molecular relations using 2D and 3D Quantitative
Structure-Activity Relationships.

25
•To create compounds with not only a high affinity for the target but also optimal
pharmacokinetic properties.
•To predict and evaluate all fundamental characteristics necessary for a molecule to
become a drug-using structure-based and ligand-based drug designing.[28]

26
.

CHAPTER 2
REVIEW OF LITERATURE

27
● Alzheimer's disease (AD), a common chronic neurodegenerative disease, has
become a major public health concern. Despite years of research, therapeutics for
AD are limited. Overexpression of secretory glutaminyl cyclase (sQC) in AD brain
leads to the formation of a highly neurotoxic pyroglutamate variant of amyloid-beta,
pGlu-Aβ, which acts as a potential seed for the aggregation of full-length Aβ.
Preventing the formation of pGlu-Aβ through inhibition of sQC has become an
attractive disease-modifying therapy in AD.[29]

● With limited understanding regarding the cause of disease, it is commonly identified

by extracellular deposit of amyloid β (Aβ) peptides as senile plaques. Pyroglutamate
Aβ is identified from the brain of AD patients and constitutes the majority of total
Aβ present. The formation of Pyroglutamate Aβ could be hindered by the use of
Glutaminyl cyclase inhibitors and could efficiently improve the symptoms of
Alzheimer’s.

● A diverse range of N-terminally truncated and modified forms of amyloid-β (Aβ)

oligomers have been discovered in Alzheimer's disease brains, including the
pyroglutamate-Aβ (AβpE3). AβpE3 species are shown to be more neurotoxic when
compared with the full-length Aβ peptide. Findings visibly suggest that glutaminyl
cyclase (QC) catalyzed the generation of cerebral AβpE3, and therapeutic effects are
achieved by reducing its activity. In recent years, efforts to effectively develop QC
inhibitors have been pursued worldwide. The inhibitory activity of current QC
inhibitors is mainly triggered by zinc-binding groups that coordinate Zn2+ ions in
the active site and other common features. Herein, we summarised the current state
of discovery and evolution of QC inhibitors as a potential Alzheimer's
disease-modifying strategy.[30]

● Human glutaminyl cyclase (hQC) appeared as a promising new target with its
inhibitors attracting much attention for the treatment of Alzheimer’s disease (AD) in

28
 recent years. But so far, only a few compounds have been reported as hQC
inhibitors. To find novel and potent hQC inhibitors, a high-specificity ZBG
(zinc-binding groups)-based pharmacophore model comprising customized ZBG
feature was first generated using HipHop algorithm in Discovery Studio software
for screening out hQC inhibitors from the SPECS database. After purification by
docking studies and drug-like ADMET properties filters, four potential hit
compounds were retrieved. Subsequently, these hit compounds were subjected to
30-ns molecular dynamic (MD) simulations to explore their binding modes at the
active side of hQC.

● MD simulations demonstrated that these hit compounds formed a chelating

interaction with the zinc ion, which was consistent with the finding that the
electrostatic interaction was the major driving force for binding to hQC confirmed
with MMPBSA energy decomposition. Higher binding affinities of these
compounds were also verified by the binding free energy calculations compared
with the references. Thus, these identified compounds might be potential hQC
candidates and could be used for further investigation.[31]

● Glutaminyl cyclase (QC) and isoGlutaminyl cyclase (isoQC) are two

metalloenzymes that catalyze the intramolecular cyclization of Aβ-N-terminal
glutamine and CCL2 into pyroglutamic acid (pGlu) exhibiting more severe
neurotoxicity than normal Aβ and CCL2. Both pGlu- modified Aβ-peptides and
pE-CCL2 are discussed to be the crucial species involved in the early onset of AD.
So far, most QC inhibitors have demonstrated a sub- or low micromolar IC50. QC
represents a novel target for the development of AD treatments.[32]

● The literature revealed the competence of quantitative structure-activity/property

relationship studies in drug discovery. The present work explores the efficiency of
Monte Carlo-based QSAR modeling studies on a dataset of 125 Glutaminyl cyclase
inhibitors with pKi taken as the endpoint for QSAR analysis. The dataset is divided

29
 into training, subtraining, calibration and validation sets resulting in the generation
of five random splits.

● The validation is performed in accordance with the Organisation of Economic

Cooperation and Development principles. The values of R2, Q2, index of the
ideality of correlation, concordance correlation coefficient, av. r2m and delta r2m of
the calibration set of the best split are found to be 0.9012, 0.8775, 0.9479, 0.9435,
0.8347, and 0.0847, respectively. The structural features responsible for increasing
the inhibitory activity are identified. These structural features are added to a base
compound from the dataset to design six novel molecules. These new molecules
possess improved inhibitory activity as compared to the base compound. The results
are further supported by docking studies.[33]

● The inhibition of glutaminyl cyclase (QC) may provide a promising strategy for the
treatment of early Alzheimer's disease (AD) by reducing the amount of the toxic
pyroform of β-amyloid (AβΝ3pE) in the brains of AD patients. It was identified as
potent QC inhibitors with subnanomolar IC50 values that were up to 290-fold
higher than that of PQ912, which is currently being tested in Phase II clinical trials.
Among the tested compounds, the cyclopentyl methyl derivative (214) exhibited the
most potent in vitro activity (IC50 = 0.1 nM), while benzimidazole (227) showed
the most promising in vivo efficacy, selectivity and druggable profile. 227
significantly reduced the concentration of pyroform Aβ and total Aβ in the brain of
an AD animal model and improved the alternation behavior of mice during Y-maze
tests. The crystal structure of human QC (hQC) in complex with 214 indicated tight
binding at the active site, supporting that the specific inhibition of QC results in
potent in vitro and in vivo activity. Considering the recent clinical success of
donanemab, which targets AβΝ3pE, small molecule-based QC inhibitors may also
provide potential therapeutic options for early-stage AD treatment.[34]

30
● In a current study, through a pharmacophore assisted high throughput virtual
screening, it was reported that a novel sQC inhibitor (Cpd-41) with a
piperidine-4-carboxamide moiety (IC50 = 34 μM). Systematic molecular docking,
MD simulations and X-ray crystallographic analysis provided atomistic details of
the binding of Cpd-41 in the active site of sQC. The unique mode of binding and
moderate toxicity of Cpd-41 make this molecule an attractive candidate for
designing high affinity sQC inhibitors.[35]

● On the basis of a putative active conformation of the prototype inhibitor 1, a series

of N-substituted thiourea, urea, and α-substituted amide derivatives were developed.
The structure-activity relationship analyses indicated that conformationally
restrained inhibitors demonstrated much improved QC inhibition in vitro compared
to nonrestricted analogues, and several selected compounds demonstrated desirable
therapeutic activity in an AD mouse model. The conformational analysis of a
representative inhibitor indicated that the inhibitor appeared to maintain the Z-E
conformation at the active site, as it is critical for its potent activity.[36]

● Pyroglutamate-modified amyloid β peptides (pGlu-Aβ) are highly neurotoxic and

promote the formation of amyloid plaques. The pGlu-Aβ peptides are generated by
glutaminyl cyclase (QC), and recent clinical studies indicate that QC represents an
alternative therapeutic target to treat Alzheimer's disease (AD). A recent study
focused on the structure-activity relationship (SAR) of analogues with modifications
in the D-region and evaluated their biological activity. Most compounds in this
series exhibited potent activity in vitro, and our SAR analysis and the molecular
docking studies identified compound 202 as a potential candidate because it forms
an additional hydrophobic interaction in the hQC active site. Overall, the study
provides valuable insights into the Arg-mimetic pharmacophore that will guide the
design of novel QC inhibitors as potential treatments for AD.[37]

31
 CHAPTER 3

MATERIALS AND METHODS

32
3.1.CHEMSKETCH

Advanced Chemistry Department (ACD)/Chem Sketch freeware is a drawing package that

allows us to draw chemical structures including organics, organometallics, polymers, and
Markush structures. This software can also be used to generate names from molecular
structure, calculate molecular properties from the chemical structure, and for 2D and 3D
structure drawing and viewing. From button bars, users may choose an array of bond types.
Left-hand buttons consist of chemical element symbols to select any element of the
periodic table. On the right-hand side of the drawing area are buttons to select substructures
such as rings, chains of various lengths, common groups such as COOH and amino
acids.[38]

3.2. DATA SET COLLECTION

A set comprising fifty-three human Glutamyl Cyclase (QC) inhibitors as anti-Alzheimer's

agents was utilized for QSAR analysis. Inhibitory concentrations (EC50) of compounds
included in a data set varied and were changed over into corresponding pEC50 values
utilizing the formula given underneath.[39]
pEC50 = -log10 [EC50]

3.3. SMILES

SMILES (Simplified Molecular Input Line Entry System) is a chemical notation that
allows a user to represent a chemical structure in a way that can be used by the computer. It
is an easily learned and flexible notation. Smiles has five basic syntax rules that must be
followed. The notation consists of a series of characters containing no spaces. [40]There
are five generic Smiles encoding rules, corresponding to the specification of atoms, bonds,
branches, ring closures, and disconnection. Fifty-three human glutamyl cyclase (QC)
inhibitors with anti-Alzheimer's inhibitory activity were converted to Smiles notation.

33
3.4 DESCRIPTOR CALCULATION USING pkCSM

pkCSM is a novel method for predicting and optimizing small-molecule pharmacokinetic

and toxicity properties that rely on distance-based graph signatures. pkCSM is a
user-friendly web server that enables researchers to freely predict ADME properties for
molecules of interest.[41]The ADME properties and descriptors of the ligands were
determined using pkCSM. Log P, Surface area, CaCO2 permeability,water-solubility,
parachor, index of refraction, surface tension, density, and polarizibility were calculated.

3.5 2D QSAR MODEL DEVELOPMENT

The biological activity of dataset molecules i.e., pEC50 of the dataset alongside their
calculated descriptors were imported into the Build QSAR spreadsheet, and this 2D QSAR
model was developed utilizing Build QSAR software.[42] A correlation analysis is carried
out between the calculated descriptors (an independent variable, X) and biological activity
(dependent variable, Y) to comprehend the relation between them both.

3.6 2D QSAR MODEL DEVELOPMENT USING MLR

The dataset of the molecule was partitioned into training and test set molecule after which
only the training set of molecules was used for MLR analysis wherein biological activity,
pEC50 is considered as the dependent variable and different molecular descriptors were
used as the independent variable.[43] A multi-linear regression was carried out by selecting
different descriptor combinations. The best model was chosen depending upon the
statistical parameters such as correlation coefficient, R, and regression coefficient Q2.

34
3.7 CALCULATION OF PREDICTED ACTIVITY

The best QSAR model was built using the Multiple Linear Regression (MLR) method
choosing 9 out of 17 different molecular descriptors that include Log P, Surface area,
CaCO2 permeability,water-solubility, parachor, index of refraction, surface tension,
density, and polarizability.Fifty three molecules were divided into two sets - 31 molecules
in training set and 14 molecules in test set and rest others removed as outliers.This equation
was used to calculate the predicted activity of test set molecules.[44]

3.8 CALCULATION OF SD (Standard Deviation)

Standard deviation is a statistic that measures the dispersion of a dataset relative to its
mean and is calculated as the square root of variance. SD of the test set was
determined.[45]
• STEP 1- The mean activity of the training set was calculated using the formula- =
sum(pEC50)/the Total number of molecules in the training set
• STEP 2- The SD of the test set was calculated using the formula- (Activity of test set –
Mean of the training set)2
• STEP 3- The sum of SD of test set molecules was calculated.[46] SD = Σ (actual activity
of test set – mean activity of training set) 2

3.9 CALCULATION OF PRESS

The PRESS value of the test set was calculated using the formula-
• (Predicted Activity – Actual Activity)2
The sum of PRESS of test molecules was calculated.[47]
Press = Σ (predicted activity of test set – actual activity of test set) 2

3.10 CALCULATION OF R2

The extent to which the proposed relationship could explain the variance of biological
activity is presented with R2, the coefficient of determination. [48]sR2 value of the test set
molecules was calculated using the formula- = sum(sum of SD – the sum of PRESS) / sum
of SD R2 = (SD-Press)/SD

35
 CHAPTER 4

RESULTS AND DISCUSSION

36
4.1. 2D QSAR

Two dimensional QSAR studies were carried out to determine the relationship between

molecular properties of molecules with inhibitory activity. All the properties calculated

using pkCSM were initially considered for analysis. Correlation analysis was carried out

for all the molecular properties and biological activity (pEC50), the molecular properties

that gave good correlation with biological activity were selected for QSAR model

generation.[49].Fifty three molecules were divided into two sets - 31 molecules in training

set and 14 molecules in test set and rest others removed as outliers. A total of 9 out of 17

descriptors were used to obtain the best fit. The best QSAR model built using the multiple

linear regression (MLR) method is represented by the following equation.

Figure 5. QSAR equation using MLR method

Where n = number of observations, R = correlation coefficient ,F = Fischer’s statistic ,P =

statistical significance >99.9% 30 ,Q2 = cross-validated correlation coefficient ,SPress =

standard deviation of sum of squared error of prediction ,SDEP = standard deviation of

error

of prediction.[50]

37
4.2.CORRELATION ANALYSIS:

In the correlation analysis of chemical data, simple or multiple regression is the most

common mathematical tool. Correlation analysis is used to quantify the degree to which

two variables are related. Through the correlation analysis, you evaluate the correlation

coefficient that tells you how much one variable changes when the other one does.

Correlation analysis provides you with a linear relationship between two variables.[51] A

high correlation means that two or more variables have a strong relationship with each

other, while a weak correlation means that the variables are hardly related.

A graph describing correlation observed verses correlation calculated is given below in

figure 6.

Figure 6.Correlation Analysis Graph

38
The analysis of variance and fitting parameters of the set are specified in figure 7.

Figure 7.Analysis of Variance and Fitting Parameters

4.3.RESIDUAL VALUE:

A residual value is a measure of how much a regression line vertically misses a data point.

Residuals are the differences between observed and predicted values of data.[52] They are

diagnostic measure used when assessing the quality of a model. They are also known as

errors. The residual table listing the Y (obs) and Y (calc) is given in figure 8.

39
 Figure 8.Residual table of the set

4.4.COEFFICIENT ANALYSIS:

Coefficients in regression analysis work together to tell you which relationships in your

model are statistically significant and the nature of those relationships. The coefficients

describe the mathematical relationship between each independent variable and the

40
dependent variable.[53] The coefficient analysis of the opted predictors or descriptors is

given below in figure 9.

Figure 9.Coefficient Analysis of Predictors.

4.5.CORRELATION MATRIX:

The correlation matrix is a table that shows the correlation of one descriptor with another.

Using the data in this table, you can examine relationships among descriptors. You also can

use the data to modify and improve your QSAR equations. [54] The correlation matrix of

the

selected predictors is mentioned in figure 10.

41
 Figure 10.Correlation matrix of chosen descriptors.

• The full analysis of the dataset comprising the desirable molecules is put forth in

figure 11.

Figure 11. Full analysis of molecules.

42
4.6. SCATTER GRAPHS/ CHARTS

The scatter chart of actual activity verses predicted activity is given below in figure 12.

Figure 12. Actual activity Vs Predicted activity of test set.

● The SD value is found to be 5.65959, PRESS is 2.69465, and the R2 value is

calculated and found to be 0.52388.

43
Table 1.Human Glutamyl Cyclase (QC) inhibitors - 45 molecules

S. MOLECU STRUCTURE EC50 EXPERI PREDICTE

NO LE (nm) MENTA D VALUE
. NO. L
VALUE

1 170 5.3 8.276 7.764215

2 171 13.8 7.861 7.825830

3 173 6.9 8.162 8.207527

4 177 3.7 8.43 8.256507

5 178 40.8 7.39 7.887953

6 179 41.2 7.4 na

44
7 180 39.4 7.41 7.709899

8 181 17.5 7.76 7.807688

9 182 5.7 8.23 7.872602

10 183 18.5 7.73 na

11 184 17.5 7.75 na

12 185 24.7 7.61 na

13 186 13.4 7.873 na

14 187 12.5 7.9 na

45
15 188 12.5 7.9 na

16 189 14.1 7.85 na

17 190 9.2 8.03 na

18 191 0.7 9.15 7.945178

19 192 11.4 7.95 na

20 193 4.8 8.32 7.910369

21 194 7.3 8.14 na

46
22 195 11.7 7.94 na

23 196 8.2 8.09 na

24 197 13.8 7.87 na

25 198 7.2 8.15 7.907732

26 199 7.8 8.11 7.888901

27 200 4.6 8.34 8.012736

28 201 20.2 7.7 8.011996

47
29 202 12.3 7.92 na

30 203 7.7 8.12 na

31 204 9.0 8.05 na

32 205 8.3 8.09 na

33 206 23.5 7.63 na

34 207 14.4 7.85 na

48
35 208 9.1 8.05 na

36 209 19.8 7.71 na

37 210 5.5 8.26 na

38 211 10.7 7.98 na

39 212 4.5 8.35 na

40 213 19.0 7.73 na

49
41 215 15.3 7.82 na

42 216 19.3 7.72 na

43 217 35.5 7.45 na

44 219 20.5 7.69 na

45 220 18.4 7.74 na

50
CHAPTER 5

CONCLUSION

51
The computational ( Ligand-based drug design) approach was applied to obtain an insight into

the structural basis for the discovery of Potent Human Glutamyl Cyclase (QC) inhibitors

which act as anti-Alzheimer's agents. The study reveals pharmacophoric features responsible

for biological activity. Chemsketch software was utilized to draw the structures of these

molecules and also to generate SMILES notations/strings. These SMILES notations of all the

molecules helped in calculating various properties and aided in ADME prediction. 2D QSAR

analysis was performed using ―Build QSAR to provide a structural framework for

understanding the structure-activity relationship of these entities. This analysis also reveals the

QSAR equation of the desired molecules which exhibits good correlation coefficient (R2) and

cross-validated correlation coefficient (Q2) values. Therefore, the molecules belonging to this

set can be studied further for performing structure-based drug design and assist in the

preparation and production of compounds with therapeutic value.

52
CHAPTER 6

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53
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