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Targeting Enzymes for Pharmaceutical Development 1st
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Author(s): Nikolaos E. Labrou
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Methods in
Molecular Biology 2089

Nikolaos E. Labrou Editor

Targeting
Enzymes for
Pharmaceutical
Development
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK

For further volumes:

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For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
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 Targeting Enzymes
for Pharmaceutical Development

Methods and Protocols

Edited by

Nikolaos E. Labrou
Department of Biotechnology, Agricultural University of Athens, Athens, Greece
Editor
Nikolaos E. Labrou
Department of Biotechnology
Agricultural University of Athens
Athens, Greece

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-0162-4 ISBN 978-1-0716-0163-1 (eBook)
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Preface

Drug development is the process of bringing a new pharmaceutical drug to the market once
a lead compound has been identified through the process of drug discovery. Enzymes offer
unique opportunities for drug discovery, as they are one of the most important groups of
drug targets. Many of the significant biochemical processes in the cell are enzyme-catalyzed
reactions (biosynthesis and catabolism) or specific cellular signaling pathways that enzymes
are involved with. Several important diseases are often associated with the elevation or
repression of certain enzymes. The attractiveness of enzymes as targets for drug discovery
stems from their high levels of disease association (target validation) and druggability (target
tractability), which typically characterize this class of proteins. In general, drug discovery
starts by manipulating the target enzyme with a compound that usually leads to inhibition or
activation of its catalytic activity. Today, there are seventy-one human, bacterial, viral, and
fungal enzymes that have been used successfully for the development of currently marketed
clinically utilized drugs. All these drugs act as reversible or irreversible enzyme inhibitors.
This volume contains methods and detailed experimental protocols on the evaluation of
the effect of a compound or a mixture of compounds on the action of enzymes that are
significant targets in pharmaceutical industry. It examines the most reliable and robust
methods for both bench and R&D scientists and sets a standard for best practices in the
field. This volume consists of three different sections, each of which deals with different steps
in the process from target selection and compound design to inhibitor evaluation. The first
section presents biocomputing and bioinformatics protocols that have been developed
recently. It underlines the progress in this area and points out the advantages that enzymol-
ogists and medicinal chemists can exploit for new target selection, druggability assessment,
and structure-based design. The next section contains a selection of the state-of-the-art
modern biophysical, electrophoretic, and chromatographic methods and high-throughput
screening approaches that have been developed and are currently used for the assessment of
enzyme/inhibitor interaction. The subsequent section provides detailed protocols and
examples of the inhibition analysis and evaluation of selected enzymes. It contains critical
information on enzyme structure-function relationships as well as mechanistic aspects on
how these enzymes are inhibited.
This volume has been written by international scientists, who are active in biochemical
and biomedical research, with expertise in chemistry, protein biochemistry, enzymology,
molecular biology, and genetics. While it is not possible to detail and include every possible
method and protocol related to enzyme inhibition, the present volume attempts to provide
working tips with examples and analysis relevant to a wide range of more important enzyme
targets and commonly available enzyme inhibition techniques and protocols.
The present book would definitely be an ideal source of scientific information for
advanced students, junior researchers, and scientists involved in health sciences, cellular
and molecular biology, biochemistry, biotechnology, cosmetology, and other related areas in
academia. It is also aimed at professionals including academic faculty members, industrial
scientists, and anyone working in the pharmaceutical, food, and cosmetics industries.
I sincerely hope that the reader will enjoy the information provided in this book and find
its contents interesting and scientifically stimulating. I also hope that I have established a
successful compilation of chapters within the exciting area of enzymes as drug targets. I

v
vi Preface

would like to thank all the contributing authors for their enthusiasm and for the time they
spent preparing the chapters for this book. I would also like to thank Dr. John Walker, the
series editor, for his help and encouragement, and everybody at Springer for their helpful
advice and support. I would especially like to thank my family for their understanding and
patience during the editing and organization of the book chapters.

Athens, Greece Nikolaos E. Labrou

Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 In Silico Laboratory: Tools for Similarity-Based Drug Discovery . . . . . . . . . . . . . . 1
Samo Lešnik and Janez Konc
2 The In Silico Fischer Lock-and-Key Model: The Combined Use
of Molecular Descriptors and Docking Poses for the Repurposing
of Old Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Marco Tutone and Anna Maria Almerico
3 Determination of Half-Maximal Inhibitory Concentration of
an Enzyme Inhibitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Nikolaos Georgakis, Elisavet Ioannou, Christina Varotsou,
Georgios Premetis, Evangelia G. Chronopoulou,
and Nikolaos E. Labrou
4 Applications of Differential Scanning Fluorometry and Related
Technologies in Characterization of Protein–Ligand Interactions . . . . . . . . . . . . . 47
Bolormaa Baljinnyam, Michael Ronzetti, Adam Yasgar,
and Anton Simeonov
5 High-Throughput Differential Scanning Fluorimetry of
GFP-Tagged Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Alanna E. Sorenson and Patrick M. Schaeffer
6 Enzyme–Ligand Interaction Monitored by Synchrotron Radiation
Circular Dichroism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Rohanah Hussain, Charlotte S. Hughes, and Giuliano Siligardi
7 Measuring Small Molecule Binding to Escherichia coli AcrB by
Surface Plasmon Resonance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Steven W. Polyak, Rumana Mowla, and Henrietta Venter
8 Systematic Screening of Viral Entry Inhibitors Using Surface
Plasmon Resonance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Penmetcha K. R. Kumar
9 Screening of Beta-Secretase Inhibitors by Capillary
Electrophoresis-Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Jan Schejbal, Roman Řemı́nek, and Zdeněk Glatz
10 Electrophoretic Mobility Shift Assays with GFP-Tagged
Proteins (GFP-EMSA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Alanna E. Sorenson and Patrick M. Schaeffer
11 Online Enantioselective Capillary Electrophoretic Method for
Screening Cytochrome P450 3A4 Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Roman Řemı́nek, Zdeněk Glatz, and Wolfgang Thormann
12 Enzymatic Bioautographic Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
I. Ayelen Ramallo, Mario O. Salazar, and Ricardo L. E. Furlan

vii
viii Contents

13 High-Throughput Assessment of Metabolism-Induced Toxicity

of Compounds on a 384-Pillar Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Soo-Yeon Kang, Kyeong-Nam Yu, Pranav Joshi,
and Moo-Yeal Lee
14 Droplet-Based Microfluidics Methods for Detecting
Enzyme Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Abraham Ochoa, Frida Trejo, and Luis F. Olguı́n
15 Ligand Fishing: An Approach for the Discovery of Inhibitors
from Complex Biological Mixtures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
Evangelia G. Chronopoulou, Christina Varotsou, Nikolaos Georgakis,
Georgios Premetis, Elisavet Ioannou, and Nikolaos E. Labrou
16 HMG-CoA Reductase as Target for Drug Development . . . . . . . . . . . . . . . . . . . . . 245
Baskaran Gunasekaran and Mohd Yunus Shukor
17 Lipoxygenases as Targets for Drug Development . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Xiao-Yuan Mao
18 Cholinesterase as a Target for Drug Development
in Alzheimer’s Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Piyoosh Sharma, Manish Kumar Tripathi,
and Sushant Kumar Shrivastava

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
Contributors

ANNA MARIA ALMERICO • Department of Biological, Chemical and Pharmaceutical Sciences

and Technologies (STEBICEF), University di Palermo, Palermo, Italy
BOLORMAA BALJINNYAM • National Center for Advancing Translational Sciences, National
Institutes of Health, Rockville, MD, USA
EVANGELIA G. CHRONOPOULOU • Laboratory of Enzyme Technology, Department of
Biotechnology, School of Applied Biology and Biotechnology, Agricultural University of
Athens, Athens, Greece
RICARDO L. E. FURLAN • Facultad de Ciencias Bioquı́micas, Universidad Nacional de
Rosario-CONICET, Rosario, Argentina
NIKOLAOS GEORGAKIS • Laboratory of Enzyme Technology, Department of Biotechnology,
School of Applied Biology and Biotechnology, Agricultural University of Athens, Athens,
Greece
ZDENĚK GLATZ • Department of Biochemistry, Faculty of Science, Masaryk University, Brno,
Czech Republic
BASKARAN GUNASEKARAN • Faculty of Biotechnology and Biomolecular Sciences, Department
of Biochemistry, Universiti Putra Malaysia, Serdang, Selangor, Malaysia
CHARLOTTE S. HUGHES • Diamond Light Source Ltd., Chilton, Didcot, UK
ROHANAH HUSSAIN • Diamond Light Source Ltd., Chilton, Didcot, UK
ELISAVET IOANNOU • Laboratory of Enzyme Technology, Department of Biotechnology, School
of Applied Biology and Biotechnology, Agricultural University of Athens, Athens, Greece
PRANAV JOSHI • Department of Chemical and Biomedical Engineering, Cleveland State
University, Cleveland, OH, USA
SOO-YEON KANG • Department of Chemical and Biomedical Engineering, Cleveland State
University, Cleveland, OH, USA
JANEZ KONC • Faculty of Chemistry and Chemical Technology, University of Maribor,
Maribor, Slovenia; National Institute of Chemistry, Ljubljana, Slovenia; Faculty of
Mathematics, Natural Sciences and Information Technologies, University of Primorska,
Koper, Slovenia; Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia
PENMETCHA K. R. KUMAR • Biomedical Research Institute, National Institute of Advanced
Industrial Science and Technology, Tsukuba City, Ibaraki, Japan
NIKOLAOS E. LABROU • Laboratory of Enzyme Technology, Department of Biotechnology,
School of Applied Biology and Biotechnology, Agricultural University of Athens, Athens,
Greece
MOO-YEAL LEE • Department of Chemical and Biomedical Engineering, Cleveland State
University, Cleveland, OH, USA
SAMO LEŠNIK • Faculty of Chemistry and Chemical Technology, University of Maribor,
Maribor, Slovenia
XIAO-YUAN MAO • Department of Clinical Pharmacology, Xiangya Hospital, Central South
University, Changsha, People’s Republic of China; Institute of Clinical Pharmacology,
Central South University, Hunan Key Laboratory of Pharmacogenetics, Changsha, People’s
Republic of China; Engineering Research Center of Applied Technology of
Pharmacogenomics, Ministry of Education, Changsha, People’s Republic of China;

ix
x Contributors

National Clinical Research Center for Geriatric Disorders, Changsha, Hunan, People’s
Republic of China
RUMANA MOWLA • School of Pharmacy and Medical Sciences, University of South Australia,
Adelaide, SA, Australia
ABRAHAM OCHOA • Facultad de Quı́mica, Laboratorio de Biofı́sicoquı́mica, Universidad
Nacional Autonoma de México (UNAM), Ciudad de México, Mexico
LUIS F. OLGUÍN • Facultad de Quı́mica, Laboratorio de Biofı́sicoquı́mica, Universidad
Nacional Autonoma de México (UNAM), Ciudad de México, Mexico
STEVEN W. POLYAK • School of Pharmacy and Medical Sciences, University of South Australia,
Adelaide, SA, Australia
GEORGIOS PREMETIS • Laboratory of Enzyme Technology, Department of Biotechnology, School
of Applied Biology and Biotechnology, Agricultural University of Athens, Athens, Greece
I. AYELEN RAMALLO • Facultad de Ciencias Bioquı́micas, Universidad Nacional de Rosario-
CONICET, Rosario, Argentina
ROMAN ŘEMÍNEK • Faculty of Science, Department of Biochemistry, Masaryk University,
Brno, Czech Republic
MICHAEL RONZETTI • National Center for Advancing Translational Sciences, National
Institutes of Health, Rockville, MD, USA
MARIO O. SALAZAR • Facultad de Ciencias Bioquı́micas, Universidad Nacional de Rosario-
CONICET, Rosario, Argentina
PATRICK M. SCHAEFFER • Molecular and Cell Biology, College of Public Health, Medical and
Veterinary Sciences, James Cook University, Douglas, QLD, Australia
JAN SCHEJBAL • Faculty of Science, Department of Biochemistry, Masaryk University, Brno,
Czech Republic
PIYOOSH SHARMA • Pharmaceutical Chemistry Research Laboratory, Department of
Pharmaceutical Engineering and Technology, Indian Institute of Technology (Banaras
Hindu University), Varanasi, India
SUSHANT KUMAR SHRIVASTAVA • Pharmaceutical Chemistry Research Laboratory,
Department of Pharmaceutical Engineering and Technology, Indian Institute of
Technology (Banaras Hindu University), Varanasi, India
MOHD YUNUS SHUKOR • Faculty of Biotechnology and Biomolecular Sciences, Department of
Biochemistry, Universiti Putra Malaysia, Serdang, Selangor, Malaysia
GIULIANO SILIGARDI • Diamond Light Source Ltd., Chilton, Didcot, UK
ANTON SIMEONOV • National Center for Advancing Translational Sciences, National
Institutes of Health, Rockville, MD, USA
ALANNA E. SORENSON • Molecular and Cell Biology, College of Public Health, Medical and
Veterinary Sciences, James Cook University, Douglas, QLD, Australia
WOLFGANG THORMANN • Clinical Pharmacology Laboratory, Institute for Infectious
Diseases, University of Bern, Bern, Switzerland
FRIDA TREJO • Facultad de Quı́mica, Laboratorio de Biofı́sicoquı́mica, Universidad
Nacional Autonoma de México (UNAM), Ciudad de México, Mexico
MANISH KUMAR TRIPATHI • Pharmaceutical Chemistry Research Laboratory, Department of
Pharmaceutical Engineering and Technology, Indian Institute of Technology (Banaras
Hindu University), Varanasi, India
MARCO TUTONE • Department of Biological, Chemical and Pharmaceutical Sciences and
Technologies (STEBICEF), University di Palermo, Palermo, Italy
 Contributors xi

CHRISTINA VAROTSOU • Laboratory of Enzyme Technology, Department of Biotechnology,

School of Applied Biology and Biotechnology, Agricultural University of Athens, Athens,
Greece
HENRIETTA VENTER • School of Pharmacy and Medical Sciences, University of South
Australia, Adelaide, SA, Australia
ADAM YASGAR • National Center for Advancing Translational Sciences, National Institutes
of Health, Rockville, MD, USA
KYEONG-NAM YU • Department of Chemical and Biomedical Engineering, Cleveland State
University, Cleveland, OH, USA
 Chapter 1

In Silico Laboratory: Tools for Similarity-Based Drug

Discovery
Samo Lešnik and Janez Konc

Abstract
Computational methods that predict and evaluate binding of ligands to receptors implicated in different
pathologies have become crucial in modern drug design and discovery. Here, we describe protocols for
using the recently developed package of computational tools for similarity-based drug discovery. The
ProBiS stand-alone program and web server allow superimposition of protein structures against large
protein databases and predict ligands based on detected binding site similarities. GenProBiS allows
mapping of human somatic missense mutations related to cancer and non-synonymous single nucleotide
polymorphisms and subsequent visual exploration of specific interactions in connection to these mutations.
We describe protocols for using LiSiCA, a fast ligand-based virtual screening software that enables easy
screening of large databases containing billions of small molecules. Finally, we show the use of BoBER, a
web interface that enables user-friendly access to a large database of bioisosteric and scaffold hopping
replacements.

Key words Drug discovery, Binding sites, Enzyme binding, Virtual screening, Ligand homology
modeling, Protein sequence variants, Bioisosterism, Maximum clique algorithm, Scaffold hopping,
ProBiS, ProBiS-CHARMMing, GenProBiS, LiSiCA, BoBER

1 Introduction

Computational methods that predict protein-ligand binding have

become the cornerstone in the early steps of the drug discovery
process [1, 2]. In recent years, we developed a number of such
tools that can each be used separately or one complementing the
other in the drug discovery pipeline. All of the tools are based on an
efficient maximum clique algorithm [3], which enables fast detec-
tion of similarities between several different molecule descriptors.
This enables mining of large databases, such as the Protein Data
Bank (PDB) [4], for the discovery of novel protein functions [5],
for identifying novel binding sites on proteins [6], for the study of
effects of genetic variation on ligand binding and for drug

Nikolaos E. Labrou (ed.), Targeting Enzymes for Pharmaceutical Development: Methods and Protocols, Methods in Molecular
Biology, vol. 2089, https://doi.org/10.1007/978-1-0716-0163-1_1, © Springer Science+Business Media, LLC 2020

1
2 Samo Lešnik and Janez Konc

discovery, specifically for the identification of drug repurposing

opportunities [7].
First, we present the ProBiS algorithm [8–15], which enables a
fast comparison of a query protein structure against the entire
nonredundant PDB, and detects structures in this database that
share similar three-dimensional (3D) amino acid motifs with the
query protein. Next, we describe the web server implementation of
the ProBiS algorithm combined with the CHARMMing tool for
simulating molecular dynamics [16], the ProBiS-CHARMMing
web interface [17], which additionally enables user-friendly execu-
tion of the ProBiS algorithm, identification of ligands that are able
to binding to the query protein binding site and the subsequent
minimization and potential energy prediction of such predicted
protein-ligand complexes.
Then, we present the GenProBiS [6], an interactive web plat-
form, which allows the mapping of human somatic missense muta-
tions related to cancer and nonsynonymous single nucleotide
polymorphisms from genome sequences of 21 species to protein
binding sites from the PDB. GenProBiS enables detection of
sequence variants within a protein binding site and visual explora-
tion of specific protein-ligand interactions or, alternatively, loss of
such interactions due to specific missense mutations.
Next, we present LiSiCA [18], a ligand-based virtual screening
program that enables 2D and 3D screening of large databases
consisting of millions of small molecules, such as the ZINC data-
base [19], in a very short time. The program is also implemented as
a PyMOL [20] plugin to enable easy setup of the virtual screening
procedure and subsequent visualization of the obtained
molecules [21].
Finally, we present BoBER [22], a web tool for identifying new
bioisosteric and scaffold hopping replacements by mining the
whole PDB [23]. The tool enables the input of a drug molecular
structure after which it automatically fragments the molecule and
finds possible replacements for the found fragments.
All these software tools have been widely used in various drug
discovery projects by us and by other researchers. For example,
binding site comparison for ligand prediction with ProBiS was
successfully used to discover novel potent inhibitors of the InhA
enzyme of Mycobacterium tuberculosis [7]. Using ProBiS-
CHARMMing, we have successfully predicted the holo version of
the MurA bacterial enzyme of Escherichia coli, which enabled vir-
tual screening by molecular docking that led to the discovery of two
novel inhibitors of this enzyme [24]. Using LiSiCA, we successfully
identified new butyrylcholinesterase inhibitors with low binding-
efficiency indices that could potentially lead to new treatment of
Alzheimer’s disease [18]. Reigada and coworkers [25] used LiSiCA
to repurpose the acne drug isotretinoin against Trypanosoma cruzi
parasite, the causative agent of Chagas disease. We also successfully
 Similarity-Based Drug Discovery 3

used BoBER to increase the selectivity of covalent inhibitors of

monoamine oxidase (MAO) enzyme, from MAO-A toward the
MAO-B subtype (data not yet published).

2 Computational Tools

2.1 ProBiS 1. ProBiS algorithm [8–15] superimposes complete protein sur-

faces, surface motifs, or protein binding sites. It enables pair-
wise alignments of entire protein structures or selected binding
sites as well as fast database searches for similar protein binding
sites throughout large protein databases. The algorithm can
find similar binding sites even in proteins with different folds
and without prior knowledge of their location. ProBiS algo-
rithm is able to run in parallel on multiple CPU threads. It is
available as a stand-alone program for Linux operating systems
at http://insilab.org/probis-algorithm/.
2. ProBiS-CHARMMing is a web server [17] that enables sim-
ple user-friendly setup of pairwise or database searches and
superimposition of protein structures. It requires less knowl-
edge of the command line scripting as the stand-alone version
described above. This web server combines the functionality of
ProBiS and CHARMMing [16]. The ProBiS part enables the
prediction of ligands (small molecules, proteins, nucleic acids,
or ions) that may bind to a query protein. This is achieved by
comparing a protein’s surface structure against a pre-prepared
nonredundant database of protein structures and finding those
that have binding sites similar to that of the query protein.
Existing ligands found in the similar binding sites are then
transposed to the query according to the generated binding
site superimpositions by ProBiS. The CHARMMing part then
enables the optimization of the geometry of the predicted
protein-ligand complex as well as the calculation of the
protein-ligand interaction energy, both using the extensively
validated CHARMM force field [26, 27]. The web server is
freely accessible at https://probis.nih.gov/.
3. GenProBiS web server [6] maps amino acid sequence variants
from the UniProt Variants dataset [28] and somatic missense
mutations from the COSMIC database [29] to protein struc-
tures from the PDB [4] which has been enriched with the
ProBiS predicted protein–protein, protein–nucleic acid, pro-
tein–compound, and protein–metal ion binding sites. The
server allows intuitive visual exploration of comprehensively
mapped variants, such as human somatic missense mutations
related to cancer and nonsynonymous single nucleotide poly-
morphisms from a variety of biological species, within the pre-
dicted binding sites regions for about 80,000 PDB protein
4 Samo Lešnik and Janez Konc

structures visualized in 3D using fast WebGL graphics. The

GenProBiS webserver is open and free to all users and is avail-
able at http://genprobis.insilab.org/.

2.2 LiSiCA 1. LiSiCA is a software [18] that searches for 2D or 3D simila-

rities between a reference compound and a database of target
compounds and enables ligand-based virtual screening for
drug discovery. The similarities are detected based on SYBYL
atom typing. The software enables the execution on multiple
processor threads, therefore enabling quick screening of very
large (many millions) small molecule databases. LiSiCA can be
used in Linux and Windows operating systems and can be
obtained from http://insilab.org/lisica/.
2. LiSiCA plugin for PyMOL is a graphical user interface [21]
that enables efficient use of LiSiCA functionality within the
PyMOL molecular visualization program without the need to
be familiar with the command line tool. Additionally, it offers
the possibility of direct visualization of the highest scoring
molecules and their visual comparison to the superimposed
query molecules. The plugin is available at http://insilab.
org/lisica-plugin/.

2.3 BoBER BoBER web server [22] implements an interface to a database of

bioisosteric and scaffold hopping replacements. The server enables
user-friendly search for bioisosteric and scaffold hopping replace-
ments, which are obtained by mining the PDB. It uses the ProBiS
algorithm to search for local physicochemically similar surface func-
tional groups in binding sites of holo proteins, i.e., proteins with
bound ligands. The superimposition of similar binding sites subse-
quently leads to the alignment of bound ligands. Functional groups
of ligands that align well within these superimposed binding sites
are deposited to the web server as possible bioisosterically replace-
able moieties. The web server is accessible at http://bober.insilab.
org/.

3 Methods

Here, we describe the protocols for using the computational tools

for similarity-based drug discovery described in the previous sec-
tion. The stand-alone tools require a Linux operating system,
whereas the web servers are compatible with Firefox or Chrome
web browsers on any operating system. For the stand-alone meth-
ods, the commands in the following sections can be copied and
pasted into the terminal to be executed directly. Each command
starts with “$,” which stands for the terminal prompt.
 Similarity-Based Drug Discovery 5

3.1 Use of the ProBiS We show on five different examples in a step-by-step fashion how to
Stand-Alone Program use the ProBiS stand-alone program as a command line tool. First,
we show how to superimpose a pair of two whole protein structures
based on their local surface alignments. We then show how to
specify and superimpose certain binding sites within two compared
proteins. Next, we show how to compare a single whole protein to a
database containing many protein structures and also how to com-
pare a single binding site to a database of whole protein structures.
Finally, we demonstrate how to compare a binding site against
many binding sites.
1. Prerequisites
(a) Download the ProBiS precompiled program from:
http://insilab.org/files/probis-algorithm/probis. The
examples discussed here are also freely available for down-
load from the ProBiS algorithm source code repository at:
https://gitlab.com/janezkonc/probis.
(b) Files for each example are in a directory “example{num-
ber}” in the downloaded repository. In a terminal “cd” to
an example directory to begin. New terms, options, and
modifiers, introduced in each example, are explained in
more detail in Subheading 4.
2. Example 1: Superimpose a pair of protein structures (pair-
wise alignment I)
ProBiS finds all local structural alignments of the compared
protein surfaces, and superimposes the second protein’s coor-
dinates onto the first protein’s coordinates according to the
found alignments.
(a) Superimpose two proteins using the command (see Note 1):

$ ../probis -compare -super -f1 1phr.pdb -c1 A

-f2 3jvi.pdb -c2 A

This outputs files “∗.rota.pdb” contain 3jvi’s coordi-

nates superimposed onto 1phr according to the three
different alignments found in this case. The best super-
imposition, i.e., the one with highest z_score (see Note 2)
is in file “1phrA_3jviA.0.rota.pdb.” Alignment scores are
in “REMARK PROBIS” lines in the “.rota.pdb” files.
3. Example 2: Superimpose a pair of binding sites (pairwise
alignment II)
(a) Superimpose the two binding sites defined as residues
in a 3.0 Å radius around the SO4 ligands as follows (see
Note 3):

$ ../probis -compare –super –dist 3.0 -f1

1phr.pdb -c1 A –bsite1 SO4.158.A -f2 3jvi.pdb -
c2 A –bsite2 SO4.201.A
6 Samo Lešnik and Janez Konc

(b) Alternatively, you can directly select the binding site resi-
dues around SO4 using their residue numbers and chain
identifier(s) with the “-motif” modifier (see Note 3):

$ ../probis -compare –super –motif1 “[:A and

(12-19,129-131)]” –motif2 “[:A and (7-15)]” -f1
1phr.pdb -c1 A -f2 3jvi.pdb -c2 A

The output file “1phrA_3jviA.0.rota.pdb” contains

3jvi’s coordinates superimposed onto 1phr according to
the generated alignment of the two binding sites. Align-
ment scores are in the “REMARK PROBIS” lines in the
outputted “.rota.pdb” files.
4. Example 3: Compare a protein against many protein
structures
(a) Convert protein files from the .pdb format to the surface .
srf format, which allows for faster computations (see
Notes 4 and 5):
$ ../probis -extract -f1 1phr.pdb -c1 A –
srffile 1phrA.srf
$ for i in $(cat proteins.txt); do ../probis –
extract –f1 ${i:0:4}.pdb –c1 ${i:4:1} –srffile
$i.srf; done

(b) Run ProBiS on 8 processors for query protein 1phr.A.

Resulting pairwise alignments are saved in a .nosql file.
The host file “hosts” is a text file with worker hosts
specified one per line. Run the MPI version with (see
Note 6):
$ mpiexec –v –hostfile hosts –np 8 ../probis –
surfdb –sfile srfs.txt -f1 1phr.pdb -c1 A –
nosql example.nosql

(c) Alternatively, a non-MPI version can be executed using,

e.g., 8 processor threads; however, this is only possible on
a single host computer:
$ ../probis –ncpu 8 –surfdb –sfile srfs.txt -f1
1phrA.srf –c1 A –nosql example.nosql

(d) Read a “.nosql” file and convert alignments to Json for-

mat (see Note 7). Output the query protein PDB with
residues marked by degrees of structural conservation (see
∗.cons.pdb and Note 8):
$ ../probis –results –f1 1phr.pdb –c1 A –nosql
example.nosql –json example.json
 Similarity-Based Drug Discovery 7

(e) To get superimposed proteins as .pdb run:

$ for i in $(cat proteins.txt); do ../probis

align --alno 0 --nosql example.nosql --f1 1phr.
pdb
--c1 A f2 ${i:0:4}.pdb --c2 ${i:4:1}; done

Outputs are in the form of “∗.alno.rota.pdb” files

which contain translated and rotated coordinates of
aligned proteins and the original coordinates of the
query protein. Alignments numbers start with zero, there-
fore to obtain alignments number two or three use “-alno
1” or “-alno 2” options.
5. Example 4: Compare a binding site against many protein
structures
(a) Extract the SO4 ligand’s binding site and save it to a .srf
file. This binding site is defined here as all surface residues
in a radius of 3.0 Å around the SO4 ligand (see Note 9).
Use an intact PDB file, containing the whole protein and
ligands, with ProBiS. Do not cut out binding sites into a
separate file by hand, because ProBiS will do this for you
(see Note 10):
$ ../probis –extract –bsite SO4.158.A –dist 3.0
–f1 1phr.pdb –c1 A –srffile 1phrA.srf

(b) Alternatively, the SO4 binding site residues can also be

selected by their residue numbers and chain identifiers
using the “-motif” modifier:
$ ../probis –extract –motif “[:A and (12-19),
129-131)]” –f1 1phr.pdb –c1 A –srffile
1phrA.srf

(c) Convert .pdb files to .srf for faster computation:

$ for i in $(cat proteins.txt); do ../probis
–extract –f1 ${i:0:4}.pdb –c1 ${i:4:1} –srffile
$i.srf; done

(d) Run ProBiS on 8 processors for query protein 1phr.A.

Resulting pairwise alignments are saved in a .nosql file.
The host file “hosts” is a text file with hosts specified one
per line. Run the MPI version with:
$ mpiexec –v –hostfile hosts –np 8 ../probis –
surfdb -local –sfile srfs.txt -f1 1phr.pdb -c1
A –nosql example.nosql
8 Samo Lešnik and Janez Konc

Fig. 1 ProBiS stand-alone program. Multiple superimposed binding sites (red)

with co-laterally superimposed ligands that can be seen in the center

(e) Alternatively, the non-MPI version can be started using,

e.g., 8 processor threads, however this is possible only on
a single computer:
$ ../probis –ncpu 8 –surfdb -local –sfile
srfs.txt -f1 1phrA.srf –c1 A –nosql
example.nosql

(f) Read a .nosql file and convert alignments to JSON format.

Output the query protein PDB with residues marked by
degrees of structural conservation (see ∗.cons.pdb):
$ ../probis –results –f1 1phr.pdb –c1 A –nosql
example.nosql –json example.json

(g) To obtain the superimposed proteins (Fig. 1) as .pdb file

execute:

$ for i in $(cat proteins.txt); do ../probis

align –alno 0 –nosql example.nosql –f1 1phr.pdb
–c1 A f2 ${i:0:4}.pdb –c2 ${i:4:1}; done

Outputs are in the form of “∗.alno.rota.pdb” files

which contain translated and rotated coordinates of
aligned proteins and the original coordinates of the
query protein. Alignment numbers start from zero; there-
fore, to get alignments number two or three use “-alno 1”
or “-alno 2” options.
 Similarity-Based Drug Discovery 9

6. Example 5: Compare a binding site against many other

binding sites
(a) Everything is the same as in Example 4, except that a file
with ligand codes should be prepared. The ligand codes
need to follow the same format as the “bsite” modifier and
they define the binding sites deemed for comparison (see
“proteins.txt” in this example’s directory). Here, the com-
mand for converting .pdb to “srf” files is:

$ for i in $(cat proteins.txt); do ../probis –

extract –biste ${i:6} –dist 3.0 –f1
${i:0:4}.pdb –c2 ${i:4:1} –srffile
${i:0:5}.srf; done

3.2 Use We demonstrate how the ProBiS-CHARMMing web server can be

of the ProBiS- used to identify new ligands that bind to a known inhibitor’s
CHARMMing Web binding site on the example of the butyrylcholinesterase enzyme
Server complexed with a nitroquinoline inhibitor. We also demonstrate
how to perform energy minimization on the new predicted com-
plex and how to calculate the energy of ligand binding.
1. Access the ProBiS-CHARMMing web server at https://probis.
nih.gov/.
2. In the Enter Query Protein window enter “4XII” into the PDB
ID box and to the Chain ID(s) box enter “A.” This is an X-ray
structure of the human butyrylcholinesterase complexed with a
reversible inhibitor ligand (see Note 10).
3. Click on the Select Binding Site (optional) dropdown menu. In
this example, the “40V.1001.A” represents the reversible
inhibitor of the enzyme. Therefore this radio button should
be selected. At the bottom, click on the Distance (Å) drop-
down menu and select 6.0. Consequently, a binding site con-
sisting of protein amino acid residues that are within 6.0 Å of
the inhibitor ligand (see Note 11) is defined. On the right side,
an interactive preview in JSmol of the protein and the location
and size of the currently defined binding site can be seen.
4. The other optional parameters, that is, Pairwise Align Two
Proteins or Binding Sites (optional) and Advanced Settings
(optional), are not used in this example (see Note 12).
5. In the Your e-mail address (optional) box an email address can
be entered. An email will be sent to this address with the link to
the ProBiS results after the screening procedure is completed.
6. Click on the Submit Job. This will initiate a screening of the
selected binding site (or whole protein structure) against the
prepared nonredundant PDB (see Note 13). The screening
procedure should take about 20 min to complete. ProBiS
10 Samo Lešnik and Janez Konc

output page will automatically open when the screening is

finished.
7. The protein model is shown in a JSmol molecular viewer on the
left side of the output page. The level of evolutionary conser-
vation of the defined binding side is color coded, ranging from
nonconserved (blue) to conserved (red). Hold the left mouse
button and drag to rotate the view. Click the right mouse
button to show the More option dropdown menu. Hold the
left mouse button and drag up/down to zoom. Double-click
the left mouse button and drag to translate the view.
8. On the right side of the output screen we see the predicted
small molecule ligands shown (see Note 14)—arranged accord-
ing to Confidence as default, which is express using z-score
values (see Note 2) (Fig. 2).
9. Click on the View 3D button on the ligand from the “1eve”
source protein (fifth row). This will load the predicted pose of
the selected ligand into the protein’s binding site, as can be

Fig. 2 ProBiS-CHARMMing web server. On the left side of the screen is a ligand (carbons as gray spheres) that
was transposed from the 1eve protein structure to the predicted binding site on the butyrylcholinesterase
enzyme. Butyrylcholinesterase is shown as a cartoon with rockets from blue (least conserved residues) to red
(most conserved residues). On the same side of the screen also notice two “ASHLMCM” menus for the enzyme
structure and for the inhibitor. Clicking on the “M” button and then “Minimize” will bring up the “Set
Minimization Parameters” menu with the options to set the number of minimization steps, to set the tolerance
gradient and to set the use of implicit water during the minimization process. The right side of the screen
shows the predicted ligands visible from the available tabs. These ligands can be small molecules, proteins,
nucleic acids, or ions. The functionality of the ProBiS stand-alone program can also be replicated here by
clicking on the “Similar Proteins” tab
Another Random Scribd Document
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 Artificial Intelligence - Quick Reference
Fall 2025 - Program

Prepared by: Instructor Smith

Date: July 28, 2025

Discussion 1: Current trends and future directions

Learning Objective 1: Best practices and recommendations
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 2: Ethical considerations and implications
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 3: Literature review and discussion
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 4: Statistical analysis and interpretation
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 5: Case studies and real-world applications
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Key Concept: Comparative analysis and synthesis
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Problem-solving strategies and techniques
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 7: Case studies and real-world applications
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 8: Learning outcomes and objectives
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 9: Diagram/Chart/Graph]
Definition: Theoretical framework and methodology
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Unit 2: Literature review and discussion
Important: Study tips and learning strategies
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Statistical analysis and interpretation
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Historical development and evolution
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Remember: Practical applications and examples
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Practice Problem 14: Interdisciplinary approaches
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Theoretical framework and methodology
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Experimental procedures and results
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 17: Fundamental concepts and principles
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 18: Diagram/Chart/Graph]
Example 18: Ethical considerations and implications
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Practice Problem 19: Critical analysis and evaluation
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Section 3: Study tips and learning strategies
Important: Current trends and future directions
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Literature review and discussion
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Historical development and evolution
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Learning outcomes and objectives
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Current trends and future directions
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Problem-solving strategies and techniques
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Best practices and recommendations
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 27: Practical applications and examples
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 28: Ethical considerations and implications
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Key terms and definitions
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 30: Diagram/Chart/Graph]
Practice 4: Experimental procedures and results
Practice Problem 30: Statistical analysis and interpretation
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Important: Learning outcomes and objectives
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 32: Ethical considerations and implications
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Fundamental concepts and principles
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Current trends and future directions
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Best practices and recommendations
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Practice Problem 36: Interdisciplinary approaches
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 37: Diagram/Chart/Graph]
Practice Problem 37: Learning outcomes and objectives
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Problem-solving strategies and techniques
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Remember: Study tips and learning strategies
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 40: Diagram/Chart/Graph]
Discussion 5: Theoretical framework and methodology
Example 40: Comparative analysis and synthesis
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Historical development and evolution
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 42: Diagram/Chart/Graph]
Practice Problem 42: Current trends and future directions
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Example 43: Experimental procedures and results
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Example 44: Assessment criteria and rubrics
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 45: Diagram/Chart/Graph]
Remember: Ethical considerations and implications
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Definition: Fundamental concepts and principles
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Learning outcomes and objectives
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Example 48: Best practices and recommendations
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Study tips and learning strategies
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Discussion 6: Statistical analysis and interpretation
Key Concept: Theoretical framework and methodology
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 51: Diagram/Chart/Graph]
Key Concept: Literature review and discussion
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 52: Ethical considerations and implications
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Fundamental concepts and principles
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Practical applications and examples
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
[Figure 55: Diagram/Chart/Graph]
Example 55: Research findings and conclusions
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Key Concept: Ethical considerations and implications
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Theoretical framework and methodology
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Important: Experimental procedures and results
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 59: Diagram/Chart/Graph]
Definition: Interdisciplinary approaches
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Part 7: Key terms and definitions
Practice Problem 60: Learning outcomes and objectives
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 61: Study tips and learning strategies
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 62: Diagram/Chart/Graph]
Important: Study tips and learning strategies
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Note: Problem-solving strategies and techniques
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Important: Ethical considerations and implications
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 65: Key terms and definitions
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Problem-solving strategies and techniques
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Historical development and evolution
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 68: Diagram/Chart/Graph]
Remember: Experimental procedures and results
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Experimental procedures and results
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 70: Diagram/Chart/Graph]
Discussion 8: Best practices and recommendations
Key Concept: Problem-solving strategies and techniques
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 71: Diagram/Chart/Graph]
Practice Problem 71: Ethical considerations and implications
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 72: Diagram/Chart/Graph]
Key Concept: Experimental procedures and results
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Key Concept: Best practices and recommendations
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 74: Historical development and evolution
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 75: Diagram/Chart/Graph]
Important: Fundamental concepts and principles
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Example 76: Comparative analysis and synthesis
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Remember: Historical development and evolution
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 78: Interdisciplinary approaches
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Historical development and evolution
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Exercise 9: Learning outcomes and objectives
Remember: Experimental procedures and results
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 81: Case studies and real-world applications
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Experimental procedures and results
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Current trends and future directions
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Current trends and future directions
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Key Concept: Historical development and evolution
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Statistical analysis and interpretation
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Note: Case studies and real-world applications
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 88: Literature review and discussion
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 89: Diagram/Chart/Graph]
Important: Interdisciplinary approaches
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Module 10: Interdisciplinary approaches
Practice Problem 90: Case studies and real-world applications
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Practical applications and examples
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Best practices and recommendations
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Interdisciplinary approaches
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Best practices and recommendations
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Best practices and recommendations
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 96: Diagram/Chart/Graph]
Definition: Case studies and real-world applications
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Definition: Key terms and definitions
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Key Concept: Theoretical framework and methodology
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 99: Diagram/Chart/Graph]
Remember: Interdisciplinary approaches
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Abstract 11: Fundamental concepts and principles
Key Concept: Historical development and evolution
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Comparative analysis and synthesis
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Definition: Statistical analysis and interpretation
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 103: Diagram/Chart/Graph]
Practice Problem 103: Learning outcomes and objectives
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Important: Practical applications and examples
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Definition: Practical applications and examples
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 106: Diagram/Chart/Graph]
Important: Statistical analysis and interpretation
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 107: Diagram/Chart/Graph]
Remember: Historical development and evolution
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 108: Theoretical framework and methodology
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Comparative analysis and synthesis
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 110: Diagram/Chart/Graph]
Background 12: Problem-solving strategies and techniques
Practice Problem 110: Interdisciplinary approaches
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Learning outcomes and objectives
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Ethical considerations and implications
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
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