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NOVEL DEVELOPMENT AND ADVANCEMENT APPROACHES IN

STRUCTURE-BASED DRUG DISCOVERY ON MEMBRANE

INTEGRAL G-PROTEIN COUPLED CLASS- A RECEPTORS: A

SYSTEMATIC REVIEW

Chintan Chandrakant Davande1, Ankitesh Ramesh Gade

2, Saklen Dastagir Shaikh

3,

Prasad Ramesh Gaikwad4, Shoyal Ajan Shaikh

5 and Vishal Vijay Naik

6*

1,2,3,4,5

Final year B. pharmacy of Shree Saraswati Institute of Pharmacy, Tondavali, Kankavali,

Sindhudurg, Maharashtra.

6Second year B. Pharmacy of Shree Saraswati Institute of Pharmacy, Tondavali, Kankavali,

Sindhudurg, Maharashtra.

Dr. Babasaheb Ambedkar Technological University, Lonere, Raigad, Maharashtra.

ABSTRACT

G-protein coupled receptors are the larger and most important family

of integral or transmembrane receptors which is involved in most of

the physiological and environmental stimuli. Because of this

characteristic the G-protein coupled receptors are the major target for

the discovery of new drugs associated with various mammalian

diseases. Current pharmaceutical market says over one third of

marketed products having prime target is G-protein coupled receptors.

Recent studies help to understand the structural biology of G-protein

coupled receptors which includes three dimensional structure of GPCR,

functions of GPCR, Ligand binding and pharmacological actions. This

structural biology contributed in designing a new drug or therapeutic

agent. This topic highlights latest or novel advance perspectives in GPCR structure with

focus on Membrane receptor - Ligand interaction of class-A G-protein coupled receptors

family as well as structural features for their activation, allosterism and biased signaling

mechanism. Current methodologies for structure-based drug design in GPCR are also

discussed.

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 7.632

Volume 10, Issue 7, 937-958 Review Article ISSN 2278 – 4357

*Corresponding Author

Vishal Vijay Naik

Second year B. Pharmacy of

Shree Saraswati Institute of

Pharmacy, Tondavali,

Kankavali Sindhudurg,

Maharashtra.

Article Received on

10 May 2021,

Revised on 30 May 2021,

Accepted on 20 June 2021

DOI: 10.20959/wjpps20217-19378

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KEYWORDS: - GPCRs, allosterism, biased ligand, rhodopsin, classification of GPCRs,

MARTINI model, SBDD.

INTRODUCTION

As we know, one of the largest and important transmembrane receptors family known as G-

protein coupled receptors which are also known as seven transmembrane receptors (7-TM).

[45] GPCRs plays major roles in most of physiological stimulations. These membrane proteins

consist of about 800 genes in human genome to regulate several physiological, cognitive,

behavioral, mood and immune response.[1]

Most of the therapeutic agents use in various

diseases including cancer, cardiac and CNS disorders having common target is binding to

GPCRs.[2]

The wide family of GPCRs mainly divided into six classes on the basis of

sequence and functional similarities as follows[3,4,5]

Figure 1: Classification of GPCRs.[80]

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As the class-A GPCRs known as 7-TM receptors due to their structural organization consists

7-TM helices (TM 1 - TM 7) which are linked by three extracellular (ECL-3) and three

intracellular (ICL-3) loops.[figure 2]

The 7-TM consists two modules i.e. Intracellular and

Extracellular module (consists disulfide bonding) where N-terminal of extracellular module

helps to understand variety of ligands and ligand entry modulation.[6]

C-terminal and ICLs

linked with G-proteins, GPCR kinases (GPKs) and downstream signaling effectors for signal

transduction and modulation.[45,40]

C-terminal made up of 3,4 turn α-helix known as octahelix

positioned parallel to membrane.[7]

Figure 2: Structural Features of GPCRs.[45]

This fact is helpful to recognize variety of ligands which having different physicochemical

properties helps in signaling and modulatory process. When agonist bind to receptor ligand-

induced GPCR signaling takes place cause change in 7-TM structure.[46]

Upon agonist

binding GPCRs coupled with other G-protein families and generate secondary messengers

which initiates downstream signaling. When it binds with Gαs cause activation of adenylyl

cyclase leads production of cAMP.[47]

When it binds to Gαi/0 cause inhibition of adenylyl

cyclase but activate mitogen-activated protein kinase (MAPK).[48]

When it binds with Gαq/11

cause activation of phospholipase C which undergoes hydrolysis to activate inositol-1,4,5-

triphosphate (IP3) and diacylglycerol (DAG) which responsible got increase Ca++ influx.[8]

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Structural studies of Class-A Non-rhodopsin GPCRs

Methodological advances for visualization of GPCRs & signal complexes

Due to limited expression of GPCRs, this largest family of receptors remains unexplored with

respect to their structural biology and molecular interpretation.[9]

The first GPCR found in

bovinae family which are the large family of animal including goats, sheeps, cows, buffalos

and bison. The first human GPCR structure known as β2-adrenergic receptor.[49]

GPCRs

biochemistry and X-ray Crystallography are the two important novel approaches for

visualization of GPCRs and their signaling complexes. Also other methods include protein

engineering, Lipoidal Cubic Phase (LCP) crystallization method and microfocus synchrotron

beamlines are helps to determine structures of GPCRs.[4]

These are highly flexible in nature

and coupled with different type of effectors and shows diversified signaling mechanisms.

These are embedded in bilayer lipid membrane in which T4 lysosome (T4L) and B562 RIL

(BRIL) plays an important role in protein fusion.[10]

To achieve conformation flexibility, the

thermostabilization of receptor (StaR) is done which cause stability as well as enhance

protein expression.[50]

As GPCRs are integral protein they can be extracted by forming

micelles system of N-dodecyl-β-D-maltoside (DDM) and Cholesteryl Hemmisuccinate

(CHS).[10]

In crystallization membrane proteins can be crystallized in the form of vapour or

another method use for crystallization is bicelles formation or LCP methods.[4]

In comparison

with vapour crystallization bicelles formation or LCP is more amenable method. In LCP

membrane proteins embedded in lipid environment and the interaction of these two improve

crystal constant in hydrophilic and lipophilic regions.[10]

The current advance technique in

GPCR Crystallography is X-ray free electron laser (xFEL) which working on microcrystals to

give high quality results.[4]

Structural classes of Class-A Non-Rhodopsin GPCRs

The structure based study or observations will help to design and develop the novel

therapeutics with effective pharmacological agents.[45]

In recent studies shows that 120

different structure are studied and according to structural arrangements the classification of

class-A GPCRs is given in following table:

Sr. no. Main class Sub-class Examples

1) Rhodopsin 1) Bovine rhodopsin

2) Squid rhodopsin

-----

-----

2) Aminergic 1) Adrenergic

2) Dopamine

3) Histamine

β1AR, β2AR

D3R

H1R

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4) Muscarinic

Acetylcholine

5) Serotonin

M1, M2, M3, M4

5HT1B, 5HT2B

3) Nucleotide

like

1) Adenosine

2) Purinergic

A2AAR

P2Y1, P2Y12

4) Peptide like 1) Chemokine

2) Opioid

3) Neurotensin

4) Protease activated

5) Angiotensin -II

6) Orexin

7) Endothelial

CXCR4, CCR5

δ-OR, ᴋ-OR, ꭎ-OR,

NOP

NTSR1

PAR1

AT1R

OX1R, OX2R

ETB

5) Lipid like 1) Free fatty acid

2) Sphingosine-1-PO4

3) Lysophosphatidic

acid

4) Cannabinoid

FFAR1

S1P1

LPA1

CB1

6) Unclassified ------ US 28

GPCR targeting ligands

In drug discovery targeting GPCRs involved endogenous substrate which acts as GPCRs

agonist after binding to allosteric site and substrate which does not activate GPCRs after

binding known as antagonist.[11]

According to ligand action it divides into following types:

1) Positive Allosteric Modulator (More potent)

2) Negative Allosteric Modulator (Less potent)

3) Silent Allosteric Modulator (Identical)

4) Ago-allosteric (Own potency)

Some ligands give a bitopic action where ligand binds with allosteric and othrosteric site.

Biased ligand pathway is one of the advance approach for drug design. As the most of the

Class-A GPCRs are composed of hepta-helical structure in which peptidic ligands are bind to

othrosteric site.[12]

The biased agonism or biased ligands is most promising approach in drug

design or discovery. In this the ligand shows binary actions after binding to receptor. The

biased agonism activate downstream pathway and produce distinct physiological action.[13,14]

Several studies show biased GPCRs signaling targets intracellular regions of receptor. This

will help to show high resolution structure of GPCRs.[15,16]

The biased signaling change

perception of GPCR activation and also effector coupling which is a novel approach in GPCR

drug discovery. The endogenous allosteric modulator binds to allosteric region and shows

greater therapeutic candidates.[17,18,19]

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Development approaches for 3D structure modelling

Receptor structure prediction

Methodological steps of homology modeling the two main method for protein modeling or

structural prediction is used.[24]

1) Homology modeling/comparative modeling of proteins:- It is the technique which

allows to construct structural model given protein target from its amino acid sequence and

experimental 3D structure of a related homologous protein template.[20]

The two main

hypothesis of homology modeling is as follows:

i. Each amino acid sequence determines a particular 3D structure of protein.[45]

ii. Modification or evolutionary conserved proteins have similar sequence, adopting

similar tertiary structure.[45]

This hypothesis aims to predict an unknown protein structure from template structure whose

3D structure has already experimentally figured out by X-ray, NMR (nuclear magnetic

resonance), Cryo-EM (cryogenic electron microscopy).[21]

Figure 3: - Homology modeling.[79]

Approaches for accurate prediction of target structure: - Template selection is important

and the similarity between target protein sequence and the template and these related directly

with quality of model.[21]

Homology modeling is most practical, accurate for the GPCRs

structure prediction.[22]

If the length and percentage is fall into safe region of two sequence of

the identical residue the they are practically allowed to adopt a similar structure.[figure 4]

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Figure 4: Steps of homology modeling.[45]

Denovo modeling: - In this method the algorithms do not depend on homologous template

thus it only been carried out for relatively small proteins. In this structural studies by NMR

and X-ray crystallography the Nano lipoprotein particles (NPLs) method used to express

GPCRs and model proteins such as bacteriorhodopsin reconstituted into NPL.[53]

The

bacteriorhodopsin structure served as the structural template for modeling GPCR. The

resolution of more than 30 different drug gable GPCRs structure.[54]

Since 2007, has

contributed in the development of novel crystallization techniques.[55]

The TM

(transmembrane) helical are important for making functional inference for different parts of

protein sequence. This orientation if the TM helices gives topology of proteins. The presence

of high conserved structural motifs and the structural constraint impose by the TM helical

domain make this approach successful.[56]

Figure 5: De novo modeling.[79]

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Ligand-receptor recognition, there binding mode and affinity prediction

Molecular docking: - it is device for identification of protein-ligand interaction. Molecular

docking is most common application for SBDD (structure based drug design) model.[57]

In the

molecular docking the protein cam be considered as the lock and the ligand can be considered

as key.[44]

Rigid docking (Lock and Key): - In rigid docking the internal geometry of both the ligand

and receptor are treated as rigid. The docking is important in industry and academic for

rationale design of drug with better affinity, subtype selectivity or efficacy.[57,58]

The

molecular docking predicts the most likely binding confirmation of small molecule ligands at

the appropriate target binding site.[58]

Protein flexibility in docking studies:- During the biological reaction the proteins are

undergoes conformational changes and also in molecular recognition. The common

software’s used for docking purpose are as follows: [59]

1) UCSF Dock – USA (1988)

2) Auto dock – USA (1990)

3) Flex-X Germany (1996)

4) Gold – UK (1995)

The docking program ―gold‖ is performs automated dockings with partial protein flexibility

in and around active site. It has option to set side chain flexibility for several residues.[43,44]

Advancement approaches

Structural based virtual screening

Actually virtual screening is divided into two types technique one is structural based virtual

screening (SBVS) and another is ligand based virtual screening (LBVS) as shown in a

previous flow chart.[60]

SBVS implement docking of ligands into a protein and bind to the

protein molecule with high affinity. It is an important tool to access new drug

technology/novel drug deliveries like compound. It is effective and low cost technique for

evaluation new compounds libraries.[61]

The binding site of docking provides orthosteric

ligand binding pocket which is the primary binding site for a crystal structure. The other

pockets like allosteric sits are forms bond with the allosteric ligands, but it was coupling with

the site of allosteric binding pocket and satisfy the mode of action of ligand molecule. SBVS

gives the number to the docked molecules, because the evaluation and selection is based on

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the docking score or energy.[62]

This technique is depending on the amount of information

depend on particular disease targeted. The pharmacophore hypothesis generates secondary

messenger, selected hits can give activity towards bimolecular target or biophysical

approaches i.e. Surface Plasmon Resonance (SPR) or radio-ligand binding assay, to fulfill the

binding target or receptor and predict the affinity.[63]

The SPR biosensor have a central tool

for characterizing and quantifying the bimolecular interactions. In the SBVS most important

is lead compound for the generation of new drug technique to identify the SAR study and

found lead compound.[23]

Ligand based virtual screening

The ligand based virtual screening (LBVS) is based on the screening of new molecule with

particular shape which is similar to the active molecule. This molecule is shift to the specific

binding site where they fit’s hence they occupied there targeted binding site. The specific

ligand bind to the targeted protein is known as pharmacophore method.[61]

There are two

types of pharmacophore method one is 3D and another is 2D. in 3D pharmacophore

interaction of biological compounds is based on bioactive conformation.[64]

If the 3D

structure of target is available, then the SBVS is combine to work with the LBVS for the

effective screening. 3D pharmacophore includes functional and structural features including

biological activities. The 2D pharmacophore interaction is based on chemical similarity,

where scanning of database of molecule against the one or more active structure.[57,65]

Fragment based drug design approach (FBDD)

Fragment is defined as the low molecular weight, moderately lipophilic, and highly soluble

organic compound. FBDD is used as an identification of novel hits with low molecular

weight like (100-250 Da) FBDD is work along with the combination of SBDD with soluble

protein like protease and kinases.[24]

Fragment typically binds to the low affinity with

micrometer to the millimeter range. And can grow, merge and linked with other fragment to

improve their efficiency it’s also called as computational method. In FBDD having various

subsides to bind with the small molecule but in case of large molecule having the more steric

hindrance or electrostatic clashes then the fragment molecule then it has diverse chemical

space to bind.[66]

Fragments are used in a drug design because high throughput screening

(HTS) of pre-existing compounds in various pharmacological assay. This technique is mostly

used to hit the parent compound to formed a fragment and other fragment from another

parent compound this two child fragment become a new compound.[44]

Some computational

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methods are work efficiently when combinations are used. some drawbacks of computational

method were low accuracy in fragment binding, rapid accumulation of errors.

Screening of orthosteric ligand

Orthosteric ligand means the primary, unmodulated binding site of ligand. The orthosteric

sites for binding the substrate or competitive inhibitors of enzyme and agonist or competitive

antagonist of receptors.[67]

The orthosteric ligand interacts with the same binding site as the

natural endogenous agonist like neurotransmitter or hormones. Orthosteric agonist like 5-

HT2A/2C subtype which demonstrate the different intervals of inositol phosphate (IP)

accumulation and arachidonic acid (AA) release.[68]

In that 5-HT2c receptor agonist which is

3-trifluoromethylphenyl-piperazine (TFMPP) give us IP generation rather than the AA

release, whereas the lysergic acid diethylamide give us AA release rather than IP generation.

The recent advancement in drug discovery which is the determination of x-ray structures by

the GPCR’s ligand based discovery. [28]

Novel receptor adenosine receptor (A2A) which

passes signals periphery and CNS with agonistic and antagonistic. Agonist gives the anti-

inflammatory responses and antagonistic gives the neurodegenerative disease (e.g.

Parkinson’s disease). There are four types of adenosine receptors (A1, A2A, A2B, and A3.) it’s

based on the docking studies which is bi-substituted imidazole ring utilize the extra

hydrophobic binding site for the AT1 receptor.[69]

It is angiotensin-ll receptors which is based

on the vasopressor effect and regulate aldosterone secretion. It’s important effectors for

controlling blood pressure or cardiovascular system. Four compounds having high binding

affinity towards the AT1 receptor and also high antagonistic affinity equal or same as of

losartan.[70]

The virtual screenings when we performed for identification of new compounds

or binders the receptors are tasted against the ꭎ and μ-opioid receptors.[71]

Proprietary or

public database of inactive β2 agonist combined with the inverse agonist-l and compared with

the low hit of random compound (0.0 1%) the hit rate was successful for the proprietary

database 36% and satisfactory for the ZINC (Public). This structure is commercially docked

with the 1,00,000 compounds and in that selected 25 hits, which is submitted for further

evaluation.[25,26]

Discovery of subtype or confirmation selective ligand

The discovery of subtype and confirmation selective ligand is selected difficult in the

receptors subtype. Thousands of ligands are present or known but the subtypes of receptors

are few is known.[28]

The regular modelling of receptors was difficult to identify or discover.

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As we know about the adenosine receptor there is four type (A1, A2A, A2B, and A3.) evaluate

the limit of homology modeling and docking to identify the new selectivity subtype of A1

receptor subtype is tasted against the (A1, A2A, A3.) out of four types. In the A1AR is tasted

against the subtype but it is not sufficient to tasted against the few subtype.[51,27]

GPCRs are

integral transmembrane proteins that transmitted extracellular signals from neurotransmitter,

hormone, odorants and other signals across the cellular membrane. The muscarinic

acetylcholine receptors of M1-5, using the structural based docking against the M2 and M3 we

screen the 3.1 million molecules for ligands with the new subtype selectivity’s. M3 receptor

show higher selectivity against the M2 receptor.[29]

As well as the same procedure follow by

the 5-HT1B and 5-HT2B selectivity, the 5-HT1B is more selective rather than the 5-HT2B.[30]

They showed the high affinity towards identification of compounds more than 360-folds

selectivity of target than the 5-HTB. CXCR3 and CXCR4 are the two example of CXC

chemokine receptor. This receptor is forced to identified and bind with GPCR instead of

selective ligand for specific subtype. Active state structure of β2AR to model the D2R in

active confirmations.[31]

They reported the 2.7 million lead-like and 400 k fragment like

molecules from the ZINC database, yielding several full agonist and partial agonist. β2AR

which is not suitable for the D2R agonist and also it is not transferable.[45]

Screening of allosteric Modulators and Bitopic ligand:- The term allosteric derived from

the Greek word Allos means ―others‖ and stereos means ―shape‖ or ―size‖ and combined

called the ―other site‖. Conformational changes within the receptors caused by the

modulators through which the modulators affect the receptor function.[72]

The allosteric

modulators are a group of substances that bind with the receptor to change that receptors

response to stimulus. Modulators are of three types positive, negative, and neutral. Positive

type increase the response of receptor will increase the probability of an agonist will bind to

the receptor i.e. affinity, increase its ability to activate the receptor i.e. efficacy, or both.

Negative type decreases the agonist affinity and efficacy.[52]

Neutral type which do not effect

on the agonist affinity but they can stop the others modulators from the binding site. Two

negative allosteric modulators are used in clinically that is mozobil (45 plerixafor) and

selzentry (44 maraviroc).[32]

After the autologous stem cell transplantation 45 receptor used to

promote the stem cells from the bloodstream and compound 44 is high affinity towards the

receptor CCR5 and in 2007 it was used in treatment of HIV combine with the antiretroviral

agent. As the name indicate bitopic ligand the ligand must be bind with the othosteric as well

as allosteric site. In the dopamine the crystal structure D3 receptor combined with the

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antagonist 18, and it is extending towards the ECL2 and formed by the TM-1,2,7. [33]

It

obtained the two models: one is form Apo state and other one is combined with dopamine.

The basic strength of bitopic ligand is depend on the high affinity of orthosteric binding site

and high selectivity by the allosteric binding site. These bitopic ligands add new era based on

the GPCR mechanism.[73]

Advancement in simulation tools to understand Structural and Mechanistic intricacies

of class A GPCR’s

The briefly explained in the previous section, any crystal structure represents receptor or

protein that has been captured in a specific state. There are several techniques for more

complete picture of the conformational dynamics of GPCRs, such as NMR, fluorescence-

based microscopy, and electron resonance spectroscopic technique etc. computational

stimulations helps to capture the receptor motion for complement and substantiate

experimental findings.[34]

Stimulation technique helps to determine molecular mechanism and

driving forces in biological processes.[35]

In following section, we describe about

computerized techniques their suitability and limitations.

1) Multiscale simulation techniques:- The dynamics of GPCR’s consider as a ranked

process that consider in a time scale management.[34]

The fast dynamics binding into the

ligand into a femtosecond isomerization of retinal in rhodopsin. In active site the ligand

binding into the pico-to nanosecond time scale rearrangement. i.e. the activation of

conformational sites to the inactive state activate at millisecond to the second time period.

[74] And the spatiotemporal organization of GPCR’s are made in micrometer in seconds

and more. This different levels of changes required the specific length and time cycles.[45]

i) Quantum mechanical (QM) model:- QM level of molecules introduced into the

subatomic orbitals from the electron potentials of all atoms. The Schrodinger equation is

important to solve because to understanding the quantum mechanical behavior of

molecules.[34]

In the quantum model the small size of molecules must be used to

introduced the QM level. The QM model is joined with the MM model (i.e. molecular

mechanics) gives the QM/MM multiscale models that efficiently work with each other as

a subtype of quantum descriptors.[34]

ii) Atomistic model:- In this techniques every molecule as a point particle corresponds to its

surrounding particles. In this technique the particular atom is represent the specific

position and velocity at specific time interval. When we defining the atomistic properties

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like mass, charge, and connectivity (bond length and angles) bonded terms (harmonic

potential for bonds and angles) and un-bonded terms (vdW and coulombic interactions)

requires a potential function to compute with energetics and along with the parameters.[75]

And also other several force field, CHARMM, GROMOS, OPLS, and AMBER, are

basically used to study the GPCR dynamics. All atomistic simulations are made up of

membrane protein and a bound ligand is surrounded by a lipid bilayer in the water bath.

[36]

iii) Coarse-grained model:- In this model a set of atoms is mediated by a single beds and

each molecule have a set of beds to minimize the degrees of freedom of the system. This

system is more popular because its develops for the study of dynamics in large system

and a longer time scale.[76]

The one of the best suitable model of coarse graining is the

MARTINI force field. In this model head groups of lipid molecule represents beads and

tells as bond used to simulate dynamic processes of lipid bilayer MARTINI model

represents by 1-5 beads of proteins with amino acid residue.[34]

iv) Continuum electrostatic model:- it is computerized technique helps to determined

memory involvement and solvent particles by implementing average electrostatic

properties. Commonly used methods are poisson-boltzmann (PB) and generalized born

(GB) methods which influence environment of solute. Protein and ligands are represented

at an atomistic scale with additional surface area in hybrid MM/PBSA model.[34,45]

2) Molecular dynamic simulation (MD)

It is one of the best computerized technique which helps to predict the time dependent motion

of biomolecules. Firstly, the initial position and velocities of molecules are defined after that

time evolution is carried out using molecular system using classical equation of motion of

particles. Steps to focused GPCR-MD are as follows:

i) System setup:- As the GPCRs are lipid bilayer receptors the protein insertion into the

lipid bilayer is most important step.[77]

Simulation caused by two common ways which

includes replacement method i.e. pseudo atoms of lipid distributed around the protein and

replaced by lipid molecule. Another one is insertion method in which membrane proteins

is inserted into the hole which previously made in lipid bilayer and overlapping lipid gets

removed.[37]

ii) Simulation steps:- Lipid membrane, topology and force field files are required for

incorporation of receptor-ligand structure to start simulation process. Topology includes

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structure and the bonding or connecting points between atoms. Force field file is a set of

parameter describes interactions between molecules.[45]

Simulations caused in four steps

in which first one include finding of stable point to minimize potential energy of surface,

second step include heating of system gradually up to targeted temperature (usually 300

K), third step contains the dynamic run calculation by using Newton’s equation of motion

i.e. Fi = mi * ai (where, Fi = force , mi = mass and ai = acceleration of motion) and final

step include production of dynamic stage to determine thermodynamic average or

sampling new configuration.[37]

iii) Current challenges in MD simulation:- Structural modification of proteins is the major

event in MD simulation. If high energy barrier exists between low energy states, then

simulation will cross barrier and forms new low state energy state. Advance computerized

techniques improve simulations.[58]

To improve time scale simulation improved

algorithms as well as steady increasing computer power is required (e.g. cloud

computing). Advancement in force field development will contribute into new drug

discovery and optimization with accuracy and efficiency in near future.[36]

3) Investigation of dynamic event characterizing GPCR function

MD technique implement in molecular docking to include protein flexibility. Recent

advancement in computational power and rendered MD methodologies is a complimentary

tool for experimentation.[45]

i) Prediction of binding free energy:- Receptor-ligand interactions generally predicted by

docking scores. for the prediction of binding free energy of given ligand surrounding

environmental factors like receptor, membrane and solvated water molecules should be

considered.MM/PBSA (molecular mechanics Poisson-Boltzmann surface area)

approaches use for calculation of salvation free energy. Free energy perturbation (FEP)

and thermodynamic integration (TI) are more accurate methods to describe binding

affinities.[34]

ii) State Transition and Signaling complex formation:- Dynamics of GPCRs is important

aspect for their activation. Major focus of current structural studies on GPCRs is to

investigate fully active states complexes with signaling proteins, G-proteins or β-arrestin.

Computational studies help to analyze activation dynamics from inactive state of receptor

to its active state. In active state highly conserved residue of class-A GPCRs involved in

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complex formation with inter and intrahelical hydrogen bonding. In transition state

GPCRs coupled with partner proteins ternary signaling proteins.[38,39]

iii) Allosterism:- It is a condition where activity of protein is altered or modified due to

consequence of some molecular binding at different site from the active site or change in

conformation of an enzyme.[78]

Recent studies in allostery shows dynamic and

intrinsically disordered proteins. Computational methods help to explore allosteric events

at atomic scale. Many studies focus on transient allosteric site, receptor conformation and

statistical nature of interaction responsible for information transmission.[41]

iv) Detection of binding paths of GPCRs ligands:- The path ways of ligand entry into

β2AR, subsequent binding site and exit from receptor were analyze in pioneer studies.

According to that two main points for ligand entry. This process can study by using

microsecond time scale unbiased atomistic MD simulation.[42]

CONCLUSION

In past few decade evolution in the advancement techniques for structural biology of GPCRs

shows promising results. This will help to determining or predict GPCR functions, ligand

binding and pharmacological actions as well as design for new drugs. This will help in future

for predicting receptor-ligand interactions of class-A GPCRs as well as their activation

mechanism, biased signaling and allosteric mechanism. In the above topic current scenario or

computational techniques use for structure based drug design has been summarized. It also

includes collaborative work of GPCRs with structural biology, molecular pharmacology,

medicinal chemistry and computational methods which help to provide comprehensive

picture of structural and functional characteristics of GPCRs and found new drug moiety

which work more efficiently than the previous one. Although there will be increase in the

success stories in the field of SBDD but also there are some limitations. Most of the structural

features are estimated by Homology Modelling as compare to crystallography. As the GPCRs

are the dynamic in nature, it provides variety of binding sites for ligand molecules.

Conformational changes of proteins initiated at the GPCRs binding site to understand protein

function and signaling pathways. MD simulation is a new static method to provide

opportunities for studying protein dynamics and functional models of GPCRs. Association of

MD simulation with computer power technology will produce detailed information related to

structure based drug delivery. The dynamic methods provide clue related to molecular

interactions of ligands with different therapeutically activities i.e. agonism, antagonism and

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biased agonism. Structural studies also give details about binding sites such as allosteric

binding site and G-protein or β-arrestin binding sites. In most of drug discovery studies

orthrosteric site in TM region is a targeting site but currently lipid-exposed site is highly

demanding to understand in GPCR function. To understand GPCR signaling mechanism and

to achieve rational drug discovery will be the challenge.

ACKNOWLEDGEMENT

To the best of our knowledge, the material included in this topic is having original sources

are appropriately acknowledged and referred. We would like to express our sincere gratitude

to our professor Mrs. Rohini Vichare for her continuous guidance support and motivation.

We would like to thank to fellow teaching staff of Shree Saraswati institute of pharmacy. We

would also like to thank our library staff.

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