In silicon drug design towards inhibition of Lewy body formation in Parkinson

disease
 RESULTED IN THE SUCCESSFUL COMPLETION OF THIS PROJECT.

Table of
S.No. contents Page No.
Abstract and
1 Background 6
Parkinson
2 Disease 6
3 Alpha Synuclein 18
Polymerization
of Alpha
4 Synuclein 26
 Procedure of the
5 drug design 26
Step 1
(obtaining the
6 structure) 27
Step 2
(GRAMM­X
7 docking) 30
Step 3 (analysis
8 of model 8) 37
Step 4 (analysis
9 of model 4) 39
Protein
Interactions
Calculator
10 result 40
Step 5
11 (SCWRL) 42
Step 6 (Auto
12 Docking) 44
13 Step 7 (result) 49
14 Step 8 50
 (conclusion)
15 References  51

Abstract:

Aggregation of misfold proteins into amyloid oligomers or fibrils that are deposited as
pathological lesions within areas of the brain cause many neurodegenerative diseases.
Identifying agents that inhibit the onset or propagation of proteins aggregation is an
attractive   therapeutic   strategy   for   such   diseases.   Our   proteins   of   interest   are   Alpha
synuclein,   the   major   protein   component   of   lewy   bodies   associated   with   Parkinson’s
disease.

Background:

Parkinson’s disease:

First described in 1817, PD is the most common progressive movement disorder in the
elderly and is characterized by tremor, rigidity, and bradykinesia. There is increasing
evidence that PD is a multisystemic disorder showing both progressive degeneration of
the dopaminergic nigrostriatal system and widespread extranigral pathology. In PD, LB
pathology first appears in lower brainstem nuclei such as the dorsal motor nucleus of the
vagus and the olfactory system. Afterwards,ascending progression leads to changes in
the coeruleus complex, substantia nigra pars compacta, basal forebrain magnocellular
nucleus, subthalamic nucleus, and amygdala (Stages 3­4). Finally, involvement of the
neocortex may supervene (Stages 5­6).

Over   the   last   few   years   advances   in   PD   genetics   have   revealed   that   mutations   are
responsible for only a small proportion of cases, the majority being of sporadic origin.
Of the six genes responsible for Mendelian forms of PD, the first identified was the AS
(SNCA) gene, in which three pathogenic point mutations (A30P, E476K and A53T) as
well as duplications and triplications have been detected. Genes involved in PD genetics
by mutations in autosomic dominant familial cases the ubiquitin Cterminal hydrolase L1
(UCH­L1) gene, the dardarin gene, leucine­rich kinase 2 (LRRK 2) and the HtrA2/Omi
gene. DJ­1 gene, PTEN­induced putative kinase 1 (PINK1) gene and parkin (PRKN)
gene mutations are responsible for autosomal recessive Parkinson cases.
Interestingly, the vast majority of PD instances associated with PRKN mutations lack
LBs  Sporadic PD  have  been associated  to  mutations  in the  synphilin, LRRK  2  and
HtrA2/Omi genes and to the S18Y polymorphism of the UCH­L1 gene that lowers the
risk to suffer PD 

DLB   is   the   second   most   frequent   cause   of   dementia   in   the   elderly   after   Alzheimer
disease (AD) nd is clinically characterized by progressive dementia, often accompanied
by parkinsonism and psychiatric symptoms. Widespread distribution of LBs in virtually
every brain area is a typical feature of DLB, although the frontal cortex, pigmented
midbrain and brainstem  nuclei, dorsal efferent nucleus of  the vagus,  basal forebrain
nuclei, and limbic cortical regions are particularly involved. A high percentage of DLB
cases   show,   in   addition   to   LB   related   pathology,   AD   characteristic   changes,   where
higher Braak stages of AD­type pathology result in a clinical diagnosis of AD rather
than   DLB.   Accordingly,   the   misdiagnosis   of   DLB   increases   with   increasing   Braak
stages of AD associated pathology.

Four mutations, the E46K mutation on SNCA, the UCH­L1 gene I93M mutation and
two beta­synuclein mutations (V70M and P123H) have been described in four DLB
pedigrees.   In   contrast,   up   to   the   present,   no   genetic   marker   has   been   found   to   be
associated with sporadic DLB.

Parkinson's disease (also known as Parkinson disease or PD) is a degenerative disorder
of the central nervous system that often impairs the sufferer's motor skills, speech, and
other functions.

It   is   characterized   by   muscle   rigidity,   tremor,   a   slowing   of   physical   movement

(bradykinesia)   and   a   loss   of   physical   movement   (akinesia)   in   extreme   cases.   The
primary symptoms are the results of decreased stimulation of the motor cortex by the
basal ganglia, normally caused by the insufficient formation and action of dopamine,
which is produced in the dopaminergic neurons of the brain. Secondary symptoms may
include   high   level   cognitive   dysfunction   and   subtle   language   problems.   PD   is   both
chronic and progressive.

PD   is   also   called   "primary   parkinsonism"   or   "idiopathic   PD"   (classically   meaning

having no known cause although this term is not strictly true in light of the plethora of
newly   discovered   genetic   mutations).   While   many   forms   of   parkinsonism   are
"idiopathic", "secondary" cases may result from toxicity most notably of drugs, head
trauma, or other medical disorders. 

The   term   Parkinsonism   is   used   for   symptoms   of   tremor,   stiffness,   and   slowing   of
movement   caused   by   loss   of   dopamine.   "Parkinson's   disease"   is   the   synonym   of
"primary parkinsonism", i.e., isolated parkinsonism due to a neurodegenerative process
without any secondary systemic cause. In some cases, it would be inaccurate to say that
the cause is "unknown", because a small proportion is caused by genetic mutations. It is
possible   for   a   patient   to   be   initially   diagnosed   with   Parkinson's   disease   but   then   to
develop additional features, requiring revision of the diagnosis.

There are other disorders that are called Parkinson­plus diseases. These include: multiple
system   atrophy   (MSA),   progressive   supranuclear   palsy   (PSP)   and   corticobasal
degeneration   (CBD).   Some   include   dementia   with   Lewy   bodies   (DLB)   —   while
idiopathic Parkinson's disease patients also have Lewy bodies in their brain tissue, the
distribution is denser and more widespread in DLB. Even so, the relationship between
Parkinson disease, Parkinson disease with dementia (PDD), and dementia with Lewy
bodies (DLB) might be most accurately conceptualized as a spectrum, with a discrete
area   of   overlap   between   each   of   the   three   disorders.   The   cholinesterase   inhibiting
medications have shown preliminary efficacy in treating the cognitive, psychiatric, and
behavioral aspects of the disease of both PD and DLB. The natural history and role of
Lewy bodies is little understood.

These   Parkinson­plus   diseases   may   progress   more   quickly   than   typical   idiopathic
Parkinson disease. If cognitive dysfunction occurs before or very early in the course of
the movement disorder, then DLBD may be suspected. Early postural instability with
minimal   tremor,   especially   in   the   context   of   ophthalmoparesis,   should   suggest   PSP.
Early autonomic dysfunction, including erectile dysfunction and syncope, may suggest
MSA. The presence of extreme asymmetry with patchy cortical cognitive defects such
as   dysphasia   and   apraxias   (especially   with   "alien   limb"   phenomena)   should   suggest
CBD.
The usual anti­Parkinson's medications are typically either less effective or completely
ineffective in controlling symptoms; patients may be exquisitely sensitive to neuroleptic
medications like haloperidol, so correct differential diagnosis is important.

Essential tremor may be mistaken for Parkinson's disease, but lacks all other features
besides   tremor,   and   has   particular   characteristics   distinguishing   it   from   Parkinson's
disease, such as improvement with beta blockers and alcoholic beverages.

Signs and symptoms:

Four motor symptoms are considered cardinal in PD: tremor, rigidity, bradykinesia and
postural instability. Tremor is the most apparent and well­known symptom. It is most
commonly   a   rest   tremor:   maximal   when   the   limb   is   at   rest   and   disappearing   with
voluntary movement and sleep. It affects to a greater extent the most distal part of the
extremity and is typically unilateral at onset. Though around 30% of PD sufferers do not
have tremor at disease onset most of them would develop it along the course of the
disease. Rigidity is due to joint stiffness and increased muscle tone, which combined
with a resting tremor produce a ratchety, "cogwheel rigidity" when the limb is passively
moved. Rigidity may be associated with joint pain, such pain being a frequent initial
manifestation   of   the   disease.   Bradykinesia   (slowness   of   movement)   is   the   most
characteristic   clinical   feature   of   PD   and   it   produces   difficulties   not   only   with   the
execution of a movement but also with its planning and initiation. The performance of
sequential and simultaneous movements is also hindered. In the late stages of the disease
postural instability is typical, which leads to impaired balance and falls

PD  motor   symptomatology is  not  limited  to these   four  symptoms.   Gait and  posture
disturbances such as decreased arm swing, a forward­flexed posture and the use of small
steps when walking; speech and swallowing disturbances; and other symptoms such as a
mask­like face expression or a small handwriting are only examples of the ample range
of common motor problems that can appear with the disease.

Example of reported prevalences of
mood problems in PD patients with
dementia
Mood problem Prevalence
Depression  58%
Apathy  54%
Anxiety  49%
Parkinson's   disease   causes   neuropsychiatric   disturbances,   which   include   mainly
cognition, mood and behaviour problems and can be as disabling as motor symptoms.

Cognitive disturbances occur even in the initial stages of the disease in some cases. A
very high proportion of sufferers will have mild cognitive impairment as the disease
advances.   Most   common   cognitive   deficits   in   non­demented   patients   are   executive
dysfunction,   which   translates   into   impaired   set   shifting,   poor   problem   solving,   and
fluctuations   in   attention   among   other   difficulties;   Slowed   cognitive   speed,   memory
problems; specifically in recalling learned information, with an important improvement
with cues; and visuo spatial skills difficulties, which are seen when the person with PD
is   for   example   asked   to   perform   tests   of   facial   recognition   and   perception   of   line
orientation.   Deficits   tend   to   aggravate   with   time,   developing   in   many   cases   into
dementia. A person with PD has a six­fold increased risk of suffering it and the overall
rate   in   people   with   the   disease   is   around   30%.   Moreover,   prevalence   of   dementia
increases in relation to disease duration, going up to 80%. Dementia has been associated
with a reduced quality of life in disease sufferers and caregivers, increased mortality and
a higher probability of attending a nursing home

Cognitive   problems   and   dementia   are   usually   accompanied   by   behaviour   and   mood
alterations, although these kinds of changes are also more common in those patients
without   cognitive   impairment   than   in   the   general   population.   Most   frequent   mood
difficulties include depression, apathy and anxiety. Obsessive–compulsive behaviours
such as craving, binge eating, hyper sexuality, pathological gambling, or other, can also
appear in PD, and have been related to a dopamine dysregulation syndrome associated
with the medications for the disease.
Pathophysiology:

Dopaminergic   pathways   of   the   human   brain   in   normal   condition   (left)   and

Parkinson's disease (right). 

The   symptoms   of   Parkinson's   disease   result   from   the   greatly   reduced   activity   of
pigmented dopamine­secreting (dopaminergic) cells in the pars compacta region of the
substantia nigra (literally "black substance"). These neurons project to the striatum and
their loss leads to alterations in the activity of the neural circuits within the basal ganglia
that regulate movement, in essence an inhibition of the direct pathway and excitation of
the indirect pathway.

HYPERLINK "http://en.wikipedia.org/wiki/File:NIGRA2.jpg"

Black­staining granules of neuromelanin within neurons of the substantia nigra

The direct pathway facilitates movement and the indirect pathway inhibits movement,
thus   the   loss   of   these   cells   leads   to   a   hypokinetic   movement   disorder.   The   lack   of
dopamine results in increased inhibition of the ventral anterior nucleus of the thalamus,
which sends excitatory projections to the motor cortex, thus leading to hypokinesia.

There are four major dopamine pathways in the brain; the nigrostriatal pathway, referred
to   above,   mediates   movement   and   is   the   most   conspicuously   affected   in   early
Parkinson's disease. The other pathways are the mesocortical, the mesolimbic, and the
tuberoinfundibular.   Disruption   of   dopamine   along   the   non­striatal   pathways   likely
explains much of the neuropsychiatric pathology associated with Parkinson's disease.

The   mechanism   by   which   the   brain   cells   in   Parkinson's   are   lost   may   consist   of   an
abnormal   accumulation   of   the   protein   alpha­synuclein   bound   to   ubiquitin   in   the
damaged   cells.   The   alpha­synuclein­ubiquitin   complex   cannot   be   directed   to   the
proteosome.   This   protein   accumulation   forms   proteinaceous   cytoplasmic   inclusions
called Lewy bodies. The latest research on pathogenesis of disease has shown that the
death of dopaminergic neurons by alpha­synuclein is due to a defect in the machinery
that   transports   proteins   between   two   major   cellular   organelles   —   the   endoplasmic
reticulum (ER) and the Golgi apparatus. Certain proteins like Rab1 may reverse this
defect caused by alpha­synuclein in animal models.

Excessive   accumulations   of   iron,   which   are   toxic   to   nerve   cells,   are   also   typically
observed in conjunction with the protein inclusions. Iron and other transition metals
such as copper bind to neuromelanin in the affected neurons of the substantia nigra.
Neuromelanin   may   be   acting   as   a   protective   agent.   The   most   likely   mechanism   is
generation  of   reactive  oxygen  species.Iron   also  induces   aggregation  of  synuclein  by
oxidative mechanisms. Similarly, dopamine and the byproducts of dopamine production
enhance alpha­synuclein aggregation. The precise mechanism whereby such aggregates
of alpha­synuclein damage the cells is not known. The aggregates may be merely a
normal reaction by the cells as part of their effort to correct a different, as­yet unknown,
insult. Based on this mechanistic hypothesis, a transgenic mouse model of Parkinson's
has been generated by introduction of human wild­type alpha­synuclein into the mouse
genome under control of the platelet­derived­growth factor­β promoter.

A recent view of Parkinson's disease implicates specialized calcium channels that allow
substantia nigra neurons, but not most neurons, to repetitively fire in a "pacemaker" like
pattern. The consequent flooding of calcium into these neurons may aggravate damage
to mitochondria and may cause cell death. One study has found that, in experimental
animals,   treatment   with   a   calcium   channel   blocker   isradapine   had   a   substantial
protective effect against the development of Parkinson's disease.

HYPERLINK "http://upload.wikimedia.org/wikipedia/commons/c/c9/Basal_ganglia_circuits.png"

Basal ganglia circuit (Normal)
 HYPERLINK
"http://upload.wikimedia.org/wikipedia/commons/b/b0/Basal_ganglia_in_Parkinson's_disease.png"

Basal ganglia circuit (In parkinson’s disease)

Research directions

Gene therapy

Currently under investigation is gene therapy. This involves using a non­infectious virus
to shuttle a gene into a part of the brain called the subthalamic nucleus (STN). The gene
used leads to the production of an enzyme called glutamic acid decarboxylase (GAD),
which catalyses  the production of  a neurotransmitter  called GABA.GABA  acts  as  a
direct inhibitor on the overactive cells in the STN.

GDNF infusion involves the infusion of GDNF (glial­derived neurotrophic factor) into
the   basal   ganglia   using   surgically   implanted   catheters.   Via   a   series   of   biochemical
reactions,   GDNF   stimulates   the   formation   of   L­dopa.   GDNF   therapy   is   still   in
development.

Neuroprotective treatments

Neuroprotective treatments are at the forefront of PD research, but are still under clinical
scrutiny.   These   agents   could   protect   neurons   from   cell   death   induced   by   disease
presence   resulting   in   a   slower   progression   of   disease.   Agents   currently   under
investigation   as   neuroprotective   agents   include   anti­apoptotic   drugs   (CEP   1347   and
CTCT346), lazaroids, bioenergetics, antiglutamatergic agents and dopamine receptors.
Clinically   evaluated   neuroprotective   agents   are   the   monoamine   oxidase   inhibitors
selegiline and rasagiline, dopamine agonists, and the complex I mitochondrial fortifier
coenzyme Q10.

Neural transplantation

The   first   prospective   randomised   double­blind   sham­placebo   controlled   trial   of

dopamine­producing cell transplants failed to show an improvement in quality of life
although some significant clinical improvements were seen in patients below the age of
60. A significant problem was the excess release of dopamine by the transplanted tissue,
leading to dystonias. Research in African green monkeys suggests that the use of stem
cells might in future provide a similar benefit without inducing dystonias.
Alternative Treatments

Nutrients have been used in clinical studies and are used by people with PD in order to
partially treat PD or slow down its deterioration. The L­dopa precursor L­tyrosine was
shown to relieve an average of 70% of symptoms.Ferrous iron, the essential cofactor for
L­dopa biosynthesis was shown to relieve between 10% and 60% of symptoms in 110
out of 110 patients.More limited efficacy has been obtained with the use of THFA,
NADH, and pyridoxine—coenzymes and coenzyme precursors involved in dopamine
biosynthesis.Vitamin C and vitamin E in large doses are commonly used by patients in
order to theoretically lessen  the cell damage that occurs in PD. This is because  the
enzymes superoxide dismutase and catalase require these vitamins in order to nullify the
superoxide   anion,   a   toxin   commonly   produced   in   damaged   cells.   However,   in   the
randomized controlled trial, DATATOP of patients with early PD, no beneficial effect
for vitamin E compared to placebo was seen. Coenzyme Q10 has more recently been
used for similar reasons. MitoQ is a newly developed synthetic substance that is similar
in structure and function to coenzyme Q10. Most of these therapies are covered in Dr.
Laurie Mischley's Natural Therapies for Parkinson's Disease.

Alpha Synuclein:

Alpha­synuclein chain as seen in VMD

Alpha­synuclein  HYPERLINK "http://en.wikipedia.org/wiki/Primary_structure" \o "Primary

structure"primary structure is usually divided in three distinct domains:

Residues   1­60:   An  HYPERLINK "http://en.wikipedia.org/wiki/Amphiphile" \o

"Amphiphile"amphipathic   N­terminal   region   dominated   by   four   11­residue   repeats
including   the  HYPERLINK "http://en.wikipedia.org/wiki/Consensus_sequence" \o "Consensus
sequence"consensus   sequence   KTKEGV.   This   sequence   has   a   structural  HYPERLINK
"http://en.wikipedia.org/wiki/Alpha_helix" \o "Alpha helix" alpha   helix   propensity   similar   to
apolipoproteins­binding domain.
Residues   61­95:   A   central   hydrophobic   region   which   includes   the  non­amyloid
component (NAC) region, involved in protein aggregation.
Residues   96­140:   an   highly   acidic   and  HYPERLINK "http://en.wikipedia.org/wiki/Proline" \o
"Proline"proline­rich region which has no distinct structural propensity
Alpha­Synuclein (­SYN) is a small protein (14 kDa) expressed at high   levels in nervous
tissue. Three point mutations and multiplication  events in the gene encoding ­syn have
been   associated   with   rare  inherited   forms   of   Parkinson’s   disease   (PD).  These   links
between ­syn and PD led to the discovery that Lewy   bodies (LBs) and Lewy neurites
(LNs), the characteristic lesions in brains of patients with sporadic PD and dementia with
Lewy  bodies (DLB), contain ­syn fibrils . Several neurodegenerative  diseases involve
­syn deposition and are hence collectively  known as "synucleinopathy disorders". There
is   substantial   evidence   to   suggest   that   the   conversion   of   Alpha­syn   from   soluble
monomers to aggregated, insoluble forms in the   brain is a key event in the pathogenesis
of PD and related diseases. A specific PD­related problem is   the long latency between
the first damage to cells in at­risk   nuclei of the nervous system, including the substantia
nigra  in the human brainstem and the onset of clinical symptoms. The   symptoms and
signs of PD do not develop until 70–80% of  dopaminergic neurones have already been
lost. To date, there is no readily available serological or urine analysis­based  test that can
confirm   the   diagnosis   of   PD   in   already   symptomatic   patients.   The   need   for   such
diagnostic   methods   is   amply   demonstrated  by   studies   indicating   that   the   clinical
diagnosis of this disease  during life is correct in 75% of cases when made by general
neurologists,   and   in   85%   when   made   by   movement   disorder   specialists .  The
development   of   reliable   surrogate   markers   for  the   presence   and   abundance   of   ­syn
lesions   (LBs,   LNs,   and   glial  cytoplasmic   inclusions)   in   the   brain   would   naturally
facilitate  a   more   streamlined   work­up   during   the   early   care   of   movement   disorder
patients, and allow for the biologically guided evaluation  of future drug trials aimed at
neuroprotection in the synucleinopathies. 
­Synuclein (­syn) protein has been linked to the  pathogenesis of PD with the discovery
of mutations in the gene  encoding ­syn in familial cases with early­onset PD. Lewy
bodies  and Lewy neurites, which constitute the main pathological features   in the brains
of   patients   with   sporadic   PD   and   dementia   with  Lewy   bodies,   are   formed   by   the
conversion of soluble monomers of ­syn into insoluble aggregates.

Tissue expression of Alpha synuclein:
Alpha­synuclein is primarily found in neural tissue, making up to 1% of all proteins in
the cytosol. It is predominantly expressed in the neocortex, hippocampus, substantia
nigra, thalamus, and cerebellum. It is predominantly a neuronal protein, but can also be
found in glial cells. In melanocytic cells, SNCA protein expression may be regulated by
MITF.

It has been established that alpha­synuclein is extensively localized in the nucleus of
mammalian brain neurons, suggesting a role of alpha­synuclein in the nucleus.Synuclein
is however found predominantly in the presynaptic termini, in both free or membrane­
bound forms,with roughly 15% of synuclein being membrane­bound in any moment in
neurons.

At   least   three   isoforms   of   synuclein   are   produced   through   alternative   splicing.The

majority form of the protein, and the one most investigated, is the full 140 aminoacids­
long transcript. Other isoforms are alpha­synuclein­126, where exon 3 is lost and lacks
residues 41­54; and alpha­synuclein­112, which lacks residue 103­130 due to loss of
exon 5

     α­synuclein chain showing the hydrophobic 

          as well as hydrophilic sides.

The normal function of  α­synuclein is poorly understood. Although it is expressed at
high levels in the brain, relatively specifically within neurons, it is also found in other
tissues,  e.g., hematopoietic cells .  α­Synuclein can bind to lipids and, in neurons, is
associated with presynaptic vesicles and the plasma membrane, possibly via lipid rafts .
The association of α­synuclein with vesicles is modulated by synaptic activity where the
protein   dissociates   from   vesicles   after   electrical   stimulation   of   the   neuron   and   only
slowly   re­associates.   However,   α­synuclein   knockout   mice   show   only   subtle
abnormalities in neurotransmission, suggesting that  α­synuclein plays a non­essential
function at the synapse. There is some evidence that such a modulatory role may be
more   important   under   conditions   where   reactive   oxygen   species   or   nitric   oxide   are
present, although the mechanism(s) are not yet fully defined.

In the normal brain,  α­synuclein immunostaining reveals a diffuse pattern of reactivity
throughout   the   neuropil   that   would   be   consistent   with   a   predominantly   synaptic
localization. However, in PD brains,  α­synuclein antibodies strongly stain Lewy bodies
and   Lewy   neurites   .   Because   of   this   sensitivity,   α­synuclein   staining   is   now   more
commonly   used   than   eosin   or   ubiquitin   staining   for   these   structures.   Biochemical
analyses have shown that α­synuclein is a major protein component of Lewy bodies and
may be part of the fibrillar structure of these structures. The deposited, pathological
forms of  α­synuclein are aggregated and show lower solubility than the normal protein
and   may   be  modified  post­translationally   by  truncation,  nitration,  ubiquitylation   and
phosphorylation.

Therefore,   α­synuclein   protein   deposition   into   Lewy   bodies   is   a   marker   of   the   PD

disease state. However, because we require Lewy bodies for a PD diagnosis this isn't an
especially strong argument for involvement of  α­synuclein in the disease process. It is
also important to note that, although we cannot determine if Lewy bodies previously
formed in the cells that eventually died, the individual neurons where Lewy bodies are
found are the ones that have survived the disease process (though they still may be
dysfunctional). Very recently, it has been shown that Lewy bodies form in functional
dopaminergic   neurons   grafted   in   to   brains   of   people   with   PD   as   a   therapeutic
intervention,   although   this   is   not   always   seen   .   These   were   embryonic   cells   that
remained   apparently   healthy   and   were   functional   after   grafting,   which   suggests   that
there is the environment of the PD brain predisposes even young cells to form Lewy
bodies.
Alpha Synuclein aggregation pathway:

Events in α­synuclein toxicity:

The   central   panel   shows   the   major   pathway   for   protein   aggregation.   Monomeric   α­
synuclein is natively unfolded in solution but can also bind to membranes in an  α­helical
form. It seems likely that these two species exist in equilibrium within the cell, although
this is unproven. From in vitro work, it is clear that unfolded monomer can aggregate
first into small oligomeric species that can be stabilized by  β­sheet­like interactions and
then into higher molecular weight insoluble fibrils. In a cellular context, there is some
evidence that the presence of lipids can promote oligomer formation:  α­synuclein can
also form annular, pore­like structures that interact with membranes. The deposition of
α­synuclein into pathological structures such as Lewy bodies is probably a late event
that occurs in some neurons. On the left hand side are some of the known modifiers of
this process. Electrical activity in neurons changes the association of  α­synuclein with
vesicles and may also stimulate polo­like kinase 2 (PLK2), which has been shown to
phosphorylate   α­synuclein   at   Ser129.   Other   kinases   have   also   been   proposed   to   be
involved. As well as phosphorylation, truncation through proteases such as calpains, and
nitration, probably through nitric oxide (NO) or other reactive nitrogen species that are
present during inflammation, all modify synuclein such that it has a higher tendency to
aggregate. The addition of ubiquitin (shown as a black spot) to Lewy bodies is probably
a secondary process to deposition. On the right are some of the proposed cellular targets
for   α­synuclein   mediated   toxicity,   which   include   (from   top   to   bottom)   ER­golgi
transport,   synaptic   vesicles,   mitochondria   and   lysosomes   and   other   proteolytic
machinery. In each of these cases, it is proposed that α­synuclein has detrimental effects,
listed below each arrow, although at this time it is not clear if any of these are either
necessary or sufficient for toxicity in neurons.

Polymerization (Self­oligomerization) of a­synuclein:

Oligomerization and aggregation of alpha­synuclein molecules are believed to play a
major role in neuronal dysfunction and loss in Parkinson's disease (PD) and dementia
with   Lewy   bodies.   However,   alpha­synuclein   oligomerization   and   aggregation   have
been   detected   only   indirectly   in   cells   using   detergent   extraction   methods.   Alpha­
Synuclein is a component of the abnormal protein depositions in senile plaques and
Lewy bodies of Alzheimer's disease (AD) and Parkinson's disease respectively.

Two   α­synuclein   chains   showing   hydrophobic   interactions   which   leads   to

aggregation as the formation of Lewy bodies.

Procedure of the drug design:
We used two molecular modelling softwares in generating the drug design, which are 
VMD (Visual Molecular Dynamics) and Auto Dock. SCRWL was then used for 
generating a high­resolution 3D model of a protein's structure, based on the known 
structure of a related protein, in our case it was Alpha Synuclein. The mutated models of
our protein were then Auto docked using the Auto dock software and finally we reached 
to our desired goal.

Step 1 (obtaining the experimental structure of Alpha Synuclein):
We downloade the experimentally determined structure of Alpha synuclein from the
Protein   Data   Bank  HYPERLINK
"http://www.pdb.org/pdb/home/home.do"http://www.pdb.org/pdb/home/home.do 

The Protein Data Bank (PDB) archive is the single worldwide repository of information
about the 3D structures of large biological molecules, including proteins and nucleic
acids. These are the molecules of life that are found in all organisms including bacteria,
yeast, plants, flies, other animals, and humans. Understanding the shape of a molecule
helps   to   understand   how   it   works.   This   knowledge   can   be   used   to   help   deduce   a
structure's role in human health and disease, and in drug development. The structures in
the archive range from tiny proteins and bits of DNA to complex molecular machines
like the ribosome. 
The PDB archive is available at no cost to users. The PDB archive is updated each week
at the target time of Wednesday 00:00 UTC (Coordinated Universal Time). The most
recent release is times tamped and linked on every page in the top right header. 
The PDB was established in 1971 at Brookhaven National Laboratory and originally
contained   7   structures.   In   1998,   the   Research   Collaboratory   for   Structural
Bioinformatics (RCSB) became responsible for the management of the PDB. In 2003,
the HYPERLINK "http://www.wwpdb.org" \t "_blank"wwPDB was formed to maintain a single
PDB archive of macromolecular structural data that is freely and publicly available to
the global community. It consists of organizations that act as deposition, data processing
and distribution centers for PDB data. 
After obtaining the experimental structure of our proteins from the Protein Data Bank,
we also obtained the sequence of Alpha synuclein and Beta synuclein protein from the
NCBI   database   and   ran   them   against   BLAST   (HYPERLINK
"http://blast.ncbi.nlm.nih.gov/Blast.cgi"http://blast.ncbi.nlm.nih.gov/Blast.cgi ) 

The results were very important for us in order to know where both of these proteins
differ in their sequence. The sequence similarity server found out that Alpha synuclein
and Beta synuclein have difference in their sequences from residue 73 to residue 83.

These residues are the basis of our drug designing experimentation. As Beta synuclein
polymerize and DO NOT lead to the formation of lewy bodies while Alpha synuclein
polymerize at around these residues and leads to aggregation.

Alpha­synuclein sequence [Homo sapiens]:

>gi|16356657|gb|AAL15443.1|
MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGV
VHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKK
DQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA

Beta­synuclein sequence [Homo sapiens]:

>gi|2501105|sp|Q16143.1|
MDVFMKGLSMAKEGVVAAAEKTKQGVTEAAEKTKEGVLYVGSKTREGV
VQGVASVAEKTKEQASHLGGAVFSGAGNIAAATGLVKREEFPTDLKPEEV
AQEAAEEPLIEPLMEPEGESYEDPPQEEYQEYEPEA

BLAST SCORE: Identities = 76/131 (58%), Positives = 93/131 (70%), Gaps = 16/131 
(12%)

Alpha synuclein: 
MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHG
VATVAEKTK  60 MDVFMKGLS AKEGVVAAAEKTKQGV EAA 
KTKEGVLYVGSKT+EGVV GVA+VAEKTK
Beta synuclein:    
MDVFMKGLSMAKEGVVAAAEKTKQGVTEAAEKTKEGVLYVGSKTREGVVQG
VASVAEKTK  60

Alpha synuclein:   
EQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGI
LEDMPV­­  118

Beta synuclein:    EQASHLGGAVFS­­­­­­­­­­­
GAGNIAAATGLVKREEFPTDLKPEEVAQEAAEEPLIE  109

Alpha synuclein:   ­­­DPDNEAYE  126

Beta synuclein:    PLMEPEGESYE  120
Step2 (GRAMM­X docking):

Alpha synuclein is a single chain made up of Alpha helix. With the help of a Perl script
we change the ID of the PDB file of our protein from Chain ID A to Chain ID B and this
is how we get two chains of the same proteins with different chain ID’s.

After this step we will make use of the Docking server Gramm­X and dock the proteins
against itself (i.e. Chain A vs. Chain B). Gramm­X will then send us the number of
models requested (in our case 10) to our given e­mail within next one day which can be
viewed in VMD.

GRAMM­X public web server for protein­protein docking:

The   growing   needs   of   experimental   and   computational   biology   require   reliable

computational   procedures   for   modelling   of   protein   interactions.   Recent   progress   in
docking   algorithms   and   computer   hardware   makes   it   possible   to   implement   such
procedures as automated web servers, which greatly improves the utility of the docking
approaches in the biological community. Such servers also allow an objective test of the
underlying docking methodologies, unbiased by expert human intervention. In 2005, we
launched   our   docking   web   server   GRAMM­X   (HYPERLINK
"http://vakser.bioinformatics.ku.edu/resources/gramm/grammx" \t
"pmc_ext"http://vakser.bioinformatics.ku.edu/resources/gramm/grammx).   GRAMM­X
grew out of the original Fast Fourier Transformation (FFT) GRAMM methodology. It
represents a new implementation that uses a smoothed Lennard­Jones potential on a fine
grid during the global search FFT stage, followed by the refinement optimization in
continuous coordinates and rescoring with several knowledge­based potential terms.

The field of protein–protein docking is currently in the state of rapid development and
expansion. A number of existing docking methods rely on large database scans, such as
PSI­BLAST searches for evolutionary conserved residues. The minimization procedures
used in protein docking are computationally demanding, requiring parallel execution on
large   supercomputers/Linux   clusters.   These   factors   make   the   standard   model   for
research software distribution inconvenient for an average biologist (the software has to
be downloaded, installed and configured by the user). The paradigm of a web server
interface acting as the front end for the developers' own computational cluster solves the
problems   of   installation   complexity,   frequent   updates,   and   the   availability   of   large
uniformly configured computational resources.

In a few months since  its launch,  the  GRAMM­X server  has  processed  >1000 jobs

submitted   by   >250   users.   The   new   features   were   extensively   evaluated   on   the
benchmark of unbound protein pairs with known complexed structures. The server is
also regularly subjected to peer review by ~30 professional groups working in the field
of protein docking through our participation in the CAPRI blind prediction experiment
(HYPERLINK "http://capri.ebi.ac.uk" \t "pmc_ext"http://capri.ebi.ac.uk) 

GRAMM­X METHOD:

In the original GRAMM method, the intermolecular energy potential is a step function
approximating   Lennard­Jones   potential,   based   on   the   grid   representation   of   the
molecules.   The   smoothing   of   the   intermolecular   energy   landscape   is   achieved   by
increasing potential range and lowering the value of the repulsion part. Since the range
is the step of the grid, the step becomes larger and the structural representation of the
molecules is reduced to lower resolution. Such approach has an important advantage of
implementation   simplicity   and   computational   speed.   It   also   allows   the   study   of
fundamental molecular recognition characteristics focusing on underlying simplicity of
the basic principles. However, in practical docking applications aimed at maximizing the
chances of the correct prediction, the association of the potential range with the grid step
often becomes a disadvantage. The grid step association with the potential range is not
suitable for more sophisticated forms of the potential. At lower resolution, it is also
sensitive   to   the   positioning   of   the   molecules   for   the   grid   digitization,   introducing   a
significant degree of random noise. Disassociation of the interatomic energy potential
from   the   grid,   implemented   in   GRAMM­X,   provides   a   possibility   to   alleviate   these
negative factors.

The procedure uses a fine­grid projection of a softened Lennard­Jones potential function
calculated for a probe atom:

The   benchmarking   docking   showed   that   the   optimal   values   of   the   parameters   for   a
typical protein in unbound conformation are  α  = 0.4,  σ  = 0.33 nm and = 0.5. These
uniform values of σ and applied to all non­hydrogen atoms yielded better results than the
values   taken from   the AMBER   atom types.  The  docking runs  also  showed  that  the
results do not improve for translation grid steps <1.5 ?  and rotation steps <10°, which
can be explained by the subsequent minimization of the grid predictions in continuous
space.

The top 4000 grid­based predictions are subjected to a conjugate gradient minimization
in   continuous   6D   rigid   body   space   with   the   same   soft   potential.   The   minimization
accumulates many points, initially located on the grid, in a fewer local minima. One
representative   prediction   for   each   minimum   is   stored   and   the   number   of   initial
predictions falling into this minimum is marked as the volume of the minimum. The
average   radius   of   such   minima   on   our   smoothed   landscape   is   5   ? .   The   local
minimization   of   a   smoothed   landscape   can   be   viewed   as   clustering   on   the   original
rugged Lennard­Jones landscape, and helps locate the protein binding funnel. For each
minimized prediction the following terms are calculated: soft Lennard­Jones potential,
evolutionary conservation of predicted interface, statistical residue–residue preference,
volume of the minimum, empirical binding free energy and atomic contact energy. To
eliminate predictions that are likely to be located far from the correct binding site, we
apply the Support Vector Machine filter trained on a subset of the benchmark set using
the above mentioned set of potential terms. The remaining predictions are re­scored by a
weighted   sum   of   the   potential   terms.   A   detailed   description   of   the   algorithm   was
reported   earlier.   New   GRAMM­X   features   are   regularly   made   available   to   the   web
server back­end after extensive benchmarking.

The limited number of available test cases [Protein Data Bank (PDB) complexes for
which   unbound   structures   also   exist   in   PDB]   does   not   yet   allow   one   to   obtain   a
quantitative estimate for the expected accuracy of docking versus the properties of the
input structures. The main factor that affects the quality of the docking prediction is the
degree of  conformational  change  of  the input structures.  Especially important is  the
degree of such change at the interface area. There is no reliable method at this time to
estimate this without the actual knowledge of the bound conformations. An attempt to
address that problem was presented in our earlier study. Where we were substituting
bound   conformations   of   the   most   flexible   interface   side   chains   into   the   unbound
structures from the benchmark complexes. It demonstrated that the knowledge of the
conformational   change   upon   binding   of   only   three   critical   interface   side   chains   per
complex would provide a 40% improvement of the benchmarking results, and beyond
that other factors such as backbone changes and force field accuracy would dominate.
However, the critical question is how well the existing PDB benchmarks represent the
real   population   of   all   interacting   proteins.   The   Dockground   ( HYPERLINK
"http://dockground.bioinformatics.ku.edu" \t "pmc_ext"http://dockground.bioinformatics.ku.edu)
project   currently   under   development   in   our   group   aims   at   increasing   the   statistical
significance of benchmarking results by generating simulated unbound structures for
known complexes. That should increase the number of test cases by about an order of
magnitude. Such expanded benchmark will also improve our understanding of other
properties that influence docking: the type of interaction (antibody–antigen, enzyme–
inhibitor), the size of the proteins, etc. Currently, only some qualitative considerations
can   be   provided:   the   antibody–antigen   complexes   are   more   difficult   to   predict   than
enzyme–inhibitor complexes, the limited conformational change upon binding can be
tolerated with a reasonable chance of success [interface should have <3 ?  root mean
square deviation (r.m.s.d.) of conformational change], and complexes with a significant
backbone movement at the interface area are typically out of scope. The users of the
docking server should treat the output structures as potential candidates and critically
evaluate them using the available biological information.

GRAMM­X is implemented in Python and C++, thus combining fast prototyping power
of Python and numerical performance of C++ modules. The message passing interface
(MPI) library is used for parallelization. The full docking protocol completes in 2 min.
When the simulation request is submitted from the web server to our cluster of 320
CPUs, it will be queued, and the wait time will vary depending on the current load of the
cluster.
The   10   models   obtained   from   GRAMM­X   and   viewed   in   VMD   using
Ribbonsrepresentation:  

Step 3 (analysis of model 8):

From above 10 models we chose the best model which was Model 8 as shown below.

Model 8 contained the Alpha synuclein chain which we named as Chain A and the
residues 73­83 which we named as Chain B, which also worked as our Ligand.

 After docking (on Gramm­X) them together we again obtained 10 models and chose the
best fitting model in them.

In those 10 models we got two models which showed similarities and were close enough
with each other in interactions. They were model four and model eight.

Model 8

The 10 models obtained from GRAMM­X after docking the model 8 chain A and
model 8 Chain B (ligand):

Step 4 (analysis of model 4):

From all these models we chose model 4 

We will use this model 4 in PIC web server to find out the protein­protein interactions.

Interactions within a protein structure and interactions between proteins in an assembly
are essential considerations in understanding molecular basis of stability and functions
of proteins and their complexes. There are several weak and strong interactions that
render stability to a protein structure or an assembly. Protein Interactions Calculator
(PIC) is a server which, given the coordinate set of 3D structure of a protein or an
assembly, computes various interactions such as disulphide bonds, interactions between
hydrophobic   residues,   ionic   interactions,   hydrogen   bonds,   aromatic­aromatic
interactions, aromatic­sulphur interactions and cation­pi interactions within a protein or
between  proteins  in a  complex.  Interactions  are calculated   on the  basis  of  standard,
published criteria. The identified interactions between residues can be visualized using a
RasMol and Jmol interface. The advantage with PIC server is the easy availability of
inter­residue interaction calculations in a single site. It also determines the accessible
surface area and residue­depth, which is the distance of a residue from the surface of the
protein.   User   can   also   recognize   specific   kind   of   interactions,   such   as   apolar­apolar
residue interactions or ionic interactions, that are formed between buried or exposed
residues or near the surface or deep inside.
Hydrophobic Interactions within 5 Angstroms
POSITION RESIDUE CHAIN POSITION RESIDUE CHAIN
71 VAL A 85 ALA B
74 VAL A 82 VAL B
74 VAL A 85 ALA B
78 ALA A 78 ALA B
78 ALA A 82 VAL B
82 VAL A 74 VAL B
82 VAL A 78 ALA B
85 ALA A 71 VAL B
85 ALA A 74 VAL B
88 ILE A 70 VAL B
88 ILE A 74 VAL B
89 ALA A 70 VAL B
89 ALA A 71 VAL B

NO PROTEIN­PROTEIN DISULPHIDE BRIDGES ARE FOUND

NO PROTEIN­PROTEIN HYDROGEN BONDS ARE FOUND

NO   PROTEIN­PROTEIN   MAIN   CHAIN­SIDE   CHAIN   HYDROGEN

BONDS ARE FOUND
 NO   PROTEIN­PROTEIN   SIDE   CHAIN­SIDE   CHAIN   HYDROGEN
BONDS ARE FOUND

NO PROTEIN­PROTEIN IONIC INTERACTIONS ARE FOUND

NO   PROTEIN­PROTEIN   AROMATIC­AROMATIC   INTERACTIONS

ARE FOUND 4 and 6

NO PROTEIN­PROTEIN AROMATIC­SULPHUR INTERACTIONS ARE
FOUND

NO PROTEIN­PROTEIN CATION­PI INTERACTIONS ARE FOUND

   

Residues:

      

These are the residues of the peptide (73­83) forming hydrophobic interaction with the
Alpha – Synuclein; and are coloured purple.

Step 5 (SCWRL):

SCWRL is a program for adding sidechains to a protein backbone based on a backbone­
dependent rotamer library. The library provides lists of chi1­chi2 pairs for residues at
given phi­psi values, and explores these pairs to try to minimize sidechain­ backbone
clashes and sidechain­sidechain clashes. You may get output from the program at any of
three   steps   (best   library   rotamers,   no   clashes   relieved;   backbone   clashes   relieved;
backbone and sidechain­sidechain clashes relieved). Preliminary studies show prediction
of chi1 correct for all residues on native backbones is 77%. Chi3 and chi4 are built in
extended conformations.

SCWRL is based on a new algorithm and new potential function that results in improved
accuracy   at   reasonable  speed.  This  has   been  achieved   through:  1)   a  new  backbone­
dependent rotamer library based on kernel density estimates; 2) averaging over samples
of   conformations   about   the   positions   in   the   rotamer   library;   3)   a   fast   anisotropic
hydrogen bonding function; 4) a short­range, soft van der Waals atom­atom interaction
potential;   5)   fast   collision   detection   using   k­discrete   oriented   polytopes;   6)   a   tree
decomposition algorithm to solve the combinatorial problem; and 7) optimization of all
parameters by determining the interaction graph within the crystal environment using
symmetry operators of the crystallographic space group.

Preparation of required files
The input to SCWRL consists of 1) The pdb file with the structure of the template. 2) A
seqence file for your target protein.
Make a working directory (  mkdir scwrl  ). If you want to follow the example
presented here, you can copy the template pdb file and target sequence with this
command:cp /sb/demo/workshop/scwrl/* . (do include the space and the dot).
If   you   want   to   work   with   your   own   files,   take   the   following   points   into
consideration:  The seqence  file should be  in FASTA format, without the first
(identification) line, and without a terminal asterisk.
Make sure that the length of the sequence matches the length of the sequence in
your   pdb   file.   It   can   help   to   run   a   command   such   as  seq   <   template.pdb   >
template.seq to extract the sequence of the pdbfile for comparison.
The pdbfile should have no chain breaks, but missing density in the side­chains 3.
will be tolerated.

Usage:
scwrl4 −i inputpdbfilename −o outputpdbfilename > logfilename
Required 
­i   <inputfilename>   Input   file   in   PDB   format   of   required   N,CA,C,O   atoms
­o <outputfilename> Output file in PDB format including predicted side­chain
coordinates

Optional  
­f <framefilename> Input file in PDB format of ligand coordinates (see below for
format)
­s <sequencefilename> Sequence file for specifying new sequence of fixed side
chains
­p   <paramfilename>   Input   file   of   parameters
­h   Disable   output   of   hydrogen   atoms
­t   Disable   capping   of   N   and   C   terminal   residues   with   HN   and   OXT   atoms
­#   Calculate   side­chain   conformations   of   crystal

­i   <inputfilename>  
The   main   input   file   to   scwrl   should   be   a   protein   backbone,   with   or   without
sidechains, cofactors, or solvent. Residues with incomplete backbones are treated
as glycines. Residues with names that do not match the standard 20 amino acid
names are also treated as glycines. The sequence of residues is read from the first
atom in each residue. If you wish to change this sequence, the ­s flag allows you
to   enter   a   new   sequence   independently.  

­o   <outputfilename>  
The output file contains the identical backbone as the input file, with predicted
coordinates   for   the   sidechains.  

­f   <framefilename>  
This file is used to add additional steric boundaries to the sidechains. It should be
in pdb format, and might contain cofactors or metal atoms, lipid molecules, or
another   protein.   In   any   case,   it   is   held   fixed   and   used   only   for   steric   clash
checks.Radii   were   determined   from   atom­atom   distances   in   the   PDB.   All
elements   currently   observed   in   the   PDB   can   be   treated   by   scwrl.  

­s   <sequencefilename>  
This flag is followed by a sequence file. The sequence should have the same
number of residues in it as the input backbone. White space, carriage returns, and
numbers   are   ignored.   Lower­case   letters   in   the   sequence   indicate   that   the
Cartesian coordinates for the corresponding residues are to be left untouched, and
will   be   treated   as   steric   boundaries   only   for   the   other   side   chains.  

Examples: 
SDERYCNM   ­   full   SCWRL   side­chain   replacement  
SdERYCNM   ­   input   residue   (aspartate)   is   left   where   as   is.  
SxERYCNM   ­   input   residue   (aspartate)   is   left   where   as   is.  
­p   <paramfilename>  
File   that   specifies   parameters   for   SCWRL4.   The   default   file   is   set   during
installation; if it is not present in that location, SCWRL4 will look in the current
directory and in the directory where the executable is located. These options can
be   overridden   using   this   flag.  

­#  
Perform   calculation   of   side­chain   conformations   within   the   crystal.   Requires
CRYST1   record   in   inputfilename.   SCWRL4   uses   crystal   symmetry   to   build
backbone and side­chain coordinates of asymmetric units neighboring the input
coordinates.  

­h  
Disables   the   output   of   hydrogen   atom   coordinates.  

­t  
Disables adding hydrogens to the N­terminal nitrogen atom. By default, only a
residue numbered 1 will be treated as N­terminal. Disables addition of OXT atom
to C­terminal residue for each chain.

Step 6 (Auto Docking):

Finally with SCRWL we got the library of residual peptide structures which we
Auto Docked generating our drug peptides. In all those ligand­protein interactions
we   chose   the   peptide   which   was   the   best   binder   and   interacted   with   Alpha
synuclein protein chain and blocked the polymerization of two Alpha synuclein –
Alpha synuclein chains and preventing the aggregation of Lewy body formation.

Auto Docking:
AutoDock is a suite of automated docking tools. It is designed to predict how
small   molecules,   such   as   substrates   or   drug   candidates,   bind   to   a   receptor   of
known 3D structure.
AutoDock   actually   consists   of   two   main   programs:   AutoDock   performs   the
docking of the ligand to a set of grids describing the target protein; AutoGrid pre­
calculates these grids.
In addition to using them for docking, the atomic affinity grids can be visualised.
This   can   help,   for   example,   to   guide   organic   synthetic   chemists   design   better
binders.
We   have   also   developed   a   graphical   user   interface   called   AutoDockTools,   or
ADT  for  short, which amongst  other  things  helps to set  up which bonds  will
treated as rotatable in the ligand and to analyze dockings.
AutoDock   has   applications   in   X­ray   crystallography;structure­based   drug
design;lead   optimization;virtual   screening   (HTS);combinatorial   library   design;
protein­protein docking; chemical mechanism studies.

AutoDockTools, or ADT, is the free GUI for AutoDock developed by the same
laboratory that develops AutoDock. You can use it to set up, run and analyze
AutoDock dockings and isocontour AutoGrid affinity maps, as well as compute
molecular   surfaces,   display   secondary   structure   ribbons,   compute   hydrogen­
bonds, and do many more useful things.

AutoDockTools,   or   ADT,   is   the   ultimate   GUI   to   set   up,   launch   and   analyze
AutoDockruns. With ADT, you can: 
View molecules in 3D, rotate & scale in real time.
Add all hydrogens or just non­polar hydrogens.
Assign partial atomic charges to the ligand and the macromolecule (Gasteiger or
Kollman United Atom charges).
Merge non­polar hydrogens and their charges with their parent carbon atom.Set
up rotatable bonds in the ligand using a graphical version of AutoTors. Set up the
AutoGrid Parameter File (GPF) using a visual representation of the grid box, and
slider­based widgets.
Set up the AutoDock Parameter File (DPF) using forms.
Launch AutoGrid and AutoDock and View isocontoured AutoGrid affinity maps.
Read in the results of an AutoDock job and graphically display them.
Auto docking flow chart:

Result of Auto Docking(in terms of Energy Kcal/mol):
pepORG.dlg­USER    (1) Final Intermolecular Energy     =   ­4.33 kcal/mol
pepIAI.dlg­USER    (1) Final Intermolecular Energy     =   ­5.40 kcal/mol
pepIAL.dlg­USER    (1) Final Intermolecular Energy     =   ­4.39 kcal/mol
pepIAV.dlg­USER    (1) Final Intermolecular Energy     =   ­4.59 kcal/mol
 pepIII.dlg­USER    (1) Final Intermolecular Energy     =   ­3.94 kcal/mol
pepIIL.dlg­USER    (1) Final Intermolecular Energy     =   ­4.23 kcal/mol
pepIIV.dlg­USER    (1) Final Intermolecular Energy     =   ­6.19 kcal/mol
pepILI.dlg­USER    (1) Final Intermolecular Energy     =   ­4.30 kcal/mol
PepILL.dlg­USER    (1) Final Intermolecular Energy     =   ­4.07 kcal/molm
pepILV.dlg­USER    (1) Final Intermolecular Energy     =   ­3.47 kcal/mol
pepIVI.dlg­USER    (1) Final Intermolecular Energy     =   ­5.57 kcal/mol
pepIVL.dlg­USER    (1) Final Intermolecular Energy     =   ­4.75 kcal/mol
pepIVV.dlg­USER    (1) Final Intermolecular Energy     =   ­3.82 kcal/mol
pepLAI.dlg­USER    (1) Final Intermolecular Energy     =   ­5.76 kcal/mol
pepLAL.dlg­USER    (1) Final Intermolecular Energy     =   ­4.26 kcal/mol
pepLAV.dlg­USER    (1) Final Intermolecular Energy     =   ­4.87 kcal/mol
pepLII.dlg­USER    (1) Final Intermolecular Energy     =   ­5.12 kcal/mol
pepLIL.dlg­USER    (1) Final Intermolecular Energy     =   ­4.58 kcal/mol
pepLIV.dlg­USER    (1) Final Intermolecular Energy     =   ­4.96 kcal/mol
pepLLI.dlg­USER    (1) Final Intermolecular Energy     =   ­3.87 kcal/mol
pepLLL.dlg­USER    (1) Final Intermolecular Energy     =   ­4.93 kcal/mol
pepLLV.dlg­USER    (1) Final Intermolecular Energy     =   ­4.60 kcal/mol
pepLVI.dlg­USER    (1) Final Intermolecular Energy     =   ­5.62 kcal/mol
pepLVL.dlg­USER    (1) Final Intermolecular Energy     =   ­4.91 kcal/mol
pepLVV.dlg­USER    (1) Final Intermolecular Energy     =   ­4.30 kcal/mol
pepVAI.dlg­USER    (1) Final Intermolecular Energy     =   ­4.24 kcal/mol
pepVAL.dlg­USER    (1) Final Intermolecular Energy     =   ­4.70 kcal/mol
pepVAV.dlg­USER    (1) Final Intermolecular Energy     =   ­4.96 kcal/mol
pepVII.dlg­USER    (1) Final Intermolecular Energy     =   ­6.22 kcal/mol
pepVIL.dlg­USER    (1) Final Intermolecular Energy     =   ­5.66 kcal/mol
pepVIV.dlg­USER    (1) Final Intermolecular Energy     =   ­4.41 kcal/mol
pepVLI.dlg­USER    (1) Final Intermolecular Energy     =   ­5.99 kcal/mol
pepVLL.dlg­USER    (1) Final Intermolecular Energy     =   ­3.85 kcal/mol
pepVLV.dlg­USER    (1) Final Intermolecular Energy     =   ­5.19 kcal/mol
pepVVI.dlg­USER    (1) Final Intermolecular Energy     =   ­4.49 kcal/mol
pepVVL.dlg­USER    (1) Final Intermolecular Energy     =   ­4.18 kcal/mol
         pepVVV.dlg­USER    (1) Final Intermolecular Energy     =   ­3.74       kcal/mol

Step 7 (result):
Once   all   the   peptide   sequences   of   our   library   have   been   auto   docked,   we   can   now
compare them against each other to find out our drug (the best binder) which will have
the lowest intermolecular energy. Result for this can be depicted more clearly with a bar
graph.

Step 8 (conclusion):
In all the 37 peptides we found out two peptides with intermolecular energies of ­ 6.22
Kcal/mol   and   –   6.19Kcal/mol   where   as   our   original   ligand   had   ­4.33Kcal/mol   of
intermolecular   energy.   The   peptides  GVTAVIQKTIE  (­6.22   Kcal/mol)   and
GITAVIQKTVE  (­6.19Kcal/mol)   are   the   best   binders   which   can   bind   with   Alpha
synuclein chain instead and with stronger hydrophobic interactions.

References:
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HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Outeiro%20TF%22%5BAuthor%5D" Outeiro TF,
HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Nguyen%20P%22%5BAuthor%5D" Nguyen   P,
HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22McLean%20PJ%22%5BAuthor%5D" McLean PJ,
HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Hyman%20BT%22%5BAuthor%5D" Hyman BT.
MassGeneral Institute for Neurodegenerative Disease, Alzheimer's Disease Research Unit, Massachusetts General Hospital, 114 16 St.,
Charlestown, MA 02129, USA.
Department of Neurosciences, School of Medicine, University of California, San Diego, La Jolla 92093­0624.

Department of Pathology, University of California, San Diego, La Jolla, CA92093­0624, USA

Department of Neurochemistry, Tokyo Institute of Psychiatry, 2­1­8 Kamikitazawa, Setagaya, Tokyo 156­8585, Japan

Alzheimer's Disease Research Unit, Department of Neurology, Massachusetts General Hospital East, Charlestown, Massachusetts 02129,
USA.

Department of Pathology, Hospital Universitari Germans Trias i Pujol, Autonomous University of Barcelona, 08916 Badalona, 
Barcelona, Spain

Center for Bioinformatics, The University of Kansas, 2030 Becker Drive, Lawrence, KS 66047, USA
Department of Molecular Biosciences, The University of Kansas, 2030 Becker Drive, Lawrence, KS 66047, USA

Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.

PDB  HYPERLINK "http://www.pdb.org/pdb/static.do?

p=general_information/about_pdb/index.html"http://www.pdb.org/pdb/static.do?p=general_information/about_pdb/index.html
Auto Dock HYPERLINK "http://autodock.scripps.edu/"http://autodock.scripps.edu/
SCWRL https://structbio.vanderbilt.edu/twiki/bin/view/CSBTutorials/UsingSCRWL
HYPERLINK "http://dunbrack.fccc.edu/scwrl4/"http://dunbrack.fccc.edu/scwrl4/
HYPERLINK "http://en.wikipedia.org/wiki/Parkinson's_disease"http://en.wikipedia.org/wiki/Parkinson's_disease  ,
http://en.wikipedia.org/wiki/Alpha_synuclein
PAGE \* MERGEFORMAT 5