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INTRODUCTION

Drug design is the approach of finding drugs by design, based on their

biological targets. Typically a drug target is a key molecule involved in aparticular metabolic or signalling pathway that is specific to a disease

condition or pathology, or to the infectivity or survival of a microbial

pathogen.

Rational drug design is a process used in the biopharmaceutical industry to

discover and develop new drug compounds. RDD uses a variety of

computational methods to identify novel compounds, design compounds for

selectivity, efficacy and safety, and develop compounds into clinical trial

candidates. These methods fall into several natural categories structure-

based drug design, ligand-based drug design, de novo design and homology

modeling depending on how much information is available about drug

targets and potential drug compounds. Well focus on structure-based drug

design in this article and describe a few of its salient features

Structure-based drug design is one of several methods in the rational drug

design toolbox. Drug targets are typically key molecules involved in a specific

metabolic or cell signaling pathway that is known, or believed, to be related

to a particular disease state. Drug targets are most often proteins and

enzymes in these pathways. Drug compounds are designed to inhibit, restore

or otherwise modify the structure and behavior of disease-related proteins

and enzymes. SBDD uses the known 3D geometrical shape or structure of

proteins to assist in the development of new drug compounds. The 3D

structure of protein targets is most often derived from x-ray crystallographyor nuclear magnetic resonance (NMR) techniques. X-ray and NMR methods

can resolve the structure of proteins to a resolution of a few angstroms

(about 500,000 times smaller than the diameter of a human hair). At this

level of resolution, researchers can precisely examine the interactions

between atoms in protein targets and atoms in potential drug compounds

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that bind to the proteins. This ability to work at high resolution with both

proteins and drug compounds makes SBDD one of the most powerful

methods in drug design.

Bioinformatics plays an important role in the design of new drug compounds.

Drug discovery has long been a multidisciplinary effort to optimize ligands

properties (potency, selectivity, pharmacokinetics) towards a single

macromolecular target. It is estimated that, out of the 2025 000 human

genes supposed to encode for ca. 3000 druggable targets only a subset of

that pharmacological space has currently been investigated by the

pharmaceutical industry. Remarkably, medicinal chemistry followed a parallel

boost with the miniaturization and parallelization of compound synthesis,

such that over 10 million non-redundant chemical structures covers the

actual chemical space, out of which ca. 1000 have been approved as drugs.

Therefore, only a small fraction of compounds describing the current

chemical space has been tested on a fraction of the entire target space.

LEUKEMIA : AN INTRODUCTION

Leukemia is a cancer of the blood or bone marrow and is characterized by

an abnormal proliferation (production by multiplication) of blood cells,

usually white blood cells (leukocytes). It is part of the broad group of

diseases called hematological neoplasms.

Damage to the bone marrow, by way of displacing the normal bone marrow

cells with higher numbers of immature white blood cells, results in a lack of

blood platelets, which are important in the blood clotting process. This

means people with leukemia may become bruised, bleed excessively, or

develop pinprick bleeds (petechiae). White blood cells, which are involved

in fighting pathogens, may be suppressed or dysfunctional. This could cause

the patient's immune system to be unable to fight off a simple infection or to

start attacking other body cells.

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Finally, the red blood cell deficiency leads to anemia, which may cause

dyspnea. All symptoms can be attributed to other diseases; for diagnosis,

blood tests and a bone marrow examination are required.

Some other related symptoms:

Fever, chills, night sweats and other flu-like symptoms

Weakness and fatigue

Swollen or bleeding gums

Neurological symptoms (headache)

Enlarged liver and spleen

Frequent infection

Bone pain Joint pain

Dizziness

Swollen tonsils

Combining these two classifications provides a total of four maincategories:

Acute Chronic

lymphocytic

leukemia

Acute lymphocytic leukemia(also known as AcuteLymphoblastic Leukemia, or ALL)is the most common type ofleukemia in young children. Thisdisease also affects adults,especially those age 65 and older.

Chronic lymphocytic

leukemia (CLL) most oftenaffects adults over the age of55. It sometimes occurs inyounger adults, but it almostnever affects children.

myelogenous

leukemia (or"myeloid")

Acute myelogenous

leukemia (also known as AcuteMyeloid Leukemia, or AML) occursmore commonly in adults than inchildren. This type of leukemia waspreviously called "acutenonlymphocytic leukemia".

Chronic myelogenousleukemia (CML) occursmainly in adults. A very smallnumber of children alsodevelop this disease

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There is no single known cause for all of the different types of leukemia. The

different leukemias likely have different causes, and very little is certain

about what causes them. Researchers have strong suspicions about four

possible causes:

natural or artificial ionizing radiation

certain kinds of chemicals

some viruses

genetic predispositions

Acute myeloid leukemia (AML), also known as acute myelogenous leukemia,

is a cancer of the myeloid line ofwhite blood cells, characterized by the rapid

proliferation of abnormal cells which accumulate in the bone marrow and

interfere with the production of normal blood cells. AML is the most

common acute leukemia affecting adults, and its incidence increases with

age. Although AML is a relatively rare disease, accounting for approximately

1.2% of cancer deaths in the United States, its incidence is expected to

increase as the population ages.

The World Health Organization (WHO) classification of acute myeloid

leukemia attempts to be more clinically useful and to produce more

meaningful prognostic information than the FAB criteria. Each of the WHO

categories contains numerous descriptive sub-categories of interest to the

hematopathologist and oncologist; however, most of the clinically

significant information in the WHO schema is communicated via

categorization into one of the five subtypes listed below.

The WHO subtypes of AML are:

AML with characteristic genetic abnormalities, which includes

AML with translocations between chromosome 8 and 21 [t(8;21)],

inversions in chromosome 16 [inv(16)], or translocations between

chromosome 15 and 17 [t(15;17)]. Patients with AML in this category

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Transcription 3.7 kb; 2979 bp open reading frame

Protein

Description Size: 993 amino acids; 112804 Da;

FLT3 is a class III receptor tyrosine kinase (RTK) structurally

related to the receptors for platelet derived growth factor

(PDGF), colony stimulating factor 1 (CSF1), and KIT ligand

(KL).; these RTK contain five immunoglobulin-like domains in

the extracellular region and an intracelular tyrosine kinase

domain splitted in two by a specific hydrophilic insertion (kinase

insert); immunoprecipitation of the human FLT3 protein results

in the appearance of a minor band of Mr 130 000 and a majorband of Mr 155 000/160 000; the high-molecular-weight band

corresponds to the mature, N-glycosylated form, and the low-

molecular-weight band to the immature, high mannose-

containing form; N-linked glycosylations account for 50 000

daltons.

Expression FLT3 expression was described on bone marrow CD34-positive

cells, corresponding to multipotential, myeloid and B-lymphoid

progenitor cells, and on monocytic cells; FLT3 expression is

restricted to cells of the fetal liver expressing high levels of

CD34; in addition, the FLT3 protein is expressed on blast cells

from most ANLL and B-ALL.

Localisation Subcellular location: Type I membrane protein. 3D structure:

PDB id 1RJB (3D).

Function FLT3 receptor function can be defined by the activity of its

ligand (FL); FL is an early acting factor and supports thesurvival, proliferation and differentiation of primitive

hemopoietic progenitor cells. Ligand binding to FLT3 promotes

receptor dimerization and subsequent signalling through

posphorylation of multiple cytoplasmatic proteins, including

SHC, SHP-2, SHIP, Cbl, Cbl-b, Gab1 and Gab2, as well as the

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activation of several downstream signalling pathways, such as

the Ras/Raf/MAPK and PI3 kinase cascades.

Function: Receptor for the FL cytokine. Has a tyrosine-protein

kinase activity. Catalytic activity: ATP + a protein tyrosine =

ADP + protein tyrosine phosphate.

Similarity: Belongs to the Tyr protein kinase family. CSF-

1/PDGF receptor subfamily. Contains 1 immunoglobulin-like C2-

type domain.

Homology Other tyrosine kinases: KIT, PDGFRA, PDGFRB, VEGFR

Mutations

Somatic Mutations in the FLT3 gene are the most frequent geneticaberration that have been described in acute myeloid leukemia.

With 20-25% length mutations in the juxtamembrane domain are

the most frequent, followed by 7-8% mutations in the second

tyrosine kinase kinase domain, mostly point mutations in codon

835 or deletions of codon 836. Also point mutations in the juxta

membrane domain have been described and the number of new

mutations all over the total gene is still growing.

Implicated in

Entity FLT3-length mutation (FLT3-LM)

Disease Internal tandem duplications and/or insertions and, rarely,

deletions in the FLT3-gene are implicated in 20-25% of all

acute myeloid leukemias (AML). It was also described to be

involved in 5-10 % myelodysplastic syndromes (MDS)

refractory anaemia with excess of blasts (RAEB 1 and RAEB 2)and rare cases with acute lymphoblastic leukemia (ALL) The

duplicated sequence belongs to exon 11 but sometimes

involves intron 11 and exon 12. The most frequently used

nomenclature is FLT3-ITD (internal tandem duplication).

Because of the very heterogenous molecular structure the

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term FLT3-LM (length mutation) seems to be more adequate.

Prognosis An unfavourable impact on prognosis especially a high relapse

rate of the FLT3-LM has been shown by many study groups.

Patients with loss of the wildtype allele have an even worse

prognosis than the mutated with retention of the wildtype

allele. Perspective : It is of special interest that this mutation

allows to perform PCR-based minimal residual disease

detection in a high number of these high risk AML patients.

Cytogenetics FLT3-LM are highly correlated with a) normal karyotype,

b) t(15;17)(q25;q21)

c) CYTOGENETICS Perspective: It is of special interest that

this mutation allows to perform PCR-based minimal residual

disease detection in a high number of these high risk AML

patients.

Oncogenesis This mutation leads to constitutive ligand independent

autophosphorylation of the receptor. The FLT3-LM vary in size

and position in a nearly patient specific manner. Overall the

aberrant structure of the juxtamembrane domain disrupts a

negative regulatory domain, which leads to the constitutive

receptor activation. Several Groups have reported qualitative

differences in the intracellular signals provided by wild type

and mutated receptors.Mutated receptor weakly works through

MAP kinase and Akt but instead through strong and

constitutively activated STAT5.

Entity FLT3 Tyrosine Kinase Domain Mutation (FLT3-TKD)

Disease In the second tyrosine kinase domain point mutations and

small deletions mostly of codons 835 and 836, respectively,

can be found in 7-8% of all AML.

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Prognosis No independent impact on prognosis shown yet.

Cytogenetics In contrast to the FLT3-LM they do not seem to be specifically

correlated to a certain AML type.

Oncogenesis These mutations also lead to constitutive autoactivation of the

receptor. It has been suggested that TKD mutation may both

trigger the activation loop and stabilize it in the active state.

Pathways Of Formation And Action Of Flt3 protein

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Fig- Production And Action Of Flt3 protein

FLT3 is a class III receptor tyrosine kinase (RTK) structurally related to the

receptors for platelet derived growth factor (PDGF), colony stimulating factor

1 (CSF1), and KIT ligand (KL).; these RTK contain five immunoglobulin-like

domains in the extracellular region and an intracelular tyrosine kinase

domain splitted in two by a specific hydrophilic insertion (kinase insert);

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immunoprecipitation of the human FLT3 protein results in the appearance of

a minor band of Mr 130 000 and a major band of Mr 155 000/160 000; the

high-molecular-weight band corresponds to the mature, N-glycosylated form,

and the low-molecular-weight band to the immature, high mannose-

containing form; N-linked glycosylations account for 50 000 daltons.

FLT3 expression was described on bone marrow CD34-positive cells,

corresponding to multipotential, myeloid and B-lymphoid progenitor cells,

and on monocytic cells; FLT3 expression is restricted to cells of the fetal liver

expressing high levels of CD34; in addition, the FLT3 protein is expressed on

blast cells from most ANLL and B-ALL.

FLT3 receptor function can be defined by the activity of its ligand (FL); FL is

an early acting factor and supports the survival, proliferation and

differentiation of primitive hemopoietic progenitor cells. Ligand binding to

FLT3 promotes receptor dimerization and subsequent signalling through

posphorylation of multiple cytoplasmatic proteins, including SHC, SHP-2,

SHIP, Cbl, Cbl-b, Gab1 and Gab2, as well as the activation of several

downstream signalling pathways, such as the Ras/Raf/MAPK and PI3 kinase

cascades.

Function: Receptor for the FL cytokine. Has a tyrosine-protein kinase

activity. Catalytic activity: ATP + a protein tyrosine = ADP + protein tyrosine

phosphate.

Similarity: Belongs to the Tyr protein kinase family. CSF-1/PDGF receptor

subfamily. Contains 1 immunoglobulin-like C2-type domain.

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FIG. . Flt3 protein cascade

FMS-related tyrosine kinase 3 (FLT3, also called Flk2), is a member of the

type III receptor tyrosine kinase family, which includes c-Kit, PDGFR and M-CSF receptors. FLT3 is expressed on early hematopoietic progenitor cells and

supports growth and differentiation within the hematopoietic system (1,2).

FLT3 is activated after binding with its ligand FL, which results in a cascade of

tyrosine autophosphorylation and tyrosine phosphorylation of downstream

targets (3). The p85 subunit of PI3 kinase, SHP2, GRB2 and Shc are

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associated with FLT3 after FL stimulation (4-6). Tyr589/591 is located in the

juxtamembrane region of FLT3 and may play an important role in regulation

of FLT3 tyrosine kinase activity. Somatic mutations of FLT3 consisting of

internal tandem duplications (ITDs) occur in 20% of patients with acute

myeloid leukemia

REVIEW OF LITERATURE

Mizuki et-al, 2003 reported The receptor tyrosine kinase Flt3 is expressed

and functionally important in early myeloid progenitor cells and in the majority

of acute myeloid leukemia (AML) blasts. Internal tandem duplications (ITDs) in

the juxtamembrane domain of the receptor occur in 25% of AML cases.

Previously, we have shown that these mutations activate the receptor and

induce leukemic transformation. In this study, we performed genome-wide

parallel expression analyses of 32Dcl3 cells stably transfected with either wild-

type or 3 different ITD isoforms of Flt3. Comparison of microarray expression

analyses revealed that 767 of 6586 genes differed in expression between

FLT3-WT- and FLT3-ITD-expressing cell lines. The target genes of mutationally

activated Flt3 resembled more closely those of the interleukin 3 (IL-3)

receptor than those of ligand-activated Flt3. The serine-threonine kinase Pim-

2 was up-regulated on the mRNA and the protein level in Flt3-ITD-expressing

cells. Further experiments indicated that Pim-2 function was important for

clonal growth of 32D cells. Several genes repressed by the mutations were

found to be involved in myeloid gene regulation. Pu.1 and C/EBPalpha, both

induced by ligand-activation of wild-type Flt3, were suppressed in theirexpression and function by the Flt3 mutations. In conclusion, internal tandem

duplication mutations of Flt3 activate transcriptional programs that partially

mimic IL-3 activity. Interestingly, other parts of the transcriptional program

involve novel, IL-3-independent pathways that antagonize differentiation-

inducing effects of wild-type Flt3. The identification of the transcriptional

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program induced by ITD mutations should ease the development of specific

therapies.

Steffen et-al, 2005 reported the description of the molecular pathogenesis of

acute myeloid leukemias (AML) has seen dramatic progress over the last

years. Two major types of genetic events have been described that are crucial

for leukemic transformation: alterations in myeloid transcription factors

governing hematopoietic differentiation and activating mutations of signal

transduction intermediates. These processes are highly interdependent, since

the molecular events changing the transcriptional control in hematopoietic

progenitor cells modify the composition of signal transduction molecules

available for growth factor receptors, while the activating mutations in signal

transduction molecules induce alterations in the activity and expression of

several transcription factors that are crucial for normal myeloid differentiation.

The purpose of this article is to review the current literature describing these

genetic events, their biological consequences and their clinical implications. As

the article will show, the recent description of several critical transforming

mutations in AML may soon give rise to more efficient and less toxic

molecularly targeted therapies of this deadly disease.

Schessl ,et-al, 2005 work showed that The molecular characterization of

leukemia has demonstrated that genetic alterations in the leukemic clone

frequently fall into 2 classes, those affecting transcription factors (e.g., AML1-

ETO) and mutations affecting genes involved in signal transduction (e.g.,

activating mutations of FLT3 and KIT). This finding has favored a model of

leukemogenesis in which the collaboration of these 2 classes of genetic

alterations is necessary for the malignant transformation of hematopoietic

progenitor cells. The model is supported by experimental data indicating that

AML1-ETO and FLT3 length mutation (FLT3-LM), 2 of the most frequent

genetic alterations in AML, are both insufficient on their own to cause leukemia

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in animal models. Here we report that AML1-ETO collaborates with FLT3-LM in

inducing acute leukemia in a murine BM transplantation model. Moreover, in a

series of 135 patients with AML1-ETO-positive AML, the most frequently

identified class of additional mutations affected genes involved in signal

transduction pathways including FLT3-LM or mutations of KIT and NRAS.

These data support the concept of oncogenic cooperation between AML1-ETO

and a class of activating mutations, recurrently found in patients with t(8;21),

and provide a rationale for therapies targeting signal transduction pathways in

AML1-ETO-positive leukemias.

Stirewalt DL and Radich JP., 2003 work showed Normal haematopoietic

cells use complex systems to control proliferation, differentiation and cell

death. The control of proliferation is, in part, accomplished through the ligand-

induced stimulation of receptor tyrosine kinases, which signal to downstream

effectors through the RAS pathway. Recently, mutations in the FMS-like

tyrosine kinase 3 (FLT3) gene, which encodes a receptor tyrosine kinase, have

been found to be the most common genetic lesion in acute myeloid leukaemia

(AML), occurring in approximately 25% of cases. Exploring the mechanism by

which these FLT3 mutations cause uncontrolled proliferation might lead to a

better understanding of how cells become cancerous and provide insights for

the development of new drugs.

Mizuki et-al, 2000, Their studies were performed to Somatic mutations of

the receptor tyrosine kinase Flt3 consisting of internal tandem duplications

(ITD) occur in 20% of patients with acute myeloid leukemia. They are

associated with a poor prognosis of the disease. In this study, wecharacterized the oncogenic potential and signaling properties of Flt3

mutations. We constructed chimeric molecules that consisted of the murine

Flt3 backbone and a 510-base pair human Flt3 fragment, which contained

either 4 different ITD mutants or the wild-type coding sequence. Flt3

isoforms containing ITD mutations (Flt3-ITD) induced factor-independent

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growth and resistance to radiation-induced apoptosis in 32D cells. Cells

containing Flt3-ITD, but not those containing wild-type Flt3 (Flt3-WT),

formed colonies in methylcellulose. Injection of 32D/Flt3-ITD induced rapid

development of a leukemia-type disease in syngeneic mice. Flt3-ITD

mutations exhibited constitutive autophosphorylation of the immature form

of the Flt3 receptor. Analysis of the involved signal transduction pathways

revealed that Flt3-ITD only slightly activated the MAP kinases Erk1 and 2 and

the protein kinase B (Akt) in the absence of ligand and retained ligand-

induced activation of these enzymes. However, Flt3-ITD led to strong factor-

independent activation of STAT5. The relative importance of the STAT5 and

Ras pathways for ITD-induced colony formation was assessed by transfection

of dominant negative (dn) forms of these proteins: transfection of dnSTAT5

inhibited colony formation by 50%. Despite its weak constitutive activation

by Flt3-ITD, dnRas also strongly inhibited Flt3-ITD-mediated colony

formation. Taken together, Flt3-ITD mutations induce factor-independent

growth and leukemogenesis of 32D cells that are mediated by the Ras and

STAT5 pathways.

Quentmeier et-al, 2003 estimated Internal tandem duplications (ITD) and

D835 point mutations of the receptor tyrosine kinase (RTK) FLT3 are found in

a high proportion of cases with acute myeloid leukemia (AML). These genetic

aberrations may lead to the constitutive activation of the receptor, thus

providing the molecular basis for a persisting growth stimulus. We have

screened 69 AML-derived cell lines for FLT3 mutations. Four of these cell lines

showed ITD of the FLT3 gene, none carried a D835 point mutation. Two cell

lines (MUTZ-11 and MV4-11) expressed exclusively the mutated allele, the

other two cell lines (MOLM-13 and PL-21) displayed a mutated and the wild-

type version of the gene. Although mutationally activated FLT3 is supposed to

substitute for the stimulatory signal of a growth factor, one of these cell lines

(MUTZ-11) was strictly cytokine-dependent. FLT3 transcripts were found in all

four cell lines, but the constitutively phosphorylated receptor protein was

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clearly detectable only in cell line MV4-11, possibly explaining why MUTZ-11

cells were growth-factor dependent. Thus, not all FLT3 ITD-positive cells

express high levels of the active receptor protein, a finding that might be of

relevance for a possible future application of a kinase inhibitor as therapeutic

agent. It had been described that STAT-5 phosphorylation was part of the

FLT3 signalling chain and that STAT-5 molecules were constitutively

phosphorylated in FLT3 ITD-positive cells. Although we observed the

constitutive phosphorylation of STAT-5 molecules in FLT3-mutant cells, FLT3

ligand (FL) did not induce STAT-5 phosphorylation in FLT3 wild-type cells.

These results suggest that the signalling mechanisms of the mutated FL

receptor differ at least to some extent from those conferred by wild-type FLT3.

In conclusion, (1) not all cells with FLT3 ITD express significant amounts of

the mutated receptor protein; (2) signals downstream from wild-type and

mutant FLT3 receptors are not 100% identical; and (3) MV4-11 represents a

model cell line for FLT3 ITD signalling.

Choudhary et-al, 2005 sujjested that Activating mutations of Fms-like

tyrosine kinase 3 (Flt3) are the most common genetic lesions in acute

myeloid leukemia (AML) and are present in approximately one third of AML

patients. The 2 classes of Flt3 mutations are internal tandem duplications in

the juxtamembrane domain and point mutations in the tyrosine kinase

domain. In normal hematopoietic progenitor cells, Flt3 ligand induces the

activation of several downstream signal-transduction mediators, including

phosphoinositol 3-kinases, Src kinases, mitogen-activated protein kinases,

and the phosphorylation of several adaptor proteins. Oncogenic mutations in

Flt3 result in ligand-independent constitutive and deregulated activation of

these signaling pathways. In addition, however, oncogenic mutations of Flt3

also result in the activation of aberrant signaling pathways, including strong

activation of STAT5, induction of STAT target genes, and repression of

myeloid transcription factors c/EBP-3 and Pu.1. Aberrant activation of these

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signaling pathways by oncogenic Flt3 may play a critical role in mutant Flt3-

mediated leukemic transformation.

Moore, et-al, 2007 worked on human leukemogenesis by transduction of

human hematopoietic stem cells (HSC) with genes associated with leukemia

and expressed in leukemic stem cells. METHODS: Constitutive activation of

Flt3 (Flt3-ITD) has been reported in 25 to 30% of patients with acute

myeloid leukemia (AML). Retroviral vectors expressing constitutively

activated Flt3 and STAT5A were used to transduce human cord blood

CD34(+) cells and HSC cell self-renewal and differentiation were evaluated.

RESULTS: We have demonstrated that retroviral transduction of Flt3

mutations into CD34(+) cells enhanced HSC self-renewal as measured in

vitro in competitive stromal coculture and limiting-dilution week-2

cobblestone (CAFC) assays. Enhanced erythropoiesis and decreased

myelopoiesis were noted together with strong activation of STAT5A.

Consequently, transduction studies were undertaken with a constitutively

active mutant of STAT5A (STAT5A[1( *)6]) and here also a marked, selective

expansion of transduced CD34(+) cells was noted, with a massive increase in

self-renewing CAFC detectable at both 2 and 5 weeks of stromal coculture.

Differentiation was biased to erythropoiesis, including erythropoietin

independence, with myeloid maturation inhibition. The observed phenotypic

changes correlated with differential gene expression, with a number of genes

differentially regulated by both the Flt3 and STAT5A mutants. These included

upregulation of genes involved in erythropoiesis and downregulation of genes

involved in myelopoiesis. The phenotype of week-2 self-renewing CAFC also

characterized primary Flt3-ITD(+) AML bone marrow samples. Isolation of

leukemic stem cells (LSC) with a CD34(+), CD38(-), HLA-DR(-) phenotype

was undertaken with Flt3-ITD(+) AML samples resulting in co-purification of

early CAFC. Gene expression of LSC relative to the bulk leukemic population

revealed upregulation of homeobox genes (HOXA9, HOXA5) implicated in

leukemogenesis, and hepatic leukemia factor (HLF) involved in stem cell

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proliferation. CONCLUSION: Myeloid leukemogenesis is a multi-stage process

that can involve constitutively activated receptors and downstream pathways

involving STAT5, HOX genes, and HLF.

Spiekermann, et-al, 2003 indicated that Activating length mutations in the

juxtamembrane domain (FLT3-LM) and mutations in the tyrosine kinase

domain (FLT3-TKD) of FLT3 represent the most frequent genetic alterations

in acute myeloid leukemia (AML). However, the functional role of active FLT3

mutants in primary AML blast cells is not well characterized. EXPERIMENTAL

DESIGN: We analyzed the transforming potential and the signaling of FLT3-

ITD mutants in Ba/F3 cells and in primary AML blasts. RESULTS: FLT3-ITD

mutants induce an autophosphorylation of the receptor, interleukin 3-

independent growth in Ba/F3 cells, and a strong STAT5 and mitogen-

activated protein kinase (MAPK) activation. In contrast to the FLT3-ITD

mutants, the ligand-stimulated FLT3-WT receptor was unable to transduce a

fully proliferative response in Ba/F3 and monocytic OCI-AML5 cells. The

ligand-stimulated FLT3-WT receptor activated AKT and MAPK, but not STAT5.

In primary blast cells from 60 patients with AML, FLT3 was expressed in

91.9% of patients carrying a FLT3-LM/TKD mutation compared with 77.8% in

FLT3-LM/TKD-negative patients. STAT3 and STAT5 were constitutively

activated in 76 and 63% of patients, respectively. In accordance with the

results in Ba/F3 cells, a high FLT3 expression and the presence of a FLT3-LM

was strongly associated with the STAT5 but not with the STAT3 activation in

primary AML blast cells. Moreover, the constitutive tyrosine phosphorylation

of STAT5 was efficiently down-regulated by a FLT3 protein tyrosine kinase

inhibitor in AML cells expressing an active FLT3 mutant. CONCLUSIONS:

Active FLT3 receptor mutants have transforming potential in hematopoietic

cells and induce a strong activation of STAT5 in primary AML cells. The FLT3-

STAT5 pathway contributes to the malignant phenotype and represents a

promising molecular therapeutic target structure in AML.

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MATERIALS AND METHODS

Retrieval of Protein Sequence of Flt3 in Homo sapiens:

Protein sequence of Flt3 in Homo sapiens was done from National Center OfBiotechnology information(www.ncbi.nlm.nih.gov/). The sequence of

protein was in FASTA format :

>gi|406323|emb|CAA81393.1| FLT3 receptor tyrosine kinase precursor

[Homo sapiens]

MPALARDGGQLPLLVVFSAMIFGTITNQDLPVIKCVLINHKNNDSSVGKSSSYPMVSESPEDLGCALRPQ

SSGTVYERAAVEVDVSASITLQVLVDAPGNISCLWVFKHSSLNCQPHFDLQNRGVVSMVILKMTETQAGE

YLLFIQSEATNYTILFTVSIRNTLLYTLRRPYFRKMENQDALVCISESVPEPIVEWVLCDSQGESCKEES

PAVVKKEEKVLHELFGMDIRCCARNELGRECTRLFTIDLNQTPQTTLPQLFLKVGEPLWIRCKAVHVNHG

FGLTWELENKALEEGNYFEMSTYSTNRTMIRILFAFVSSVARNDTGYYTCSSSKHPSQSALVTIVEKGFI

NATNSSEDYEIDQYEEFCFSVRFKAYPQIRCTWTFSRKSFPCEQKGLDNGYSISKFCNHKHQPGEYIFHAENDDAQFTKMFTLNIRRKPQVLAEASASQASCFSDGYPLPSWTWKKCSDKSPNCTEEITEGVWNRKANRK

VFGQWVSSSTLNMSEAIKGFLVKCCAYNSLGTSCETILLNSPGPFPFIQDNISFYATIGVCLLFIVVLTL

LICHKYKKQFRYESQLQMVQVTGSSDNEYFYVDFREYEYDLKWEFPRENLEFGKVLGSGAFGKVMNATAY

GISKTGVSIQVAVKMLKEKADSSEREALMSELKMMTQLGSHENIVNLLGACTLSGPIYLIFEYCCYGDLL

NYLRSKREKFHRTWTEIFKEHNFSFYPTFQSHPNSSMPGSREVQIHPDSDQISGLHGNSFHSEDEIEYEN

QKRLEEEEDLNVLTFEDLLCFAYQVAKGMEFLEFKSCVHRDLAARNVLVTHGKVVKICDFGLARDIMSDS

NYVVRGNARLPVKWMAPESLFEGIYTIKSDVWSYGILLWEIFSLGVNPYPGIPVDANFYKLIQNGFKMDQ

PFYATEEIYIIMQSCWAFDSRKRPSFPNLTSFLGCQLADAEEAMYQNVDGRVSECPHTYQNRRPFSREMD

LGLLSPQAQVEDS

Homology Modelling :

Homology modelling is required when the exact structure of the protein is not

available. The structure of Flt3 was also unavailable, so homology modeling

was required. It is also known as comperative modelling. Here we model the

molecule (protein) from amino acid sequence by following a protocol to

model.The amino acid sequence is query or target sequence. Homology

modeling techniques depend on identificatiction of one or more stuctures

known as template, which resembles the sructure of query sequence. The

sequence alignment and template stucture are used to produce a structural

model of the target. Usually sequence similarity corresponds to high

structural similarity.

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Different softwares are used for Homology Modelling such as SWISS MODEL

SERVER.,CPH MODEL SERVER.,MODELLER etc. In this project Swiss

Model server is used for Homology Modelling. The methodology for

homology modeling with Swiss Model Server is:

BLAST

BLAST (Basic Local Alignment Search Tool),(www.ncbi.nlm.nih.gov/BLAST)is a tool

by which we can find alignment between our query in form of nucleotide or

protein sequence, against the database of BLAST. The results show us the

extent to which our query sequence matches the sequences stored in theBLAST database.In case our sequence is a novel entry ,it does not show any

results. Here I carried out protein-protein BLAST of my query sequence,

against pdb (protein data bank);which consists of amino acid sequences of

the proteins submitted in pdb .The results generated by BLAT were furthur

used for the modeling of the protein. After we get BLAST results we carry out

CLUSTAL W.

CLUSTAL W

CLUSTAL-W (www.ebi.ac.uk/clustalw/) is a multiple sequence alignment

programme for DNA or proteins. It provides multiple sequence alignment

forgiven sequence. It gives the match between the query sequences and

allows us to have the idea of best match between our target sequence and

template. This informationis furthur used in swiss model. Evolutionaryrelationships can be viewed by cladograms or phylograms.

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Swiss Model:

It is totally automated protein structure homology modeling server,

accessible via ExPASy web server or from swiss pdb viewer.

(www.swissmodel.expasy.org//SWISS-MODEL.html).

SWISS MODEL SERVER: It is used for final modeling of protein, using

results of CLUSTAL-W. Basically there are three moes os SWISS MODEL,

which are:

1. First approach mode: it only requires a single amino acid sequence

information as input data. The server automatically selects suitable template.

However the user may specify up to five template structures either fromExPDB library, or opload co-ordinate files. The process starts if atleast one

template sequence has a identity of more than 25% with submitted target

sequence. The reliability of model decreases as sequence identity decreases.

2. Alignment Mode: it is done by submitting a sequence alignment. The ser

predicts the target sequence and the one, which is structurally known protein

FROM ExPDB library. The server builds yhe model according to given

alignment.

3. Project Mode: here user submits a manually optimized modeling request

to SWISS MODEL server The starting moe is a Deep View project file. It

contains superposed template structures and alignment between target and

template. It allows template selection or gap placement in the alignment. It

can also be used to improve the output of first approach mode.

Here alignment mode of SWISS MODEL was used to predict structure of Flt3model.

There are certain steps to be followed in this process, which are:

Retrieval of protein seuence from NCBI in FASTA format.

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Protein BLAST of protein sequence obyained in last step against

pdb(protein data bank).

Selecting the second, third, fourth match results and obtaining their

FASTA format of sequence.

Puting the results obtained in last step alongwith target protein

sequence of protein in a notepad. The sequences obtained in last steo

are pot\entail te,plates.

Open CLUSTAL W page and paste the sequence obtained in last step in

window displayed and submit.

Open SWISS MODEL SERVER page and paste the sequence in window

and submit.

The results are obtained, asve the result file with (.pdb) extension, to

save a pdb file.

Open the saved file with rasmol viewer to view 3-D image of the

modeled protein.

Retrieval of inhibitor against Flt3:

Inhibitor against Flt3 protein retrieved through two major sources.

1. BRENDA (www.brenda.uni-koeln.de) is the main collection of enzyme

functional data available to the scientific community. BRENDA is maintainedand developed at the institute of Biochemistry at the University of Cologne

2. NCBI Pubchem Compound: PubChem Structure Search allows

PubChem Compound Database to be queried using a chemical structure.

Chemical structure queries may be sketched using the PubChem Sketcher.

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You may also specify the structural query input by PubChem Compound

Identifier (CID), SMILES, SMARTS, InChI, Molecular Formula, or by upload of

a supported structure file format.

This standardizing allows NCBI to compute chemical parameters and

similarity relationships between compounds. The compounds are grouped

into levels of chemical similarity from most general to most specific: same

bonding connectivity and any tautomer; same bonding connectivity; same

stereochemistry; same isotopes; and same stereochemistry and isotopes.

PubChem Compound also indexes these chemicals using 34 fields, many of

which represent computed chemical properties such as the number of chiral

centers, the number of hydrogen bond donors/acceptors, molecular formula

and weight, total formal charge, and octanol-water partition coefficients

(XlogP). These groups are provided as Entrez links that allow similar

compounds to be retrieved quickly.

Building of 3d structure (PDB file) of Inhibitors:

2D structure of potent inhibitors are obtained by submitting the CID no tothe NCBIs Pubchem compound and convert it into SDF format then convert it

into PDB format to get the 3D-structure.

Procedure of converting 2D-structure into 3D-structure

1) To select SDF format from NCBI.

Open google and enter NCBI home page.

Choose pubchem compound from search drop-down menu.

Type CID no.

When answers come ,Change display format to SDFand save it .

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To retrieve PDB file of Inhibitors:

2-D structure of protein has been obtained by put the specific CID no. in the

pubchem compound it retrieved the 2-D or SDF file of inhibitor we save it &

will convert this 2-D file in the 3-D file i.e. in the form of PDB file, using

software Babel.

Babel Molecule format Converter:

Babel is a cross-platform program designed to interconvert between many

file formats used in molecular modeling and computational chemistry and

related areas. Babel is a chemical toolbox designed to allowing anyone,

convert, analyze, or store data from molecular modeling, chemistry, solid-

state materials, biochemistry, or related areas.

Procedure to convert the 2-D file in the 3-D file or PDB file

First of all open the Babel page.

Set the parameter for input and output file i.e. SDF for input file

& PDF for output file.

Paste the data of 2-D file in the input section or upload the SDF

file.

Click on the convert file.

The result will show in the output section in the form of PDF file, copy thatdata and paste in the word pad and save that file with (.pdb) extension.

COMPND 1248

HETATM 1 O1 LIG 1 5.411 0.262 0.000 1.00 0.00 O

HETATM 2 O2 LIG 1 8.160 -1.543 0.000 1.00 0.00 O

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HETATM 3 O3 LIG 1 8.160 -3.152 0.000 1.00 0.00 O

HETATM 4 O4 LIG 1 3.579 2.966 0.000 1.00 0.00 O

HETATM 5 O5 LIG 1 2.000 2.653 0.000 1.00 0.00 O

HETATM 6 O6 LIG 1 5.717 2.011 0.000 1.00 0.00 O

HETATM 7 N7 LIG 1 3.682 -1.827 0.000 1.00 0.00 N

HETATM 8 C8 LIG 1 4.588 -1.313 0.000 1.00 0.00 C

HETATM 9 C9 LIG 1 3.682 -2.868 0.000 1.00 0.00 C

HETATM 10 C10 LIG 1 4.600 -0.313 0.000 1.00 0.00 C

HETATM 11 C11 LIG 1 5.482 -1.847 0.000 1.00 0.00 C

HETATM 12 C12 LIG 1 4.588 -3.382 0.000 1.00 0.00 C

HETATM 13 C13 LIG 1 5.482 -2.847 0.000 1.00 0.00 C

HETATM 14 C14 LIG 1 2.682 -1.823 0.000 1.00 0.00 C

HETATM 15 C15 LIG 1 3.185 -0.959 0.000 1.00 0.00 C

HETATM 16 C16 LIG 1 3.802 0.280 0.000 1.00 0.00 C

HETATM 17 C17 LIG 1 6.348 -1.347 0.000 1.00 0.00 C

HETATM 18 C18 LIG 1 6.348 -3.347 0.000 1.00 0.00 C

HETATM 19 C19 LIG 1 4.123 1.227 0.000 1.00 0.00 C

HETATM 20 C20 LIG 1 7.214 -1.847 0.000 1.00 0.00 C

HETATM 21 C21 LIG 1 5.117 1.211 0.000 1.00 0.00 C

HETATM 22 C22 LIG 1 7.214 -2.847 0.000 1.00 0.00 C

HETATM 23 C23 LIG 1 2.821 0.085 0.000 1.00 0.00 C

HETATM 24 C24 LIG 1 3.464 1.979 0.000 1.00 0.00 C

HETATM 25 C25 LIG 1 2.483 1.784 0.000 1.00 0.00 C

HETATM 26 C26 LIG 1 2.162 0.837 0.000 1.00 0.00 C

HETATM 27 C27 LIG 1 8.744 -2.347 0.000 1.00 0.00 C

HETATM 28 C28 LIG 1 2.676 3.382 0.000 1.00 0.00 C

HETATM 29 H8 LIG 1 4.054 -0.998 0.000 1.00 0.00 H

HETATM 30 1H9 LIG 1 3.071 -2.762 0.000 1.00 0.00 H

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HETATM 31 2H9 LIG 1 3.473 -3.452 0.000 1.00 0.00 H

HETATM 32 H10 LIG 1 5.149 -0.601 0.000 1.00 0.00 H

HETATM 33 1H12 LIG 1 4.194 -3.861 0.000 1.00 0.00 H

HETATM 34 2H12 LIG 1 4.993 -3.852 0.000 1.00 0.00 H

HETATM 35 1H14 LIG 1 2.680 -2.443 0.000 1.00 0.00 H

HETATM 36 2H14 LIG 1 2.062 -1.820 0.000 1.00 0.00 H

HETATM 37 3H14 LIG 1 2.684 -1.203 0.000 1.00 0.00 H

HETATM 38 1H15 LIG 1 2.647 -1.266 0.000 1.00 0.00 H

HETATM 39 2H15 LIG 1 2.877 -0.421 0.000 1.00 0.00 H

HETATM 40 3H15 LIG 1 3.724 -0.651 0.000 1.00 0.00 H

HETATM 41 H17 LIG 1 6.348 -0.727 0.000 1.00 0.00 H

HETATM 42 H18 LIG 1 6.348 -3.967 0.000 1.00 0.00 H

HETATM 43 H23 LIG 1 2.621 -0.502 0.000 1.00 0.00 H

HETATM 44 H26 LIG 1 1.554 0.716 0.000 1.00 0.00 H

HETATM 45 1H27 LIG 1 9.205 -2.762 0.000 1.00 0.00 H

HETATM 46 2H27 LIG 1 9.205 -1.933 0.000 1.00 0.00 H

HETATM 47 1H28 LIG 1 2.993 3.915 0.000 1.00 0.00 H

HETATM 48 2H28 LIG 1 2.179 3.753 0.000 1.00 0.00 H

TER 49 LIG 1

CONECT 1 10 21

CONECT 2 20 27

CONECT 3 22 27

CONECT 4 24 28

CONECT 5 25 28

CONECT 6 21 21

CONECT 7 8 9 14 15

CONECT 8 7 10 11 29

CONECT 9 7 12 30 31

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CONECT 10 1 8 16 32

CONECT 11 8 13 17 17

CONECT 12 9 13 33 34

CONECT 13 11 12 18 18

CONECT 14 7 35 36 37

CONECT 15 7 38 39 40

CONECT 16 10 19 19 23

CONECT 17 11 11 20 41

CONECT 18 13 13 22 42

CONECT 19 16 16 21 24

CONECT 20 2 17 22 22

CONECT 21 1 6 6 19

CONECT 22 3 18 20 20

CONECT 23 16 26 26 43

CONECT 24 4 19 25 25

CONECT 25 5 24 24 26

CONECT 26 23 23 25 44

CONECT 27 2 3 45 46

CONECT 28 4 5 47 48

CONECT 29 8

CONECT 30 9

CONECT 31 9

CONECT 32 10

CONECT 33 12

CONECT 34 12

CONECT 35 14

CONECT 36 14

CONECT 37 14

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CONECT 38 15

CONECT 39 15

CONECT 40 15

CONECT 41 17

CONECT 42 18

CONECT 43 23

CONECT 44 26

CONECT 45 27

CONECT 46 27

CONECT 47 28

CONECT 48 28

END

Docking Of Flexible Ligands to the Receptors

For docking the flexible ligands to the receptors following softwares can be used which

are listed below:

SN Name License Term Platform Keyword

1 Autodock Commercial UNIX,LINUX,SGI GA/LGA,MC

2 Affinity Commercial SGI Monte Carlo

method

3 Dock Vision Commercial LINUX.IRIS MC,GA

4 DOT(Daughter

of Turnip)

Free Supercomputers,UNIX

5 Flex X Commercial UNIX Fragnent Based

6 Shape E-mail request UNIX Structure andchemistry of

molecular surface

7 LEAPFROG Commercial SGI ligand design

8 Q site Commercial UNIX,LINUX,SGI Mixed

quantum and

molecular

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mechanics

9 HINT Commercial Windows

2000,SGI,LINUX

Hydropathic

interaction

10 GOLD Free evaluation UNIX GA

Cygwin: It is a collection of free software tools originally developed from

Cygnus solutionsto allow various versions of Microsoft windows to act

similarto a Linux operating system.As Autodock is programmed to run on

Linux operating system,so for those systems which run on windows,cygwin is

a must.It can be freely downloaded from the internet.

AUTODOCK: 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.AutoDockactually consists of two main

programs: AutoDock performs the docking of the ligand to a set of grids

describing the target protein; Auto Grid pre-calculates these grids. In

additions to using them for docking, the atomic affinity grids can be

visualized. This can help, for example, to guide organic synthetic chemistsdesign better binders.

1.Autogrid.

2.Autodock.

AUTOGRID:

A. Peparing a Ligand for Autodock:

In autodock page,go to ligand and in it click on input.

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In input click on open AD3.

Go to your folder and open the (.pdb) file of the inhibitor.

Go to Ligand again in autodock window and in it Torsion Tree andclick ondetect route.

In Ligand select Torsion Tree and click on Choose Torsionand click

on done.

Go to Ligand again and select Torsion Tree and select Set number

Of Torsions.The number of torsions

Set number of torsions less than or equal to 6 and click on Dismiss.

Go to Logand and in it to Output and then click on Save As

PDBQ.Then go to your folder a save your this file as (inhibitor

name.out.pdbq).

B. Preparing A Macromolecule For Autodock:

Go to Grid and in it click on Macromolecule and in it click on OpenAG3.

Open the (.pdb) file from your folder and click on OK.

Now save this file as protein name.pdbqs.

Come back to autodock window and press shift key+n to visualize the

protein on screen.

C. Preparing The Grid Parameter File:

Go to Grid and select Set Map Types and in it select Choose Ligand

AG3.

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Now select the inhibitor file.

Now click on Ligand and then click on Accept.

Go to Grid and selectGrid Box.

On the window that opens,set x,y,z co-ordinate axes so that the

macromolecule is completely covered.You can rotate the molecule by

pressing shift+right click of mouse.

On the same window,click on File and there click on Saving CurrentSetting.

Go to Grid again and in it go to Output,then click on SaveGPF(AG3)

Save this file in your folder as protein name.gpf.

Go to Grid and in it go to Edit Grid and clik it,then click on Accept.

Go to your folder and copy the path of your folder eg(C:\probir) and

open the (.gpf) file in your folder wit wordpad and paste this path

followed by \ on left of wherever you find protein name in this file.It

is to be noted that there should not be any gap between path and

protein name.

Click on Save in this window after you finish.

AUTODOCK:

A. Startimg Autodogrid:

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Go to Run and in it click on Run Autogrid.

On the window that opens on the first Browse option click and select

autogrid.exe file.

Then in second Browse option click and go to your folder and open

the (.gpf) file.

Back to autogrid, select the entire bottom line(i.e. the path) of the

Browse window and press cntrl+c.

Open Cygwin and in it go to Edit and Paste the path copied and

press Enter.

B. Preparing A docking Parameter For Autodock:

Click on Docking on the the autodock window.

In it select on Macromolecule and in it click on Choose AD3.

Select your protein in the window that opens and click on OK.

Click on Select Macromolecule and click on window that opens twice.

Go to Docking and in it go to Ligand and in it click on Choose

(AD3).

On the window that opens click and select the ligand.

Click on Select Ligand and click on Accept on the window.

Go to docking again and selectSearch Parameterand in it click on

Genetic Algorithm.Click Accept on yhe window that opens.

Go to Docking again and in it click on Docking Parameters.Click on

Accept on the window that opens.

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Go to Docking again and in it select Output and in it click on

Lamarkian GA(AD3).Then go to your folder and save the file as

(inhibitor name.dpf).

Go to your folder and copy the path of (.dpf) file.

Open this (.dpf) file in wordpad and paste the copied path

everywhere you find inhibitor name,followed by \ i.e.path+\,on left

of the inhibitor name.Continue till you reach on yhe line with move

and here do the same.

Save the page.

C. Starting Autodock:

Back to autodock,click on Run and in it click on Run Autodock.

On the window that opens on first Browse option,click it and open the

autodock.exe file.

On the second Browse option,click and go to your folder and open the

the (.dpf) file.

Come back to Browse window and copy the path at the bottom by

selecting it and then pressing cntrl+c.

Open Cygwin and go to Edit and click on Paste .

Press Enter to run Autodock.

D. Analysing Autodock Results:

Click on Analyse on the autodock window and in it click on Docking.

Go to your folder and open the (.dlg) file. And click ok.

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Now go to Macromolecule in Analyse and go to your folder and open

the (.pdbqs) file of the target.

Press Shift+n to visualize the macromolecule on the screen.

Go to Conformation in Analyse and in it click on Load,a box

appears.

Go to Conformation again and click on Play,another window opens.

In the Play window,click on (&) sign ,a new window opens.

Now on the first window that came on pressing Load go to its second

line and click

It shows the docking energy and various other docking

parameters.Note it.The more negative dock energy,the better inhibitor

is;positive dock energies(if found) are neglected as it is not a proper

result.

Now go to the window which came in Play,in it click on the direction

buttons to analyse each of the ten active sites.It gives information

about various parameters on a particular active site.Here we also

search for hydrogen bonds which are shown on the bottom of that

window and we can also find the amino acid to which the ligand binds.

Expand the bottom of the box which we got on clicking Load.

Click on Write Current Coords and go to your folder and save this file

as (inhibitor.docked.pdbq).

Record the results.

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PMV (Python Molecular Viewer):

Python Molecular Viewer is a tool to view the binding of hydrogen bonds in

the target molecule.It helps to visualise and analyse the hudrogen bonds.The

process of operation of PMV is enlisted below:

Procedure For Operation Of PMV:

Open PMV.

Go to File and click on Browse Command.

In the window that opens,click on pmv

In the adjacent window click on trace command,then click on

Load.Again in the adjacent window of pmv click on hbond command

and load this too.

Go to file in PMV and click on Read Molecule nad go to your folder and

open (protein name.pdbqs) file.

Go to Compute and in it go to Trace.

Here click on Compute Extrude Trace.

Go to color and click on choose color.

In the that opens click on CAT race and click ok.

In the next window which opens, choose any color of your choice and

click on Dismiss.

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Go to Display of PMV and click on Display.

In the next window click on undisplay and click ok.

Go to Select and I it click on Select From String.

In the window that opens in the box where Residue Number is written

enter the residue with its number which was noted from Analyse of

autodock. In the place where atom type is given type *.

Click on Add.

Click on Dismiss.

Go to Display and in it click on cpk.

In the window that opens adjust the Scale Factor to 0.7 and Sphere

Quality to 15,by moving the mouse across the wheels.

Click ok.

Go to Color and click on By Atom Type.

Click cpk in the window that opens.

Click ok.

Go to File and in it go to Read Molecule.

By last step go to your folder and open the (inhibitor.docked.pdbq) file

and open it.

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Go to Select and in it click on Direct Select.

In the window that opens click on Molecule and in it click on your

macromolecule.

Click on Molecule again and now click on the ligand or the inhibitor.

Click on Dismiss.

Go to Display.

In it click on Stick and Balls.

In the window that opens set Stick Quality to 15 and set Ball

Quality to 15.

Click ok.

Go to Color and click on By Atom Type.

In the next window click on Stick and Balls.

Click ok.

Go to Hydrogen Bond.

In it go to Build.

In it click on Set Parms+Build.

In the window that opens click on specify two sets.

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In the next window click on,in the top list under Molecule List click it

and select macromolecule.

I the bottom Molecule List,click it and select the ligand.

Click ok.

Go to H bond and in it click on Display.

In it click on as lines.

Click Dismiss in the window that opens.

Again go to Display and click on Cylinders.

In the window that opens adjust bond length and bond radius of the

hydrogen bond by using mouse,to a suitable size.

Go to Dj vu GUI and click on camera in the lower window.

On the window which lengthens, click on Set Background Color.

Click on SW and then click on S.

Go to File of PMV and click on save as.

In the window that opens click on Browse and go to your folder and

save the picture as (protein.tif).

RESULTS AND DISCUSSION

Retrieval of protein sequence of Flt3

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>gi|406323|emb|CAA81393.1| FLT3 receptor tyrosine kinase precursor

[Homo sapiens]

MPALARDGGQLPLLVVFSAMIFGTITNQDLPVIKCVLINHKNNDSSVGKSSSYPMVSESPEDLGCALRPQ

SSGTVYERAAVEVDVSASITLQVLVDAPGNISCLWVFKHSSLNCQPHFDLQNRGVVSMVILKMTETQAGE

YLLFIQSEATNYTILFTVSIRNTLLYTLRRPYFRKMENQDALVCISESVPEPIVEWVLCDSQGESCKEES

PAVVKKEEKVLHELFGMDIRCCARNELGRECTRLFTIDLNQTPQTTLPQLFLKVGEPLWIRCKAVHVNHG

FGLTWELENKALEEGNYFEMSTYSTNRTMIRILFAFVSSVARNDTGYYTCSSSKHPSQSALVTIVEKGFI

NATNSSEDYEIDQYEEFCFSVRFKAYPQIRCTWTFSRKSFPCEQKGLDNGYSISKFCNHKHQPGEYIFHA

ENDDAQFTKMFTLNIRRKPQVLAEASASQASCFSDGYPLPSWTWKKCSDKSPNCTEEITEGVWNRKANRK

VFGQWVSSSTLNMSEAIKGFLVKCCAYNSLGTSCETILLNSPGPFPFIQDNISFYATIGVCLLFIVVLTL

LICHKYKKQFRYESQLQMVQVTGSSDNEYFYVDFREYEYDLKWEFPRENLEFGKVLGSGAFGKVMNATAY

GISKTGVSIQVAVKMLKEKADSSEREALMSELKMMTQLGSHENIVNLLGACTLSGPIYLIFEYCCYGDLL

NYLRSKREKFHRTWTEIFKEHNFSFYPTFQSHPNSSMPGSREVQIHPDSDQISGLHGNSFHSEDEIEYEN

QKRLEEEEDLNVLTFEDLLCFAYQVAKGMEFLEFKSCVHRDLAARNVLVTHGKVVKICDFGLARDIMSDS

NYVVRGNARLPVKWMAPESLFEGIYTIKSDVWSYGILLWEIFSLGVNPYPGIPVDANFYKLIQNGFKMDQ

PFYATEEIYIIMQSCWAFDSRKRPSFPNLTSFLGCQLADAEEAMYQNVDGRVSECPHTYQNRRPFSREMD

LGLLSPQAQVEDS

Retrieval of structure of FLT3 protein.

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BLAST Result:-

Distribution of 174 Blast Hits on the Query Sequence

424
http://www.ncbi.nlm.nih.gov/blast/newoptions.html#graphical-overviewhttp://www.ncbi.nlm.nih.gov/blast/newoptions.html#graphical-overview


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Fig4.1: BLAST RESULT

ClustalW Results:

Results of search

Number of sequences 4

434
http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#6435671http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#6435671http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#119389607http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#119389607http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#158431054http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#158431054http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#126030689http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#126030689http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#88192844http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#88192844http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#109157762http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#109157762http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#30749935http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#30749935http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#93279684http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#93279684http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#126030685http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#126030685http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#30749934http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#30749934http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#126030694http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#126030694http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#149241245http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#149241245http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#114794378http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#114794378http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#158431485http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#158431485http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#109157754http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#109157754http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#158430354http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#158430354http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#10835731http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#10835731http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#30750130http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#17943043http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#146386625http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#20663951http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#28373614http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#114794791http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#114794791http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#34811267http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#40889699http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#114794793http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#114794793http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#2780855http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#114794789http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#114794789http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#157831492http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#13399500http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#145580114http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#15988250http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#158429514http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#158429514http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#22218646http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#22218646http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#158429549http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#158429549http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#158429593http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#158429593http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#158429562http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#158429562http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#158429508http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#158429587http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#158429510http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#158429233http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#158429233http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#158429585http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#50513700http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#50513700http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#50513701http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#50513701http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#34810084http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#34810084http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#119390020http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#119390020http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#126031591http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#126031591http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#2392334http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#158429479http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#7546569http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#75765648http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#145580440http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#145580439http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#71041982http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#119390010http://www.ncbi.nlm.nih.gov/blast/Blast.cgi#42543565


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gi|119390010|pdb|2I0V|A --------------------------------------------------

gi|71041982|pdb|1Y6A|A --------------------------------------------------

gi|406323|emb|CAA81393.1| QTPQTTLPQLFLKVGEPLWIRCKAVHVNHGFGLTWELENKALEEGNYFEM 300

gi|42543565|pdb|1RJB|A --------------------------------------------------

gi|119390010|pdb|2I0V|A --------------------------------------------------

gi|71041982|pdb|1Y6A|A --------------------------------------------------

gi|406323|emb|CAA81393.1| STYSTNRTMIRILFAFVSSVARNDTGYYTCSSSKHPSQSALVTIVEKGFI 350

gi|42543565|pdb|1RJB|A --------------------------------------------------

gi|119390010|pdb|2I0V|A --------------------------------------------------

gi|71041982|pdb|1Y6A|A --------------------------------------------------

gi|406323|emb|CAA81393.1| NATNSSEDYEIDQYEEFCFSVRFKAYPQIRCTWTFSRKSFPCEQKGLDNG 400

gi|42543565|pdb|1RJB|A --------------------------------------------------

gi|119390010|pdb|2I0V|A --------------------------------------------------

gi|71041982|pdb|1Y6A|A --------------------------------------------------

gi|406323|emb|CAA81393.1| YSISKFCNHKHQPGEYIFHAENDDAQFTKMFTLNIRRKPQVLAEASASQA 450

gi|42543565|pdb|1RJB|A --------------------------------------------------

gi|119390010|pdb|2I0V|A --------------------------------------------------

gi|71041982|pdb|1Y6A|A --------------------------------------------------

gi|406323|emb|CAA81393.1| SCFSDGYPLPSWTWKKCSDKSPNCTEEITEGVWNRKANRKVFGQWVSSST 500

gi|42543565|pdb|1RJB|A --------------------------------------------------

gi|119390010|pdb|2I0V|A --------------------------------------------------

gi|71041982|pdb|1Y6A|A --------------------------------------------------

gi|406323|emb|CAA81393.1| LNMSEAIKGFLVKCCAYNSLGTSCETILLNSPGPFPFIQDNISFYATIGV 550

gi|42543565|pdb|1RJB|A --------------------------------------------------

gi|119390010|pdb|2I0V|A --------------------------------------------------

gi|71041982|pdb|1Y6A|A --------------------------------------------------

gi|406323|emb|CAA81393.1| CLLFIVVLTLLICHKYKKQFRYESQLQMVQVTGSSDNEYFYVDFREYEYD 600

gi|42543565|pdb|1RJB|A -------------HKYKKQFRYESQLQMVQVTGSSDNEYFYVDFREYEYD 37

gi|119390010|pdb|2I0V|A ----------GVDYKYKQKPKYQVRWKIIESYEG--NSYTFIDPTQLPYN 38gi|71041982|pdb|1Y6A|A --------------MDPDELPLDEHCERLPYDAS---------------- 20

.: : : : : .

gi|406323|emb|CAA81393.1| LKWEFPRENLEFGKVLGSGAFGKVMNATAYGISKTGVSIQVAVKMLKEKA 650

gi|42543565|pdb|1RJB|A LKWEFPRENLEFGKVLGSGAFGKVMNATAYGISKTGVSIQVAVKMLKEKA 87

gi|119390010|pdb|2I0V|A EKWEFPRNNLQFGKTLGAGAFGKVVEATAFGLGKEDAVLKVAVKMLKSTA 88

gi|71041982|pdb|1Y6A|A -KWEFPRDRLKLGKPLGRGAFGQVIEADAFGIDKTATCRTVAVKMLKEGA 69

******:.*::** ** ****:*::* *:*:.* . *******. *

gi|406323|emb|CAA81393.1| DSSEREALMSELKMMTQLGSHENIVNLLGACT-LSGPIYLIFEYCCYGDL 699

gi|42543565|pdb|1RJB|A DSSEREALMSELKMMTQLGSHENIVNLLGACT-LSGPIYLIFEYCCYGDL 136

gi|119390010|pdb|2I0V|A HADEKEALMSELKIMSHLGQHENIVNLLGACT-HGGPVLVITEYCCYGDL 137

gi|71041982|pdb|1Y6A|A THSEHRALMSELKILIHIGHHLNVVNLLGACTKPGGPLMVIVEFCKFGNL 119

.*:.*******:: ::* * *:******** .**: :* *:* :*:*

gi|406323|emb|CAA81393.1| LNYLRSKREKFHRTWTEIFKEHNFSFYPTFQSHPNSSMPGSREVQIHPDS 749

gi|42543565|pdb|1RJB|A LNYLRSKREKFS-------------------------------------- 148gi|119390010|pdb|2I0V|A LNFLRRKR------------------------------------------ 145

gi|71041982|pdb|1Y6A|A STYLRSKRNEFVPYKTKGARFRQGKDYVGAIPVDLKRRLDSITSSQSSAS 169

.:** **

gi|406323|emb|CAA81393.1| DQISGLHGNSFHSEDEIEYENQKRLEEEEDLNVLTFEDLLCFAYQVAKGM 799

gi|42543565|pdb|1RJB|A -------------EDEIEYENQKRLEEEEDLNVLTFEDLLCFAYQVAKGM 185

gi|119390010|pdb|2I0V|A -------------PPGLEYSYNPSHNPEE---QLSSRDLLHFSSQVAQGM 179

gi|71041982|pdb|1Y6A|A SG--------FVEEKSLSDVEEEEAPEDLYKDFLTLEHLICYSFQVAKGM 211

:. : : *: ..*: :: ***:**

gi|406323|emb|CAA81393.1| EFLEFKSCVHRDLAARNVLVTHGKVVKICDFGLARDIMSDSNYVVRGNAR 849

454


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gi|42543565|pdb|1RJB|A EFLEFKSCVHRDLAARNVLVTHGKVVKICDFGLARDIMSDSNYVVRGNAR 235

gi|119390010|pdb|2I0V|A AFLASKNCIHRDVAARNVLLTNGHVAKIGDFGLARDIMNDSNYIVKGNAR 229

gi|71041982|pdb|1Y6A|A EFLASRKCIHRDLAARNILLSEKNVVKICDFGLARDIYKDPDYVRKGDAR 261

** :.*:***:****:*::. :*.** ******** .*.:*: :*:**

gi|406323|emb|CAA81393.1| LPVKWMAPESLFEGIYTIKSDVWSYGILLWEIFSLGVNPYPGIPVDANFY 899

gi|42543565|pdb|1RJB|A LPVKWMAPESLFEGIYTIKSDVWSYGILLWEIFSLGVNPYPGIPVDANFY 285

gi|119390010|pdb|2I0V|A LPVKWMAPESIFDCVYTVQSDVWSYGILLWEIFSLGLNPYPGILVNSKFY 279

gi|71041982|pdb|1Y6A|A LPLKWMAPETIFDRVYTIQSDVWSFGVLLWEIFSLGASPYPGVKIDEEFC 311

**:******::*: :**::*****:*:********* .****: :: :*

gi|406323|emb|CAA81393.1| KLIQNGFKMDQPFYATEEIYIIMQSCWAFDSRKRPSFPNLTSFLGCQLAD 949

gi|42543565|pdb|1RJB|A KLIQNGFKMDQPFYATEEIYIIMQSCWAFDSRKRPSFPNLTSFLGCQLAD 335

gi|119390010|pdb|2I0V|A KLVKDGYQMAQPAFAPKNIYSIMQACWALEPTHRPTFQQICSFLQEQAQE 329

gi|71041982|pdb|1Y6A|A RRLKEGTRMRAPDYTTPEMYQTMLDCWHGEPSQRPTFSELVEHLGNLLQA 361

: :::* :* * ::. ::* * ** :. :**:* :: ..*

gi|406323|emb|CAA81393.1| AEEAMYQNVDGRVSECPHTYQNRRPFSREMDLGLLSPQAQVEDS 993

gi|42543565|pdb|1RJB|A AEEAMYQNV----------------------------------- 344

gi|119390010|pdb|2I0V|A DRRERD-------------------------------------- 335

gi|71041982|pdb|1Y6A|A NAQQD--------------------------------------- 366

SWISS MODEL Result: _________________

CLUSTAL W(1.81) multiple sequence alignment

Target/1-495 MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTD

Template/1-495 ------------------------------------------------------------

Target/1-495 PGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLV

Template/1-495 ---------------------------------------YESQLQMVQVTGSSDNEYFYV

.. : : * . : * *

Target/1-495 DRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYH

Template/1-495 DFREYEYDLKWEFPRENLEFGKVLGSGAFGKVMNATAYGISKTGVSIQVAVKMLKER---

* * : : : * * :* . . * :. . .: . : :*

Target/1-495 RLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVS-VSKASYLLREGEEFTVTCTIKDVS

Template/1-495 ---EALMSELKMMTQLGSHENIVN-----------LLGACTLSGPIYLIFEYCCYGDLLN

: : : *..: : :. .: * * . :.

Target/1-495 SSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSA

Template/1-495 YLRSKREKFL---------------------TFEDLLCFAYQVAKGMEFLEFKS--CVHR

. * * : :* .. *: : .

Target/1-495 NVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKW

Template/1-495 DLAARNVLVTHGKVVKICDFGLARDIMS-DSNYVVRGNARLPVKWMAPESLFEGIYTIKS

:::: :* :* : : :. : : . :. :: :* :: :* *

Target/1-495 EDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYD

Template/1-495 DVWSYG---------------------------------ILLWEIFSLGVNPYP------

: :. . : *.: **. *

Target/1-495 RLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSID

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TARGET 398 DVNAAIAFNV YVNTKPEILT YDRLVNGMLQ CVAAGFPEPT IDWYFCPGTE

1rjbA 876 -illweifsl gvnpyp---- ---------- ----gipvda nfykliqngf

TARGET hhhhh sss sss hhhhh

1rjbA hhhhhhh hhhhhhhh

TARGET 448 QRCSASVLPV DVQTLNSSGP PFGKLVVQSS IDSSAFKHNG TVECKAY

1rjbA 907 kmdqpfyate eiyiimqscw afdsrkrpsf pnltsflg-- ----cql-

TARGET h hhhhhhhh h hhhhhhh

1rjbA h hhhhhhhhh h hhhhhhhh hh

INHIBITOR TABLE:

Table 4.1 : List of inhibitors against Flt3 protein

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Inhibi

torname

Chemical

formula

Molecul

arweight

Chemical structure IUPAC

name

Ag1296

C16H14N2O2 266.29456g/mol

6,7-dimethoxy-2-phenylquinoxaline

Cep701

C26H21N3O4 439.46264g/mol

Cep701.

Mln518

C31H42N6O4 562.70298g/mol

4-(6-methoxy-7-(3-piperidin-1-ylpropoxy)quinazolin-4-yl)piperazine-1-carboxylic acid(4-isopropoxyphenyl)amide

494
http://%20void%20window.open%28%27../image/structurefly.cgi?cid=3038522&width=400&height=400%27,%20%27StructureFly%27,%20%27resizable=yes,%20scrollbars=yes,%20WIDTH=620,%20HEIGHT%20=%20620%27)http://%20void%20window.open%28%27../image/structurefly.cgi?cid=126565&width=400&height=400%27,%20%27StructureFly%27,%20%27resizable=yes,%20scrollbars=yes,%20WIDTH=620,%20HEIGHT%20=%20620%27)http://%20void%20window.open%28%27../image/structurefly.cgi?cid=2049&width=400&height=400%27,%20%27StructureFly%27,%20%27resizable=yes,%20scrollbars=yes,%20WIDTH=620,%20HEIGHT%20=%20620%27)


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Pkc412

C35H30N4O4

570.637g/mol

Pkc412

Su5614

C15H13ClN2O 272.72952

g/mol

5-Chloro-3-[(3,5-

dimethylpyrrol-2-yl)methylene]-2-

indolinone

Sui C21H20NO6+

382.3866

g/mol

6-(6,6-

dimethyl-7,8-dihydro-5H-[1,3]dioxolo[4,5-g]isoquinolin-6-ium-5-yl)-6H-furo[4,3-g][1,3]benzodioxol-8-one

Table4.4 shows the chemical formula, molecular weight, chemical structure and IUPAC

name of different inhibitors which show interaction with Flt3 protein. The IUPAC name

of the inhibitor is further used in making pdb file of that inhibitor.

Docking of Ligand to Receptor

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Fig :Docking result Between Flt3 Proteins Active Site and Inhibitor AG1296

Python Molecular Viewer (PMV) Results:

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Fig :Docking result Between Flt3 Proteins Active Site and Inhibitor CEP701


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Fig : Docking result Between Flt3 Proteins Active Site and Inhibitor MLN518


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Fig4:Docking result Between Flt3 Proteins Active Site and Inhibitor PKC412.


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Fig:Docking result Between Flt3 Proteins Active Site and Inhibitor Su5614.


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Rational Drug Designing Strategies reduce a lot of time, money and energy

as compared to other hit and trial methods.According to recent trends

mathematical modelling has become very valuable.The use of sophasticated

softwares and tools greatly help in this process, helping furthur development

in research and development in this field. The main concern in AutoDock is

computation of docking energy, which essentially should be less than

zero.The more negative the docking energy, the better it is.

Graphical Representation of Autodoc Result

Fig : Shows the relative docked energies of various inhibitors with the target protein.

From the figure we can conclude that Su5614 has the minimum docked energy,hence

the best inhibitor.

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CONCLUSION

After the project work on rational drug design for flt3,the conclusion is that

out of all the inhibitors chosen for the docking,su5614 emerged to be be the

best inhibitor for the protein flt3.The precise reason for it was its docking

energy which was the lowest(docked energy=-14.74),among all other

inhibitors used.Hence the conclusion is that su5614 is the best inhibitor,for

flt3.Hence the task was completed successfully.

Cancer is a major threat to the worlds health. There are many reasons and

factors responsible for induction of cancer. flt3 is also one of those factors

responsible for the induction of cancer.Basically flt3 is an enzyme present inour body which is an integral part of the inflamatory responses of our

immune system.If due to any reason the secretion of this enzyme crosses a

perticular threshold,it can cause tumor formation.This tumor may under the

influence of mitogens and other carcinogens can cause cancer.The threat if

cancer being cause by flt3 has spread globally and many bio-pharma giants

have launched several medicines eg: Standard induction therapy for acute

myeloid leukemia includes two drugs: An anthracycline (such as daunorubicin

or idarubicin) in combination with the nucleoside analogue, cytosine

arabinoside. The main concern is to reduce the over-expression of flt3 and

not to terminate its secretion completely.There are many inhibitors which are

used for the inhibition of over secretion of flt3.Out of many inhibitors,some

are rejected due to their side effects,as the chemotherapy drugs(such as

daunorubicin or idarubicin) will kill normal bone marrow and leukemic cells

equally, so the most significant side effects besides nausea and vomiting are

a temporary reduction of normal white blood cells, red blood cells, and

platelets. The lack of white cells results in lowered immunity and a high

likelihood of infections. A low platelet count may result in easy bruisability

and spontaneous bleeding. A decrease in the red cell count, termed anemia,

may result in fatigue, shortness of breath, and lack of energy.However there

is no inhibihitor which is fully perfect and without any

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sideeffects.Nevertheless we try to reduce the burden on the general health of

the patient to the maximum extent possible.Hence ,newer drugs are required

which have the same efficacy as the older one but are having fewer side

effects.The intial phase of discovering a new drug nowadays is by using

CADD.This method has greately reduced the time ,energy and money

involved in the traditional methods.After a drug has been designined in-

silico,its furthur verification is done,as stated earlier in laoratories.This

method of using computer to design the drugs has indeed hastened the

process of drug discovery.

Rational Drug Designing Strategies reduce a lot of time ,money and energy

as compared to other hit and trial methods.According to recent trends

mathematical modelling has become very valuable recently.The use of

sophasticated softwares and tools greatly help in this process,helping furthur

development in research and development in this field.

In my project I found su5614 (docking energy= -14.74) as to be the best

leukemia project

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