p.e Seminar Updated

8/8/2019 p.e Seminar Updated

1/21


2/21

y Introduction:. What are CSFS?. What is G-CSF?. What is HG-CSF?

. Biological function of G-CSF. Therapeutic uses of G-CSF

Binding and action of G-CSF.y Structure of KW-2228.y Comparisons and differences between wild type. G-CSF and KW-2228.y Stability and activity of KW-2228.y Conclusion .


3/21

y Colony-stimulating factors (CSFs) are a family ofglycoproteins that stimulate the proliferation anddifferentiation of hematopoietic cells in vivo.

TYPES OF CSFS:y Granulocyte colony-stimulating factor (G-CSF),y Granulocyte-macrophage colony-stimulating

factor (GM-CSF),y Macrophage colony-stimulating factor (M-CSF),y Interleukins and Erythropoietin.


4/21

y Granulocyte colony-stimulating factor (G-CSF orGCSF) is a colony-stimulating factor hormone.

y It is a 19.6-kDa glycoprotein consisting of 174 amino

acid residues, produced by a number of differenttissues to stimulate the bone marrow to producegranulocytes and stem cells.

y G-CSF then stimulates the bone marrow to releasethem into the blood.

y It also stimulates the survival, proliferation,differentiation, and function of neutrophil precursorsand mature neutrophils.


5/21

y G-CSF is produced by endothelium, macrophages, and anumber of other immune cells.

y The natural human glycoprotein exists in two forms, a 174-and 180-amino-acid-long protein of molecular weight19,600 grams per mole.

y The more-abundant and more-active 174-amino acid formhas been used in the development of pharmaceuticalproducts by recombinant DNA (rDNA) technology.

y The G-CSF-receptor is present on precursor cells in thebone marrow, and, in response to stimulation by G-CSF,initiates proliferation and differentiation into maturegranulocytes.

y G-CSF is a also a potent inducer ofHSCs(HematopoeiticStem Cells) mobilization from the bone marrow into thebloodstream.


6/21

y Human G-CSF (hG-CSF) is a 19.6-kDa glycoproteinconsisting of 174 amino acid residuesy Human granulocyte colony stimulating factor

(hG-CSF) is a hematopoietic cytokine that acts on

cells of the neutrophil lineage causingproliferation and differentiation of committedprecursor cells and activation of matureneutrophils.

y KW-2228 is a tailored human granulocyte colony-

stimulating factor (hG-CSF) which has morepotent granulopoietic activity and is more stablethan wild-type hG-CSF.


7/21

y More than 100 novel hG-CSFs have beenengineered with the aim of identifying novelproteins with enhanced biological activity and

physicochemical properties .y One of these engineered proteins, designatedKW-2228, has more potent granulopoietic activitythan that of wild-type hG-CSF, both in vitro and in

vivo .y KW-2228 (Neu-up) is currently being used

clinically and is yielding very satisfactory results.


8/21

y G-CSF stimulates the production of white blood cells (WBC).

y In oncology and hematology, a recombinant form of G-CSF is used withcertain cancer patients to accelerate recovery from neutropenia afterchemotherapy, allowing higher-intensity treatment regimens.

y Chemotherapy can cause myelosuppression and unacceptably low levelsof white blood cells, making patients prone to infections and sepsis.

y G-CSF is also used to increase the number of hematopoietic stemy cells in the blood of the donor before collection by leukapheresis for use

in hematopoietic stem cell transplantation.y It may also be given to the receiver, to compensate for conditioning

regimens.y G-CSF is used to treat heart degeneration by injecting it into the blood-

stream, plus SDF(stromal cell-derived factor) directly to the heart.

y Recently, the application of G-CSF against infectious diseases has beenconsidered, and some animal experiments have been carried out tosupport such an application on human infectious diseases.


9/21

y

Sweet's syndrome is a known side effect of usingthis drug.y It was first marketed by Amgen with the brand

name Neupogen.y The recombinant human G-CSF synthesised in an

E. coli expression system is called filgrastim.y Filgrastim (Neupogen) and PEG-filgrastim

(Neulasta) are two commercially-available formsof rhG-CSF (recombinant human G-CSF).

y Another form of recombinant human G-CSFcalled lenograstim is synthesised in ChineseHamster Ovary cells (CHO cells).


10/21

y The binding of G-CSF to its receptor on pluripotent stemcells triggers terminal differentiation of the cells.

y Understanding the binding mechanism is a prerequisite todesigning an improved G-CSF.

y The residues that contribute to G-CSF receptor binding arelocated on the surface of G-CSF.y They predicted that residues 2046 and the C-terminus

bind to the G-CSF receptor.y Residues 138142 are recognized by common antibodies.

y Based on the structural and biological informationobtained to date, we predict that residues 4346 and 138140and/or those near them are involved in G-CSF receptorbinding .


11/21

y The main structural feature of KW-2228 is an antiparallel four--helix bundle (Fig. 1 and Fig. 2) which has a left-handed twist withoverall dimensions of approximately 452525 3.

y The -helices, which are designated A through D, consist of

residues 1039, 7392, 99123, and 142171, respectively.

y The overall topology of the bundle is very similar to thoseobserved in bovine, canine and wild-type hG-CSFs and very closeto that of an ideal left-handed antiparallel four--helix bundle.

y The four helices are connected by three loops designated AB, BCand CD. The AB and CD loops are long overhand connections,and the shortest loop, BC, joins helix B to helix C. In addition tothe major helices there is a short extended 310 helix designated Ewithin the AB loop which consists of residues 4352.


12/21

Fig. 1. The schematic drawing of the secondary structure elementsof KW-2228. The rectangles mark -helices. A, B, C, D and E helices

are labeled according to Scheme 1


13/21

Fig. 2. Stereoscopic ribbon drawing of KW-2228. Helices A, B, C, D and E are shown in green, cyan, yellow, red

and blue, respectively.


14/21

In G-CSF (which is the G-CSF structure mostaccurately determined so far), the coordinates ofresidues 17, 129135 and 174 are not located clearlyand the structures of the N-terminal region and

the middle of the CD loop are not definitive.y In the structure of KW-2228, however, the electron

density is clearly defined and the positions of allof the residues except 1, 2, 130 and 131 are

definitive.y Thr1, Leu3, Gly4, Pro5 and Cys17 in wild-type hG-CSF

are replaced by Ala, Thr, Tyr, Arg and Ser,respectively, in KW-2228.


15/21

y Although G-CSF is expected to be structurallyflexible due to the presence of the remarkablylong overhand loops, the substitution of amino

acids at residues 1, 3, 4, 5 and 17 in KW-2228 hasfrozen the conformation and allowed us todetermine almost the entire structure of hG-CSFfor the first time.


16/21

y There are two disulfide bridges in hG-CSF. TheCys64Cys74 disulfide bridge is buried in theinterior of the molecule, but the Cys36Cys42

bridge is on the surface of the molecule. Thesedisulfide bridges restrict the conformation of thelong overhand AB connection.

y

There is a specific hydrophobic network in theinterior of the bundle which is markedly rich inleucine residues.


17/21

y The characteristic leucine-rich hydrophobicnetwork obviously plays a very important role instabilization of the four helices in the bundleconfiguration.

y In G-CSF there are particularly long overhandconnections which are potentially flexible.Therefore the leucine-rich hydrophobic network

may function as a scaffold to stabilize the bundleand adjust the positions of specific residuesfavorable for binding of receptors.


18/21

y The biological activity and stability of KW-2228 inhuman serum are markedly enhanced compared withthose of wild-type hG-CSF.

y The residues which are engineered in KW-2228 arelocated near the N-terminus.

y The positively charged atom of Arg5 is located near thecarbonyl oxygen of Ala172

y The specific network of interactions which involveArg5 contribute stabilization of the conformation ofthe N- and C-terminal regions and fix the middle ofthe long overhand AB connection in KW 2228.


19/21

y Cys17 is substituted by Ser in KW-2228.

y

The hydroxyl group of Ser17

forms a hydrogen bondwith the carbonyl group of Leu14 in kw-2228.

y This substitution may contribute to the stability of theprotein.

In short,y The mutations introduced in KW-2228 contribute to

the stabilization of the conformation of the moleculeincluding the two long overhand connections.

y The significant enhancement of the physicochemicaland biological stability and biological activity of KW-2228 is undoubtedly due to its stabilized activeconformation.


20/21

yThe present analysis has clearlyrevealed that substituted residues play

important roles in substantialenhancement of the biological activityand stability.


21/21

y

linkinghub.elsevier.com/retrieve/pii/S0014579397005383 y www.ncbi.nlm.nih.gov/

y www.youtube.com/watch

y Isao Fujii, Yoshitomo Nagahara, Motoo Yamasak, Yoshiharu Yokoo, Seiga Itohand Noriaki Hirayama .Department of BiologicalScience and Technology, TokaiUniversity, 317 Nishino, Numazu, Shizuoka 410-03, Japan

Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., 3-6-6 Asahimachi,Machida, Tokyo 194, Japan.

y www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T36-3RB8YT6-2B&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1002387513&_rerunOrigin=google&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=9db5ac1c32dd28a99acfaae06c1deb4e.

y Jb.oxfordjournals.org

y

www.google.comy www.springerlink.org

y www.sciencemag.org

Date post:	10-Apr-2018
Category:	Documents
Upload:	mohit-philip-mathew
View:	218 times
Download:	0 times

p.e Seminar Updated

Documents