protein kinase c βii in diabetic complications: survey of structural, biological and computational...

1. Introduction

2. The role of PKC-bII in diabetes

mellitus

3. Structural and biological

aspects of PKC-bII

4. Inhibitors and

structure--activity relationship

(SAR) study

5. Computational studies

6. Work in progress:

(Unpublished data)

7. Concluding remarks

8. Expert opinion

Review

Protein kinase C bII in diabeticcomplications: survey ofstructural, biological andcomputational studiesM Elizabeth Sobhia†, Baljinder K Grewal, Jyotsna Bhat, Shishir Rohit &Vijay PuniaNational Institute of Pharmaceutical Education and Research (NIPER), Department of

Pharmacoinformatics, Punjab, India

Introduction: PKC-bII is a conventional isoform of PKC. It is overexpressed in

hyperglycemic conditions and is known to trigger various diabetic complica-

tions, mainly cardiovascular complications and to a certain extent nephropathy,

neuropathy, retinopathy etc. Selective inhibition of this enzyme will be one of

the favorable approaches to treat diabetes-mellitus-related complications. Due

to high sequence similarities among PKC isoforms, selective inhibition of

PKC-bII is difficult and yet to be achieved successfully.

Areas covered: This review discusses the studies carried out in various aspects

of PKC-bII. The biological aspects, crystal structure data, structure--activity

relationship study (SAR) and in silico studies related to PKC-bII such as

homology modeling, molecular docking, molecular dynamics, quantitative

structure--activity relationship (QSAR) studies and pharmacophore modeling

etc. are summarized.

Expert opinion: PKC-bII is a potential target for treating diabetes-related com-

plications. Selective inhibitors of this enzyme are under clinical trials but to

date, success has not been achieved. Thus, extensive research is essential in

this direction; the contribution of in silico tools in designing and optimizing

selective inhibitors of PKC-bII is valuable.

Keywords: diabetes, homology modeling, in silico, molecular dynamics, pharmacophore

modeling, PKC-bII, QSAR, SAR

Expert Opin. Ther. Targets (2012) 16(3):325-344

1. Introduction

Protein kinases are a group of enzymes which catalyze phosphorylation reaction andtransduce a variety of signals in eukaryotic cells [1]. They play a vital role in variouscellular processes, functions, deregulations and mutations [2]. PKC is a member ofthe kinases family and belongs to the class of serine/threonine kinases as they cata-lyze the phosphorylation of serine and threonine residues of proteins [3]. Since thediscovery of PKC in 1977, by Nishizuka et al., more than 12 isoforms of PKCare reported [4]. On the basis of homology and sensitivity to activators, PKCisozymes are divided into three subfamilies: Conventional/classical isozymes, novelisozymes and atypical isozymes [5]. Conventional isozymes consist of PKC-a,PKC-bI, PKC-bII and PKC-g . These isozymes are expressed by separate genesbut PKC-bI and PKC-bII are derived from the alternative splicing of the samegene and thus, they differ in their composition involving 50 amino acids at theC-terminal end [6]. All conventional isozymes require diacylglycerol (DAG), Ca2+,and phosphatidylserine (PS) for activation. Novel isozymes consist of PKC-d,PKC-", PKC-h/l and PKC-q and they require DAG and PS for activation but

10.1517/14728222.2012.667804 © 2012 Informa UK, Ltd. ISSN 1472-8222 325All rights reserved: reproduction in whole or in part not permitted

Exp

ert O

pin.

The

r. T

arge

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Suss

ex L

ibra

ry o

n 10

/26/

12Fo

r pe

rson

al u

se o

nly.

remain insensitive towards Ca2+. Atypical isozymes consistof PKC-z and PKC-i/l and they require only PS fortheir activation.PKC-bII, a classical PKC isoform is widely distributed in

kidney, CNS, heart, vascular smooth muscle and retina. It isoverexpressed in cardiac hypertrophy and fibrosis [7], andhas been implicated as a key player in the pathogenesis of dia-betic microangiopathy and macroangiopathy [5]. It is prefer-entially activated in hyperglycemia, as a result, extensiveresearch is being carried out to study the role of PKC-bII inthe pathogenesis of diabetes mellitus I and II. Microangiop-athy complications lead to nephropathy, retinopathy and neu-ropathy, whereas macroangiopathy-related complications canresult in ischemic heart disease, cerebrovascular disease andperipheral vascular disease.Considering the role of PKC-bII in diverse disease condi-

tions, a potent and novel PKC-bII inhibitor will be a majoradvancement in drug discovery and development for diabe-tes and its complications. A few representative molecules,developed by pharmaceutical companies as inhibitors ofPKC-bII, are in different stages of clinical trials or research.A few of these inhibitors are as shown in Figure 1. Ruboxis-taurin (LY333531), developed by Eli Lilly, is a specificPKC-bII inhibitor [8]. Clinical studies on orally adminis-tered LY333531, suggested that it may be effective inameliorating retinopathy, progression and proliferation,and retinal vascular leakage [9]. Central PharmaceuticalResearch Institute [10], Johnson and Johnson Pharmaceutical

Research and development have also developed potentPKC-bII inhibitors [11].

The aim of this review is to bring to light the role ofPKC-bII in diabetes-induced complications and to underlinethe different structural and biological aspects of PKC-bII.The research also suggests the different possible domains orregions of PKC-bII that can be targeted for its inhibition.Computational studies of PKC-bII reported in the literatureare also summarized.

2. The role of PKC-bII in diabetes mellitus

2.1 PKC-bII in diabetic complicationsDifferent kinds of metabolic pathways are activated duringhyperglycemia and the de novo synthesis of DAG from phos-phatidic acid, which is stimulated by glucose transportertype 1 (GLUT-1) seems to play an important role in diabeticcomplications [2]. An increased level of DAG activates differ-ent classes of PKC, but in macrovascular tissues the activationof PKC-bII is predominantly found. It is interesting to notethat inositol triphosphate (IP3), formed during DAGde novo synthesis, stimulates Ca2+ release from sarcoplasmicreticulum and the released Ca2+ leads to preferential activa-tion of PKC-bII [7]. Moreover, the study conducted byPinton et al. demonstrated that nutrient stimulation ofb-cells of pancreas causes spatial and temporal complexchanges in the subcellular localization of PKC-bII, possiblydue to the generation of Ca2+ microdomains [12]. Thus, theselocalized changes in the PKC-bII activity may have a role inspatial control of insulin release. Also, Inoguchi et al. sug-gested preferential activation of PKC-bII in the aorta andheart of hyperglycemic rats [13]. Besides these, immunoblot-ting studies revealed an increased level of PKC-bII isoformsin corpus cavernousum smooth muscle, more than PKC-aand PKC-bI [13,14]. Guruswamy et al. have demonstratedthat in transgenic hyperglycemic mice, the exacerbation ofcardiac hypertrophy and fibrosis are positively correlatedwith the enhanced expression of PKC-bII. It causes excessphosphorylation of troponin-1, which leads to decreasedmyofilament response to Ca2+ [15]. In diabetic retinopathy,the role of PKC-bII is noteworthy; studies conducted ondiabetes-induced bovine retinal endothelial and bovine retinalpericytic cell culture showed an increased activity ofPKC-bII [16]. PKC-bII activation is associated with the over-expression of endothelium-derived vasoactive factor such asendothelin-1, which leads to decrease in retinal bloodflow [7]. Shiba et al. verified that injection of phorbol ester, ageneral PKC activator in the vitreous humor can reduce reti-nal blood flow. They observed an increased expression ofPKC-a, PKC-bI, PKC-bII and PKC-" isoforms in the retinalmembrane. However, the PKC bI/II isoforms exhibited sig-nificant increases in the membrane fraction of all vasculartissues [17]. Selective inhibition of PKC-b with ruboxistaurinhas been reported to normalize this retinal abnormality [18].Takagi et al. investigated the role of endogenous

Article highlights.

. Recently PKC-bII has emerged as an importanttherapeutic target for the treatment of various diabeticcomplications viz. diabetic cardiomyopathy, diabeticnephropathy. This articles provides the therapeuticimportance, various structural and biological aspects,SAR study, and computational studies reported forPKC-bII.

. The reported PKC-bII crystal structures, and inhibitors, inaddition to reported biological and computational studywould provide rational approach to develop a potentialPKC-bII lead.

. Reported crystal structures of PKC-bII provideinformation about catalytic and regulatory domains,features unique for PKC-bII, and also throw light on theflexible regions which can be targeted for designingPKC-bII inhibitors.

. The structure–activity relationship studies and QSARstudies facilitate modification of the chemical structureof bioactive compounds to enhance their selectivity andpotency towards PKC-bII inhibition. Thus, suggests arational way to design and synthesize PKC-bII selectiveinhibitors.

. Various computational studies viz. docking, moleculardynamics, pharmacophore modeling are illustrated toenhance the potency and selectivity PKC-bII inhibitors.

This box summarizes key points contained in the article.

Protein kinase C bII in diabetic complications: survey of structural, biological and computational studies

326 Expert Opin. Ther. Targets (2012) 16(3)

Exp

ert O

pin.

The

r. T

arge

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Suss

ex L

ibra

ry o

n 10

/26/

12Fo

r pe

rson

al u

se o

nly.

endothelin-1 (ET-1) in the development of abnormal retinalhemodynamics in diabetes mellitus and showed that theaction of ET-1 is associated with PKC-bII activation [19].Activation of PKC also regulates gene expression of proteinsinvolved in vascular contractibility such as fibronectin,type 4 collagen, caldesmon and NOS via stimulation ofMAPK, thus PKC is the main mediator of diabetic vascularcomplications. VEGF induces angiogenesis as well as increasesendothelial cell permeability. An increased level of VEGF hasbeen reported in ocular fluid of patient with proliferativeretinopathy [13]. The decrease in retinal blood flow causedby PKC-bII activation may result in local hypoxia which isa potent inducer of VEGF [20]. VEGF and TGF-b involvedin vascular permeability and scar tissue formation are regu-lated at the level of gene transcription by PKCs [16]. VEGFbinds to the vascular endothelium and triggers the releaseof DAG, which activates PKC-bII. Thus activation ofVEGF and activation of PKC-bII are interdependent andare associated with microangiopathy and retinopathy.

McCarty et al. explained the correlation of glomerulonecro-sis with overactivation of PKC-bII. They found that hypergly-cemia is associated with increased lipid peroxidation, whichactivates phospholipase C (PLC). PLC increases the release ofintracellular free Ca2+ and DAG synthesis, thus stimulates theactivity of PKC. Such activation of glomerular PKC leads todiabetic nephropathy [21]. A diabetes-induced increase in thepolyol pathway leads to the depletion of neuronal myoinositol,DAG and PKC. This reduces the phosphorylation of Na+-K+-ATPase, and in turn reduces nerve conduction and furtherleads to nerve degeneration. It shows that hyperglycemicPKC-bII contributes to the development of diabetic neuropa-thy [5]. Also, Igwe et al. studied the hyperalgesia caused byadjuvant-induced inflammation in rats and it has been foundthat increased activity of PKC-bII is associated withhyperalgesia [22].

PKC-bII plays a significant role in many etiologicalconditions which are associated with atherosclerosis andmacrovascular complications. PKC-bII activation also

influences the dysfunction of eNOS and activation of vascularNAD(P)H oxidase; all leading to endothelial dysfunctions [5].

PKC-bII directly interacts with different proteins and thusinfluences different functions. It interacts with Gravin, whichacts as isozyme selective scaffold in neuronal cells. It alsointeracts with insulin receptors, which results in inhibitionof insulin receptor activity in adipocytes [23].

2.2 PKC-bII in insulin resistanceInsulin resistance is a physiological state where fat and muscletissues show diminished response to insulin. During this statethese tissues absorb relatively lower concentrations of bloodglucose, and a higher concentration of insulin is needed toattain the standard rate of glucose absorption [24]. PKC-bIIis found to be one of the factors associated with myocellularinsulin resistance. Intra-myocellular PKC-bII gets overacti-vated due to increase in lipid intermediates such as DAG,fatty acyl-CoA and ceramides. PKC-bII phosphorylates ser-ine/threonine residues of insulin receptor, which results ininsulin resistance.

Itani et al. demonstrated that lipid-induced insulin resis-tance in human muscles is associated with an increase inDAG concentration and enhanced translocation of thePKC-b from the cytosol to the cell membrane. Which furtherresults in an increase in total membrane-associated PKCactivity [25]. Cortright et al. emphasized the role of PKC-bin insulin resistance and experimentally verified that a PKCactivator can cause insulin resistance which is reversible by aPKC inhibitor [26]. Osterhoff et al. carried out a search forthe functional polymorphisms among already known geneticvariants in the PKC-b promoter and investigated their rela-tionship to glucose metabolism in humans. They showedthat the promoter polymorphism at position --546 (CG) inthe PKC-b gene, might alter the expression and the functionof PKC-b and thus influence insulin sensitivity [27].

Insulin resistance is also associated with chronic kidney dys-function and the cellular mechanism behind this dysfunctionwas determined by Mima et al. They carried out comparative

HN

OO

ONH2

HN

HN

N

OO

CH2OH

HN

N

OO

N

N

B CA

Figure 1. Representative molecules of PKC-bII inhibitors developed by pharmaceutical companies. These molecules were

developed by A. Eli Lilly; B. Central Pharmaceutical Research Institute; C. Johnson & Johnson.

Sobhia, Grewal, Bhat, Rohit & Punia

Expert Opin. Ther. Targets (2012) 16(3) 327

Exp

ert O

pin.

The

r. T

arge

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Suss

ex L

ibra

ry o

n 10

/26/

12Fo

r pe

rson

al u

se o

nly.

analysis of insulin signaling and cellular actions between renalglomeruli and tubules in control, insulin-resistance and dia-betic states. They demonstrated that the renal tubules are pro-tected from the loss of insulin action but, insulin signalingand its actions in the renal glomeruli are significantly inhibi-ted. The insulin-induced phosphorylation of insulin receptorsubstrate-1 (IRS1), Akt (PKB), eNOs, and GSK-3a wereselectively inhibited in the glomeruli but not in the renaltubules of both respective models. Treatment with thePKC-b inhibitor, ruboxistaurin, enhanced insulin actionsand elevated IRS1 expression. Thus, PKC-b inhibition canprevent kidney dysfunction or improve renal function indiabetic and insulin-resistant states [28].Kawai et al. demonstrated that PKC-b plays an important

role in glucocorticoid-induced insulin resistance as inhibitionof PKC-b improves glucocorticoid-induced insulin resistancein rat adipocytes. They showed that glucocorticoid inhibitorswere not able to restore the glucose uptake in adipocytes butPKC-b specific inhibitor restored the glucose uptake. It provesthat glucocorticoid itself does not cause alteration in insulinactivity but it provokes the activation of PKC-b which furtheraffects the insulin activity. Though results were not distinguish-ing involvement of bI or bII isoforms specifically but as overex-pression of PKC-bII in adipocyte culture was found, theyemphasized the involvement of bII in insulin resistance [29].Huang et al. explained the role of PKC-b in obesity-induced

insulin resistance in mice. Elevation in the intracellular fattyacid metabolites, such as fatty acyl CoA and DAG causetissue-specific activation of specific PKC-b that lead to theimpairment of insulin signaling and activity [30].

3. Structural and biological aspects of PKC-bII

3.1 Structure of PKC- bIIAs shown in Figure 2, PKC-bII is a single polypeptide chainwith four conserved domains (C1 -- C4) interspaced by fivevariable regions (V1 -- V5). The pseudosubstrate (PS)domain, C1, C2, V1, V2 and a part of V3 constitute the reg-ulatory domain, present at the NH2 terminal side. C3, C4,V4 and V5 form the catalytic domain at the COOH terminalside and these two domains are connected by a proteolysis-sensitive hinge region. PS occupies the tip of the C1 domainand mediates autoinhibition of enzyme. It has sequence iden-tity with PKC substrates with alanine replacing the serine/threonine phosphoacceptor site. Thus, in the inactive stateof the enzyme, the PS domain blocks the substrate-binding cavity and during activation it gets released fromactive site and causes the full activation of the enzyme [5].The C1 domain (approximately 70 amino acids) is compact,cysteine-rich and comprised of five short b-strands an a-helixand two Zn2+ ions which form the DAG and PD bindingsites. The C1 domain is divided into two parts namely C1aand C1b, and shares a high degree of sequence similarity.Pericentrin (a centrosomal protein) and RING finger proteinthat interacts with C kinase (RINCK) are the two proteins

which interact with the C1a region [31]. The former is respon-sible for spindle formation and cytokinesis; disruption of thisinteraction results in inhibition of cell division, while overex-pression of the latter results in ubiquitination and degradationof PKC-bII. Thus, by binding to C1 domain of PKC-bII,these proteins can alter the subcellular localization and activityof enzyme [31]. The C2 domain is formed of eight anti-parallel b-strands connected by loops, which are lined by mul-tiple aspartic acid residues that coordinate with three Ca2+

ions, which act as a bridge between the C2 domain and phos-pholipid head group. A protein known as receptor of activatedC kinase (RACK)-1(36 kDa) consists of seven repeats of theWD40 motif arranged in a propeller like structure, interactswith PKC-bII and stabilizes its active form, thus increasesthe efficiency of substrate phosphorylation. The C3 domainis recognized as a highly conserved site for ATP binding(XGXGX2GX16KX), and the sequence for this domain issame for bI and bII isozymes [32]. A point mutation at theATP binding site abolishes the kinase activity. TheC4 domain is substrate-binding site which also takes part inthe phosphoryl transfer. It is a conserved region among allPKCs with a conserved sequence of 105 -- 108 amino acids.This sequence starts at the end of the ATP-binding site andcontinues till the beginning of the phosphate transfer group.The V5 region, made up of 50 -- 70 amino acids, plays animportant role in localization and phosphorylation of PKCs.It has diverse effect son the functions of different isoforms;in the case of PKC-bI and bII, the V5 region causes localiza-tion of enzymes in a different way in different tissues [33].Kiley et al. analyzed this V5 region effect on humanU937 monocytic cell line. They reported that PKC-bI islocalized to the microtubules, whereas PKC-bII is localizedin part to the secretory granules [34]. Blobe et al. demonstratedthis localization effect using the MOLT-4 T-lymphoblastoidcell line. They observed that PKC-bII, and not bI, translo-cates to actin microfilaments upon phorbol 12-myristate13- acetate (PMA) treatment of cells [35].

The hinge region is defined by residues 422 -- 425 whichconnects the regulatory and catalytic region. It gets destabi-lized by proteolytic enzymes, and has been identified as a tar-get for caspase-dependent cleavage, tyrosine phosphorylationand protein--protein interaction [32].

3.2 Reported crystal structures of PKC-bIITo date, only three crystal structures of different domains ofPKC-bII are reported. Grodsky et al. have reported a crystalstructure of catalytic domain of PKC-bII in complex withan ATP-competitive inhibitor (2-methyl-1H-indol-3-yl-BIM-1); with the resolution of 2.6 A. Figure 3 shows that,the protein is captured in its intermediate conformation,which lies in between the typical open and closed conforma-tion. The novel a-helix (629 -- 636) is a unique feature ofPKC-bII that was not observed previously in other PKCkinase domain structures. It is flexible in nature and plays avery important role in the activation and inactivation of



Exp

ert O

pin.

The

r. T

arge

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Suss

ex L

ibra

ry o

n 10

/26/

12Fo

r pe

rson

al u

se o

nly.

PKC-bII [36]. Leonard et al. recently published a crystal struc-ture of full length PKC-bII (4A resolution) confined in closedconformation as shown in Figure 4 [37]. The study explains thenovel regulatory mechanism of allosteric activation ofPKC-bII which involves binding of DAG to the C1a andC1b domains. The NFD loop (Asn-628, Phe-629, Asp-630)is a part of the initial residues of the novel a-helix and playsan important role in PKC-bII activation. It is the most

conserved motif that is present in all PKCs and it is highlyflexible, it remains in the helical form in the inactiveconformation and extends in the active conformation [37].

3.3 Activation and localization of PKC-bIIIn the membrane, newly synthesized PKC-bII is present inthe open state conformation in such a way that itsC-terminus is exposed to PDK-1 and PKC-bII gets attached

Regulatory domain Catalytic domain

V1 C1a C1b V2 C2 V3 C3 V4C4

P P P

V5

Hydrophobicmotif

Turnmotif

Activationloop

HingePseudosubstrate(PS)

Figure 2. Domain structure of PKC-bII. The protein has a single polypeptide chain with four conserved (C1 -- C4) domains

interspaced by five variable (V1 -- V5) regions. The pseudosubstrate (purple) occupies the N terminal end, further connected to

the C1 domain (orange). The C1 domain has two subdomains C1a and C1b. V1, PS, C1, V2 and C2 collectively form the

regulatory domain. C3, V4, C4, V5 collectively form the catalytic domain occupying the C-terminal end. The C4 domain has

three phosphoryltion sites; the activation loop, turn motif and hydrophobic motif; which get phosphorylated during

activation of the proteinprotein. Regulatory and catalytic domains are connected by the proteolytic hinge region.

Val-423

Glu-421

Thr-404

Figure 3. The kinase domain of PKC-bII complexed with 2-methyl-1H-indol-3-yl-BIM-1 (PDB ID-2i0e) and ligand interaction

with residues in the ATP binding site.



Exp

ert O

pin.

The

r. T

arge

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Suss

ex L

ibra

ry o

n 10

/26/

12Fo

r pe

rson

al u

se o

nly.

to it. Figure 5 shows the primary activation of PKC-bII.During activation, PKC-bII undergoes primary phosphory-lation at three sites and gets properly positioned. Theattached PDK-1 carries out transphosphorylation of Thr-500 present on the activation loop and this is essential forthe maturation of the isozymes [32]. In order to maintainthe stabilized state; PKC-bII undergoes two autophosphory-lations at its turn motif (Thr-641) and hydrophobic motif(Ser-660).The mature PKC-bII at this stage is still inactive due to

the binding of the pseudosubstrate which prevents the bind-ing of substrate at the active site. This mature but inactiveisozyme is released in the cytosol. DAGs are the secondarymessengers that allosterically activate PKC-bII. Glucoseincreases DAG and PKC signaling in some cell types butthe initial burst in DAG production during insulin actionis due to phosphatidylcholine hydrolysis in the plasma mem-brane. After the initial burst de novo phosphatidic acid syn-thesis in the endoplasmic reticulum acts as a source of DAG.Generation of these extracellular signals increase the level ofDAG and Ca2+ which preferentially activate PKC-bII overother PKC isoforms [38]. Before the allosteric activation,the enzyme is in the intermediate partially active conforma-tion. The allosteric activation is initiated by binding of Ca2+

to the C2 domain of the Ca2+ binding region (CBR), andthis causes translocation of PKC-bII towards the membrane.Due to this translocation of protein, C1 can interact withDAG and binds to DAG. Figure 6 demonstrates the

allosteric activation of PKC-bII. DAG first binds to theC1a and this initiates the release of pseudosubstrate fromthe catalytic cleft, thus relieving the autoinhibition mediatedby pseudosubstrate. C1a promotes binding of phosphatidyl-serine to C2 and localizes the protein in the membraneregion; however, the structure is not yet in its fully activatedstate. The NFD loop remains in contact with the C1bdomain forming clamp and this conformation is known asthe out conformation. Phe-629, one of the residues of theNFD loop interacts with C1b when the NFD loop has ahelix conformation and resides away from the ATP bindingsite. Interaction of this Phe-629 phenyl ring and adeninering of the ATP (present in the ATP binding site) is neces-sary for the proper substrate binding and the phosphoryla-tion of the substrate. Thus, this clamp formation preventsadenine and Phe-629 interaction resulting in autoinhibition.Another molecule of DAG binds to the C1b and causes dis-engagement of the NFD clamp and relieves NFD-mediated autoinhibition. Disengagement of the NFD helixthus triggers the phosphorylation of the substrate [37].

In response to PKC-bII activation, the protein RACK1translocates from cytosol to the same site in the membraneas an activated protein. The C2 domain has the RACK bind-ing site and RACK-like sequence known as pseudoRACK. Inthe inactive close conformation, pseudoRACK binds to theC2 domain and stabilizes the inactive state of the enzyme.During activation, Ca2+ ions interact with protein andinitiate the release of pseudosubstrate and pseudoRACK.

C1b Domain

C2 Domain

Kinasedomain

Figure 4. Full length crystal structure of PKC-bII complexed with ATP analogue (PDB ID- 3pfq).



Exp

ert O

pin.

The

r. T

arge

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Suss

ex L

ibra

ry o

n 10

/26/

12Fo

r pe

rson

al u

se o

nly.

T500

T641

S660Kinase

domain C4

C3 Domain

DAGPhosphatidylserine

PSNFD

Kinasedomain C4

T641S660

DAG

C1b C1a

DAG

PS

NFD

C2Domain

Ca++

Ca++

T500

C1aC2

Domain

C1b

Figure 6. Allosteric activation of PKC-bII. In the membrane the enzyme is in closed conformation and in an inactive state. DAG

allosterically activates the enzyme. DAG first binds to the C1a, this causes release of pseudosubstrate (PS) from the catalytic

cleft, thus relieving the autoinhibition mediated by pseudosubstrate. The NFD sequence remains in contact with the C1b

domain and forms a clamp this conformation is also called the out conformation, resulting in autoinhibition. Another

molecule of DAG binds to the C1b and causes disengagement of the NFD clamp and relieves NFD- mediated autoinhibition.

T500

T500

T641S660

ATP

Autophosphorylation

Kinasedomain C4

Kinasedomain C4

Kinasedomain C4

ATP

C1bC1b

C1b

NFD

NFD N

FDC1a

C1a

C1a

C1b

NFDC1a

T500T641S660

Kinasedomain C4

Extracellular Ca++ entersin to cell and interacts

with C2 domain

C2Domain

C2Domain

PDK1

C2Domain

MembranePhosphatidylserine

Ca++

C2Domain

Figure 5. Primary activation and localization of PKC-bII. In the membrane PDK1 carries out transphosphorylation at Thr-

500 present on the activation loop; PKC-bII gets released from membrane and undergoes two autophosphorylations at Thr-

641 and Ser-660 present on the turn motif and hydrophobic motif respectively. After these three primed phosphorylations

extracellular Ca2+ enters in to cytoplasm and interacts with the C2 domain and PKC-bII translocates again towards the

membrane and binds to phosphatidylserine and gets localized in membrane region.



Exp

ert O

pin.

The

r. T

arge

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Suss

ex L

ibra

ry o

n 10

/26/

12Fo

r pe

rson

al u

se o

nly.

Subsequently, the protein attains its active state and bothRACK1 and substrate get access to their binding sites [39].PKC-bII after binding to RACK1 acts as a shuttling proteinand the active form of PKC-bII is rapidly dephosphorylatedwhich becomes destabilized [31]. A protein known asHsp70 (chaperone) binds to the turn motif of the dephos-phorylated enzyme to stabilize it and allow it to rephosphory-late, the enzyme then reenters the signaling cascade. Absenceof Hsp70 binding or chronic activation of PKC-bII resultsin accumulation of dephosphorylated enzyme and its conse-quent degradation [40]. Expression of another protein RINCKresults in ubiquitination and degradation of PKC-bII [31].Alteration in the phosphorylation state, conformation orlocalization of PKC-bII can disrupt the signaling events,leading to altered physiological states that are observed indiabetic complications.

4. Inhibitors and structure--activityrelationship (SAR) study

PKC inhibitors targeting regulatory domain or catalytic domainand dual inhibitors are known. The regulatory-domain inhibi-tors may target the phospholipid or phorbol ester binding siteof the PKC structure whereas catalytic-domain inhibitors bindto the substrate site or the ATP binding site [41]. Some of thereported PKC inhibitors are mentioned in Table 1, viz. Midos-taurin (PKC412, CGS41251, N-benzoyl staurosporine),UCN-01 (KW-2401, NSC-638850), Lestaurtinib (CEP-701,KT-5555), Ro-31 -- 8220 and Go6976 [41-46].All these are nonspecific PKC inhibitors thus, have severe

side effects. UCN-01 was developed for cancer but was with-drawn from clinical trials due to its side effects [7]. UCN-01and Midostaurin [CGP41251 (PKC412)] inhibits the activi-ties of cPKC (PKC-bII) to a greater extent than nPKCs.GP41251 inhibits the response mediated by the activation

of the VEGF receptor, platelet-derived growth factor receptoror stem cell receptor [47].

Staurosporine is the most potent PKC inhibitor describedin literature so far, but it lacks the ability to selectivelyinhibit PKCs. As a result various research groups are tryingto develop a PKC inhibitor which is both potent and selec-tive. Subsequently, various staurosporine analogs werereported with improved selectivity for ‘conventional’ PKCisoenzymes over ‘novel’ and ‘atypical’ ones. Selective PKCinhibitors, like indolcarbazole or bisindoylmaleimide aretargeted mainly to the ATP-binding site of the catalyticdomain. Various inhibitors of PKC, a and b inhibitors arestudied in vitro and in vivo in detail, for prevention ofchronic hyperglycemia-induced damage in diabetic kidney.The PKC-bII-selective inhibitor LY333531 has shown byfar the most promising effects of preventing of diabeticvascular complications.

In 1991, Toullec et al. synthesized various bisindolylma-leimide derivatives based on staurosporine structural modifi-cations and reported GF-109203X as a potent and selectiveinhibitor of PKC both in vitro and in intact cells [48]. In1992, Davis et al. reported a series of 2,3-bisarylmaleimidesand simultaneously substituted bisindolylmaleimides(BIMS) with improved potency and selectivity. Their serieswas based on the structural modification of indolocarbazolesand staurosporine. They removed the bond between the twoindoles present in staurosporine and added another carbonylgroup. Due to steric repulsion between the two indole rings,the resulting molecule attained a conformation whichincreases its selectivity towards PKC over PKA maintainingthe same potency as that of staurosporine [49,50]. In 1993,Bit et al. reported bisindolylmalemides with improvedpotency and selectivity for PKC. They were guided bymolecular graphics and conformational restriction of thecationic side chain. They showed that introduction of cat-ionic side chain into these compounds resulted in inhibitorsof greater potency and selectivity and to improve thepotency of these amines, they adopted conformationalrestriction approach [51]. In 1996, Jirousek et al. reportedthe SAR study of LY333531, a novel class of macrocyclicbisindolylmaleimdes isozyme, selective inhibitors ofPKC-bII. A panel of eight cloned human PKC isozymeswas used to identify the series and optimize SAR.LY333531 inhibits PKC-bI (IC50 = 4.7 nM) and PKC-bII(IC50 = 5.9 nM) isozymes. It was 76- and 61-fold selectivein inhibiting PKC-bI and PKC-bII in comparison withPKC-a, respectively [18]. In 2003, Faul et al. reported syn-thesis and SAR of N-(azacycloalkyl) bisindolymaleimides,an acyclic derivative of staurosporine. The representativecompound of this series (illustrated in Table 2) exhibits anIC50 of 40 -- 50 nM against the human PKC-bI andPKC-bII, and selectively inhibits PKC-b over other PKCisozymes [10,52]. In 2005, Zhang et al. synthesized and exam-ined novel indolylindazolylmaleimides for kinase inhibition.They reported the SAR study and cardiovascular safety of

Table 1. Reported PKC-inhibitors with their target sites

and their clinical trial status.

Target Inhibitor Status

Regulatory domain(Phospholipid orphorbol esterbinding site)

Bryostatin-1 Phase II discontinuedSafingol Phase I discontinuedAprinocarsen Phase III discontinuedCalphostin C --Sphingosine --

ATP binding site Midastourin Phase IIUCN-01 Phase II discontinuedLestaurtinib Phase II discontinuedRo-31-8220 Pre-clinicalG 06976 Pre-clinicalSangivamtycin --Isoquininoline H7 --Staurosporine --

Targeting bothsites mentionedabove

Acridine yellow gold --



Exp

ert O

pin.

The

r. T

arge

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Suss

ex L

ibra

ry o

n 10

/26/

12Fo

r pe

rson

al u

se o

nly.

Table 2. Representative molecule reported in SAR studies with their structure and activities.

Chemical class Molecule Activity IC50 (mM) Ref.

PKC inhibitorsAminoalkyl bisindolylmaleimidesGF 109203X3-(1-(3-(dimethylamino)propyl-(1H-indol-3-yl)-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione

HN

NH

N

O O

N

0.010 ± 0.005 [48]

2, 3-Bisindolylmaleimides derivatives3-(5-chloro-1-methyl-1H-indol-3-yl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione

HN

NN

OO

Cl

0.11 ± 0.04 [49]

Phenylindolyl-maleimides3-(1-methyl-1H-indol-3-yl)-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione

HN

N

OO

NO2

0.67 ± 0.19 [49]

Bisindolylmaleimides derivatives3-(3-(4-(1-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)-1H-indol-1-yl)propylcarbamimidothioate

HN

NN

O O

S

H2N NH

0.010 ± 0.003 [50]



Exp

ert O

pin.

The

r. T

arge

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Suss

ex L

ibra

ry o

n 10

/26/

12Fo

r pe

rson

al u

se o

nly.

Table 2. Representative molecule reported in SAR studies with their structure and activities (continued).


Bisindolylmaleimides derivatives3-(8-(aminomethyl)-6,7,8,9-tetrahydropyrido[1,2-a]indol-10-yl)-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione

HN

NH

N

O O

NH2

0.0076 ± 0.002 [51]

PKC-b inhibitorsLY333531(S)-13-[(Dimethylamino)methyl]-10,11,14,15-tetra-hydro-4,9:16,21-dimethono-1H,13H-dibenzo[e,k]pyrrolo[1,4,13]oxadiazacyclohexadecene-1,3(2H)-dione

HN

NN

O O

O

N

0.0059 [18]

N-(azacycloalkyl)bisindolylmaleimides)3-(1-methyl-1H-indol-3-yl)-4-(1-(4-(pyridin-2-yl)cyclohexyl)-1H-indol-3-yl)-1H-pyrrole-2,5-dione

HN

NN

O O

N

0.03 [52]



Exp

ert O

pin.

The

r. T

arge

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Suss

ex L

ibra

ry o

n 10

/26/

12Fo

r pe

rson

al u

se o

nly.



Indolylindazolylmaleimides derivatives3-(1-(3-(dimethylamino)propyl)-1H-indol-3-yl)-4-(1-(thiophen-2-yl)-1H-indol-3-yl)-1H-pyrrole-2,5-dione

HN

NN

O O

N

S

0.010 [11]

3-anilino-4-(3-indolyl) maleimides3-(2-(aminomethyl)-2,3-dihydro-1H-pyrrolo[1,2-a]indol-9-yl)-4-(phenylamino)-1H-pyrrole-2,5-dione

HN

HN

N

O O

H2N

0.0014 [53]

Anilino-monoindolylmaleimides3-(1-(3-(1H-imidazol-1-yl)propyl)-1H-indol-3-yl)-4-(phenylamino)-1H-pyrrole-2,5-dione

HN

HN

N

OO

NN

0.005 [10]

Bisindolylmaleimides derivatives3-(1-methyl-1H-indol-3-yl)-4-(3-(4-methylpiperazin-1-yl)naphthalen-1-yl)-1H-pyrrole-2,5-dione

HN

N

OO

N

N

0.0018 ± 0.002 [54]



Exp

ert O

pin.

The

r. T

arge

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Suss

ex L

ibra

ry o

n 10

/26/

12Fo

r pe

rson

al u

se o

nly.



Macrocyclic bisindolymaleimide derivative HN

NN

OO

N

N

O

0.009 ± 0.001 [56]

3-(7-Azaindolyl)-4-arylmaleimides3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-(3-methoxyphenyl)-1H-pyrrole-2,5-dione

HN

NN

OO

O

OH

0.004 ± 0.001 [57]

Bis(indolyl)maleimide pyridinophanes derivatives HN

NN

OO

N

NH2

0.011 [58]



Exp

ert O

pin.

The

r. T

arge

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Suss

ex L

ibra

ry o

n 10

/26/

12Fo

r pe

rson

al u

se o

nly.

novel indolylmaleimides as inhibitors of PKC-bII. Theyidentified low nanomolar inhibitors of PKC-b with goodto excellent selectivity versus other PKC isozymes andGSK-3b. In the cardiovascular assessment, representativelead compounds bound to the ether-a-go-go-related genetype H (hERG) channel with high affinity, potently inhib-ited ion current in a patch-clamp experiment, and causeda dose dependent increase of QTc in guinea pigs [11]. In2006, Tanaka et al. reported conformationally restricted3-anilino-4-(3-indolyl) maleimide derivatives acting asPKC-b selective inhibitors and possessing oral bioavailabil-ity. Among these, compounds having a fused five-membered ring at the indole 1,2-position exhibited activityat a very low concentration and had ameliorative effects ina rat diabetic retinopathy via the oral route and showedgood oral bioavailability [10,53]. In 2009, Wagner et al.reported a series of indolylquinazolylmaleimides, which arenovel maleimide-based derivatives. Molecule AEB071 wasreported as a potent, selective inhibitor of classical andnovel PKC isotypes. It was also claimed to be an efficientimmune-modulator that acts by inhibiting early T cell acti-vation. Modifications of the structure of staurosporineresulted in multiple types of partially isoform-specific PKCinhibitors. A series of novel maleimide-based inhibitorswere designed, synthesized, and evaluated in 2009 whichalso showed selectivity towards PKC-bII [54]. In 2011,Li et al. reported initial SAR studies on a series of novelpyrrolopyrazole-based PKC-bII inhibitors from highthroughput screening [55]. Table 2 lists the moleculesreported in SAR studies [10,11,18,48-54,56-58].

Ruboxistaurin and enzastaurin are two potent inhibitorsselective for PKC-b and they are specific for ATP-binding site; both are in Phase III clinical trials. Enzastaurinhas shown anti-apoptotic and anti-proliferative properties. Itmainly acts on tumor cells; multiple myeloma tumor ofanimal models and in cells of lung, colon, renal andglioma tumors. Ruboxistaurin also exhibited potential clinicalpromise for diabetic retinopathy and nephropathy [8,59].

5. Computational studies

Computational studies aid drug discovery processes by reduc-ing chemical space to be searched to identify new potentiallead. They use various in silico approaches viz. homologymodeling, molecular docking, molecular dynamics and quan-titative SAR (QSAR) to model or mimic the behavior ofdrugs. These computational methods are used to investigatethe structure, dynamics, surface properties and thermodynam-ics of inorganic and biological systems. They are also applica-ble in analyzing conformational changes and stability ofprotein. A few of these in silico methods are explained herein brief.

Protein structures are more conserved than proteinsequences. Homologous proteins have similar structuresthough they don’t have identical sequences. We can predict

the possible structure of a protein if its sequence and thecrystal structure of a homologous protein (> 20% sequenceidentity) is known. When crystal structural information isnot available for particular protein, we can generate thestructural data by aligning sequences generated using FASTAsoftware to the target sequence to the template protein(homologous protein). This process is called homologymodeling [60].

Molecular docking is a method of predicting possible con-formation of one molecule in complex with other molecule.During the docking process, the ligand and the receptor adopta conformation that will have a minimal free energy. It isassumed that this minimal energy conformation is a bioactivestate of a complex. Different scoring functions are used in dif-ferent docking methods. These scoring functions determinethe binding energies of different conformations and rankthem by following certain criteria. Molecular docking isused to predict the preferred binding affinity and the bestmolecular orientation of a ligand, when it is bound to thereceptor [61].

Molecular dynamics simulation deals with dynamics, thatis motion, of a molecular system with respect to time. Posi-tion and velocity of a molecule is given at different time stepsby applying Newton’s laws of motion. At each step totalenergy is calculated, force is calculated from total energy,acceleration and velocity are calculated. By considering accel-eration and velocity, the position of a system at the next step iscalculated [62].

QSARs are mathematical models or regression models thatare used to predict activities such as physicochemical proper-ties, biological activities, toxicity etc. based on physical char-acteristics of the structure of chemicals. It uses linearstatistical methods or non-linear methods to generate amathematical model that correlates experimental measureswith a set of chemical descriptors. QSAR models are helpfulto predict the biological activities of untested compoundsdepending on their structural properties [63].

Pharmacophore is a 3D model which defines the essentialmolecular features (mainly steric and electrostatic) whichenables the biological activity of a ligand. It has applicationin both the presence and absence of crystal structure of areceptor. In case of unavailability of crystal structure of areceptor, a set of ligands together with their measured bind-ing affinities towards the receptor help to form a pharmaco-phore model which explains how structurally diverse ligandscan bind to a common receptor site. It has applicability inde novo design to develop totally novel drugs that satisfy thepharmacophore requirements and it is useful in leadoptimization [64].

5.1 Homology modeling and molecular dockingTo determine the selectivity of ruboxistaurin towards PKC-bamong other conventional PKCs, comparative studies werecarried out by Tang et al. As crystal structural data was notavailable for kinase domains of PKC-a, -bI and -z, they



Exp

ert O

pin.

The

r. T

arge

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Suss

ex L

ibra

ry o

n 10

/26/

12Fo

r pe

rson

al u

se o

nly.

derived the homology models of these kinase domains.PKC-q, PKC-t, PKB were used as template structures dueto their high sequence identity with the respective isoforms.These homology models were further studied to identifyruboxistaurin--protein interactions, which are responsible forruboxistaurin specificity towards PKC-b isozymes. A mole-cular docking study on PKC-a, PKC-bI and PKC-z was car-ried out simultaneously. Comparison of docking results ofdifferent isozymes revealed different binding sites and bindingconformations. In the case of PKC-z, two conformations werefound; one was bound to the ATP-binding site and the otherone was bound at a site adjacent to the ATP-binding site,termed the allosteric site. Such two sites are also present inother isozymes of PKC, but they are placed far apart, thussuch binding did not occur with other isozymes. In this dock-ing study, ruboxistaurin showed favorable interactions withPKC-bI, but did not show the desired interactions with otherisozymes [65]. Hence, this finding supports ruboxistaurinselectivity towards PKC-bI. Zhang et al. synthesized theindolylindazolylmaleimide molecule shown in Figure 7 [11],and docked it into the ATP binding sites of a homologymodel of PKC-bII, constructed on the basis of PKA (PDBcode 1STC). The compound with maximum selectivitytowards PKC-bII shows some specific interactions. It formstwo hydrogen bonds with the carbonyl group of Glu-91 andthe amide hydrogen of Val-93. The amine group in thelong tail forms another hydrogen bond with Asp-140.An aromatic-stacking interaction between the benzothienylgroup and Tyr-100 is unique for PKC-bII, and a pocketsurrounded by the hinge allows the benzothienyl group toaccommodate well.

5.2 Molecular dynamicsBanci et al. carried out a molecular dynamics study for charac-terization of the C2 domain of PKC-b. PKC isozymes havespecific anchoring proteins termed RACK proteins. C2domain of PKC-b contains a part of a RACK binding site

and it also has a RACK-like sequence in it, termed pseudoRACK. They proposed that this pseudo-RACK site mediatesintermolecular interaction with one of the RACK-bindingsites in the C2 domain itself, stabilizing the inactive confor-mation of PKC-b. Upon activation with Ca2+ and PS,PKC-b binds to RACK1. So, they studied the conformationalchanges in the C2 RACK-binding sites, in the absence andpresence of Ca2+, using computer simulation techniques ofmolecular dynamics (MD). The studies suggested a changein conformation within the C2 domain, perhaps due tocalcium and PS binding. Ca2+ stabilizes the b-sandwich struc-ture of the C2 domain and affects two of the three RACK-binding sites within the C2 domain. Removal of Ca2+ didnot show a modification in the interactions between the thirdRACK binding site and the pseudo-RACK site. On that basis,they predicted that the pseudo-RACK site within theC2 domain masks a RACK-binding site in another domainof PKC-b1, possibly the V5 domain. Also molecular dyna-mics modeling showed that two Ca2+ ions are able to interactwith two molecules of O-phospho-L-serine. This datasuggested that Ca2+ ions may be directly involved inPKC binding to phosphatidylserine, thus involved in PKCactivation [39].

Another molecular dynamics simulation was performed byTang et al. on the energy-minimized ruboxistaurin--PKC-acomplex and ruboxistaurin--PKC-bI complex, to predict theselectivity of ruboxistaurin towards PKC isoenzyme.

The thermodynamic integration method was used to cal-culate the binding energy of ruboxistaurin with that of activesite of PKC-a and PKC-bI. Relative free energy of PKC-aand of PKC-bl showed that ruboxistaurin complex withPKC-a is less favored by 5 kcal/mol free energy and this issupported by its experimental IC50 values. Thus, thisanalysis estimated that ruboxistaurin shows 6 l fold greaterselectivity towards PKC-bl [65].They employed molecularmechanic/Poisson-Boltzmann surface area (MM-PBSA)protocol to compute the absolute binding affinity for rubox-istaurin bound to the kinase domains of PKC-a, -b and -z.MM-PBSA calculations computed the nonentropic bindingenergy (DEMM-PBSA), sums of van der Waals contributions(DEvdw), polar (DGPB) and nonpolar (DGSA) desolvationenergies for the complexes of ruboxistaurin with PKC-aand bI. On the basis of these calculated energies, they con-cluded that PKC-bI has the most favored electrostatic energycontribution (DEelec), lowest DEMM-PBSA as well as the leastdesolvation penalty (DGPB). Thus, ruboxistaurin PKC-bIcomplex is the most favored complex in terms of energy val-ues and shows selective inhibition of PKC-b [65]. They usedMM-GBSA methodology to investigate the residue-levelinteractions of ruboxistaurin with PKCs. In the case ofPKC-bI, Lys-467 forms favorable interactions, and Asp-465 exhibited unfavorable interactions, for the binding ofruboxistaurin. The polar residues in the binding site of thekinase domain of PKC-bI viz. Asp-426, Asp-465, Asp-469and Asp-483 exhibited unfavorable interactions for the

N

N

NN

OO

H

N(CH3)2S

Figure 7. Structure of compound 8j.



Exp

ert O

pin.

The

r. T

arge

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Suss

ex L

ibra

ry o

n 10

/26/

12Fo

r pe

rson

al u

se o

nly.

binding of ruboxistaurin. Lys-467 and Asp-483 contributedthe most to the ligand binding according to residue energydecomposition. The ruboxistaurin--PKC-bI complex exhibi-ted the largest number of hydrogen bonds among all otherisozyme complexes. Thus, this residue interaction analysisshows that ruboxistaurin makes favorable interaction withPKC-b isozymes [65].

5.3 QSARKumar et al. had carried out hologram QSAR (HQSAR)and comparative molecular similarity indices analysis(CoMSIA) studies to explore the structural requirementsof PKC-bII inhibitors. Figure 8 describes the contourmap analysis of the generated HQSAR and CoMSIA mod-els which suggested some useful points in designing noveland more active molecules for PKC-bII [66]. As per theirresults, a terminal substitution with electronegative atomat the indole or azaindole ring is essential for PKC-bIIinhibition, while substitution of the bulkier group in thelinker connecting two heteroaryl rings diminishes the

activity. Sri.Divya et al. carried out 2D and 3D QSARanalysis; anilino-monoindolylmaleimides, acting as aPKC-bII selective inhibitor was studied to identify favor-able substitution sites to increase their activity [67]. TheirCoMSIA analysis suggested the sites of favorable substitu-tions of indolymalemides derivatives and that alkyl aminesubstitution on the pharmacophore would be a favorablesubstitution for improving the biological activity. TheHQSAR showed that the presence of a tertiary acceptorgroup may probably be responsible for the increase inthe activity, whereas, the occurrence of cyclic ring struc-tures would lead to a decrease in the activity. These mod-els can also be used for predicting a molecule similar tothis dataset for PKC-bII inhibition.

5.4 Pharmacophore modelingJain et al. designed and validated selective peptide inhibitorsfor PKC-bII based on the RACK 1 region using in silicoapproaches. The RACK-1 sequence, specific for PKC-bIIisoform was exploited to design peptide inhibitors with

Purple

Y

G

Y

A. B.

C. D.

Y

Y

Y

P

B

R

BRR

P

RCyan

R

Figure 8. CoMSIA Contour representations on most active molecule: Y(yellow), G(green), P(pink), B(blue), R(red). A. Steric

contour with favorable green region and unfavorable yellow region. B. Hydrophobic favored areas in yellow, disfavored in

white. C. Hydrogen bond donor favored areas in cyan, disfavored areas in purple. Hydrogen-bond-acceptor favored areas in

magenta, disfavored areas contribution in red. D. Electrostatic contour showing positive-charge favored areas in blue,

negative charge favored areas in red.The authors have been granted permission to re-publish the figure from [66].



Exp

ert O

pin.

The

r. T

arge

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Suss

ex L

ibra

ry o

n 10

/26/

12Fo

r pe

rson

al u

se o

nly.

good selectivity, and the designed peptides were depicted tohave good binding affinity [68]. They proposed five probabletype 3 peptidomimetic inhibitors of PKC-bII, which mayact as isozyme-selective inhibitors. Jain et al. studied the bind-ing mode of PKC-bII inhibitors enzastaurin and ruboxis-taurin with the active site residues of PKB and PKC-bII. Apharmacophore model for both PDK-1 and PKC-bII wasdeveloped and used for virtual screening of chemicaldatabases [69]. Figure 9 illustrates the PKC-bII pharmacophoremodel comprised of five chemical features, namely, onehydrophobic (HY), one hydrogen bond acceptor (HBA),one hydrogen bond donor (HBD), and two ring aromaticdomains. Also PKB has four chemical features namely onehydrophobic (HY), one hydrogen bond acceptor (HBA),one hydrogen bond donor (HBD), and one ring aromatic(RA). The pharmacophore mapping approach helped inunderstanding the requirement for important chemical fea-tures in molecules acting as dual inhibitors of PKB andPKC-bII. The virtual screenings, using these pharmacophoresproduced few hits, which may act as dual inhibitors of PKBand PKC-b II. Leads can be designed by accommodatingthese features established for the two targets, and therebyefficiently preventing the binding of substrates and theeffects thereof.

6. Work in progress: (Unpublished data)

Our research group has designed peptide inhibitors, based onthe auto-inhibitory domain, as this domain has a recognitionmotif specific for each isoform to resolve the specificity issue,and the designed peptide inhibitors were validated by molec-ular docking. Peptides designed from these binding regionshave lower binding affinities than the proteins as such. Butthese peptides can act as inhibitors of the respective proteininteraction by acting as a block between the two interactingproteins. This rationale prompted us to design peptides

from regions of pseudosubstrate and troponin interactingwith PKC-bII. Troponin was selected to design peptidesbecause of the fact that phosphorylation of cardiac Tn-T and Tn-I by PKC was found to be an important regulatorymechanism in cardiac pathophysiology. Validation of thepeptide inhibitors was carried out in silico by molecular dock-ing. The designed peptide inhibitors might act as selectivePKC-bII inhibitors and can be used to develop peptidomi-metics. The active site of PKC-bII includes residues of activa-tion loop and alpha C helix. A pair wise alignment usingEMBOSS::Water (Local) was performed between two pro-teins, 2I0E (PKC-bII) and 1CDK (PKA in complex withMn2+) which have sequence identity and sequence similarityof 37.9 and 56.9% respectively. An analysis after the align-ment was carried out to identify the corresponding residuesof PKC-bII interacting with the side chains of substrateresidues. A 3D superposition of 2I0E and 1CDK was per-formed in Sybyl7.1 using the module Biopolymer. RMSDof 2.19A was obtained after aligning 2I0E and 1CDK. Ourresearch group is further trying to explore mechanism ofallosteric activation of PKC-bII with the help of the fullenzyme structure.

7. Concluding remarks

PKC-bII is preferentially activated in hyperglycemia-inducedde novo synthesis of DAG and various lines of experimentalevidence show that this activation plays a pivotal role in thepathogenesis of diabetic complications. Therefore, specificinhibition of the PKC-bII isoform does appear to be a promi-sing approach for the treatment of these complications; how-ever, comparatively limited reports are available especiallyfor PKC-bII inhibitors. Computational tools have shownsuccess in designing safe and effective drug molecules withtarget specificity, a similar approach appears to be helpful intargeting PKC-bII.

8. Expert opinion

Splendid work has been performed on ‘PKC-bII as a ther-apeutic target’ in diabetes-induced complications. Variousin vitro and in silico experiments have been carried outto reveal the involvement of PKC-bII in diabetic complica-tions. In vitro studies provide a detailed insight into thesignaling mechanism of enzymes in hyperglycemic condi-tions. Activation and localization of enzymes in differenttissues and in different physiological and pathological con-ditions have been studied in detail. Also, studies related toactivation of PKC-bII and its consequences associated withdiabetic complications formed a base for selectively target-ing PKC-bII. Two crystal structures of the kinase domainof PKC-bII are available which help us in understandingvarious unique structural features of PKC-bII that can beused for designing specific PKC-bII inhibitors. Variousin silico methods have been utilized to determine the best

5.83Å5.86Å

5.96Å8.75Å

5.04

6.45Å2.81Å

6.75Å

7.96Å

4.78Å

Figure 9. 3D spatial relationship and geometric parameters

of Hypo1 of PKC-bII.The authors have been granted permission to re-publish the figure from [68].



Exp

ert O

pin.

The

r. T

arge

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Suss

ex L

ibra

ry o

n 10

/26/

12Fo

r pe

rson

al u

se o

nly.

possible approach for inhibiting PKC-bII efficiently.Various specific inhibitors have been designed and syn-thesized; ruboxistaurin is one of those that have reachedclinical trials.

Successful inhibition of PKC-bII will help us to reducediabetic complications, which is the ultimate goal of allthese studies. To date, no success has been achieved indeveloping a drug that a selective inhibitor of PKC-bIIand has a safe toxicological profile. We need to focus onfactors causing toxicity, with selectivity being anotherimportant factor. Our research group has worked on design-ing peptidomimetic inhibitors but synthesis and in vitrotesting of these molecules is essential. This peptidomieticapproach may solve the toxicity issue. Collaborative applica-tion of in silico and in vitro approaches will definitely helpus to deal with selectivity and toxicity problems in targetingPKC-bII. Considering structural features of the enzyme,various domains can act as potential targets for inhibitionpurpose. The ATP binding site has been mainly exploitedfor inhibition of the enzyme but other domains from theregulatory region are of equal importance. C2, C1a andC1b domains and the pseudosubstrate region plays a vitalrole in the activation and autoinhibition of PKC-bII andmore study is still required. Localization of the enzymeplays an essential role in its tissue-specific action,which involves enzyme--membrane interactions. Activation,deactivation and autoinhibition of the enzyme involve

open--close conformational changes and all theseconformational changes are yet to be explored.

Selective inhibition of PKC-b isoforms is a controversialissue. The general strategy in dealing with enzyme inhibi-tion involves specific inhibition of particular isoforms ofa protein, so as to avoid adverse effects and off-targettoxicity issues. Ghoreschi et al. conveyed a new app-roach in kinase targeting. They explained that broad-spectrum kinase inhibition is not as problematic as waspreviously expected. With the help of a study based oninhibitors of Janus kinase, they have explained the advan-tages and disadvantages of selectively inhibiting this classof kinases [70]. Liu et al. have demonstrated that action ofruboxustaurin in the recovery of cardiomyopathy is associ-ated with inhibition of PKC-a and not PKC-b or g .Ruboxistaurin shows equal selectivity towards b and a iso-forms of PKC [71]. Thus, ruboxistaurin action actuallydepends on inhibition of not only PKC-b but also otherclassical isoforms like PKC-a and g . This broad-spectrum targeting strategy needs to be thoroughlystudied and it may lead to a successful remedy fordiabetic complications.

Declaration of interest

The authors state no conflict of interest and have received nopayment in preparation of this manuscript.



Exp

ert O

pin.

The

r. T

arge

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Suss

ex L

ibra

ry o

n 10

/26/

12Fo

r pe

rson

al u

se o

nly.

BibliographyPapers of special note have been highlighted as

either of interest (�) or of considerable interest(��) to readers.

1. Newton AC. Protein kinase C: structure,

function, and regulation. J Biol Chem

1995;270:28495-8

2. Koya D, King GL. Protein kinase C

activation and the development of

diabetic complications. Diabetes

1998;47:859-66

3. Idris I, Gray S, Donnelly R. Protein

kinase C activation: isozyme-specific

effects on metabolism and cardiovascular

complications in diabetes. Diabetologia

2001;44:659-73

4. Nishizuka Y. Studies and perspectives of

protein kinase C. Science

1986;233:305-12

5. Avignon A, Sultan A. PKC-epsilon

inhibition: a new therapeutic approach

for diabetic complications?

Diabetes Metab 2006;32:205-13

6. Kubo K, Ohno S, Suzuki K. Primary

structures of human protein kinase C

betaI and betaII differ only in their

C-terminal sequences. FEBS Lett

1987;223:138-42

7. Das Evcimen N, King GL. The role of

protein kinase C activation and the

vascular complications of diabetes.

Pharmacol Res 2007;55:498-510

8. Wheeler GD. Ruboxistaurin (Eli Lilly).

IDrugs 2003;6:159-63

9. Aiello LP. The potential role of PKC

beta in diabetic retinopathy and macular

edema. Surv Ophthalmol

2002;47:S263-9

10. Tanaka M, Sagawa S, Hoshi J, et al.

Synthesis of

anilino-monoindolylmaleimides as potent

and selective PKC beta inhibitors.

Bioorg Med Chem Lett 2004;14:5171-4

11. Zhang HC, Derian CK, McComsey DF,

et al. Novel indolylindazolylmaleimides

as inhibitors of protein kinase C-beta

synthesis, biological activity, and

cardiovascular safety. J Med Chem

2005;48:1725-8

12. Pinton P, Tsuboi T, Ainscow EK, et al.

Dynamics of glucose-induced membrane

recruitment of protein kinase C betaII in

living pancreatic islet beta-cells.

J Biol Chem 2002;277:37702-10

13. Inoguchi T, Battan R, Handler E, et al.

Preferential elevation of protein kinase C

isoform betaII and diacylglycerol levels in

the aorta and heart of diabetic rats:

differential reversibility to glycemic

control by islet cell transplantation.

Proc Natl Acad Sci 1992;89:11059-63

14. Kikkawa R, Haneda M, Uzu T, et al.

Translocation of protein kinase Calpha

and zeta in rat glomerular mesangial cells

cultured under high glucose conditions.

Diabetologia 1994;37:838-41

15. Gurusamy N, Watanabe K, Ma M, et al.

Inactivation of 14-3-3 protein exacerbates

cardiac hypertrophy and fibrosis through

enhanced expression of protein kinase C

beta2 in experimental diabetes.

Biol Pharm Bull 2005;28:957-62

16. Idris I, Donnelly R. Protein kinase C

inhibition: a novel therapeutic strategy

for diabetic microangiopathy. Diab Vasc

Dis Res 2006;3:172-8

17. Shiba T, Inoguchi T, Sportsman JR,

et al. Correlation of diacylglycerol level

and protein kinase C activity in rat retina

to retinal circulation. Am J Physiol

Endocrinol Metab 1993;265:E783-93

18. Jirousek MR, Gillig JR, Gonzalez CM,

et al. (S)-13-[(dimethylamino)methyl]-

10,11,14,15-tetrahydro-4,9:16, 21-

dimetheno-1H, 13H-dibenzo[e,k]pyrrolo

[3,4-h][1,4,13]oxadiazacyclohexadecene-

1,3(2H)-dione (LY333531) and related

analogues: isozyme selective inhibitors of

protein kinase C beta. J Med Chem

1996;39:2664-71

19. Takagi C, Bursell SE, Lin YW, et al.

Regulation of retinal hemodynamics in

diabetic rats by increased expression and

action of endothelin-1.

Invest Ophthalmol Vis Sci

1996;37:2504-18

20. Aiello LP, Avery RL, Arrigg PG, et al.

Vascular endothelial growth factor in

ocular fluid of patients with diabetic

retinopathy and other retinal disorders.

N Engl J Med 1994;331:1480-7

21. McCarty MF. A central role for protein

kinase C overactivity in diabetic

glomerulosclerosis: implications for

prevention with antioxidants, fish oil,

and ACE inhibitors. Med Hypotheses

1998;50:155-65

22. Igwe OJ, Chronwall BM. Hyperalgesia

induced by peripheral inflammation is

mediated by protein kinase C betaII

isozyme in the rat spinal cord.

Neuroscience 2001;104:875-90

23. Piontek J, Brandt R. Differential and

regulated binding of cAMP-dependent

protein kinase and protein kinase C

isoenzymes to gravin in human model

neurons. J Biol Chem 2003;278:38970-9

24. Hulver MW, Dohm GL. The molecular

mechanism linking muscle fat

accumulation to insulin resistance.

Proc Nutr Soc 2004;63:375-80

25. Itani S, Ruderman N, Schmieder F,

Boden G. Lipid-induced insulin

resistance in human muscle is associated

with changes in diacylglycerol, protein

kinase C, and IkB-alpha. Diabetes

2002;51:2005-11

26. Cortright RN, Azevedo Jr JL, Zhou Q,

et al. Protein kinase C modulates insulin

action in human skeletal muscle. Am J

Physiol Endocrinol Metab

2000;278:E553-62

27. Osterhoff MA, Heuer S, Pfeiffer M,

et al. Identification of a functional

protein kinase Cbeta promoter

polymorphism in humans related to

insulin resistance. Mol Genet Metab

2008;93:210-15

28. Mima A, Ohshiro Y, Kitada M, et al.

Glomerular-specific protein kinase

C-beta-induced insulin receptor

substrate-1 dysfunction and insulin

resistance in rat models of diabetes and

obesity. Kidney Int 2011;79:883-96

29. Kawai Y, Ishizuka T, Kajita K, et al.

Inhibition of PKCbeta improves

glucocorticoid-induced insulin resistance

in rat adipocytes. IUBMB Life

2002;54:365-70

30. Huang W, Bansode R, Mehta M,

Mehta KD. Loss of protein kinase Cbeta

function protects mice against diabetes

induced obesity and development of

hepatic steatosis and insulin resistance.

Hepatology 2009;49:1525-36

31. Gould CM, Newton AC. The life and

death of protein kinase C.

Curr Drug Targets 2008;9:614-25

32. Steinberg SF. Structural basis of protein

kinase C isoform function. Physiol Rev

2008;88:1341-78.. This review covers information

regarding the structural basis of

individual PKC isoforms, the

regulation, maturation, activation,

signaling function etc. of these

enzymes. It also includes detailed

descriptions of isoform-specific post-



Exp

ert O

pin.

The

r. T

arge

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Suss

ex L

ibra

ry o

n 10

/26/

12Fo

r pe

rson

al u

se o

nly.

translational modification patterns,

protein--protein interactions and

subcellular targeting. This information

is significant for further

isoform-specific inhibitor design.

33. Mellor H, Parker PJ. The extended

protein kinase C superfamily. Biochem J

1998;332:281-92

34. Kiley SC, Parker PJ. Differential

localization of protein kinase C isozymes

in U937 cells: evidence for distinct

isozyme functions during monocyte

differentiation. J Cell Sci

1995;108:1003-16

35. Blobe GC, Stribling DS, Fabbro D,

et al. Protein kinase C betaII specifically

binds to and is activated by F-actin.

J Biol Chem 1996;271:15823-30

36. Grodsky N, Li Y, Bouzida D, et al.

Structure of the catalytic domain of

human protein kinase C betaII

complexed with a bisindolylmaleimide

inhibitor. Biochemistry (Mosc)

2006;45:13970-81. This study describes the crystal

structure of the catalytic domain of

PKC-bII complexed with an inhibitor

at 2.6 A resolution. It is the first

structural description of any

conventional PKC kinase domain, to

our knowledge. This structure has

been used as a template for the

computational studies and for rational

design of inhibitors as potential

therapeutic agents.

37. Leonard TA, Rozycki B, Saidi LF, et al.

Crystal structure and allosteric activation

of protein kinase C betaII. Cell

2011;144:55-66. This study describes the crystal

structure of full-length PKC-bII at 4.0

A. It has revealed the intermediate

conformation of PKC-bII in the

activation pathway and. It is the only

structure of PKC-bII which has kinase

and regulatory domains linked to

each other.

38. Standaert ML, Musunuru K, Yamada K,

et al. Insulin-stimulated

phosphatidylcholine hydrolysis,

diacylglycerol/protein kinase C signalling,

and hexose transport in pertussis

toxin-treated BC3H-1 myocytes.

Cell Signal 1994;6:707-16

39. Banci L, Cavallaro G, Kheifets V, et al.

Molecular dynamics characterization of

the C2 domain of protein kinase C-beta.

J Biol Chem 2002;277:12988-97

40. Newton AC. Regulation of the ABC

kinases by phosphorylation: protein

kinase C as a paradigm. Biochem J

2003;370:361-71

41. Shen GX, Way KJ, Jacobs JR, King GL.

Applications of inhibitors for protein

kinase C and their isoforms.

Methods Mol Biol 2003;233:397-422

42. Davies SP, Reddy H, Caivano M,

Cohen P. Specificity and mechanism of

action of some commonly used protein

kinase inhibitors. Biochem J

2000;351:95-105

43. Cabanis A, Gressier B, Brunet C, et al.

Effect of the protein kinase C inhibitor

GF 109 203X on elastase release and

respiratory burst of human neutrophils.

Gen Pharmacol Vasc Syst

1996;27:1409-14

44. Wang Y, Yin OQP, Graf P, et al.

Dose-and time-dependent

pharmacokinetics of midostaurin in

patients with diabetes mellitus.

J Clin Pharmacol 2008;48:763-75

45. Wang Y, Yang H, Liu H, et al. Effect of

staurosporine on the mobility and

invasiveness of lung adenocarcinoma

A549 cells: an in vitro study.

BMC Cancer 2009;9(1):174

46. Goekjian PG. Staurosporine as an early

lead:an overview of kinase inhibitors

inspired by the iindolocarbazole

alkaloids. Dosis 2006;22:124-51

47. Alakananda B. The potential of protein

kinase C as a target for anticancer

treatment. Pharmacol Ther

1993;59:257-80

48. Toullec D, Pianetti P, Coste H, et al.

The bisindolylmaleimide GF 109203X is

a potent and selective inhibitor of

protein kinase C. J Biol Chem

1991;266:15771-81

49. Davis PD, Hill CH, Lawton G, et al.

Inhibitors of protein kinase C. 1. 2,

3-bisarylmaleimides. J Med Chem

1992;35:177-84

50. Davis PD, Elliott LH, Harris W, et al.

Inhibitors of protein kinase C. 2.

Substituted bisindolylmaleimides with

improved potency and selectivity.

J Med Chem 1992;35:994-1001

51. Bit RA, Davis PD, Elliott LH, et al.

Inhibitors of protein kinase C. 3. Potent

and highly selective bisindolylmaleimides

by conformational restriction.

J Med Chem 1993;36:21-9

52. Faul MM, Gillig JR, Jirousek MR, et al.

Acyclic N-(azacycloalkyl)

bisindolylmaleimides: isozyme selective

inhibitors of PKCbeta. Bioorg Med

Chem Lett 2003;13:1857-9

53. Tanaka M, Sagawa S, Hoshi J, et al.

Synthesis, SAR studies, and

pharmacological evaluation of 3-anilino-

4-(3-indolyl) maleimides with

conformationally restricted structure as

orally bioavailable PKCbeta-selective

inhibitors. Bioorg Med Chem

2006;14:5781-94

54. Wagner J, von Matt P, Sedrani R, et al.

Discovery of 3-(1 H-Indol-3-yl)-4-[2-(4-

methylpiperazin-1-yl) quinazolin-4-yl]

pyrrole-2, 5-dione (AEB071), a potent

and selective inhibitor of protein kinase

C isotypes. J Med Chem

2009;52:6193-6

55. Li H, Hong Y, Nukui S, et al.

Identification of novel pyrrolopyrazoles

as protein kinase C betaII inhibitors.


56. Zhang HC, White KB, Ye H, et al.

Macrocyclic bisindolylmaleimides as

inhibitors of protein kinase C and

glycogen synthase kinase-3. Bioorg Med

Chem Lett 2003;13:3049-53

57. Zhang HC, Ye H, Conway BR, et al. 3-

(7-Azaindolyl)-4-arylmaleimides as

potent, selective inhibitors of glycogen

synthase kinase-3. Bioorg Med

Chem Lett 2004;14:3245-50

58. Zhang HC, Bonaga LVR, Ye H, et al.

Novel bis (indolyl) maleimide

pyridinophanes that are potent, selective

inhibitors of glycogen synthase kinase-3.


59. Graff JR, McNulty AM, Hanna KR,

et al. The protein kinase Cbeta--selective

inhibitor, enzastaurin (LY317615. HCl),

suppresses signaling through the AKT

pathway, induces apoptosis, and

suppresses growth of human colon cancer

and glioblastoma xenografts. Cancer Res

2005;65:7462-9

60. Chothia C, Lesk AM. The relation

between the divergence of sequence and

structure in proteins. EMBO J

1986;5:823-6

61. Lengauer T, Rarey M. Computational

methods for biomolecular docking.

Curr Opin Struct Biol 1996;6:402-6

62. Allen MP. Introduction to molecular

dynamics simulation. Comput Soft



Exp

ert O

pin.

The

r. T

arge

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Suss

ex L

ibra

ry o

n 10

/26/

12Fo

r pe

rson

al u

se o

nly.

Matter Synthetic Polymers Proteins

2004;23:1-28

63. Selassie CD. History of quantitative

structure-activity relationships. Burger’s

Med Chem Drug Discov 2003;1:1-48

64. Dror O, Shulman-Peleg A, Nussinov R,

Wolfson HJ. Predicting molecular

interactions in silico: I. A guide to

pharmacophore identification and its

applications to drug design.

Curr Med Chem 2004;11:71-90

65. Tang S, Xiao V, Wei L, et al. Protein

kinase C isozymes and their selectivity

towards ruboxistaurin. Proteins Struct

Funct Bioinform 2008;72:447-60.. This paper demonstrates the selectivity

of ruboxistaurin towards PKC-b with

the help of in silico studies. This study

includes homology modeling, docking,

molecular dynamics,

MM-PBSA calculation and molecular

mechanics/generalized Born surface

area (MM-GBSA) analysis to verify the

selective inhibition of PKC-b.

66. Kumar H, Kumar R, Grewal BK,

Sobhia ME. Insights into the structural

requirements of PKC betaII inhibitors

based on HQSAR and CoMSIA analyses.

Chem Biol Drug Des 2011;78:283-8

67. Sri Divya P, Grewal B,

Elizabeth Sobhia M. 2D and 3D QSAR

analyses to predict favorable substitution

sites in anilino-monoindolylmaleimides

acting as PKCbetaII selective inhibitors.

Med Chem Res 2011;20:1188-99

68. Jain K, Sobhia ME. Targeting PKC-beta

II by peptides and peptidomimetics

derived from RACK 1: an in silico

approach. Mol Inform 2011;30:45-62

69. Jain K, Ajay D, Sobhia ME. Targeting

PKC-betaII and PKB connection: design

of dual inhibitors. Mol Inform

2011;30:329-44

70. Ghoreschi K, Laurence A, O’Shea JJ.

Selectivity and therapeutic inhibition of

kinases: to be or not to be?

Nat Immunol 2009;10:356-60

71. Liu Q, Chen X, MacDonnell SM, et al.

Protein kinase Calpha, but not PKCbeta

or PKCgamma, regulates contractility

and heart failure susceptibility. Circ Res

2009;105(2):194-200

AffiliationM Elizabeth Sobhia†, Baljinder K Grewal,

Jyotsna Bhat, Shishir Rohit & Vijay Punia†Author for correspondence

National Institute of Pharmaceutical Education

and Research (NIPER), Department of

Pharmacoinformatics, Sector 67, S.A.S. Nagar,

Punjab 160062, India

Tel: +91 172 2211343;

Fax: +91 172 2214692;

E-mail: [email protected]



Exp

ert O

pin.

The

r. T

arge

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Suss

ex L

ibra

ry o

n 10

/26/

12Fo

r pe

rson

al u

se o

nly.

protein kinase c βii in diabetic complications: survey of structural, biological and computational...

Documents