REVIEW

Allosteric regulation of pathologic angiogenesis: potentialapplication for angiogenesis-related blindness

Dong Hyun Jo • Jin Hyoung Kim • Kyu-Won Kim •

Young-Ger Suh • Jeong Hun Kim

Received: 14 October 2013 / Accepted: 24 December 2013 / Published online: 7 January 2014

� The Pharmaceutical Society of Korea 2014

Abstract Angiogenesis-related blindness (ARB) includes

age-related macular degeneration, diabetic retinopathy, and

retinopathy of prematurity, all of which are based on

pathologic angiogenesis. Current treatment options such as

surgery, laser photocoagulation, and steroid have shown

limitations because they do not directly resolve the path-

ologic events in the retina. Furthermore, recently approved

and developed therapeutic drugs only focus on direct

inhibition of growth factors and suppression of downstream

signaling molecules of activated receptor proteins by

orthosteric ligands. In this regard, allosteric regulation of

receptors and ligands can be a valuable mechanism in the

development of novel drugs for ARB. In this review, we

briefly address the clinical significance of ARB for further

discussion on allosteric regulation of pathologic angio-

genesis for ARB. Interestingly, key molecules in the

pathogenesis of ARB can be targets for allosteric regula-

tion, sharing characteristics as allosteric proteins. With

investigation of allostery by introducing well-established

models for allosteric proteins and currently published novel

allosteric modulators, we discuss the potential of allosteric

regulation for ARB. In conclusion, we hope that allosteric

regulation of pathologic angiogenesis in ARB can open

new opportunities for drug development.

Keywords Allosteric regulation � Pathologic

angiogenesis � Age-related macular degeneration �Diabetic retinopathy � Retinopathy of prematurity

Introduction: allostery gives insights into drug

development for angiogenesis-related blindness (ARB)

In 1963, Monod, Changeux, and Jacob reported an elegantly

written paper in Journal of Molecular Biology, introducing

the concept of ‘allostery’ in the action of proteins regarding

cellular control systems (Monod et al. 1963). Simply,

allosteric proteins indicate proteins that have allosteric

binding sites other than orthosteric ones where orthosteric

ligands can bind to induce effector functions of specific

proteins. The authors of the monumental article suggested

glutamic-dehydrogenase, acetyl-CoA carboxylase from the

adipose tissue, muscle phosphorylase b, and hemoglobin as

representative examples of allosteric proteins (Monod et al.

1963). The first 3 proteins are enzymes, whereas hemo-

globin represents non-enzymic proteins of important bio-

logical functions. During the 50-year period after the

publication of the paper by Monod et al. allosteric regula-

tion have been extended to cell surface receptors and pro-

teins in signal transduction and transcription regulation,

giving insights into understanding the general principles of

various allosteric proteins (Changeux 2012).

In the field of drug development, past a few decades

were blossomed with advancement accompanying timely

D. H. Jo � J. H. Kim � J. H. Kim (&)

Fight Against Angiogenesis-Related Blindness (FARB)

Laboratory, Clinical Research Institute, Seoul National

University Hospital, Seoul 110-744, Korea

e-mail: steph25@snu.ac.kr

D. H. Jo � J. H. Kim

Department of Biomedical Sciences, College of Medicine,

Seoul National University, Seoul 110-799, Korea

K.-W. Kim � Y.-G. Suh

College of Pharmacy, Seoul National University, Seoul 151-742,

Korea

J. H. Kim

Department of Ophthalmology, College of Medicine,

Seoul National University, Seoul 110-744, Korea

123

Arch. Pharm. Res. (2014) 37:285–298

DOI 10.1007/s12272-013-0324-y

discovery of novel orthosteric agonists or antagonists. Lead

compounds targeting orthosteric binding sites of receptor

proteins or effector proteins have shown promising pre-

clinical and clinical results. Even more, monoclonal anti-

bodies against orthosteric ligands suppress the activation of

receptor proteins and further downstream signaling mole-

cules (Rodrigues et al. 2009). With the modulation of

binding of orthosteric ligands to orthosteric binding sites,

we can control proliferation of malignant cells, block

aberrant signaling cascades, and suppress transcription of

various disease-related genes. As for ARB, anti-vascular

endothelial growth factor (VEGF) agents are used to

address vision-threatening complications from pathologic

angiogenesis mediated by VEGF (Jo et al. 2010). After the

introduction of anti-VEGF agents to everyday clinical

settings, visual outcomes are improved, as other orthosteric

drugs revolutionize the treatment of various diseases.

Unfortunately, there are limitations of orthosteric ago-

nists or antagonists. Direct suppression of receptors and

their downstream signaling pathways induce unbearable

side effects and certain groups of patients and disease

components show refractoriness to therapeutic agents. This

is the same with current therapeutic agents targeting VEGF

pathway (Chen and Cleck 2009; Miller et al. 2012). Fur-

thermore, the more orthosteric binding sites and mecha-

nisms excavated for utilization as drug targets, the less

potential target sites and mechanisms remained. As with

therapeutic agents targeting G protein-coupled receptors

(GPCR), the rate of novel drug discovery based on mech-

anisms in the axis of orthosteric ligand-orthosteric site can

be in decline in the field of drug development for ARB

(Booth and Zemmel 2004; Keov et al. 2011). In order to

overcome complications of ARB, novel targets and

modalities addressing those targets are desperately required.

Interestingly, allosteric regulation of GPCR is widely

studied to show potentials in the treatment of psychological

and cognitive disorders (Gregory et al. 2011; Keov et al.

2011; May et al. 2007). Allosteric regulators can affect the

functions of specific proteins by modulating conformation

or free energy of them (Tsai et al. 2008). In short, allosteric

regulation can enhance the binding affinity of orthosteric

ligands to orthosteric binding sites, suppress signaling

pathways evoked by orthosteric ligands, or switch the

response or susceptibility of receptor or effector proteins to

orthosteric ligands. We expect that allosteric regulation can

expand therapeutic options for various diseases in which

allosteric proteins play an important role in the pathogen-

esis of them.

In this review, we aim to investigate the potentials of

approaches utilizing the concept of allostery in the devel-

opment of candidate therapeutics for ARB. For further

discussion, ARB is characterized by clinical features,

common mechanisms, and key molecules regarding the

pathogenesis of diseases. Then, we give an overview of

current treatment options with distinct achievement and

drawbacks. In this regard, allosteric regulation can be a

novel ‘antithesis’ for the treatment options for ARB, dif-

ferent from current paradigm focusing direct modulation of

key molecules and receptors in the pathogenesis of ARB.

Interestingly, they share characteristics to be targets for

allosteric modulation. With introduction of potential ther-

apeutic agents that utilize allosteric effects on key mole-

cules and receptors in the pathogenesis of ARB, we hope

that this review can break new ground for active utilization

of allosteric regulation in the development of therapeutic

agents for ARB, decreasing the risk of vision deterioration.

ARB

ARB includes representative vision-threatening diseases:

age-related macular degeneration (AMD), diabetic reti-

nopathy (DR), and retinopathy of prematurity (ROP). As

their names imply, each disease is related with aging,

diabetes, and preterm birth, respectively. We coined the

term, ARB, to emphasize the common mechanism of these

vision-threatening disorders, pathologic angiogenesis

(Jo et al. 2010). In order to overcome visual impairment by

complications of these diseases, it is important to suppress

pathologic angiogenesis. In this regard, many researchers

and pharmaceutical companies struggle to find novel tar-

gets and improve available therapeutics for the treatment of

ARB (Zhang et al. 2012).

Clinical features of ARB

AMD is divided into dry and wet AMD according to the

presence of choroidal neovascularization (CNV) (Lim et al.

2012a). In the retina with dry AMD, there are drusen,

pigmentary changes including depigmentation and hypo-

pigmentation, and atrophic changes. Some groups of

patients of early dry AMD develop the advanced form of

dry AMD, geographic atrophy, and CNV; whereas, CNV

can occur de novo in the retina with no definite evidence of

drusen or pigmentary changes (Clemons et al. 2005). In the

United States, the estimated prevalence of AMD was 6.5 %

and that of advanced AMD, defined as geographic atrophy

and wet AMD, was 0.8 % among adults aged 40 years and

older (Klein et al. 2011). Generally, wet AMD draws more

attention from clinical and scientific societies, because

most of cases with visual deterioration in AMD comes

from this type. In wet AMD, CNV induces fluid collection

in subretinal or intraretinal areas with leakage of plasma

contents from it. Furthermore, the rupture of CNV results

in intraretinal or subretinal hemorrhage, obscuring effec-

tive delivery of light signals to photoreceptor cone and rod

286 D. H. Jo et al.

123

cells. Particularly, as its name imply, AMD is intrinsically

the disease of the macula, the central area of the retina,

which is the most important area in vision. In this regard,

AMD is one of the leading causes of blindness among

adults, especially in the elderly (Congdon et al. 2004;

Gehrs et al. 2006).

DR is the most common microvascular complications of

diabetes (Antonetti et al. 2012; Frank 2004). From the

pooled analysis of 8 pupulation-based studies, the esti-

mated prevalence of DR and vision-threatening of DR were

40.3 and 8.2 % in the adults who were 40 years and older

in the United States (Kempen et al. 2004). Similar to AMD,

DR is divided into 2 distinct clinical entities according to

the presence of retinal neovascularization: non-prolifera-

tive DR (NPDR) and proliferative DR (PDR). In the retina

with NPDR, classical findings include microaneurysms,

cotton-wool spots of accumulation of exudative materials,

and intraretinal mirovascular abnormalities. NPDR can be

classified as mild, moderate, and severe ones by the

severity and number of these pathologic signs (Antonetti

et al. 2012). In PDR, retinal neovascularization develops in

the inner layers of the retina, protruding into the vitreous

cavity filled with extracellular matrix (ECM) proteins (Lim

et al. 2012b). Interestingly, differently from AMD in which

most cases with visual deterioration are related with wet

type, patients with NPDR can suffer from vision loss due to

macular edema, fluid collection in the macular area. Nev-

ertheless, the importance of pathologic angiogenesis in the

development of DR cannot be underestimated in that vit-

reous hemorrhage from ruptured retinal new vessels and

tractional retinal detachment from fibrovascular mem-

branes regarding retinal neovascularization result in severe

visual impairment.

ROP is a condition that manifests as a result of impaired

normal vascular development and increased pathologic

neovascularization. The incidence of ROP was 0.17 % in

34 million live births and that of ROP was 15.58 % in

preterm infants with more than 28 days of hospital stay

based on a national database in the United States (Lad et al.

2009). Low birth weight and gestational age are repeatedly

reported risk factors for higher incidence and more severe

ROP. Interestingly, these risk factors remain unchanged

between studies conducted about 10 and 20 years apart

(Good et al. 2005; Hoogerwerf et al. 2010; Palmer et al.

1991). In ROP, pathologic findings are evident at the

junction between vascularized and unvascularized retina.

Retinal neovascularization extending to vitreous cavity

forms a distinct structure named extraretinal fibrovascular

proliferation (EFP). In more advanced forms of diseases,

retinal neovascularization increases vitreous traction on the

retina, resulting in tractional retinal detachment even

involving the fovea, the epicenter of the macular area

(Chen et al. 2011).

Pathologic angiogenesis as a common mechanism

and manifestation of ARB

As previously mentioned, all 3 diseases consisting of ARB

are based on pathologic angiogenesis in nature. In general,

angiogenesis is a complex process of new vessel formation

from preexisting vessels in which a variety of cells,

mediators, and matrices play specific roles (Carmeliet and

Jain 2011). Detailed discussion over step-by-step angio-

genesis processes and dynamics in sprouting vessels is

above the scope of this review. Briefly, certain groups of

endothelial cells from preexisting vessels are divided into

tip and stalk cells upon activation with angiogenic factors

such as VEGF. With detachment of periendothelial cells

such as pericytes and disruption of basement membrane,

composition and organization of surrounding ECM are

rearranged in favor of migration of sprouting endothelial

cells. Tip cells suppress the responsiveness of stalk cells to

the sprouting activity of VEGF through activation of

NOTCH signaling in stalk cells by upregulated DLL4

(Carmeliet and Jain 2011). In this way, tip cell selection is

reinforced by lateral inhibition of stalk cells, and elonga-

tion through proliferation of endothelial cells is limited to

stalk cells (Potente et al. 2011). With tip cell guidance by

concentration gradient of various angiogenic factors, vessel

sprouting, branching, and further anastomoses and lumen

formation can be completed.

Pathologic angiogenesis shares certain characteristics of

physiologic angiogenesis such as dependence on angio-

genic factors, despite differences to a greater or lesser

gradient, and involvement of further migration, prolifera-

tion, and tube formation of endothelial cells (Carmeliet

2005). However, there are distinction points between them.

First, new blood vessels in pathologic angiogenesis do not

have proper support by periendothelial cells such as peri-

cytes. In the process of angiogenesis, the physical contact

between endothelial cells and pericytes gives a stable, non-

sprouting phenotype to endothelial cells (Adams and

Alitalo 2007; Armulik et al. 2005; Bergers 2005). How-

ever, vessels formed by pathologic angiogenesis generally

lack stable interaction between endothelial cells and peri-

cytes, disturbing maturation of blood vessels. In tumor

vasculature, abnormal structure and function make oxygen

delivery inefficient to induce hypoxic condition around the

tumor (Jain 2005). Also in DR, platelet-derived growth

factor signaling is reduced in pericytes, contributing to the

disruption of barrier functions by endothelial cells (Anto-

netti et al. 2012). Mice lacking viable pericytes also show

brain vascular damage by diminished capillary perfusion

and blood–brain barrier breakdown (Bell et al. 2010).

Second, immature vessels formed by pathologic angio-

genesis are leaky to fluid and plasma proteins. These

characteristics are partly due to failed maturation of

Allostery and pathologic angiogenesis 287

123

endothelial cells and otherwise increased vascular perme-

ability via paraendothelial and transendothelial routes (Jo

et al. 2012). Third, in this regard, pathologic angiogenesis

results in fluid collection in surrounding tissues. In the

retina, pathologic angiogenesis in AMD and DR induce

subretinal or intraretinal fluid collection and macular

edema, respectively (Antonetti et al. 2012; Lim et al.

2012a; Jo et al. 2010). At the same time, fragile retinal

neovascularization and CNV tend to be easily ruptured to

break into vitreous, intraretinal, and subretinal hemorrhage.

Interestingly, 3 diseases consisting of ARB show dif-

ferent patterns regarding pathologic angiogenesis, pre-

senting another consideration point into the pathogenesis

and treatment of these diseases. They can be divided into 2

groups: pathologic angiogenesis from retinal vasculature

and choroidal vasculature. As its name implies, CNV in

wet AMD comes from choroidal vasculature and extends

into subretinal layer through defects of Bruch’s membrane.

In this regard, complications such as fluid collection and

hemorrhage occur in subretinal areas or outer retinal layers

in most of cases (de Jong 2006; Lim et al. 2012a). In

contrast, pathologic neovascularization in DR and ROP

origins at retinal vasculature in inner retinal layers.

Therefore, early microvascular changes in DR including

microaneurysms and cotton-wool spots occur in inner ret-

inal layers (Byeon et al. 2012). In ROP, as previously

mentioned, EFP that is a complex structure of retinal

neovascularization extending with vitreous cavity and

fibrous components is a characteristic finding. In both ROP

and DR, endothelial cells from retinal neovascularization

extends into vitreous cavity and induce further complica-

tions by interacting with other types of cells and ECM

proteins which are abundant in the vitreous (Lim et al.

2012b). Clinical features and the patterns of pathologic

angiogenesis in the retina with AMD, DR, and ROP are

summarized in Fig. 1.

Key players in the pathogenesis of ARB

In ARB, pathologic angiogenesis is characterized by

involvement of mediators secreted by various cells such as

astrocytes, endothelial cells, macrophages, and pericytes

and their corresponding receptors in endothelial cells

(Jo et al. 2010). Upon activation by various mediators,

endothelial cells can increase the activity of angiogenesis

to induce neovascularization. In this section, we describe

the structure and function of important growth factors or

cell membrane receptors in ARB in consideration with

their potentials as targets for allosteric modulation.

Monod et al. suggested certain characteristics of allosteric

proteins and further studies have modified and added the

criteria of proteins that can be targets for allosteric regulation

(Christopoulos 2002; May et al. 2007; Monod et al. 1963) (1)

Allosteric proteins tend to have oligomeric structure.

Fig. 1 Pathologic angiogenesis in ARB is demonstrated with clinical

pictures and schematic diagrams of representative diseases: AMD,

DR, and ROP. a In AMD, CNV occurs from choroidal vasculature in

the macula, further resulting in formation of subretinal hemorrhage or

fluid collection. b In DR, retinal neovascularization develops from

retinal vasculature in the inner retinal layers and extends into the

vitreous cavity. Complications from retinal neovascularization in DR

include vitreous hemorrhage from ruptured new vessels and tractional

retinal detachment. c In ROP, fibrovascular proliferation occurs at the

junction between avascular and vascularized retina. CNV choroidal

neovascularization, RNV retinal neovascularization, SRF subretinal

fluid, SRH subretinal hemorrhage

288 D. H. Jo et al.

123

Simply, binding of allosteric ligand on one side of dimer can

affect binding affinity of orthosteric ligand to the other side

of dimer or the overall efficacy of the function of target

proteins (Binet et al. 2004). Furthermore, the formation of

dimer can enhance the probability of allosteric modulation

(May et al. 2007). This characteristic is directly related with

the second criteria. (2) The presence of multiple binding sites

for endogenous ligands may be one of necessary conditions

for allosteric proteins. The structure of binding pockets for

endogenous ligands can also be good binding sites for allo-

steric ligands. Of course, both monomeric and oligomeric

proteins can be allosteric proteins and small molecules can

bind to the target protein at other sites than known allosteric

sites to induce conformational change of the target protein

(Peracchi and Mozzarelli 2011). However, these criteria

definitely help to find novel targets of allosteric regulation,

especially in diseases that allosteric regulation has been

rarely investigated. Do growth factors and cell membrane

receptors in the pathogenesis of ARB meet these criteria for

allosteric proteins?

VEGF pathway

Regarding ARB and other pathologic angiogenesis during

tumors, wounds, and chronic inflammatory disorders,

VEGF-A is the main component, and binding of it to

VEGF receptor (VEGFR)-2 induces downstream signaling

pathway to result in increased angiogenesis (Ferrara 2009;

Miller et al. 2012; Nagy et al. 2007). In this regard, VEGF-

A induces proliferation, migration, and tube formation of

endothelial cells (Kim et al. 2009). In patients with PDR

and ROP, the vitreous level of VEGF is increased com-

pared to that in normal controls (Adamis et al. 1994; Sato

et al. 2009). Likewise, CNV membranes from patients with

AMD are strongly immunoreactive for VEGF (Lopez et al.

1996). Furthermore, clinical efficacy of anti-VEGF treat-

ment also demonstrates the importance of VEGF pathway

in the pathogenesis of ARB.

VEGF-A is often found as a homodimer linked by

disulfide bond, of which molecular weight is 34–42 kDa

(Hoeben et al. 2004). Of 3 VEGF tyrosine kinase receptors,

VEGFR-1, VEGFR-2, and VEGFR-3, VEGFR-2 is known

to be the most important receptor in VEGF-induced angi-

ogenesis and permeability (Waltenberger et al. 1994). As

one of receptor tyrosine kinases, VEGFR-2 undergoes

dimerization by binding of VEGF to VEGFR-2 (Schles-

singer 2000). Heteromerization of VEGFR is also reported

(Nilsson et al. 2010), but VEGFR-2 homodimerization

after binding of VEGF-A appears to be a prerequisite for

further activation of VEGFR-2 and downstream signaling

pathways (Ferrara 2009; Schlessinger 2000).

VEGFR consists of an extracellular component contain-

ing 7 immunoglobulin (Ig)-like domains, a transmembrane

segement, a juxtamembrane segment, an intracellular tyro-

sine kinase domain, and a carboxyterminal tail (Roskoski

2008). Of 7 Ig-like extracellular domains, only domains

(D) 2 and 3 are known to be binding sites for VEGF (Fuh et al.

1998; Hyde et al. 2012). Interestingly, designed ankyrin

repeat proteins targeting D4 or D7 inhibit receptor signaling

and VEGF-induced proliferation, migration, and chemotaxis

of endothelial cells without affecting dimerization of VEG-

FR-2 (Hyde et al. 2012). VEGFR-2 meets the criteria for

allosteric proteins in that it forms homodimer or heterodimer

and has multiple binding sites other than orthosteric binding

sites for VEGF-A. Furthermore, large size (*230 kDa in the

mature form of VEGFR-2) and the possibility of binding of

small molecules on other sites than known binding sites

increase the potential of VEGFR-2 as a target for allosteric

regulation.

Fibroblast growth factor (FGF) pathway

FGF is one of angiogenic factors and FGF–FGF receptor

(FGFR) binding increases migration and tube formation of

endothelial cells (Beenken and Mohammadi 2009).

Although current concerns are focused on modulation of

VEGF pathway for the treatment of ARB, FGF pathway

can also be a good target. In patients with diabetic macular

edema, the level of VEGF is significantly higher in aqueous

humor samples that that in normal controls (Jonas and

Neumaier 2007). Interestingly, downregulation of FGF

pathway by scavenging of FGF or a dominant inhibitor of

FGF receptors induce the loss of endothelial cells and

impairment of endothelial barrier functions (Murakami

et al. 2008). Furthermore, basal FGF stimulation appears to

be required for maintenance of VEGFR-2 expression in

endothelial cells and further responsiveness to VEGF

(Murakami et al. 2011).

FGF induces various biological functions by binding

FGFR and forming FGF-FGFR-heparan sulphate glycos-

aminoglycan (HSGAG) complexes (Beenken and Mo-

hammadi 2009). Interestingly, likewise interaction between

VEGF and VEGFR, binding of FGF and HSGAG to FGFR

induces dimerization and further activation of receptor

tyrosine kinase (Ornitz et al. 1992; Schlessinger et al.

2000). Also in FGFR, D2 and D3 of 3 extracellular Ig

domains act as binding sites for orthosteric ligands. In total,

3 extracellular Ig domains, a transmembrane domain, and

an intracellular tyrosine kinase domain form FGFR (Mo-

hammadi et al. 2005). In this regard, FGFR also shares

characteristics of allosteric proteins: (1) oligomeric struc-

ture and (2) multiple binding sites. Homodimerization

stabilized by the specific ligands in the active form

increases the possibility of FGFR and VEGFR as targets

for allosteric modulation (Changeux 2012).

Allostery and pathologic angiogenesis 289

123

Integrin pathway

Integrins are heterodimeric membrane glycoproteins com-

posed of a and b subunits (Avraamides et al. 2008). With

ligation of various types of ECM proteins, integrins are

involved in various biological processes including angio-

genesis, development, and inflammatory response (Arnaout

et al. 2005). Integrin subunits show variable levels of

binding affinity to ECM proteins. For example, integrins

av, a3, a5, b1, and b3 demonstrate high binding affinity to

fibronectin (Silva et al. 2008). In this way, various com-

binations of integrin subunits can modulate interaction

between endothelial cells and surrounding ECM. Integrins

expressed in endothelial cells include a1b1, a2b1, a3b1,

a4b1, a5b1, a6b1, a6b4, a9b1, avb1, avb3, avb5, and

avb8 integrins (Avraamides et al. 2008; Carmeliet and Jain

2011; Lim et al. 2012b; Silva et al. 2008). In surgically

excised PDR membranes from patients, the expression of

avb3 integrin is evident and colocalized with endothelial

cells (Ning et al. 2008). Significance of integrin pathways

in the pathogenesis of ARB can be reinforced by the fact

that the certain type of ECM proteins is increased in the

course of diseases. Extra domain-B containing fibronectin,

an alternatively spliced form of fibronectin which appears

to be involved in angiogenesis, is significantly elevated in

vitreous of PDR patients (George et al. 2009; Khan et al.

2005). We also demonstrated that small molecules inhib-

iting interaction between fibronectin and integrin a3b1

suppress retinal neovascularization in the animal model of

DR and ROP (Lim et al. 2012b).

Integrins consist of extracellular domains binding ECM

proteins, a transmembrane domain, and a relatively short

intracellular domain (Arnaout et al. 2005; Hynes 2002).

Interestingly, it is thought that integrins change their con-

formation upon binding of ligands, inducing binding of

cytoplasmic proteins and further intracellular signaling

pathways (Hynes 2002). These characteristics also raise a

possibility of integrins as allosteric proteins in that ligand

binding preferentially selects a definite conformational

status, favoring biological action of proteins (Changeux

2012; Monod et al. 1963).

Current treatment options for ARB

Unfortunately, we do not fully make use of our current

knowledge about pathogenesis in the development of

therapeutic options for ARB. Until the introduction of

bevacizumab (Avastin, Genetech Inc., South San Fran-

cisco, CA, USA), an anti-VEGF antibody, as an off-label

drug in the treatment of CNV induced by pathologic

myopia (Nguyen et al. 2005), clinicians could only resort to

systemic treatment, surgery, and local treatment. Together

with recently approved anti-VEGF agents, these treatment

options have limitations because they address the patho-

logic conditions indirectly or only by scavenging VEGF

that can be utilized as survival factors for normal vessels

and neuronal cells (Heo et al. 2012; Jo et al. 2012).

Control of blood glucose and blood pressure is one of

the mainstays in the treatment of DR. Two large prospec-

tive clinical trials conducted in the United States and

United Kingdom provided clinical rationales for these

treatment approaches (The Diabetes Control and Compli-

cations Trial Research Group 1993; UK Prospective Dia-

betes Study Group 1998). Intensive treatment of diabetes

with maintaining blood glucose levels near to the normal

range reduces the risk for the development of DR and

slows down the progression of DR along with other

microvascular complications of diabetes (The Diabetes

Control and Complications Trial Research Group 1993).

Simply, correction of risk factors such as hyperglycemia in

DR and low birth weight and gestational age in ROP is

thought to improve their complications effectively. This is

partly true. However, in DR, there is evidence showing that

pathologic angiogenesis or clinical signs of DR progresses

despite intensive therapy on blood glucose level (White

et al. 2008). Furthermore, the phenomenon of higher risks

of development and progression of DR in the conventional

treatment group than in the intensive treatment group after

the discontinuation of different treatment strategies leads to

the concept of ‘‘metabolic memory’’, which is also

undermined at the molecular level (Tewari et al. 2012;

White et al. 2008; Zhong and Kowluru 2011). Interestingly,

hyperglycemia insult induces mitochondrial damage such

as epigenetic changes in mitrochondrial superoxide dis-

mutase, resulting in progression of DR even with good

glycemic control (Tewari et al. 2012; Zhong and Kowluru

2011). Likewise, in ROP, the gestational age of 40 weeks

or more does not guarantee the regression of neovascu-

lar change unless retinal vasculature is fully developed

(Sapieha et al. 2010).

In this regard, local treatment of ARB is important to

prevent vision-threatening complications such as vitreous

hemorrhage and tractional retinal detachment. Local treat-

ment options include intravitreal injection of bevacizumab,

ranibizumab (Lucentis, Genentech Inc.), pegaptanib (Mac-

ugen, Eyetech Pharmaceuticals, Inc., Lexington, MA, USA),

and aflibercept (Eyelea, Regeneron Pharmaceuticals Inc.,

Tarrytown, NY, USA), laser photocoagulation, cryotherapy,

photodynamic therapy, and surgery (Table 1). Particularly,

intravitreal injection of anti-VEGF agents show consider-

able efficacy in the treatment of ARB, revolutionizing

everyday clinical settings (Jo et al. 2010, 2012). Adminis-

tration of anti-VEGF agents has a rationale that direct inhi-

bition of VEGF in the vitreous cavity or subretinal spaces

reverses or blocks pathologic progression of VEGF-induced

complications in ARB. As expected, anti-VEGF agents

290 D. H. Jo et al.

123

demonstrate improved visual outcomes significantly. In

AMD, intravitreal administration of ranibizumb, a recom-

binant, humanized, monoclonal antibody Fab fragment,

prevents vision loss and improves mean visual acuity in

patients with choroidal neovascularization (Brown et al.

2006; Rosenfeld et al. 2006). In DR, intravitreal ranibizumab

reduces the risk of progression of DR in eyes with macular

edema and some of patients experience improvement in the

Table 1 Therapeutics for ARB

currently utilized and in

development process

C complement, COX

cyclooxygenase, mTOR

mammalian target of

rapamycin, nAchR nicotinic

acetylcholine receptor, PDGF

platelet-derived growth factor,

PKC protein kinase C, siRNA

small interfering RNA; TORC

target of rapamycin complex

Agent name Target Molecular platform Current status in

development

process

Aflibercept VEGF Fusion protein In market

Bevacizumab VEGF Monoclonal antibody In market (off-label)

Pegaptanib VEGF RNA aptamer In market

Ranibizumab VEGF Antibody fragment In market

Triamcinolone acetonide Inflammation Chemical In market

Anecortave acetate Inflammation Chemical Phase 3

Cand5 VEGF siRNA Phase 3

Celecoxib COX-2 Chemical Phase 3

Fluocinolone acetonide Inflammation Chemical Phase 3

Infliximab TNF-a Monoclonal antibody Phase 3

KH902 VEGF Fusion protein Phase 3

Ruboxitaurin PKC-b Chemical Phase 3

Squalamine lactate Plasma membrane Chemical Phase 3

Adalimumab TNF-a Monoclonal antibody Phase 2

AG-013958 VEGFR Chemical Phase 2

AGN211745 VEGFR siRNA Phase 2

ALG-1001 Integrin Peptide Phase 2

Aliskiren Renin Chemical Phase 2

ATG-003 nAchR Chemical Phase 2

Combretastatin A4

phosphate

b-tubulin Chemical Phase 2

Daclizumab CD25 Monoclonal antibody Phase 2

E10030 PDGF RNA aptamer Phase 2

ESBA1008 VEGF Antibody fragment Phase 2

Everolimus mTOR Chemical Phase 2

LFG316 C5 Monoclonal antibody Phase 2

OT-551 Anti-oxidant Chemical Phase 2

Pazopanib VEGFR Chemical Phase 2

PF-04523655 RTP801 gene siRNA Phase 2

Sirolimus mTOR Chemical Phase 2

TG100801 VEGFR Chemical Phase 2

Vatalanib VEGFR Chemical Phase 2

ACZ885 IL-1b Monoclonal antibody Phase 1

ARC1905 C5 RNA aptamer Phase 1

Efalizumab CD11a Monoclonal antibody Phase 1

MP0112 VEGF Antibody mimetic

protein

Phase 1

Palomid 529 TORC1, TORC2 Chemical Phase 1

POT-4 C3 Chemical Phase 1

Sonepcizumab Sphingosine

1-phosphate

Monoclonal antibody Phase 1

Volociximab a5b1 integrin Monoclonal antibody Phase 1

JSM6427 a5b1 integrin Peptide Phase 1

Allostery and pathologic angiogenesis 291

123

severity of DR with the treatment (Ip et al. 2012; Nguyen

et al. 2012). In ROP, intravitreal bevacizumab shows a sig-

nificant benefit in the control of ROP (Mintz-Hittner et al.

2011). However, there are certain groups of patients who do

not respond to anti-VEGF treatment (Brown et al. 2006; Ip

et al. 2012; Nguyen et al. 2012; Rosenfeld et al. 2006). Even

worse, anti-VEGF agents can induce complications in the

retina and all over the body (Chen and Cleck 2009; Lee et al.

2012; Manousaridis and Talks 2012; Miller et al. 2012; Patel

et al. 2012). There is a possibility of worsening ischemia in

the macular area with the treatment of anti-VEGF agents

(Manousaridis and Talks 2012), and in some patients with

ROP, atypical traction membrane is formed during regres-

sion of fibrovascular components of diseases, leading to

vision-threatening retinal detachment (Lee et al. 2012; Patel

et al. 2012). Even though the dosage is much less in oph-

thalmology than in treating cancer patients, systemic com-

plications such as hypertension, thromboembolism, and

cardiomyopathy are also expected to require close moni-

toring (Chen and Cleck 2009; Miller et al. 2012).

We speculate that anti-VEGF agents can also affect

retinal neurons and even physiologic vascular develop-

ment. VEGF is not only a prominent mediator of patho-

logic angiogenesis and hyperpermeability but also a

survival factor for retinal neuronal cells and endothelial

cells (Nishijima et al. 2007). We also demonstrate that the

treatment of retinoblastoma cells with bevacizumab affects

neuronal differentiation even at the concentration of no

definite effect on cellular viability (Heo et al. 2012). Taken

together, current local treatment options lack the feasibility

of addressing the diseases in a pathogenesis-specific way

and fine-tuning pathologic conditions in ARB. Novel

approaches to grasp these 2 goals are desperately required

to treat pathologic angiogenesis in ARB to overcome

vision-threatening complications.

Allosteric regulation of pathologic angiogenesis

We expect that allosteric regulation of effector proteins in

the pathogenesis of ARB can revolutionize therapeutic

approaches once again after the introduction of anti-VEGF

agents in clinical settings. In this section, we briefly sum-

marize the general principle and mechanisms of allosteric

regulation. Then, with introduction of candidate allosteric

modulators of VEGF, FGF, and integrin pathways, we

demonstrate the potential of allosteric regulation as a novel

approach in the development of therapeutic drugs for ARB.

Allostery: a general principle in regulation of proteins

Allosteric transition indicates the change in conformation

induced by binding of allosteric modulators, endogenous

or exogenous, to allosteric sites of proteints to affect

metabolic activity of them (Monod et al. 1963). Site-to-site

communication among the allosteric site, the orthosteric

site, and the effector site give molecular control mecha-

nisms for cells or organisms to adjust environmental

alterations rapidly and precisely (Goodey and Benkovic

2008). Allosteric modulators bind to allosteric sites of

certain proteins, modify the conformation of them, and

further switch their functions effectively by inducing

alterations in binding affinity of orthosteric ligands, effi-

cacy of target proteins, and overall distribution of confor-

mations of them (Fig. 2) (Changeux 2012; Goodey and

Benkovic 2008; Monod et al. 1963). This flowline of

interaction among allosteric modulators, orthosteric

ligands, and target proteins reflects the concept that the

structure indicates the function and property of the pro-

teins. As previously mentioned, allostery, a general prin-

ciple in regulation of proteins such as enzymes and

signaling proteins, governs the metabolic activity and sig-

nal transduction (Changeux 2012; Christopoulos 2002;

Monod et al. 1963). Canonical examples from Monod et al.

include enzymes, glutamic-dehydrogenase, acetyl-CoA

carboxylase, and muscle phosphorylase b, and non-enzy-

mic protein hemoglobin (Monod et al. 1963). Currently,

GPCR is one of the most widely-studied targets in term of

allostery, and many candidate allosteric modulators of

potential are developed to treat disorders in the central

nervous system including psychological disorders in which

GPCR plays an important role (Conn et al. 2009; Keov

et al. 2011; May et al. 2007).

The presence of allosteric effect even without confor-

mational change adds another layer of the landscape of

allostery, introducing the concept of ‘‘dynamic allostery’’

(Cooper and Dryden 1984; del Sol et al. 2009; Popovych

et al. 2006; Tsai et al. 2008). This concept is based on the

phenomenon that the change in the conformation of the

protein is not always related with that in the function. In

contrary, allosteric modulators can alter the free energy of

proteins by reorienting and rewiring side chains of proteins

without involvement of change in the backbone shape (Tsai

et al. 2008). In this regard, allostery expands its denotation

to involve the phenomenon that allosteric ligands can

change the thermodynamic status, and frequencies and

amiplitudes of macromolecular thermal fluctuation, by

binding to the allosteric sites of the target proteins (Cooper

and Dryden 1984; del Sol et al. 2009). Binding of the first

cAMP to the dimeric catabolite activator protein is a good

example showing that changes in protein motions can

induce allosteric effect (Popovych et al. 2006).

Explanatory models of allosteric regulation

Binding of allosteric and orthosteric ligands can induce

conformational (enthalpy) or thermodynamic (entropy)

292 D. H. Jo et al.

123

change respectively and cooperatively (Changeux 2012;

Tsai et al. 2008). The Monod-Wyman-Changeux (MWC)

model, the Koshland–Nemethy–Filmer (KNF) model, and

the Ensemble allosteric model (EAM) were devised to

understand the interaction in the ternary complex of the

allosteric ligand, the orthosteric ligand, and the allosteric

protein (Hilser et al. 2012; Koshland 1958; Monod et al.

1965). These models reflect observed phenomena with

allosteric proteins in which binding of allosteric ligands

affect the binding affinity of orthosteric ligands and/or the

efficacy of allosteric proteins. KNF model postulates that

the ligand induces the conformational change of the bind-

ing pockets of the target protein (induced fit) (Weikl and

von Deuster 2009). That is, ligand binding results in the

change from the unbound form to the active and bound

form of high-affinity (Koshland 1958, 1959). In contrary,

MWC model postulates that there is a pre-existing equi-

librium of ground and excited states of the protein and

ligand binding selects and stabilizes a conformational sta-

tus from the equilibrium of status (selected fit or confor-

mational selection) (Weikl and von Deuster 2009). This

model can explain the rapid adaptation to the certain status

of proteins with binding of allosteric ligands.

EAM helps to understand the phenomenon of allostery

in the context of the ensemble of conformational free

energies of proteins (Hilser et al. 2012). In fact, the protein

does not exist only in the forms of unbound and bound, but

in the combinations of various energy statuses (Peracchi

and Mozzarelli 2011). The allosteric ligand is thought to

bind to the protein to change the width of the conforma-

tional distribution and this model provides a framework to

investigate allostery in terms of energetic determinants

(Hilser et al. 2012).

Advantages of allosteric regulation in suppression

of pathologic angiogenesis

Suppression of pathologic angiogenesis without disturbing

physiologic angiogenesis and neural homeostasis is one of

ideal prerequisites for therapeutic agents for ARB. Inter-

estingly, allosteric regulation shows characteristics that

meet this prerequisite to enhance the therapeutic value of

allosteric modulators in the treatment of pathologic angi-

ogenesis in ARB.

First, allosteric regulators are thought to have a limit in

their efficacy even though the dosage gets higher (Birdsall

Fig. 2 Modes of action of

allosteric modulators on

allosteric proteins. Allosteric

modulators can induce a change

in the binding affinity of

orthosteric ligands to target

proteins, b change in the

efficacy of target proteins, and

c change in the distribution of

conformational or

thermodynamic statuses of

target proteins. These modes of

action can be utilized

independently or concomitantly

Allostery and pathologic angiogenesis 293

123

et al. 1999; May et al. 2007). This advantage is partly due

to the phenomenon that allosteric regulators have no

intrinsic effects but cooperatively up-regulate or down-

regulate the efficacy of the target proteins induced by

binding of orthosteric ligands. Allosteric activators can

enhance the binding affinity and/or the action of orthosteric

ligands up to the maximal effect of orthosteric ligands

themselves. Allosteric inhibitors also do not suppress the

action of target proteins extremely in the presence of or-

thosteric ligands. These so called ‘‘ceiling effect’’ provides

enough safety of allosteric modulators against overdose to

be utilized in the treatment of various diseases (May et al.

2007). Particularly, for the treatment of ARB, local

administration such as intravitreal injection is possible to

require much less dosage than intravenous injection.

Therefore, allosteric regulators in the intravitreal form

guarantee much more safety.

Second, allosteric modulators ‘‘fine-tune’’ physiologic

action of target proteins such as cell surface receptors in the

presence of endogenous ligands (Conn et al. 2009; Peracchi

and Mozzarelli 2011). In the situation when allosteric

ligands do not exist, the efficacy of target proteins is

determined only by the concentration of orthosteric ligands

and interaction between target proteins and orthosteric

ligands. However, allosteric ligands provide another factor

to this landscape by modulating the binding affinity of

orthosteric ligands to target proteins and/or the efficacy of

the target proteins with change in conformational or ther-

modynamic status (Peracchi and Mozzarelli 2011; Pop-

ovych et al. 2006). In this regard, allosteric modulators are

expected to reverse pathologic upregulation of the action of

VEGF or other increased extracellular factors by delicate

modulation of responses of cell surface receptors without

inducing undesirable side effects induced by excessive

downregulation of physiologic actions of them.

Furthermore, the interaction between allosteric ligands

and target proteins tend to be more specific than that

between orthosteric ligands and target proteins (Christo-

poulos 2002; May et al. 2007). In the view of evolution,

there is a tendency of less conservation in the allosteric

sites than orthosteric sites (Hauske et al. 2008). Compari-

son studies between species demonstrate higher homology

in the orthosteric site domains. In contrast, higher sequence

divergence between subtypes and species also contributes

the specificity of the action of allosteric ligands (Christo-

poulos 2002). In this regard, allosteric ligands can affect

target proteins with more specificity, not disturbing the

action of proteins of different subtypes. Another important

factor adding the specificity of allosteric modulators is the

selective cooperativitiy of allosteric modulators according

to the orthosteric ligands (Kenakin 2005; Lazareno et al.

1998, 2004; May et al. 2007). Interestingly, the same

allosteric modulator can act as an inhibitor or an enhancer

depending on the type of orthosteric ligands (Lazareno

et al. 1998, 2004). In the control of pathologic angiogenesis

of ARB, enhanced specificity for target proteins improves

therapeutic potential of allosteric modulators.

Potential allosteric modulators of pathologic

angiogenesis

In this section, we discuss the potential allosteric modu-

lators of key players in pathologic angiogenesis in ARB:

VEGF pathway, FGF pathway, and integrin pathway. Until

now, these candidate drugs do not aim at addressing the

pathologic angiogenesis in ARB, but in tumor vasculature.

However, we speculate that these candidates can be

investigated in the treatment of ARB because tumor vas-

culature and pathologic angiogenesis in ARB share com-

mon pathologic pathways (Fens et al. 2010). Definitely,

bevacizumab and aflibercept are good excellent precedents

showing how anticancer drugs can be repositioned to treat

ophthalmic diseases (Browning et al. 2012; Mintz-Hittner

et al. 2011; Nguyen et al. 2005). We performed a search for

candidate therapeutic agents through PubMed (http://www.

ncbi.nlm.nih.gov/pubmed; search date: October 1, 2013)

and ASD, a comprehensive database of allosteric proteins

and modulators (Huang et al. 2011), evidencing several

potential allosteric modulators of the key pathologic

pathways in the pathogenesis of ARB. Interestingly, most

of current potential allosteric modulators target cell surface

receptors (Table 2). This tendency might be due to oligo-

mer formation and large sizes of VEGFR-2, FGFR, and

integrin that can meet the criteria for allosteric proteins.

VEGF pathway

As previously mentioned, suppression of VEGF-A/VEG-

FR-2 axis is a valuable target in the development of ther-

apeutic agents for ARB. However, because direct

inhibition of VEGF can induce undesirable effect, we

expect that allosteric modulation of this axis may help

more stable control of pathologic angiogenesis induced by

VEGF. GU40C4, a peptoid agent of antiangiogenic effect,

Table 2 Potential allosteric modulators of pathologic angiogenesis

Affected molecule Allosteric modulators References

VEGFR-2 GU40C4 Udugamasooriya

et al. (2008a, b)

Analogues of serotonin

derivatives

Buttner et al.

FGF-2 KIN59 Liekens et al.

Integrin a2b1 Analogues of arylamide

derivatives

Yin et al.

Decorin Fiedler et al.

294 D. H. Jo et al.

123

and VEGF do not compete for binding to the extracellular

domain of VEGFR-2 (Udugamasooriya et al. 2008a).

Interestingly, the binding of GU40C4 is thought to result in

inhibition of the conformational change of the VEGFR-2 to

activate downstream signals induced by VEGF. This agent

is an example of allosteric inhibitors that affect the efficacy

of target proteins without definite change in binding affinity

of orthosteric ligands. In this regard, GU40C4 suppresses

VEGF-induced phosphorylation of VEGFR-2, further

inhibiting tumor growth in the mouse model of subcutane-

ous injected sarcoma (Udugamasooriya et al. 2008b).

In a study with several analogues of serotonin deriva-

tives, they show inhibitory effects on various receptor

kinases including epidermal growth factor receptor, insu-

lin-like growth factor-1 receptor, VEGFR-2, and VEGFR-3

via a non-ATP-competitive mechanism, suggesting their

actions are based on allostery (Buttner et al. 2010). Sero-

tonin derivatives of inhibitory activity possess a free amino

group and a phenylethyl substituent in 5-position, respec-

tively, and demonstrates antiproliferative activity against

human umbilical vein endothelial cells, epithelial breast

and colorectal cancer cell lines, and a fibroblast cell line.

FGF pathway

The ternary complex of FGF-FGFR-HSGAG increases the

susceptibility of this complex as a target for allosteric

modulators. KIN59, the thymidine phosphorylase inhibitor

5’-O-tritylinosine, inhibits the binding of FGF-2 to FGFR-

1, further suppressing the formation of effective FGF-

FGFR-HSGAG complexes (Liekens et al. 2012). In this

study, KIN59 inhibits endothelial proliferation, activation

of FGFR-1, and a downstream signaling pathway of Akt

induced by FGF-2, however, there is no change in bio-

logical responses stimulated by VEGF. This specific ther-

apeutic agent against FGF pathway can be utilized not only

as a monotherapy but also in combination with currently

available anti-VEGF agents.

Integrin pathway

A group of arylamide derivatives are shown to inhibit the

binding of type I collagen to integrin a2b1 (Yin et al.

2006). The locations of affective residues by arylamide

derivatives demonstrate that the targeting site is an allo-

steric site that is distinct from the collagen-binding metal

ion-dependent adhesion site of integrin. The proteoglycan

decorin also binds to an allosteric site different from the

collagen I-binding A-domain, and enhances the binding

activity of collagen I to integrin a2b1 (Fiedler et al. 2008).

Due to this effect, decorin promotes integrin a2b1 depen-

dent endothelial cell migration on fibrillar collagen I. These

candidate drugs are examples of allosteric enhancers.

Conclusions and future directions

In the treatment of ARB, we cannot afford to miss definite

goals of effective suppression of pathologic angiogenesis

and sustainable maintenance of vision. Novel therapeutic

agents utilizing the concept of allostery can expand our

armaments against vision-deteriorating complications from

ARB. Allosteric modulators might improve specificity and

safety of therapeutic agents for ARB, further optimizing

treatment options for patients. Nevertheless, the studies for

allosteric modulators to control pathologic angiogenesis

are still closer to the beginning, and much of efforts are

dedicated in the treatment of malignant disorders. We

expect that the design of allosteric modulators can give

opportunities to researchers on the development of novel

therapeutic options in the treatment of ARB.

Acknowledgments This study was supported by National Research

Foundation (NRF) grant funded by the Korea government (MEST)

(2012-0006019), the Seoul National University Research Grant (800-

20130338), the Pioneer Research Program of NRF/MEST (2012-

0009544), and the Bio-Signal Analysis Technology Innovation Pro-

gram of NRF/MEST (2009-0090895).

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