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Rational design of LEDGINs as firstallosteric integrase inhibitors for thetreatment of HIV infectionBelete A. Desimmie, Jonas Demeulemeester, Frauke Christ, Zeger Debyser*Laboratory for Molecular Virology and Gene Therapy, KU Leuven, Leuven 3000, Flanders, Belgium

Drug Discovery Today: Technologies Vol. 10, No. 4 2013

Editors-in-Chief

Kelvin Lam – Blue Sky Biotech, Inc., Worcester, MA

Henk Timmerman – Vrije Universiteit, The Netherlands

Modulation of protein–protein interactions

The interaction between lens epithelium-derived

growth factor (LEDGF/p75) and HIV-1 integrase (IN)

is an attractive target for antiviral development

because its inhibition blocks HIV replication. Develop-

ing novel small molecules that disrupt the LEDGF/p75–

IN interaction constitutes a promising new therapeutic

strategy for the treatment of HIV. Here we will high-

light recent advances in the design and development of

small-molecule inhibitors binding to the LEDGF/p75

binding pocket of IN, referred to as LEDGINs.

Introduction

Protein–protein interactions (PPIs) represent an attractive

group of biologically relevant targets for the development

of small molecule inhibitors, classified as SMPPIIs (small

molecule protein–protein interaction inhibitors) [1–3]. How-

ever, protein–protein interfaces often have flat, weakly

defined and large hydrophobic surfaces that are not ideal

for small molecules to bind to. Therefore, obtaining valid

starting points for the design and optimization of SMPPIIs has

been difficult [3]. Moreover, modulation of PPIs to develop

therapeutics is defined not only by the physicochemical

properties of the protein–protein interface but also by the

biological properties of the identified inhibitors.

The human immunodeficiency virus (HIV) relies on host

cellular machinery to complete its replication cycle. HIV

hijacks several biological processes and protein complexes

*Corresponding author.: Z. Debyser (zeger.debyser@med.kuleuven.be)

1740-6749/$ � 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ddtec.2012

Section editor:Christian Ottman – Max Planck Society, Dortmund,Germany.

of the host cell through distinct virus–host PPIs [4]. Because

these host–pathogen interactions directly mediate viral repli-

cation and disease progression, their specific disruption can

provide alternative targets for therapeutic intervention.

Herein, we discuss the recent success in the application of

structure-based drug design in the discovery and develop-

ment of allosteric HIV-1 integrase inhibitors, the LEDGINs

[5]. LEDGINs are characterized by their binding to the

LEDGF/p75 binding site on the core domain of integrase

and inhibit the interaction between lens epithelium-derived

growth factor/p75 (LEDGF/p75) and HIV-1 integrase (IN). We

will briefly discuss the role of the LEDGF/p75–IN interaction

in HIV-1 replication, followed by a discussion on how LED-

GINs block HIV replication.

HIV infection and the quest for novel antiretroviral

drugs

HIV infection remains a substantial public health as well as a

socioeconomic problem worldwide [6]. Although highly

active antiretroviral therapy (HAART) profoundly increases

survival by chronically suppressing viral replication to below

detection limits, it has not been possible to achieve a cure.

Interruption of HAART typically results in a rebound of viral

replication. This is primarily because HIV ingeniously escapes

from the continuous immune surveillance in a small pool of

latently infected cells that are not susceptible to drug therapy.

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Drug Discovery Today: Technologies | Modulation of protein–protein interactions Vol. 10, No. 4 2013

(a)

(b)

IN

PWWP

LEDGF/p75

AT hooks

IBD

Chromosomal DNA

IN

I365

D366

L368

IN IN

Intasome

Drug Discovery Today: Technologies

Figure 1. LEDGINs bind to the LEDGF/p75 binding pocket on HIV-1

integrase. (a) LEDGF/p75 has an N-terminal chromosomal DNA

binding region including a PWWP domain and AT hooks. The C-

terminus contains the well-characterized integrase binding domain

(IBD) and acts as a protein interaction playground. The interaction of

the catalytic core domain (CCD) of HIV integrase with the IBD of

LEDGF/p75 is crucial to facilitate the tethering of the HIV intasome on

the chromatin. (b) Cartoon representation of the IN CCD dimer

(pale green and pale yellow) with LEDGIN 6 superposed with the IBD

(PDB entry 2B4J, grey) reveals mimicry of the protein–protein

interaction. LEDGIN 6 phenyl, acid and chlorine groups substitute for

LEDGF/p75 residues I365, D366 and L368 side chains, respectively.

These latently infected cells reside in reservoirs where the

distribution of antiretroviral (ARV) drugs is extremely vari-

able and often lower than the expected maximal inhibitory

concentration [7–10]. Moreover, the rapid replication rate

and the generation of extensive genetic diversity support the

emergence of drug resistant viral strains, resulting in treat-

ment failure. Therefore, there is a continuous demand to

search for novel and better ARVs to better control the HIV

pandemic with the hope of eventually inducing permanent

remission of the disease.

In recent years our understanding of the HIV–host inter-

action has dramatically increased. Not surprisingly, there

are numerous interactions between HIV and cellular pro-

teins involved in all stages of virus replication, opening a

window for the discovery of novel therapeutic classes [4,11–

13]. In principle, any distinct interaction between virus

encoded proteins and host co-factors has the potential to

be a target for drug design. The CCR5 antagonist, maraviroc,

was approved as the first ARV targeting a host factor [14].

Maraviroc binds to the CCR5 co-receptor on the surface of

cells and prevents interaction with the Gp120 envelope

protein of the virus [15]. Targeting virus–host PPIs demon-

strates that HIV-1 therapeutic targets are not limited to

virus-encoded enzymes and that understanding of the

virus–host interactome can be the basis for effective anti-

HIV drugs [4,16]. In theory, this pharmacological strategy is

expected to make it more difficult for the virus to develop

resistance. Because the host factor is genetically conserved

in a biologically relevant host–virus interaction, resistance

is less likely to occur, increasing the clinical potential of

these drugs.

The LEDGF/p75–IN interaction as novel antiviral

target

LEDGF/p75 is implicated in the regulation of stress response

proteins. The protein is 530 amino acids long and a strong

binding partner of HIV-1 IN [17]. LEDGF/p75 is characterized

by an N-terminal chromatin and DNA interacting region and

a C-terminal integrase binding domain (IBD) (Fig. 1a). HIV

integrase is an oligomeric enzyme that orchestrates the inser-

tion of the viral genome into the host chromatin [18,19]. It is

composed of three domains: the N-terminal domain (NTD),

the catalytic core domain (CCD, containing the active site)

and the C-terminal domain (CTD). The LEDGF/p75 binding

pocket on integrase is only evident from at least a dimeric

enzyme [20]. The crystal structure of the IBD in complex with

the CCD of IN was a major advance in defining the structural

properties and the stoichiometry of the IBD–CCD complex

[20]. The nature of the interaction between LEDGF/p75 and

HIV-1 IN proteins has been firmly established by genetic and

biochemical studies (for a comprehensive review see Ref.

[21]). Additionally, the importance of LEDGF/p75 in HIV

replication was extensively studied via mutagenesis, RNAi,

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transdominant overexpression of the IBD of LEDGF/p75 and

knock-out studies [22–30].

The feasibility of inhibiting the LEDGF/p75–IN interaction

was initially demonstrated by De Rijck et al. [28] who showed

that overexpression of the IBD of LEDGF/p75 in cells reduced

HIV replication 100-fold. Serial passaging of the virus in IBD

overexpressing cells yielded a resistant virus with IN muta-

tions A128T and E170G, located in the LEDGF/p75 binding

pocket [29]. Al-Mawsawi et al. [31] subsequently showed that

a LEDGF/p75-derived oligopeptide containing the IN inter-

acting residues Ile355 and Asp366 blocked the interaction

between LEDGF/p75 and IN [31]. Even though peptides and

natural products have been shown to modulate PPIs in several

therapeutic areas, their physicochemical properties make

them less amenable for drug development [1]. Therefore,

identification of small molecule inhibitors that bind to the

LEDGF/p75 binding pocket in HIV-1 IN was suggested as

Vol. 10, No. 4 2013 Drug Discovery Today: Technologies | Modulation of protein–protein interactions

novel therapeutic strategy [22]. Du et al. [32] reported a

benzoic acid derivative small molecule inhibitor, D77 that

disrupts the LEDGF/p75–IN interaction and inhibited HIV

replication. Subsequently, structure-based drug design

resulted in the identification of small molecules (CHIBA-

3002 and its analogs) that weakly inhibit the LEDGF/p75–

IN interaction [33]. However, the first potent inhibitors of

HIV replication that act by disrupting the LEDGF/p75–IN

interaction were only reported in 2010. We applied rational

drug design to identify LEDGINs, small molecule inhibitors

that bind to the LEDGF/p75 binding pocket in HIV-1 IN

(Fig. 1b) and inhibit HIV integration [5].

Rational design of LEDGF/p75–IN interaction

inhibitors

Different approaches have been employed to design and

identify small-molecule inhibitors of the LEDGF/p75–IN

interaction. These include in vitro high-throughput screen-

ing of compound libraries [32,34], in silico virtual screening

of libraries and structure-based de novo design [5,33]. High-

throughput screening of large libraries of compounds

against a biological target is still the prevailing method

for the identification of new hit compounds in modern

drug discovery. Alternatively, virtual screening is based

on a knowledge-driven, computer-aided survey of virtual

libraries. This approach usually results in a limited subset of

small molecules, possessing certain features defined by the

screening algorithm and which are predicted to have the

desired activity on a target. Subsequently only this relative

small selection of molecules is evaluated for biological

activity.

To obtain bona fide LEDGF/p75–IN interaction inhibitors,

we embarked on structure-based drug design in 2007 [5]. The

development of LEDGINs required a multi-disciplinary effort

integrating expertise in molecular modeling, medicinal

chemistry, crystallography and virology [5]. Different crystal

structures of the HIV-1 IN CCD [35], a co-crystal structure

with the IBD of LEDGF/p75 [20] and a co-crystal structure

with a ligand (tetraphenyl arsonium) bound to the CCD [36]

were superposed to deduce a consensus pharmacophore

model for the LEDGF/p75 binding pocket. This model repre-

sents a series of steric and electronic features in 3D space

predicted to be crucial for binding to the LEDGF/p75 binding

pocket located at the dimer interface of the HIV-1 IN CCD.

Around 200,000 commercially available and structurally

diverse compounds were subjected to a set of 2D rule-based

filters describing SMPPII chemical space. The compounds

selected through filtering were fitted to the pharmacophore

query and the best scoring hits were submitted to docking

analysis. After consensus scoring, the highest ranking com-

pounds were inspected manually and 25 compounds were

selected for biological evaluation in an LEDGF/p75–IN inter-

action assay.

In principle, any drug discovery project requires design,

prioritization, analysis and interpretation of results from

consecutive experiments to ultimately facilitate the develop-

ment of new therapeutic compounds. It is the combination of

methods, rather than a single experiment that moves a drug

discovery project forward. Therefore information from the

first round of rational design is usually used to re-evaluate and

optimize the initial pharmacophore model, which leads to

multiple sequential rounds of in silico design and biological

testing. The scheme of the rational drug design workflow used

during the discovery and hit-to-lead optimization process of

LEDGINs is depicted in Fig. 2.

Activity and optimization of hit compounds

Our primary assay was a direct LEDGF/p75–IN interaction

assay built on the AlphaScreen platform, a bead-based assay

technology able to detect biomolecular interactions

[5,34,37]. Of the 25 molecules retained from the initial

screening, four hit molecules moderately inhibited the

LEDGF/p75–HIV-1 IN interaction. One of the hit molecules,

LEDGIN 1, inhibited the PPI by 36% at 100 mM (Table 1) [5].

Based on this initial activity, LEDGINs 2 and 3 were selected

from commercial databases, which marked the beginning of

structure activity relationship (SAR) investigations aimed at

the identification of more potent analogs. Co-crystals of

LEDGIN 3 with the IN CCD were obtained and validated

the pharmacophore model: a clear mimicry was observed of

LEDGF/p75 residues I365, D366 and L368 by the LEDGIN

phenyl, acid and chlorine groups, respectively. Each of these

three substituents satisfied a crucial feature of the initial

pharmacophore hypothesis. Further medicinal chemistry

optimization, fueled by structural input from the LEDGIN-

soaked HIV-1 CCD crystals, generated analogs of LEDGIN 3

(including LEDGIN 6 and 7) with improved biological activity

(Table 1).

Integrase strand transfer inhibitors (INSTIs) bind to the

active site of IN [38]. Unlike INSTIs which recognize the

conformational changes of IN catalytic site after assembly

on a specific viral DNA ends, LEDGINs bind to IN irrespective

of its assembly on viral DNA ends. In addition these first-in-

class anti-HIV compounds are potent antivirals in cell culture

and are active against a broad-spectrum of HIV-strains with a

high selectivity index.

Of note Hou et al. [34] identified several compounds inhi-

biting the LEDGF/p75–IN interaction through high-through-

put screening of a compound library of more than 700,000

small molecules using AlphaScreen assay. However, the nat-

ure of these compounds and their antiviral activity spectrum

is yet to be revealed. Nevertheless, LEDGINs are the first

examples of potent and specific inhibitors of HIV-1 replica-

tion which have been extensively evaluated for their ther-

apeutic potential and mechanism of action in cell-based

antiviral assays (including in primary cells) [5].

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Drug Discovery Today: Technologies | Modulation of protein–protein interactions Vol. 10, No. 4 2013

Medicinal chemistry

Co-crystallization

Biological evaluationMTT/MT4 antiviral assay

Hit-to-lead optimization

EC50

CC50

HIV-infected cells

Mock-infected cells

ControlsCompound concentration (µM)

Cl OH

ON

Molecular modelling

Drug Discovery Today: Technologies

Figure 2. Rational design of LEDGINs; the workflow. LEDGINs result from a rational drug discovery strategy involving a multidisciplinary effort. A 3D

pharmacophore query was constructed to virtually screen around 200,000 molecules from commercial libraries. After docking, multiply scoring and

filtering, 25 of the highest-ranking molecules were purchased and tested in the in vitro AlphaScreen assay. A hit compound emerging from the screen was

optimized by reiterative chemical refinement and biological profiling in AlphaScreen and a cell-based antiviral assay: MTT/MT4. Structure–activity

relationships were deduced and used together with co-crystals of IN and LEDGINs to guide synthesis of analogues with enhanced activity as depicted in

Table 1. The resulting early lead compounds were then further optimized while the molecular mechanism of action was comprehensively investigated in cell

culture, including a time of addition (TOA) analysis. In the AlphaScreen subset of the biological evaluation the letters D and A stand for a donor and acceptor

beads, respectively. EC50; effective concentration required to reduce HIV-1 induced cytopathic effect by 50% in MT-4 cells and CC50; cytotoxic

concentration reducing MT-4 cell viability by 50%.

LEDGINs as HIV-1 therapeutics

A crucial evaluation of the mechanism of action and ther-

apeutic potential of LEDGINs requires evaluation of the

following drug characteristics: (a) a high binding affinity

and specificity to HIV IN, (b) potent and broad-spectrum

anti-HIV activities in cell-based antiviral assays, and (c) lack

of toxicity. To date, more potent LEDGIN congeners meet

these criteria and are in advanced preclinical development.

Like INSTIs, LEDGINs inhibit the integration step of HIV-1

replication as shown by quantitative PCR (Q-PCR) [5]. Inte-

gration inhibitors are characterized by Q-PCR analyses of the

copy number of integrated provirus and 2-LTR circles. The

latter are dead-end by-products of non-integrated viral DNA

and are used to corroborate a defect in integration without

significant effects on the preceding steps [38]. Classical INSTIs

such as raltegravir, but also LEDGINs, significantly reduce the

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number of integrated proviral DNA copies and consequently

induce accumulation of 2-LTR circles [5]. Importantly, LED-

GINs do not show any cross-resistance with INSTIs, implying

that they might serve as second-generation inhibitors if they

meet the pharmacokinetic and pharmacodynamic require-

ments needed for further clinical development.

A resistant strain was selected against LEDGIN 6 which

carries an A128T substitution in integrase. In the native

complex, IN A128 makes substantial Van der Waals interac-

tions with LEDGF/p75 residues I365 and L368 – both impor-

tant features in the pharmacophore model – but also with

F406 and V408 which extend from the second interfacial IBD

loop [20]. LEDGIN 6, at least in part, mimics these interac-

tions and is in close contact with A128. Correspondingly, the

A128T substitution reduced the binding of LEDGINs to IN

and caused loss of their antiviral activity, confirming the

Vol. 10, No. 4 2013 Drug Discovery Today: Technologies | Modulation of protein–protein interactions

Table 1. Assay results of hits and their biological activity

LEDGIN Structure LEDGF–IN interaction

IC50 (mM)

Antiviral activity

EC50 (mM)

1a 36% ND

2 27.27 ND

3 12.2 � 3.4 41.9 � 1.1

4 9.24 � 0.79 10.8 � 1.1

5 13.2 � 2.8 12.4 � 1.2

6 1.4 � 0.4 2.35 � 0.3

7 0.85 � 0.3 0.76 � 0.08

ND, not determined.a Compound showed 36% inhibition of LEDGF/p75–IN interaction at 100 mM.

antiviral target. However, it did not induce any cross-resis-

tance towards INSTIs, substantiating the novel mode of

action of LEDGINs [5].

There are some obvious advantages of drugs targeting the

LEDGF/p75–IN interaction. First, LEDGINs show a divergent

resistance development pathway to that of INSTIs and lack

cross-resistance with other classes of ARVs. Another attractive

advantage of LEDGINs is their broad-spectrum anti-HIV-1

activity. Discovery of LEDGINs is a good example of rational

drug design targeting well-defined and biologically relevant

protein–protein interactions.

Conclusions

This review highlights both the importance of LEDGF/p75–

IN interactions as a key component of HIV replication and

the rational design of LEDGINs as novel antivirals. Because

PPIs have pivotal roles in virtually all physiological and

disease-related intracellular macromolecular complexes,

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Drug Discovery Today: Technologies | Modulation of protein–protein interactions Vol. 10, No. 4 2013

selection of a tractable protein–protein interaction is impor-

tant. Moreover, targeting the PPIs is more challenging than

drug targets that naturally bind small molecules. While the

example discussed here is particularly relevant to the field of

virology, application of the screening and characterization

protocols that were implemented to discover LEDGINs will

offer a powerful technology to other fields as our knowledge

on the role of PPIs in human diseases expands.

Acknowledgments

Our work is funded by the European Commission (FP6/FP7)

through the European Consortia TRIoH (LSHB-CT-2003-

503480) and THINC (HEALTH-F3-2008-201032) and K.U.

Leuven BOF. B.A.D. is a DBOF fellow of the K.U. Leuven,

F.C. is an IOF fellow and JD is an FWO fellow.

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