Rational design of LEDGINs as first allosteric integrase inhibitors for the treatment of HIV infection

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 DRUG DISCOVERY

TODAY

TECHNOLOGIES

Modulation of protein–protein interactions

Rational design of LEDGINs as first allosteric integrase inhibitors for the treatment of HIV infection Belete A. Desimmie, Jonas Demeulemeester, Frauke Christ, Zeger Debyser* Laboratory for Molecular Virology and Gene Therapy, KU Leuven, Leuven 3000, Flanders, Belgium

The interaction between lens epithelium-derived growth factor (LEDGF/p75) and HIV-1 integrase (IN) is an attractive target for antiviral development

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

because its inhibition blocks HIV replication. Developing novel small molecules that disrupt the LEDGF/p75– IN interaction constitutes a promising new therapeutic strategy for the treatment of HIV. Here we will highlight 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]. However, 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 ([email protected]) 1740-6749/$ ß 2012 Elsevier Ltd. All rights reserved.

of the host cell through distinct virus–host PPIs [4]. Because these host–pathogen interactions directly mediate viral replication 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 development 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 LEDGINs 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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These latently infected cells reside in reservoirs where the distribution of antiretroviral (ARV) drugs is extremely variable 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 treatment 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 interaction has dramatically increased. Not surprisingly, there are numerous interactions between HIV and cellular proteins 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 demonstrates 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 antiHIV 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 insertion 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, e518

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(a) Intasome

IN p75

GF/

LED

WP

PW

AT

IN IN

IN

BD

I

oks

ho

Chromosomal DNA (b)

I365

D366 L368

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 Cterminus 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.

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 mutations 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 interacting 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

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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 (CHIBA3002 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].

In principle, any drug discovery project requires design, prioritization, analysis and interpretation of results from consecutive experiments to ultimately facilitate the development 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.

Rational design of LEDGF/p75–IN interaction inhibitors

Activity and optimization of hit compounds

Different approaches have been employed to design and identify small-molecule inhibitors of the LEDGF/p75–IN interaction. These include in vitro high-throughput screening of compound libraries [32,34], in silico virtual screening of libraries and structure-based de novo design [5,33]. Highthroughput 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 represents 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 compounds were inspected manually and 25 compounds were selected for biological evaluation in an LEDGF/p75–IN interaction assay.

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 LEDGINsoaked 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-inclass 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 inhibiting the LEDGF/p75–IN interaction through high-throughput screening of a compound library of more than 700,000 small molecules using AlphaScreen assay. However, the nature 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 replication which have been extensively evaluated for their therapeutic potential and mechanism of action in cell-based antiviral assays (including in primary cells) [5]. www.drugdiscoverytoday.com

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Medicinal chemistry

OH

Cl

Molecular modelling

N

Co-crystallization

O

Biological evaluation MTT/MT4 antiviral assay EC50

HIV-infected cells

CC50

Mock-infected cells

Compound concentration (µM) Controls

Hit-to-lead optimization

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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 therapeutic 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]. Integration 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 e520

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number of integrated proviral DNA copies and consequently induce accumulation of 2-LTR circles [5]. Importantly, LEDGINs do not show any cross-resistance with INSTIs, implying that they might serve as second-generation inhibitors if they meet the pharmacokinetic and pharmacodynamic requirements 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 interactions with LEDGF/p75 residues I365 and L368 – both important 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 interactions 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

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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-resistance 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, www.drugdiscoverytoday.com

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selection of a tractable protein–protein interaction is important. 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-2003503480) 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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