p75 integrase interaction, a novel target for anti-HIV therapy

Virology 435 (2013) 102–109

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Review

The LEDGF/p75 integrase interaction, a novel target for anti-HIV therapy Frauke Christ n, Zeger Debyser Laboratory for Molecular Virology and Gene Therapy, Division of Molecular Medicine, KU Leuven, Kapucijnenvoer 33, 3000 Leuven, Belgium

a r t i c l e i n f o

Keywords: HIV LEDGF/p75 Integrase LEDGINs Drug discovery Co-factor INSTI Peptides Structure based design

a b s t r a c t To accomplish their viral life cycle, lentiviruses such as HIV highjack host proteins, the so-called cellular co-factors of replication. Lens Epithelium-derived Growth factor (LEDGF/p75), a transcriptional co-activator, is a co-factor of HIV-integrase (IN) and is required for the tethering and correct integration of the viral genome into the host chromatin. Due to its important role in HIV-replication the LEDGF/ p75–IN interaction is an attractive antiviral novel target for the treatment of HIV/AIDS. Intensive drug discovery efforts over the past years have validated the LEDGF/p75–IN interaction as a drugable target for antiviral therapy and have resulted in the design and synthesis of LEDGINs, small molecule inhibitors binding to the dimer interface of HIV-integrase and inhibiting viral replication with a dual mechanism of action: potent inhibition of the LEDGF/p75–IN protein–protein interaction and allosteric inhibition of the catalytic function. Furthermore they inhibit both early and late steps of the replication cycle which increases their potential for further clinical development. In this review we will highlight the research validating the LEDGF/p75–IN interaction as a target for anti-HIV drug discovery and the recent advances in the design and development of LEDGINs. & 2012 Elsevier Inc. All rights reserved.

Contents LEDGF/p75 and lentiviral integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Targeting HIV-IN in anti-HIV therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Does the LEDGF/p75–IN interaction qualify as a drug target for anti-HIV therapy? . . . . . . . . . . . . . . . . . . . . . . . . . . . Peptides inhibiting the LEDGF/p75–IN interaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LEDGINs, small molecules binding to the LEDGF/p75 binding pocket of integrase inhibit diverse integrase functions 2-(tert-butoxy)-2-substituted acetic acid derivatives, LEDGINs with antiviral activities in the nanomolar range . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

LEDGF/p75 and lentiviral integration Integration of the viral DNA into the host cell genome is a critical step during HIV replication. A stably inserted provirus enables productive infection and permanently archives the genetic information of HIV in the host. Due to its limited genome, HIV needs to rely on cellular co-factors for provirus establishment and efficient replication in the host cell (Van Maele et al., 2006). LEDGF/p75, a transcriptional co-activator (Ganapathy et al., 2003; Ge et al., 1998; Singh et al., 2000a), was initially identified

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Corresponding author. E-mail address: [email protected] (F. Christ).

0042-6822/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.virol.2012.09.033

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as an integrase co-factor by co-immunoprecipitation from cells overexpressing HIV-IN (Cherepanov et al., 2003). LEDGF/p75 is a member of the hepatoma-derived growth factor family (HDGF), composed of chromatin-associated proteins sharing certain structural features. The crucial role of LEDGF/p75 in HIV replication was evidenced via mutagenesis, RNAi-mediated depletion, transdominant overexpression of the integrase binding domain (IBD) of LEDGF/p75 and knock out studies (Busschots et al., 2007; Cherepanov et al., 2003; Ciuffi et al., 2005; De Rijck et al., 2006; Emiliani et al., 2005; Hombrouck et al., 2007a; Llano et al., 2006a; Schrijvers et al., 2012; Shun et al., 2007a, 2007b; Vandekerckhove et al., 2006). LEDGF is encoded by the PSIP1 (PC4- and SFRSinteracting protein 1) gene on the human chromosome 9 and is expressed as two splice variants the LEDGF/p52 and LEDGF/p75

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Fig. 1. Domain organization of LEDGF and HIV-integrase and crystal structure of their interaction. (A) LEDGF/p75 binding to DNA is mediated by the NLS and the nearby located AT-hook DNA binding motives whereas the N-terminal PWWP motive and charged regions (CR1-3) are critical for chromatin recognition (Hendrix et al., 2011; Llano et al., 2006b; McNeely et al., 2011; Turlure et al., 2006). The C-terminal IBD is essential for integrase binding (Cherepanov et al., 2004, 2005b) and for the interaction with cellular proteins that bind LEDGF/p75 (Bartholomeeusen et al., 2007, 2009; Maertens et al., 2006; Yokoyama and Cleary, 2008). (B) The 325 N-terminal residues of LEDGF/p52 are identical with LEDGF/p75 and therefore both splice variants share their chromatin/DNA binding preferences. The p52 isoform though harbors a unique eight amino acid sequence at its C-terminus (Singh et al., 2000a). (C) HIV-IN consists of three distinct domains: the N-terminal domain (NTD) with the conserved HHCC zinc finger motives, the catalytic core domain (CCD) housing the transposase specific catalytic triade (D64, D116, E152) and the C-terminal domain (CTD) involved in multimerization and DNA-binding. (D) The co-crystal structure of LEDGF/p75–IBD (magenta) and the CCD dimer of integrase (green and blue) reveals that the integrase CCD dimer-interface forms a cavity in which the connecting loops of the IBD 5 a-helixes bundle protrudes. Highlighting the catalytic triad in yellow visualizes that the LEDGF/p75 interaction pocket in IN is distinct from the catalytic site (PDB accession code 2B4J; Cherepanov et al., 2005a).

proteins (Fig. 1; Singh et al., 2000b). Both share their N-terminal region (aa 1–325) and therefore the nuclear localization and chromatin-binding elements defined by the PWWP (Pro–Trp– Trp–Pro) domain (Dietz et al., 2002; Izumoto et al., 1997; Stec et al., 2000), the nuclear localization signal (NLS), the A/T hook like elements (Aravind and Landsman, 1998) and three charged regions (CR1, CR2 and CR3) (Llano et al., 2006a; Turlure et al., 2006). The C-terminal region though differs and is much extended in p75 (Fig. 1A). The larger splice variant p75 therefore exclusively contains the integrase binding domain (IBD, aa347–429). Although this domain was originally characterized by its association with HIV-IN (Cherepanov et al., 2004) later studies have shown that different cellular binding partners interact with LEDGF/p75 through this domain (Bartholomeeusen et al., 2007, 2009; Maertens et al., 2006; Yokoyama and Cleary, 2008). LEDGF/ p52 (Fig. 1B) contains eight unique aa in its C-terminus (Ge et al., 1998) and fails to interact with HIV-IN (Maertens et al., 2003; Shun et al., 2007b). Co-localization studies mapped the LEDGF/ p75 interaction site on IN (Fig. 1C) to the catalytic core domain and to a less extent to the N-terminal domain (Maertens et al., 2003). Through its classical NLS LEDGF/p75 is predominantly located in the nucleus where it associates with chromatin through its PWWP domain. During HIV replication it thereby tethers IN associated with the viral genome to the host chromatin facilitating the integration into HIV-preferred sites (Ciuffi et al., 2005; Gijsbers et al., 2011; Maertens et al., 2003; Marshall et al., 2007; Nishizawa et al., 2001; Shun et al., 2007b; Tsutsui et al., 2011). Next to its tethering function LEDGF/p75 protects IN from proteolytic degradation and stimulates the catalytic activity of IN in vitro as well as in in vivo (Cherepanov et al., 2003; Hendrix et al., 2011; Llano et al., 2004; Maertens et al., 2003; Turlure et al., 2006). Evidence from integration sites analysis in human cells depleted of LEDGF/p75 by RNAi, in embryonic fibroblasts derived from LEDGF knockout mice or in human B-cell lines with a

specific LEDGF/p75 knock out corroborated this role of LEDGF/ p75 as tethering and targeting factor of HIV (Ciuffi et al., 2005; Gijsbers et al., 2011; Schrijvers et al., 2012; Shun et al., 2007a).

Targeting HIV-IN in anti-HIV therapy Since the first description of the acquired immune deficiency syndrome (AIDS) in 1981, worldwide more than 25 million people have fallen victim to HIV infections. Despite the enormous efforts in developing new effective antivirals and the introduction of highly active antiretroviral therapy (HAART), the incidence of HIV infections remains a major problem in public health. Although HIV replication can be chronically suppressed with proper HAART, no cure is in sight. Therefore the quest for novel antivirals to complement existing treatment strategies remains one of the major goals in HIV drug discovery. Classical drugs target the viral enzymes reverse transcriptase, protease and integrase. Raltegravir (Isentress, MK-518) interferes with the strand transfer reaction of viral integrase and has been approved for clinical use (Summa et al., 2008). Raltegravir is the prototypical integrase strand transfer inhibitor (INSTI). Inhibition of integration by raltegravir is accompanied by extremely fast and strong reduction in viral load (Murray et al., 2007). However, in contrast to prior predictions based on in vitro experimentation, raltegravir resistance evolves readily in the clinic (Malet et al., 2008) even in the presence of optimized highly active antiretroviral treatment (HAART) regiments (Baldanti et al., 2010), boosting the efforts to develop second generation integrase inhibitors. Since the development of raltegravir, there has been a strong interest in developing true second generation IN inhibitors lacking crossresistance. Nevertheless in the past years it has been proven difficult to develop novel antivirals with a beneficial resistance profile over raltegravir or an improved pharmacokinetics (PK) in

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patients allowing administration of single instead of twice daily doses. Elvitegravir (Shimura et al., 2008; Zolopa et al., 2010), a Phase III INSTI, can be administered once daily if combined with a pharmacological booster, but is cross-resistant with raltegravir and therefore is no treatment option for patients failing on INSTIs. S/ GSK1349572 (Dolutegravir) yet another Phase III clinical trial INSTI displays superior characteristics to raltegravir, but partially shares the resistance pathways (Garrido et al., 2011; Min et al., 2011). Due to the limited chemical space available for designing inhibitors of the catalytic activity of IN inevitable overlap of resistance for second generation integrase strand transfer inhibitors is likely; therefore efforts have moved toward novel mechanism of action such as the design of inhibitors with an allosteric mechanism of action or inhibitors of interactions with essential cellular co-factors of integration, such as the LEDGF/p75–IN interaction.

Does the LEDGF/p75–IN interaction qualify as a drug target for anti-HIV therapy? In 2005 the crystal structure of IBD in complex with a dimer of the integrase catalytic core domain was reported (Fig. 1D; PDB code: 2B4J; Cherepanov et al., 2005a), identifying the amino acid residues of LEDGF/p75 mediating the interaction with IN (Lys364, Ile365, Asp366, Phe406, Val408). The IBD structure is composed of a right handed compact bundle of 4 a-helices. The amino acid residues contacting HIV-IN are located on the interhelical loop regions of the structure. In integrase two regions of the catalytic core domain are in direct contact with LEDGF/p75, namely the region around Trp131 and Trp132 and the region extending from Ile161 to Glu170 (Busschots et al., 2007; Cherepanov et al., 2005a; Emiliani et al., 2005). The interface is located in a pocket formed by the two subunits of the IN-core dimer (a1 and a3 of one monomer and the six residues from the a4/5 connector in the other monomer). Residues located in the a4/5 connector and a hydrophobic pocket formed by the other subunit engage tightly with the two inter-helical loops of LEDGF/p75–IBD. Specifically the side chain of LEDGF/p75 Ile365 contacts the hydrophobic pocket formed by Leu102, Ala128, ALA129, Trp132 of one IN subunit and Thr174 and Met178 of the other subunit. Furthermore Ile365 establishes a hydrogen bond with the backbone carbonyl group of IN Gln168 whereas Asp366 of LEDGF/p75 forms a hydrogen bond with Glu170 (Cherepanov et al., 2005a). The importance of aa Ala128, His170, Thr174, Trp131, Trp132, Gln168 and Glu170 have been confirmed by mutagenesis studies. Mutation of these residues on integrase renders the protein defective or deficient for LEDGF/p75 interaction (Busschots et al., 2007; Cherepanov et al., 2005a; Emiliani et al., 2005; Hombrouck et al., 2007a). Vice versa substitution of aa residues Ile365, Asp366, Phe406 and Val408 decreased or abolished binding of LEDGF/p75 to integrase (Cherepanov et al., 2005b). Therefore the protein– protein interaction surface of LEDGF/p75 and IN provides a welldefined pocket, limited in its extension and with multiple hydrophobic and hydrogen bond interactions indicating that its disruption by small molecules is a feasible endeavor. Depletion of LEDGF/p75 from cells by RNAi or knock-out techniques significantly reduced infectivity of HIV in those cells (Llano et al., 2006a; Schrijvers et al., 2012; Shun et al., 2007b; Vandekerckhove et al., 2006). Together with the overexpression of the IBD of LEDGF/p75 in human cells (De Rijck et al., 2006), these findings provided proof-of-concept that the LEDGF/p75-IN interaction might be a feasible and druggable target for anti-HIV therapy. IBD, lacking the chromatin binding domain, could efficiently compete with the endogenous cofactor and inhibited HIV replication and integration more than 100-fold (De Rijck et al., 2006). Moreover by serial passaging of HIV in cells

Fig. 2. The LEDGF/p75–IN interaction is mediated by several tight interactions between both protein binding partners. (PDB accession code 2B4J; Cherepanov et al., 2005a) Cartoon representation of the CCD–IBD complex. IN CCD molecules are shown in green and blue, whereas the LEDGF/p75 IBD is colored in magenta. Residues critical for the interaction and identified through biochemical characterization and resistance selection (Busschots et al., 2007; Hombrouck et al., 2007b) are shown in stick representation and highlighted (L368, I365, D366 and K364 from LEDGF/p75 and A128, H171 and E170 from HIV-IN).

overexpressing this LEDGF/p75 fragment, a virus strain resistant to this phenotype was selected (Hombrouck et al., 2007a). Interestingly two mutations in integrase were required to render IN resistant: A128T and E170G, confirming the mutagenesis data and the validity of the IBD/IN catalytic core domain structure (Fig. 2; Busschots et al., 2007; Cherepanov et al., 2005a). Further support was granted by the reports of Al-Mawsawi et al. (2008) and Hayouka et al. (2007) demonstrating that short peptides derived from LEDGF/p75 block the interaction between LEDGF/p75 and IN. Even though peptides are not the compounds of choice for targeting intracellular targets and drug development, this work provided further support for the notion that the LEDGF/p75–IN interaction is a likely target for anti-HIV drug development. Since then different groups in academia and industry have embarked on targeting this virus–host interaction for anti-HIV drug discovery. Different approaches have been employed to design and identify small-molecule inhibitors of the LEDGF/p75–IN interaction. These include high throughput screening (HTS) of diverse chemical libraries, computational database screening of virtual small molecule libraries and structure-based design of de novo small molecules on the basis of pharmacophore models.

Peptides inhibiting the LEDGF/p75–IN interaction Although peptides pose notorious problems when it comes to stability and bioavailability, the design of small synthetic peptides interacting with one of the binding partners of a protein–protein interaction is a valid starting point to facilitate the development of peptidomimetics or genuine small molecule inhibitors. Due to the linearity of the interacting aa sequence of the IBD initial work focused on peptides derived from the LEDGF/p75 sequence (Al-Mawsawi et al., 2008; Hayouka et al., 2010a; Rhodes et al., 2011). Hayouka et al. (2007) described the design and synthesis of three LEDGF/p75 derived peptides (LEDGF/p75 353–378, 361–370 and 402–411). As LEDGF/p75 was reported earlier to preferentially bind to the tetrameric state of IN (Cherepanov et al., 2003) the authors reasoned that small peptides derived from LEDGF/ p75, interacting with the LEDGF/p75 binding pocket on IN should

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change the oligomerization state and as a consequence affect the catalytic activity of IN. Indeed they observed that the peptides shifted the oligomerization state of IN toward the tetrameric form resulting in inhibition of IN–DNA binding and as a consequence of both catalytic activities namely the 30 processing and the strand transfer. In this report though inhibition of the LEDGF/p75–IN interaction by the described peptides was not presented and the authors attributed the observed anti-viral effect solely to the inhibition of the IN catalytic activity. One year later it was reported that a similar peptide (LEDGF/p75 355–377) was capable of competing with LEDGF/p75 for the binding to integrase and therefore inhibiting the cofactor-IN interaction with an IC50 of 25 mM (Al-Mawsawi et al., 2008) in a alphascreen based interaction assay (Christ et al., 2010; Hombrouck et al., 2007b; Hou et al., 2008). The inhibition of the catalytic activity (both 30 processing and strand transfer) was less pronounced for this peptide and was lost when the IN–DNA complex was assembled prior to addition of the peptide (Al-Mawsawi et al., 2008) which led to the hypothesis that the peptide might disrupt initial DNA-binding of integrase and as such exerts its effect on the catalytic activity, namely the 30 processing reaction. The mechanism of action of shorter peptides (LEDGF/p75 361–370) was studied in detail by biophysical, biochemical and cellular assays (Hayouka et al., 2010b). A peptide as short as LEDGF/p75 365–369 was described to bind to the catalytic core domain of HIV-IN as demonstrated by fluorescence anisotrophy, but it was not sufficient to inhibit IN function. The authors identified LEDGF/p75 361–370 as the minimal inhibitory peptide. This LEDGF/p75 derived peptide was then cyclized in a later study (Hayouka et al., 2010a, 2010b) increasing its IN inhibitory activity. LEDGF/p75 derived cyclic peptides have also been used by Rhodes et al. (2011) in order to identify new interactions in the LEDGF/p75–IN interaction interface. In this study a cyclic peptide was formed using the LEDGF/p75 362–367 sequence fused to three additional amino acids (H–SLKIDNLDVNS–OH). High resolution crystallography of in total 13 cyclic peptides constructed from this original sequence bound to the LEDGF/p75 binding pocket in integrase revealed that seven out of the eight aa residues present in LEDGF/p75 make contacts with the binding pocket in integrase covering a surface area of 420 A˚ and stabilizing the conformation of the peptide by extensive intramolecular interactions. Not all contacts described in this study were known before. The previously undescribed interactions observed in this study, in particular a hydrogen bond interaction with IN–Glu168, gave valuable input for structurebased design efforts to develop novel small molecules inhibiting the LEDGF/p75–IN interaction as the authors have shown in a follow-up structure based design study reviewed below. Recently our group has published a novel reciprocal peptide approach (Desimmie et al., 2012). In contrast to the earlier work using peptides derived from the LEDGF/p75–IBD primary sequence binding to the dimer interface of IN, here peptides were designed to bind to LEDGF/p75 and as a consequence to inhibit the LEDGF/p75 integrase interaction from the side of the cellular co-factor. As the LEDGF/p75 binding pocket in integrase is not linear and therefore cannot guide development of IN-derived inhibitory peptides, a phage display strategy was employed to select for peptides with affinity for the integrase interaction side of IBD. From three different linear and cyclic phage display libraries multiple peptides were selected with moderate affinity for LEDGF/p75. Interestingly the sequence motive VM/XGHPL/XW was repeatedly found. Synthesis of cyclic peptides containing this sequence motive indeed yielded small cyclic peptide inhibitors of the LEDGF/p75–IN interaction. As peptides are notoriously difficult to be delivered to cells, stable lentiviral expression of selected active and mutant peptides was chosen. As expected, expression of active peptides led to potent inhibition of HIV-replication.

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Biochemical and biophysical studies as well as antiviral profiling demonstrated that the selected peptides indeed inhibit HIV-replication through their binding to the cellular co-factor LEDGF/p75 (Desimmie et al., 2012). It was reassuring to observe that although the peptides bind to the IBD of LEDGF/p75 no cellular toxicity was observed. This observation is in accord with previous reports describing that although IN shares the overall binding site (IBD) on LEDGF/p75 with different cellular interacting partners such as PogZ and JPO2, the detailed architecture of the interaction is distinct (Bartholomeeusen et al., 2007, 2009; Maertens et al., 2006). Due to the interaction with a cellular cofactor instead of a viral protein resistance selection failed. Taken together the study by Desimmie et al. provides proof-of concept that intracellular co-factors such as LEDGF/p75 are drug targets for antiviral therapy which might come at the benefit of a higher barrier toward resistance selection. As the described consensus sequence (Desimmie et al., 2012) is limited in size the cyclic peptides might serve as templates for genuine peptidomimetic drugs.

LEDGINs, small molecules binding to the LEDGF/p75 binding pocket of integrase inhibit diverse integrase functions The flatness of protein–protein interfaces often hampers the identification of small molecule protein–protein interaction inhibitors (SMPPIIs). LEDGF/p75 though binds to a defined pocket in the dimer interface of the HIV-IN catalytic core domain. Due to the tight interface of the LEDGF/p75–IN interaction (Cherepanov et al., 2005a) the design and/or selection of small molecules binding to this pocket is a feasible endeavor. Although a HTS approach has been described (Hou et al., 2008), most efforts so far have emanated from in silico virtual screening of compound libraries and structure-based de novo design (Christ et al., 2012, 2010; De Luca et al., 2009, 2010; Du et al., 2008; Fan et al., 2011; Peat et al., 2012). The knowledge-driven, computer aided approach usually yields a limited subset of small molecules, with chemical features defined by the applied screening algorithm. This limited selection of molecules is then evaluated for their biological activity. Albeit different classes of LEDGINs with high variability in their biological activity have been described so far, they all share certain characteristics. In contrast to INSTIs, which bind to the catalytic site after integrase has assembled on its DNA substrate (long terminal repeat sequences, LTRs; Espeseth et al., 2000; Hare et al., 2010), LEDGINs bind to the dimer interface of integrase irrespective of the assembly with LTRs, as demonstrated by crystallography (Christ et al., 2010; Peat et al., 2012) or modeling (De Luca et al., 2009, 2008; Du et al., 2008; Fan et al., 2011; Hu et al., 2012). In addition, if the tested molecules reach a critical level in potency, they exert a dual mechanism of action blocking the LEDGF/p75–IN interaction and simultaneously modulating the multimerization state of IN resulting in the allosteric inhibition of the integrase catalytic activity (Christ et al., 2012, 2010; Kessl et al., 2012; Tsiang et al., 2012). Even before the identification and validation of LEDGF/p75 as a co-factor of HIV integration tetraphenyl arsonium was identified by crystallography to bind to the dimer interface of the catalytic core domain (Molteni et al., 2001). Although this molecule does inhibit neither integrase activity nor the interaction with LEDGF/p75, it provided valuable information for later structure-based design efforts (Christ et al., 2010). Du et al. (2008) described a commercially available benzoic acid derivative, D77 (Fig. 3A). Molecular docking studies in combination with site-directed mutagenesis and surface plasmon resonance (SPR) identified this molecule as a potential binder to

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Fig. 3. Different classes of LEDGINS, inhibiting the LEDGF/p75–IN interaction. (A) Selected chemical structures of LEDGINs (D77, Du et al., 2008; BI-1001, Kessl et al., 2012; CHIBA3053, De Luca et al., 2009; CX05045, Christ et al., 2010; CX14442, Christ et al., 2012). (B) Cartoon representation of the co-crystal structure of compound 6 (yellow stick superimposed with the IBD–IN core complex structure, gray) bound in the LEDGF/p75 binding pocket of HIV-IN (green and yellow) (PDB 3LPU) reveals mimicry of the protein–protein interaction by compound 6 (Christ et al., 2010).

the LEDGF/p75 binding pocket. Intriguingly D77 did not affect dimerization of HIV-IN, an effect consistently observed for other potent LEDGINs (see below), pointing toward a divergent mechanism of action. Although D77 failed to inhibit LEDGF/ p75–IN interaction in Alphascreen based assays, it inhibited the LEDGF/p75–IN interaction in a yeast two-hybrid based cellular reporter assay. Minor antiviral activity in MT-4 cells was described, but the activity versus toxicity (selectivity index¼SI) window reported was rather narrow. Recently the same group reported a new structure based approach to identify inhibitors of the LEDGF/p75–IN interaction (Hu et al., 2012). In this study 26 known drugs were selected based on the GlideSP program allowing for flexible docking of the ligands (Friesner et al., 2004). Of the initially selected drugs eight were indeed capable of inhibiting the LEDGF/p75–IN interaction with moderate IC50 values ranging from 6.54 mM for Carbidopa to 36.85 mM for Eprosartan. Of note one of the selected compounds, Atorvastatin (IC50 8.9 mM), has been used in HIV patients to reduce cholesterol levels and an effect on HIV-replication has been reported. Whether the in vitro and antiviral activities indeed are linked with each other is still hypothetical though and requires further investigation. On the basis of a pharmacophore model, comprised of 14 distinct features derived from the IBD–IN–CCD co-crystal structure and including potential steric restrictions, a virtual screening approach was performed (De Luca et al., 2010). The contacts of LEDGF–Ile365 with IN–Gln168 and Asp366 with IN–Glu170/171 formed the basis of the model. Best fitting was obtained for CHIBA-3002, a small molecule which was previously described as an IN strand transfer inhibitor, suggesting a dual function of this small molecule. Indeed modest inhibition of the LEDGF/p75–IN interaction was observed in the Alphascreen assay. Docking of more potent congeners of these benzylindole derivatives highlighted that the CHIBA compounds likely form hydrogen bonds with the main chains of IN–Glu170 and His171 while the diketo acid moiety creates a hydrogen bond with Gln95 of the other IN– CCD subunit. Follow-up studies explored the chemical space further toward the LEDGF–Phe406 contact with Trp131 and generated a more potent congener of the compounds, CHIBA3053 (Fig. 3A), with an IC50 in the lower micromolar range.

Further optimization though is needed to reach antiviral activity for this class of small molecules. Recent molecular docking studies by De Luca et al. (2011) suggest that inhibitors previously identified as INSTIs (CHI-1043), which are based on the classical diketoacid functionality, might serve as LEDGF/p75–IN inhibitors. Indeed they observed that CHI-1043 which potently inhibits HIVreplication (De Luca et al., 2008) is a weak inhibitor of the LEDGF/ p75–IN interaction. Whether this weak SMPPII mechanism contributes to the antiviral activity is uncertain and inhibitors with more equal activities on both functionalities would be necessary to dissect the detailed mechanism of action. Fan et al., 2011 used a scaffold hopping approach to design yet another set of small molecules capable of inhibiting the catalytic site as well as the LEDGF/p75–IN interaction. As a starting point the pharmacophores of salicylic acid and catechol were fused and therefore a molecule was generated capable of chelating the catalytic Mg2 þ ions in the active site and of binding into the hydrophobic pocket formed by the dimer interface in the IN–CCD. Four different classes of compounds were described with the most potent compound (compound 5u) reaching moderate activity for the inhibition of the 30 processing and strand transfer reaction of IN (IC50 53 mM and 19 mM). The compound did not induce any cytotoxicity in H630 cells, but no antiviral activity was reported either. Further optimization of the described structures is required to reach higher activities in vitro as well as antiviral activity. Whether the strategy of designing compounds that bind both the catalytic site of integrase but also the hydrophobic LEDGF/p75 interaction site and therefore inhibit both functionalities of HIV-IN simultaneously, will be a valid strategy to potently inhibit integration with a reduced risk of resistance selection, or will lead to undesired side effects due to unequal affinity for both inhibitory sites remains to be investigated. The so far described efforts of LEDGIN development started from molecular modeling approaches based on the co-crystal structure of LEDGF/p75 IBD and HIV-IN. As described above an intensive crystallization effort had been undertaken to identify yet unknown contacts between small cyclic LEDGF/p75 derived peptides with the catalytic core domain of integrase (Rhodes et al., 2011). In this effort a hydrogen bonding interaction with IN–Gln168 was identified not exploited in any of the other

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reported LEDGINs. Therefore the authors performed a follow up study using fragment based screening to identify small molecules making use of this contact which eventually might lead to the development compounds with a higher affinity and/or a higher barrier to resistance (Peat et al., 2012). Initially 500 fragments were screened using SPR, NMR and crystallography identifying hits with good density in the LEDGF/p75 binding site of integrase. Synthesis of small molecules based on the hits of the initial fragment based screening led to compound 11 with an IC50 at 8.1 mM in the Alphascreen based LEDGF/p75-IN interaction assay. Crystallography indeed demonstrated that compound 11 penetrates deep into the hydrophobic pocket on the IN-dimer interface directly interacting with IN–Gln168 and therefore indeed targeting the 167–173 interaction site for LEDGF/p75 in IN. Compound 11 has moderate antiviral activity with an EC50 of 29 mM and no apparent toxicity and is not cross-resistant with raltegravir resistance mutants such as IN Q148H/G140S and N155H/E92Q. Future resistance selection and cross-resistance testing with known LEDGINs resistant mutants will demonstrate whether expansion deeper into the dimer interface will raise the barrier toward resistance selection for this class of LEDGINs.

2-(tert-butoxy)-2-substituted acetic acid derivatives, LEDGINs with antiviral activities in the nanomolar range In 2010 we reported the structure-based design of CX04238 and CX05045 both belonging to the class of 2-(tert-butoxy)-2substituted acetic acid derivatives (Christ et al., 2010; Fig. 3). This work was founded by the construction of a consensus pharmacophore based on different available crystal structures of the CCD of HIV-IN (Cherepanov et al., 2005a; Maignan et al., 1998; Molteni et al., 2001), representing a series of steric and electronic features predicted to be critical for LEDGF/p75 binding in the hydrophobic pocket at the IN dimer interface. Around 200,000 commercially available diverse compounds were filtered, fitted to the pharmacophore model and best scoring hits were cherry picked and subjected to biological evaluation. Four compounds weakly inhibited the LEDGF/p75–IN interaction and constituted the basis for a detailed structure–activity relationship (SAR) investigation aiming at developing more potent analogs with beneficial medicinal chemistry characteristics. Co-crystals of the identified hit compounds with the IN–CCD were obtained and confirmed the initial pharmacophore. The LEDGIN phenyl, acid and chlorine groups were indeed mimicking LEDGF/p75 Ile365, Asp366 and Leu368. This structural information was crucial to design and synthesize more potent LEDGINs with improved biological activities, such as CX05045 (Christ et al., 2010) and CX014442 (Christ et al., 2012; Fig. 3A), allowing for a complete antiviral profiling of this compound class. CX014442 is the first LEDGIN reported to display antiviral activity in the low nanomolar range, EC50 IIIB ¼69 73 nM and high selectivity, SI ¼1391. To date the 2(tert-butoxy)-2-substituted acetic acid derivatives are the best studied LEDGINs and congeners of these compounds are in advanced preclinical development. Unlike strand transfer inhibitors, LEDGINs inhibit the strand transfer and 30 processing reactions to the same extent. Complete inhibition of the integrase catalytic activities by LEDGINs can only be achieved when the compounds are added to integrase before the DNA substrate (Christ et al., 2012; Kessl et al., 2012; Tsiang et al., 2012). This is in stark contrast with the uncompetitive mode of inhibition of INSTIs that require prior binding and 30 processing of viral DNA ends (Espeseth et al., 2000; Hare et al., 2010). The inhibition of both catalytic activities of integrase suggests that LEDGIN binding influences the active site of integrase. Indeed we and others provided evidence that LEDGINs

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modulate the multimeric state of integrase upon their binding (Christ et al., 2012; Kessl et al., 2012; Tsiang et al., 2012). LEDGIN binding to the integrase dimer interface leads to a stabilization of the dimer, restricting integrase oligomeric flexibility and as a consequence affecting the productive formation of the intasome. LEDGF/p75 in turn likely modulates the integrase multimerization required for enzymatic activity (Kessl et al., 2011). Hence LEDGF/p75 can be considered an allosteric effector of integrase activity and LEDGINs as allosteric enzymatic inhibitors. Thus next to their SMPPII function LEDGINs are genuine allosteric inhibitors (Christ et al., 2012; Kessl et al., 2012; Tsiang et al., 2012). Tsiang et al. (2012) demonstrated that in cell culture LEDGINs induce a significant decrease of deletions at the 2-LTR junctions in 2-LTR circles produced during HIV-replication consistent with an antiviral mechanism involving the inhibition of 30 processing and thus consistent with the observations of the biochemical characterizations. Both, the PPI and allosteric mechanisms are relevant for their biological activity, cannot be uncoupled and lead to the inhibition of the integration reaction. In the discussion whether one mechanism should be considered more important than the other, one should keep in mind that in vivo LEDGINs will always encounter LEDGF/p75 bound to the dimer interface of integrase and therefore are required to displace LEDGF/p75 which is essential for HIV replication (Schrijvers et al., 2012). Alike INSTIs, LEDGINs inhibit the integration step of HIVreplication as shown by quantitative PCR (Q-PCR) and Time-ofaddition (TOA) studies (Christ et al., 2012, 2010). Importantly though LEDGINs are by no means cross-resistant with INSTIs. Initial resistance selection with less potent LEDGINs demonstrated that a single point mutation is sufficient to render HIV resistant to the action of LEDGINs (Christ et al., 2010). Integrase Ala128 makes substantial Van der Waals contacts with LEDGF/ p75–Ile365 and Leu368, both important features in the pharmacophore model. Hence the finding that an Ala128 to Threonin substitution reduced LEDGIN binding to integrase did not come at a surprise. The more recent report that more potent LEDGINs though are active against A128T and that at least one additional resistance mutation is required to render integrase resistant against LEDGIN action (Christ et al., 2012) proves that the expansion of the chemical space raises the barrier towards resistance development, an observation, which will be important for further clinical development of this compound class. Importantly LEDGINs retain their activity in primary cells and are active against a broad spectrum of clades and clinical isolates (Christ et al., 2012, 2010). Combination experiments demonstrate that LEDGINs and INSTIs do not antagonize each other but act in an additive or even slightly synergistic way, implementing a possible design of LEDGIN/INSTI combination therapy within the HAART treatment schemes (Christ et al., 2012). The detailed virological characterization of cyclic peptides (CPs) binding to LEDGF/p75 and therefore inhibiting HIVreplication when expressed in cell culture, provided the first evidence that inhibition of the LEDGF/p75–IN interaction results in impaired infectivity of viral particles produced in presence of this peptide inhibitor (Desimmie et al., 2012). The most recent report on the mechanism of action of LEDGINs confirms this multimodal inhibition pathway (Christ et al., 2012). Presence of LEDGINs during virus production indeed not only blocks provirus integration but affects as well the infectivity of the residual progeny virus. This observation is unique for LEDGINs and is not part of the mechanism of other integration inhibitors such as raltegravir or other classes of early replication inhibitors, such as entry blockers, NRTIs (nucleoside reverse transcriptase inhibitors) or NNRTIs (non-nucleoside reverse transcriptase inhibitors). The finding that LEDGINs not only block the integration of the viral genome but additionally impair the infectivity of viral particles,

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when present during production, makes them highly interesting candidates further clinical development.

Conclusions In the past decade the identification, validation and targeting of LEDGF/p75 for antiviral therapy represents one of most thrilling research lines in the field of HIV virology. Intensive medicinal chemistry efforts by multiple groups have proven the feasibility of targeting the LEDGF/p75–IN interaction for anti-HIV drug development and as such might also pave the way for exploiting other virus-host interaction involved in integration, but also in other steps of the viral replication cycle, for drug discovery. While the development of LEDGINs has experienced a promising start, a long winding road still needs to be taken to reach the clinic in the future. The fate of this novel class of inhibitors will not be solely depending on the antiviral activity but more importantly on pharmacokinetics and tolerability. However their multimodal mode of action is unique in the field of HIV-drug discovery holding great promise for the treatment of HIV/AIDS. The divergent resistance pathway of LEDGINs in comparison with INSTI and the lack of cross-resistance with any known class of ARVs make them candidates for future HAART, especially for patients failing on current therapy. More importantly the severe hampering of infectivity of viruses produced in presence of LEDGINs raises particularly the interest for further development. Although final proof will only be given when LEDGINs are an integral part of antiviral therapy, it is easy to imagine that the combined early (integration) and late effect (infectivity) not only increases the steepness of the slope in the inhibition of HIVreplication but will ultimately raise the barrier of resistance development in patients. Combined early and late effects define LEDGINs as unique within all classes of HIV-drugs described so far and might predestinate them for use in prevention and first in line therapy.

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