The A128T Resistance Mutation Reveals Aberrant Protein Multimerization as the Primary Mechanism of Action of Allosteric HIV-1 Integrase Inhibitors

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are a very promising new class of anti-HIV-1 agents that exhibit a multimodal mechanism of action by allosterically modulating IN multimerization and interfering with IN-lens epithelium-derived growth factor (LEDGF)/p75 binding. Selection of viral strains under ALLINI pressure has revealed an A128T substitution in HIV-1 IN as a primary mechanism of resistance. Here, we elucidated the structural and mechanistic basis for this resistance. The A128T substitution did not affect the hydrogen bonding between ALLINI and IN that mimics the IN-LEDGF/p75 interaction but instead altered the positioning of the inhibitor at the IN dimer interface. Consequently, the A128T substitution had only a minor effect on the ALLINI IC₅₀ values for IN-LEDGF/p75 binding. Instead, ALLINIs markedly altered the multimerization of IN by promoting aberrant higher order WT (but not A128T) IN oligomers. Accordingly, WT IN catalytic activities and HIV-1 replication were potently inhibited by ALLINIs, whereas the A128T substitution in IN resulted in significant resistance to the inhibitors both in vitro and in cell culture assays. The differential multimerization of WT and A128T INs induced by ALLINIs correlated with the differences in infectivity of HIV-1 progeny virions. We conclude that ALLINIs primarily target IN multimerization rather than IN-LEDGF/p75 binding. Our findings provide the structural foundations for developing improved ALLINIs with increased potency and decreased potential to select for drug resistance.     

A Critical Role of the C-terminal Segment for Allosteric Inhibitor-induced Aberrant Multimerization of HIV-1 Integrase

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are a promising class of antiretroviral agents for clinical development. Although ALLINIs promote aberrant IN multimerization and inhibit IN interaction with its cellular cofactor LEDGF/p75 with comparable potencies in vitro, their primary mechanism of action in infected cells is through inducing aberrant multimerization of IN. Crystal structures have shown that ALLINIs bind at the IN catalytic core domain dimer interface and bridge two interacting subunits. However, how these interactions promote higher-order protein multimerization is not clear. Here, we used mass spectrometry-based protein footprinting to monitor surface topology changes in full-length WT and the drug-resistant A128T mutant INs in the presence of ALLINI-2. These experiments have identified protein-protein interactions that extend beyond the direct inhibitor binding site and which lead to aberrant multimerization of WT but not A128T IN. Specifically, we demonstrate that C-terminal residues Lys-264 and Lys-266 play an important role in the inhibitor induced aberrant multimerization of the WT protein. Our findings provide structural clues for exploiting IN multimerization as a new, attractive therapeutic target and are expected to facilitate development of improved inhibitors.     

LEDGF/p75-Independent HIV-1 Replication Demonstrates a Role for HRP-2 and Remains Sensitive to Inhibition by LEDGINs

Lens epithelium–derived growth factor (LEDGF/p75) is a cellular cofactor of HIV-1 integrase (IN) that interacts with IN through its IN binding domain (IBD) and tethers the viral pre-integration complex to the host cell chromatin. Here we report the generation of a human somatic LEDGF/p75 knockout cell line that allows the study of spreading HIV-1 infection in the absence of LEDGF/p75. By homologous recombination the exons encoding the LEDGF/p75 IBD (exons 11 to 14) were knocked out. In the absence of LEDGF/p75 replication of laboratory HIV-1 strains was severely delayed while clinical HIV-1 isolates were replication-defective. The residual replication was predominantly mediated by the Hepatoma-derived growth factor related protein 2 (HRP-2), the only cellular protein besides LEDGF/p75 that contains an IBD. Importantly, the recently described IN-LEDGF/p75 inhibitors (LEDGINs) remained active even in the absence of LEDGF/p75 by blocking the interaction with the IBD of HRP-2. These results further support the potential of LEDGINs as allosteric integrase inhibitors.     

A highly potent and safe pyrrolopyridine-based allosteric HIV-1 integrase inhibitor targeting host LEDGF/p75-integrase interaction site

Allosteric integrase inhibitors (ALLINIs) are a class of experimental anti-HIV agents that target the noncatalytic sites of the viral integrase (IN) and interfere with the IN-viral RNA interaction during viral maturation. Here, we report a highly potent and safe pyrrolopyridine-based ALLINI, STP0404, displaying picomolar IC₅₀ in human PBMCs with a >24,000 therapeutic index against HIV-1. X-ray structural and biochemical analyses revealed that STP0404 binds to the host LEDGF/p75 protein binding pocket of the IN dimer, which induces aberrant IN oligomerization and blocks the IN-RNA interaction. Consequently, STP0404 inhibits proper localization of HIV-1 RNA genomes in viral particles during viral maturation. Y99H and A128T mutations at the LEDGF/p75 binding pocket render resistance to STP0404. Extensive in vivo pharmacological and toxicity investigations demonstrate that STP0404 harbors outstanding therapeutic and safety properties. Overall, STP0404 is a potent and first-in-class ALLINI that targets LEDGF/p75 binding site and has advanced to a human trial.     

Design of HIV-1 integrase inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75: a scaffold hopping approach using salicylate and catechol groups

HIV-1 integrase (IN) is a validated therapeutic target for antiviral drug design. However, the emergence of viral strains resistant to clinically studied IN inhibitors demands the discovery of novel inhibitors that are structurally as well mechanistically different. Herein, we describe the design and discovery of novel IN inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75, which is essential for the HIV-1 integration as an IN cofactor. By merging the pharmacophores of salicylate and catechol, the 2,3-dihydroxybenzamide (5a) was identified as a new scaffold to inhibit the strand transfer reaction efficiently. Further structural modifications on the 2,3-dihydroxybenzamide scaffold revealed that the heteroaromatic functionality attached on the carboxamide portion and the piperidin-1-ylsulfonyl substituted at the phenyl ring are beneficial for the activity, resulting in a low micromolar IN inhibitor (5p, IC(50)=5 μM) with more than 40-fold selectivity for the strand transfer over the 3'-processing reaction. More significantly, this active scaffold remarkably inhibited the interaction between IN and LEDGF/p75 cofactor. The prototype example, N-(cyclohexylmethyl)-2,3-dihydroxy-5-(piperidin-1-ylsulfonyl) benzamide (5u) inhibited the IN-LEDGF/p75 interaction with an IC(50) value of 8 μM. Using molecular modeling, the mechanism of action was hypothesized to involve the chelation of the divalent metal ions inside the IN active site. Furthermore, the inhibitor of IN-LEDGF/p75 interaction was properly bound to the LEDGF/p75 binding site on IN. This work provides a new and efficient approach to evolve novel HIV-1 IN inhibitors from rational integration and optimization of previously reported inhibitors.     

Fragment hopping approach directed at design of HIV IN-LEDGF/p75 interaction inhibitors

We recently identified a series of indole derivatives as active inhibitors of IN-LEDGF/p75 interaction through structure-based pharmacophore models generated from the crystal structure of dimeric catalytic core domain (CCD) of HIV-1 IN in complex with the LEDGF integrase binding domain (IBD). In this paper we used the fragment hopping approach to design small molecules able to prevent the IN-LEDGF/p75 interaction. By means of the proposed approach, we designed novel non-peptidyl compounds that mimic the biological function of some IBD residues and in particular the LEDGF hot spot residues Ile365 and Asp366. The biological results confirmed the importance of several structural requirements for the inhibitory effects of this class of compounds.     

Identification of the LEDGF/p75 binding site in HIV-1 integrase

Lens epithelium-derived growth factor (LEDGF)/p75 is an important cellular co-factor for human immunodeficiency virus (HIV) replication. We originally identified LEDGF/p75 as a binding partner of integrase (IN) in human cells. The interaction has been mapped to the integrase-binding domain (IBD) of LEDGF/p75 located in the C-terminal part. We have subsequently shown that IN carrying the Q168A mutation remains enzymatically active but is impaired for interaction with LEDGF/p75. To map the integrase/LEDGF interface in more detail, we have now identified and characterized two regions within the enzyme involved in the interaction with LEDGF/p75. The first region centers around residues W131 and W132 while the second extends from I161 up to E170. For the different IN mutants the interaction with LEDGF/p75 and the enzymatic activities were determined. IN(W131A), IN(I161A), IN(R166A), IN(Q168A) and IN(E170A) are impaired for interaction with LEDGF/p75, but retain 3' processing and strand transfer activities. Due to impaired integration, an HIV-1 strain containing the W131A mutation in IN displays reduced replication capacity, whereas virus carrying IN(Q168A) is replication defective. Comparison of the wild-type IN-LEDGF/p75 co-crystal structure with that of the modelled structure of the IN(Q168A) and IN(W131A) mutant integrases corroborated our experimental data.     

D77, one benzoic acid derivative, functions as a novel anti-HIV-1 inhibitor targeting the interaction between integrase and cellular LEDGF/p75

Integration of viral-DNA into host chromosome mediated by the viral protein HIV-1 integrase (IN) is an essential step in the HIV-1 life cycle. In this process, Lens epithelium-derived growth factor (LEDGF/p75) is discovered to function as a cellular co-factor for integration. Since LEDGF/p75 plays an important role in HIV integration, disruption of the LEDGF/p75 interaction with IN has provided a special interest for anti-HIV agent discovery. In this work, we reported that a benzoic acid derivative, 4-[(5-bromo-4-{[2,4-dioxo-3-(2-oxo-2-phenylethyl)-1,3-thiazolidin-5-ylidene]methyl}-2-ethoxyphenoxy)methyl]benzoic acid (D77) could potently inhibit the IN-LEDGF/p75 interaction and affect the HIV-1 IN nuclear distribution thus exhibiting antiretroviral activity. Molecular docking with site-directed mutagenesis analysis and surface plasmon resonance (SPR) binding assays has clarified possible binding mode of D77 against HIV-1 integrase. As the firstly discovered small molecular compound targeting HIV-1 integrase interaction with LEDGF/p75, D77 might supply useful structural information for further anti-HIV agent discovery.     

FRET analysis reveals distinct conformations of IN tetramers in the presence of viral DNA or LEDGF/p75

A tetramer of HIV-1 integrase (IN) stably associates with the viral DNA ends to form a fully functional concerted integration intermediate. LEDGF/p75, a key cellular binding partner of the lentiviral enzyme, also stabilizes a tetrameric form of IN. However, functional assays have indicated the importance of the order of viral DNA and LEDGF/p75 addition to IN for productive concerted integration. Here, we employed Förster Resonance Energy Transfer (FRET) to monitor assembly of individual IN subunits into tetramers in the presence of viral DNA and LEDGF/p75. The IN–viral DNA and IN–LEDGF/p75 complexes yielded significantly different FRET values suggesting two distinct IN conformations in these complexes. Furthermore, the order of addition experiments indicated that FRET for the preformed IN–viral DNA complex remained unchanged upon its subsequent binding to LEDGF/p75, whereas pre-incubation of LEDGF/p75 and IN followed by addition of viral DNA yielded FRET very similar to the IN–LEDGF/p75 complex. These findings provide new insights into the structural organization of IN subunits in functional concerted integration intermediates and suggest that differential multimerization of IN in the presence of various ligands could be exploited as a plausible therapeutic target for development of allosteric inhibitors.     

Identification of old drugs as potential inhibitors of HIV-1 integrase – human LEDGF/p75 interaction via molecular docking

Integration of viral-DNA into host chromosome mediated by the viral protein HIV-1 integrase (IN) is an essential step in the HIV-1 life cycle. In this process, human protein Lens epithelium-derived growth factor (LEDGF/p75) is discovered to function as a cellular co-factor for integration. LEDGF/p75-HIV-1 IN interaction represents an attractive target for anti-HIV therapy. In this study, approved drugs were investigated for the finding of potential inhibitors on this target. Via molecular docking against the LEDGF/p75-binding pocket of HIV-1 IN, 26 old drugs were selected from the DrugBank and purchased for bioassays. Among them, eight, namely Atorvastatin, Bumetanide, Candesartan, Carbidopa, Diclofenac, Diflunisal, Eprosartan, and Sulindac, were identified as potential inhibitors of LEDGF/p75- HIV-1 IN interaction, whose IC₅₀ values ranged from 6.5?μM to 36.8?μM. In addition, Atorvastatin was previously reported to block HIV-1 replication and may have an important implication for the treatment of AIDS. Our results suggested a mechanism of action for the anti-HIV effects of Atorvastatin. This work provides a new example of inhibitors targeting protein-protein interaction and confirmed that old drugs were valuable sources for antiviral drug discovery.     

Allosteric Inhibition of Human Immunodeficiency Virus Integrase: LATE BLOCK DURING VIRAL REPLICATION AND ABNORMAL MULTIMERIZATION INVOLVING SPECIFIC PROTEIN DOMAINS*

HIV-1 replication in the presence of antiviral agents results in evolution of drug-resistant variants, motivating the search for additional drug classes. Here we report studies of GSK1264, which was identified as a compound that disrupts the interaction between HIV-1 integrase (IN) and the cellular factor lens epithelium-derived growth factor (LEDGF)/p75. GSK1264 displayed potent antiviral activity and was found to bind at the site occupied by LEDGF/p75 on IN by x-ray crystallography. Assays of HIV replication in the presence of GSK1264 showed only modest inhibition of the early infection steps and little effect on integration targeting, which is guided by the LEDGF/p75·IN interaction. In contrast, inhibition of late replication steps was more potent. Particle production was normal, but particles showed reduced infectivity. GSK1264 promoted aggregation of IN and preformed LEDGF/p75·IN complexes, suggesting a mechanism of inhibition. LEDGF/p75 was not displaced from IN during aggregation, indicating trapping of LEDGF/p75 in aggregates. Aggregation assays with truncated IN variants revealed that a construct with catalytic and C-terminal domains of IN only formed an open polymer associated with efficient drug-induced aggregation. These data suggest that the allosteric inhibitors of IN are promising antiviral agents and provide new information on their mechanism of action.     

Allosteric HIV‐1 integrase inhibitors promote aberrant protein multimerization by directly mediating inter‐subunit interactions: Structural and thermodynamic modeling studies

Allosteric HIV‐1 integrase (IN) inhibitors (ALLINIs) bind at the dimer interface of the IN catalytic core domain (CCD), and potently inhibit HIV‐1 by promoting aberrant, higher‐order IN multimerization. Little is known about the structural organization of the inhibitor‐induced IN multimers and important questions regarding how ALLINIs promote aberrant IN multimerization remain to be answered. On the basis of physical chemistry principles and from our analysis of experimental information, we propose that inhibitor‐induced multimerization is mediated by ALLINIs directly promoting inter‐subunit interactions between the CCD dimer and a C‐terminal domain (CTD) of another IN dimer. Guided by this hypothesis, we have built atomic models of inter‐subunit interfaces in IN multimers by incorporating information from hydrogen‐deuterium exchange (HDX) measurements to drive protein‐protein docking. We have also developed a novel free energy simulation method to estimate the effects of ALLINI binding on the association of the CCD and CTD. Using this structural and thermodynamic modeling approach, we show that multimer inter‐subunit interface models can account for several experimental observations about ALLINI‐induced multimerization, including large differences in the potencies of various ALLINIs, the mechanisms of resistance mutations, and the crucial role of solvent exposed R‐groups in the high potency of certain ALLINIs. Our study predicts that CTD residues Tyr226, Trp235 and Lys266 are involved in the aberrant multimer interfaces. The key finding of the study is that it suggests the possibility of ALLINIs facilitating inter‐subunit interactions between an external CTD and the CCD‐CCD dimer interface.     

New class of HIV-1 integrase (IN) inhibitors with a dual mode of action

tert-Butoxy-(4-phenyl-quinolin-3-yl)-acetic acids (tBPQA) are a new class of HIV-1 integrase (IN) inhibitors that are structurally distinct from IN strand transfer inhibitors but analogous to LEDGINs. LEDGINs are a class of potent antiviral compounds that interacts with the lens epithelium-derived growth factor (LEDGF) binding pocket on IN and were identified through competition binding against LEDGF. LEDGF tethers IN to the host chromatin and enables targeted integration of viral DNA. The prevailing understanding of the antiviral mechanism of LEDGINs is that they inhibit LEDGF binding to IN, which prevents targeted integration of HIV-1. We showed that in addition to the properties already known for LEDGINs, the binding of tBPQAs to the IN dimer interface inhibits IN enzymatic activity in a LEDGF-independent manner. Using the analysis of two long terminal repeat junctions in HIV-infected cells, we showed that the inhibition by tBPQAs occurs at or prior to the viral DNA 3'-processing step. Biochemical studies revealed that this inhibition operates by compound-induced conformational changes in the IN dimer that prevent proper assembly of IN onto viral DNA. For the first time, tBPQAs were demonstrated to be allosteric inhibitors of HIV-1 IN displaying a dual mode of action: inhibition of IN-viral DNA assembly and inhibition of IN-LEDGF interaction.     

Mechanism of action of the HIV-1 integrase inhibitory peptide LEDGF 361-370

The HIV-1 integrase protein (IN) mediates integration of the viral cDNA into the host genome and is a target for anti-HIV drugs. We have recently described a peptide derived from residues 361-370 of the IN cellular partner protein LEDGF/p75, which inhibited IN catalytic activity in vitro and HIV-1 replication in cells. Here we performed a comprehensive study of the LEDGF 361-370 mechanism of action in vitro, in cells and in vivo. Alanine scan, fluorescence anisotropy binding studies, homology modeling and NMR studies demonstrated that all residues in LEDGF 361-370 contribute to IN binding and inhibition. Kinetic studies in cells showed that LEDGF 361-370 specifically inhibited integration of viral cDNA. Thus, the full peptide was chosen for in vivo studies, in which it inhibited the production of HIV-1 RNA in mouse model. We conclude that the full LEDGF 361-370 peptide is a potent HIV-1 inhibitor and may be used for further development as an anti-HIV lead compound.     

Overexpression of the lens epithelium-derived growth factor/p75 integrase binding domain inhibits human immunodeficiency virus replication

We initially identified lens epithelium-derived growth factor/p75 (LEDGF/p75) as a binding partner of human immunodeficiency virus type 1 (HIV-1) integrase. To investigate the role of LEDGF/p75 in HIV replication and its potential as a new antiviral target, we stably overexpressed two different fragments containing the integrase binding domain (IBD) of LEDGF/p75 fused to enhanced green fluorescent protein (eGFP). HIV-1 replication was severely inhibited by overexpression of the eGFP-IBD fusion proteins, while no inhibition was observed in cell lines overexpressing the interaction-deficient D366A mutant. Quantitative PCR pinpointed the block to the integration step, whereas nuclear import was not affected. Competition of the IBD fusion proteins with endogenous LEDGF/p75 for binding to integrase led to a potent defect in HIV-1 replication in both HeLaP4- and MT-4-derived cell lines. A previously described diketo acid-resistant HIV-1 strain remained fully susceptible to inhibition, suggesting that this strategy will also work in patients who harbor strains resistant to the current experimental integrase inhibitors. These data support LEDGF/p75 as an important cofactor for HIV replication and provide proof of concept for the LEDGF/p75-integrase interaction as a novel target for treating HIV-1 infection.