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.     

HRP2 determines the efficiency and specificity of HIV-1 integration in LEDGF/p75 knockout cells but does not contribute to the antiviral activity of a potent LEDGF/p75-binding site integrase inhibitor

The binding of integrase (IN) to lens epithelium-derived growth factor (LEDGF)/p75 in large part determines the efficiency and specificity of HIV-1 integration. However, a significant residual preference for integration into active genes persists in Psip1 (the gene that encodes for LEDGF/p75) knockout (KO) cells. One other cellular protein, HRP2, harbors both the PWWP and IN-binding domains that are important for LEDGF/p75 co-factor function. To assess the role of HRP2 in HIV-1 integration, cells generated from Hdgfrp2 (the gene that encodes for HRP2) and Psip1/Hdgfrp2 KO mice were infected alongside matched control cells. HRP2 depleted cells supported normal infection, while disruption of Hdgfrp2 in Psip1 KO cells yielded additional defects in the efficiency and specificity of integration. These deficits were largely restored by ectopic expression of either LEDGF/p75 or HRP2. The double-KO cells nevertheless supported residual integration into genes, indicating that IN and/or other host factors contribute to integration specificity in the absence of LEDGF/p75 and HRP2. Psip1 KO significantly increased the potency of an allosteric inhibitor that binds the LEDGF/p75 binding site on IN, a result that was not significantly altered by Hdgfrp2 disruption. These findings help to rule out the host factor-IN interactions as the primary antiviral targets of LEDGF/p75-binding site IN inhibitors.     

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.     

A New Class of Multimerization Selective Inhibitors of HIV-1 Integrase

The quinoline-based allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are promising candidates for clinically useful antiviral agents. Studies using these compounds have highlighted the role of IN in both early and late stages of virus replication. However, dissecting the exact mechanism of action of the quinoline-based ALLINIs has been complicated by the multifunctional nature of these inhibitors because they both inhibit IN binding with its cofactor LEDGF/p75 and promote aberrant IN multimerization with similar potencies in vitro. Here we report design of small molecules that allowed us to probe the role of HIV-1 IN multimerization independently from IN-LEDGF/p75 interactions in infected cells. We altered the rigid quinoline moiety in ALLINIs and designed pyridine-based molecules with a rotatable single bond to allow these compounds to bridge between interacting IN subunits optimally and promote oligomerization. The most potent pyridine-based inhibitor, KF116, potently (EC₅₀ of 0.024 µM) blocked HIV-1 replication by inducing aberrant IN multimerization in virus particles, whereas it was not effective when added to target cells. Furthermore, KF116 inhibited the HIV-1 IN variant with the A128T substitution, which confers resistance to the majority of quinoline-based ALLINIs. A genome-wide HIV-1 integration site analysis demonstrated that addition of KF116 to target or producer cells did not affect LEDGF/p75-dependent HIV-1 integration in host chromosomes, indicating that this compound is not detectably inhibiting IN-LEDGF/p75 binding. These findings delineate the significance of correctly ordered IN structure for HIV-1 particle morphogenesis and demonstrate feasibility of exploiting IN multimerization as a therapeutic target. Furthermore, pyridine-based compounds present a novel class of multimerization selective IN inhibitors as investigational probes for HIV-1 molecular biology.     

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.     

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 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.     

Dynamic modulation of HIV-1 integrase structure and function by cellular lens epithelium-derived growth factor (LEDGF) protein

The mandatory integration of the reverse-transcribed HIV-1 genome into host chromatin is catalyzed by the viral protein integrase (IN), and IN activity can be regulated by numerous viral and cellular proteins. Among these, LEDGF has been identified as a cellular cofactor critical for effective HIV-1 integration. The x-ray crystal structure of the catalytic core domain (CCD) of IN in complex with the IN binding domain (IBD) of LEDGF has furthermore revealed essential protein-protein contacts. However, mutagenic studies indicated that interactions between the full-length proteins were more extensive than the contacts observed in the co-crystal structure of the isolated domains. Therefore, we have conducted detailed biochemical characterization of the interactions between full-length IN and LEDGF. Our results reveal a highly dynamic nature of IN subunit-subunit interactions. LEDGF strongly stabilized these interactions and promoted IN tetramerization. Mass spectrometric protein footprinting and molecular modeling experiments uncovered novel intra- and inter-protein-protein contacts in the full-length IN-LEDGF complex that lay outside of the observable IBD-CCD structure. In particular, our studies defined the IN tetramer interface important for enzymatic activities and high affinity LEDGF binding. These findings provide new insight into how LEDGF modulates HIV-1 IN structure and function, and highlight the potential for exploiting the highly dynamic structure of multimeric IN as a novel therapeutic target.     

Structure-based virtual screening toward the discovery of novel inhibitors for impeding the protein-protein interaction between HIV-1 integrase and human lens epithelium-derived growth factor (LEDGF/p75)

HIV-1 integrase is a unique promising component of the viral replication cycle, catalyzing the integration of reverse transcribed viral cDNA into the host cell genome. Generally, IN activity requires both viral as well as a cellular co-factor in the processing replication cycle. Among them, the human lens epithelium-derived growth factor (LEDGF/p75) represented as promising cellular co-factor which supports the viral replication by tethering IN to the chromatin. Due to its major importance in the early steps of HIV replication, the interaction between IN and LEDGF/p75 has become a pleasing target for anti-HIV drug discovery. The present study involves the finding of novel inhibitor based on the information of dimeric CCD of IN in complex with known inhibitor, which were carried out by applying a structure-based virtual screening concept with molecular docking. Additionally, Free binding energy, ADME properties, PAINS analysis, Density Functional Theory, and Enrichment Calculations were performed on selected compounds for getting a best lead molecule. On the basis of these analyses, the current study proposes top 3 compounds: Enamine-Z742267384, Maybridge-HTS02400, and Specs-AE-848/37125099 with acceptable pharmacological properties and enhanced binding affinity to inhibit the interaction between IN and LEDGF/p75. Furthermore, Simulation studies were carried out on these molecules to expose their dynamics behavior and stability. We expect that the findings obtained here could be future therapeutic agents and may provide an outline for the experimental studies to stimulate the innovative strategy for research community.     

Small molecules targeting the interaction between HIV-1 integrase and LEDGF/p75 cofactor

The search of small molecules as protein–protein interaction inhibitors represents a new attractive strategy to develop anti-HIV-1 agents. We previously reported a computational study that led to the discovery of new inhibitors of the interaction between enzyme HIV-1 integrase (IN) and the nuclear protein lens epithelium growth factor LEDGF/p75.¹Herein, we describe new findings about the binding site of LEDGF/p75 on IN employing a different computational approach. In this way further structural requirements, helpful to disrupt LEDGF/p75-IN binding, have been identified. The main result of this work was the exploration of a relevant hydrophobic region. So we planned the introduction of suitable and simple chemical modifications on our previously reported ‘hit’ and the new synthesized compounds were subjected to biological tests.The results obtained demonstrate that the hydrophobic pocket could play a key role in improving inhibitory efficacy thus opening new suggestions to design active ligands.