Pharmacological advances in anti-retroviral therapy for human immunodeficiency virus-1 infection: A comprehensive review.

The discovery of anti-retroviral (ARV) drugs over the past 36 years has introduced various classes, including nucleoside/nucleotide reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitor, fusion, and integrase strand transfer inhibitors inhibitors. The introduction of combined highly active anti-retroviral therapies in 1996 was later proven to combat further ARV drug resistance along with enhancing human immunodeficiency virus (HIV) suppression. As though the development of ARV therapies was continuously expanding, the variation of action caused by ARV drugs, along with its current updates, was not comprehensively discussed, particularly for HIV-1 infection. Thus, a range of HIV-1 ARV medications is covered in this review, including new developments in ARV therapy based on the drug's mechanism of action, the challenges related to HIV-1, and the need for combination therapy. Optimistically, this article will consolidate the overall updates of HIV-1 ARV treatments and conclude the significance of HIV-1-related pharmacotherapy research to combat the global threat of HIV infection.

Expansion microscopy allows high resolution single cell analysis of epigenetic readers.

Interactions between epigenetic readers and histone modifications play a pivotal role in gene expression regulation and aberrations can enact etiopathogenic roles in both developmental and acquired disorders like cancer. Typically, epigenetic interactions are studied by mass spectrometry or chromatin immunoprecipitation sequencing. However, in these methods, spatial information is completely lost. Here, we devise an expansion microscopy based method, termed Expansion Microscopy for Epigenetics or ExEpi, to preserve spatial information and improve resolution. We calculated relative co-localization ratios for two epigenetic readers, lens epithelium derived growth factor (LEDGF) and bromodomain containing protein 4 (BRD4), with marks for heterochromatin (H3K9me3 and H3K27me3) and euchromatin (H3K36me2, H3K36me3 and H3K9/14ac). ExEpi confirmed their preferred epigenetic interactions, showing co-localization for LEDGF with H3K36me3/me2 and for BRD4 with H3K9/14ac. Moreover addition of JQ1, a known BET-inhibitor, abolished BRD4 interaction with H3K9/14ac with an IC50 of 137 nM, indicating ExEpi could serve as a platform for epigenetic drug discovery. Since ExEpi retains spatial information, the nuclear localization of marks and readers was determined, which is one of the main advantages of ExEpi. The heterochromatin mark, H3K9me3, is located in the nuclear rim whereas LEDGF co-localization with H3K36me3 and BRD4 co-localization with H3K9/14ac occur further inside the nucleus.

GS-9822, a preclinical LEDGIN candidate, displays a block-and-lock phenotype in cell culture.

The ability of HIV to integrate into the host genome and establish latent reservoirs is the main hurdle preventing an HIV cure. LEDGINs are small-molecule integrase inhibitors that target the binding pocket of LEDGF/p75, a cellular cofactor that substantially contributes to HIV integration site selection. They are potent antivirals that inhibit HIV integration and maturation. In addition, they retarget residual integrants away from transcription units and towards a more repressive chromatin environment. As a result, treatment with the LEDGIN CX14442 yielded residual provirus that proved more latent and more refractory to reactivation, supporting the use of LEDGINs as research tools to study HIV latency and a functional cure strategy. In this study we compared GS-9822, a potent, pre-clinical lead compound, with CX14442 with respect to antiviral potency, integration site selection, latency and reactivation. GS-9822 was more potent than CX14442 in most assays. For the first time, the combined effects on viral replication, integrase-LEDGF/p75 interaction, integration sites, epigenetic landscape, immediate latency and latency reversal was demonstrated at nanomolar concentrations achievable in the clinic. GS-9822 profiles as a preclinical candidate for future functional cure research.

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

[This corrects the article DOI: 10.1371/journal.ppat.1002558.].

Design, synthesis, in silico studies, and antiproliferative evaluations of novel indolin-2-one derivatives containing 3-hydroxy-4-pyridinone fragment.

Keeping in view the pharmacological properties of indolinones as promising scaffold as kinase inhibitors, herein, a novel series of 3-hydrazonoindolin-2-one derivatives bearing 3-hydroxy-4-pyridinone moiety were synthesized, studied by molecular docking, and fully characterized by spectroscopic techniques. All the prepared compounds were evaluated for their cytotoxicity attributes against a panel of tumor cell lines, including non-small cell lung cancer (A549), breast carcinoma (MCF-7), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML). They displayed moderate to promising antiproliferative effects toward A549 and MCF-7 cells but remarkable results against AML and CML. Especially, compound 10k was found to be more potent against AML (EC50 = 0.69 µM) compare to the other halogen-substituted derivatives. FMS-like tyrosine kinase 3 (FLT3) is known to be expressed in AML cancer cells. The molecular docking studies demonstrated that our prepared compounds were potentially bound to AML active site through essential H-bond and other vital interactions with critical binding residues.

The chromatin landscape at the HIV-1 provirus integration site determines viral expression.

HIV-1 persists lifelong in memory cells of the immune system as latent provirus that rebounds upon treatment interruption. Therefore, the latent reservoir is the main target for an HIV cure. Here, we studied the direct link between integration site and transcription using LEDGINs and Barcoded HIV-ensembles (B-HIVE). LEDGINs are antivirals that inhibit the interaction between HIV-1 integrase and the chromatin-tethering factor LEDGF/p75. They were used as a tool to retarget integration, while the effect on HIV expression was measured with B-HIVE. B-HIVE tracks insert-specific HIV expression by tagging a unique barcode in the HIV genome. We confirmed that LEDGINs retarget integration out of gene-dense and actively transcribed regions. The distance to H3K36me3, the marker recognized by LEDGF/p75, clearly increased. LEDGIN treatment reduced viral RNA expression and increased the proportion of silent provirus. Finally, silent proviruses obtained after LEDGIN treatment were located further away from epigenetic marks associated with active transcription. Interestingly, proximity to enhancers stimulated transcription irrespective of LEDGIN treatment, while the distance to H3K36me3 only changed after treatment with LEDGINs. The fact that proximity to these markers are associated with RNA expression support the direct link between provirus integration site and viral expression.

Capsid-Labelled HIV To Investigate the Role of Capsid during Nuclear Import and Integration.

The HIV-1 capsid protein performs multiple roles in virus replication both during assembly and particle release and during virus trafficking into the nucleus. In order to decipher the roles of capsid protein during early replication, a reliable method to follow its intracellular distribution is required. To complement existing approaches to track HIV-1 capsid during early infection, we developed an HIV-1 imaging strategy, relying on viruses incorporating enhanced green fluorescent protein (eGFP)-tagged capsid (CA-eGFP) protein and mCherry-tagged integrase (IN-mCherry). Wild-type infectivity and sensitivity to inhibition by PF74 point to the functionality of CA-eGFP-containing complexes. Low numbers of CA-eGFP molecules were located inside the viral core and imported into the nucleus without significant loss in intensity. Less than 5% of particles carrying both CA-eGFP and IN-mCherry retained both labelled proteins after nuclear entry, implying a major uncoating event at the nuclear envelope dissociating IN and CA. Still, 20% of all CA-eGFP-containing complexes were detected in the nucleus. Unlike for IN-mCherry complexes, addition of the integrase inhibitor raltegravir had no effect on CA-eGFP-containing complexes, suggesting that these may be not (yet) competent for integration. Our imaging strategy offers alternative visualization of viral capsid trafficking and helps clarify its potential role during integration.IMPORTANCE HIV-1 capsid protein (CA) builds a conical shell protecting viral genomic RNA inside the virus particles. Upon entry into host cells, this shell disassembles in a process of uncoating, which is coordinated with reverse transcription of viral RNA into DNA. After uncoating, a portion of CA remains associated with the viral DNA and mediates its nuclear import and, potentially, integration into host DNA. In this study, we tagged CA with eGFP to follow its trafficking in host cells and address potential CA roles in the nucleus. We found that while functional viruses import the tagged CA into the nucleus, this capsid protein is not part of integration-competent complexes. The roles of nuclear CA thus remain to be established.

The mutation of Transportin 3 gene that causes limb girdle muscular dystrophy 1F induces protection against HIV-1 infection.

The causative mutation responsible for limb girdle muscular dystrophy 1F (LGMD1F) is one heterozygous single nucleotide deletion in the stop codon of the nuclear import factor Transportin 3 gene (TNPO3). This mutation causes a carboxy-terminal extension of 15 amino acids, producing a protein of unknown function (TNPO3_mut) that is co-expressed with wild-type TNPO3 (TNPO3_wt). TNPO3 has been involved in the nuclear transport of serine/arginine-rich proteins such as splicing factors and also in HIV-1 infection through interaction with the viral integrase and capsid. We analyzed the effect of TNPO3_mut on HIV-1 infection using PBMCs from patients with LGMD1F infected ex vivo. HIV-1 infection was drastically impaired in these cells and viral integration was reduced 16-fold. No significant effects on viral reverse transcription and episomal 2-LTR circles were observed suggesting that the integration of HIV-1 genome was restricted. This is the second genetic defect described after CCR5Δ32 that shows strong resistance against HIV-1 infection.

Post-mitotic BET-induced reshaping of integrase quaternary structure supports wild-type MLV integration.

The Moloney murine leukemia virus (MLV) is a prototype gammaretrovirus requiring nuclear disassembly before DNA integration. In the nucleus, integration site selection towards promoter/enhancer elements is mediated by the host factor bromo- and extraterminal domain (BET) proteins (bromodomain (Brd) proteins 2, 3 and 4). MLV-based retroviral vectors are used in gene therapy trials. In some trials leukemia occurred through integration of the MLV vector in close proximity to cellular oncogenes. BET-mediated integration is poorly understood and the nature of integrase oligomers heavily debated. Here, we created wild-type infectious MLV vectors natively incorporating fluorescent labeled IN and performed single-molecule intensity and Förster resonance energy transfer experiments. The nuclear localization of the MLV pre-integration complex neither altered the IN content, nor its quaternary structure. Instead, BET-mediated interaction of the MLV intasome with chromatin in the post-mitotic nucleus reshaped its quaternary structure.

LEDGINs, Inhibitors of the Interaction Between HIV-1 Integrase and LEDGF/p75, Are Potent Antivirals with a Potential to Cure HIV Infection.

A permanent cure remains the greatest challenge in the field of HIV research. In order to reach this goal, a profound understanding of the molecular mechanisms controlling HIV integration and transcription is needed. Here we provide an overview of recent advances in the field. Lens epithelium-derived growth factor p75 (LEDGF/p75), a transcriptional coactivator, tethers and targets the HIV integrase into transcriptionally active regions of the chromatin through an interaction with the epigenetic mark H3K36me2/3. This finding prompted us to propose a "block-and-lock" strategy to retarget HIV integration into deep latency. A decade ago, we pioneered protein-protein interaction inhibitors for HIV and discovered LEDGINs. LEDGINs are small molecule inhibitors of the interaction between the integrase binding domain (IBD) of LEDGF/p75 and HIV integrase. They modify integration site selection and therefore might be molecules with a "block-and-lock" mechanism of action. Here we will describe how LEDGINs may become part in the future functional cure strategies.

Affinity switching of the LEDGF/p75 IBD interactome is governed by kinase-dependent phosphorylation.

Lens epithelium-derived growth factor/p75 (LEDGF/p75, or PSIP1) is a transcriptional coactivator that tethers other proteins to gene bodies. The chromatin tethering function of LEDGF/p75 is hijacked by HIV integrase to ensure viral integration at sites of active transcription. LEDGF/p75 is also important for the development of mixed-lineage leukemia (MLL), where it tethers the MLL1 fusion complex at aberrant MLL targets, inducing malignant transformation. However, little is known about how the LEDGF/p75 protein interaction network is regulated. Here, we obtained solution structures of the complete interfaces between the LEDGF/p75 integrase binding domain (IBD) and its cellular binding partners and validated another binding partner, Mediator subunit 1 (MED1). We reveal that structurally conserved IBD-binding motifs (IBMs) on known LEDGF/p75 binding partners can be regulated by phosphorylation, permitting switching between low- and high-affinity states. Finally, we show that elimination of IBM phosphorylation sites on MLL1 disrupts the oncogenic potential of primary MLL1-rearranged leukemic cells. Our results demonstrate that kinase-dependent phosphorylation of MLL1 represents a previously unknown oncogenic dependency that may be harnessed in the treatment of MLL-rearranged leukemia.

Design of reverse transcriptase-specific nucleosides to visualize early steps of HIV-1 replication by click labeling.

Only a small portion of human immunodeficiency virus type 1 (HIV-1) particles entering the host cell results in productive infection, emphasizing the importance of identifying the functional virus population. Because integration of viral DNA (vDNA) is required for productive infection, efficient vDNA detection is crucial. Here, we use click chemistry to label viruses with integrase coupled to eGFP (HIVIN-eGFP) and visualize vDNA. Because click labeling with 5-ethynyl-2'-deoxyuridine is hampered by intense background staining of the host nucleus, we opted for developing HIV-1 reverse transcriptase (RT)-specific 2'-deoxynucleoside analogs that contain a clickable triple bond. We synthesized seven propargylated 2'-deoxynucleosides and tested them for lack of cytotoxicity and viral replication inhibition, RT-specific primer extension and incorporation kinetics in vitro, and the capacity to stain HIV-1 DNA. The triphosphate of analog A5 was specifically incorporated by HIV-1 RT, but no vDNA staining was detected during infection. Analog A3 was incorporated in vitro by HIV-1 RT and human DNA polymerase γ and did enable specific HIV-1 DNA labeling. Additionally, A3 supported mitochondria-specific DNA labeling, in line with the in vitro findings. After obtaining proof-of-principle of RT-specific DNA labeling reported here, further chemical refinement is necessary to develop even more efficient HIV-1 DNA labels without background staining of the nucleus or mitochondria.

Correction to: HIV-1 IN/Pol recruits LEDGF/p75 into viral particles.

An amendment to this paper has been published and can be accessed via the original article.

Correction to: LEDGINs inhibit late stage HIV-1 replication by modulating integrase multimerization in the virions.

An amendment to this paper has been published and can be accessed via the original article.

LEDGF/p75 is dispensable for hematopoiesis but essential for MLL-rearranged leukemogenesis.

Mixed lineage leukemia (MLL) represents a genetically distinct and aggressive subset of human acute leukemia carrying chromosomal translocations of the MLL gene. These translocations result in oncogenic fusions that mediate aberrant recruitment of the transcription machinery to MLL target genes. The N-terminus of MLL and MLL-fusions form a complex with lens epithelium-derived growth factor (LEDGF/p75; encoded by the PSIP1 gene) and MENIN. This complex contributes to the association of MLL and MLL-fusion multiprotein complexes with the chromatin. Several studies have shown that both MENIN and LEDGF/p75 are required for efficient MLL-fusion-mediated transformation and for the expression of downstream MLL-regulated genes such as HOXA9 and MEIS1 In light of developing a therapeutic strategy targeting this complex, understanding the function of LEDGF/p75 in normal hematopoiesis is crucial. We generated a conditional Psip1 knockout mouse model in the hematopoietic compartment and examined the effects of LEDGF/p75 depletion in postnatal hematopoiesis and the initiation of MLL leukemogenesis. Psip1 knockout mice were viable but showed several defects in hematopoiesis, reduced colony-forming activity in vitro, decreased expression of Hox genes in the hematopoietic stem cells, and decreased MLL occupancy at MLL target genes. Finally, in vitro and in vivo experiments showed that LEDGF/p75 is dispensable for steady-state hematopoiesis but essential for the initiation of MLL-mediated leukemia. These data corroborate the MLL-LEDGF/p75 interaction as novel target for the treatment of MLL-rearranged leukemia.

Host factors for retroviral integration site selection.

To achieve productive infection, retroviruses such as HIV stably integrate their reverse transcribed RNA genome into a host chromosome. Each retroviral family preferentially integrates near a unique subset of genomic features. HIV integrase (IN) is targeted to the body of active transcription units through interaction with lens epithelium-derived growth factor (LEDGF/p75). We describe the successful effort to develop inhibitors of the interaction between IN and LEDGF/p75, referred to as LEDGINs. Gammaretroviruses display a distinct integration pattern. Recently, BET (bromo- and extraterminal domain) proteins were identified as the LEDGF/p75 counterparts that target the integration of gammaretroviruses. The identification of the chromatin-readers LEDGF/p75 and BET as cellular cofactors that orchestrate lentiviral or gammaretroviral integration opens new avenues to developing safer viral vectors for gene therapy.

Impact of LEDGIN treatment during virus production on residual HIV-1 transcription.

BACKGROUND: Persistence of latent, replication-competent provirus is the main impediment towards the cure of HIV infection. One of the critical questions concerning HIV latency is the role of integration site selection in HIV expression. Inhibition of the interaction between HIV integrase and its chromatin tethering cofactor LEDGF/p75 is known to reduce integration and to retarget residual provirus to regions resistant to reactivation. LEDGINs, small molecule inhibitors of the interaction between HIV integrase and LEDGF/p75, provide an interesting tool to study the underlying mechanisms. During early infection, LEDGINs block the interaction with LEDGF/p75 and allosterically inhibit the catalytic activity of IN (i.e. the early effect). When present during virus production, LEDGINs interfere with proper maturation due to enhanced IN oligomerization in the progeny virions (i.e. the late effect). RESULTS: We studied the effect of LEDGINs present during virus production on the transcriptional state of the residual virus. Infection of cells with viruses produced in the presence of LEDGINs resulted in a residual reservoir that was refractory to activation. Integration of residual provirus was less favored near epigenetic markers associated with active transcription. However, integration near H3K36me3 and active genes, both targeted by LEDGF/p75, was not affected. Also in primary cells, LEDGIN treatment induced a reservoir resistant to activation due to a combined early and late effect. CONCLUSION: LEDGINs present a research tool to study the link between integration and transcription, an essential question in retrovirology. LEDGIN treatment during virus production altered integration of residual provirus in a LEDGF/p75-independent manner, resulting in a reservoir that is refractory to activation.

Propargylated Purine Deoxynucleosides: New Tools for Fluorescence Imaging Strategies.

In vivo imaging of biological processes is an important asset of modern cell biology. Selectively reacting fluorophores herein are an important tool and click chemistry reactions take a large share in these events. 5-Ethynyl-2'-deoxyuridine (EdU) is well known for visualizing DNA replication, but does not show any selectivity for incorporation into DNA. Striving for specific visualization of virus replication, in particular HIV replication, a series of propargylated purine deoxynucleosides were prepared aiming for selective incorporation by HIV reverse transcriptase (RT). We here report on the synthesis and preliminary biological effects (cellular toxicity, HIV inhibitory effects, and feasibility of the click reaction) of these nucleoside analogues.

N-terminal half of transportin SR2 interacts with HIV integrase.

The karyopherin transportin SR2 (TRN-SR2, TNPO3) is responsible for shuttling specific cargoes such as serine/arginine-rich splicing factors from the cytoplasm to the nucleus. This protein plays a key role in HIV infection by facilitating the nuclear import of the pre-integration complex (PIC) that contains the viral DNA as well as several cellular and HIV proteins, including the integrase. The process of nuclear import is considered to be the bottleneck of the viral replication cycle and therefore represents a promising target for anti-HIV drug design. Previous studies have demonstrated that the direct interaction between TRN-SR2 and HIV integrase predominantly involves the catalytic core domain (CCD) and the C-terminal domain (CTD) of the integrase. We aimed at providing a detailed molecular view of this interaction through a biochemical characterization of the respective protein complex. Size-exclusion chromatography was used to characterize the interaction of TRN-SR2 with a truncated variant of the HIV-1 integrase, including both the CCD and CTD. These experiments indicate that one TRN-SR2 molecule can specifically bind one CCD-CTD dimer. Next, the regions of the solenoid-like TRN-SR2 molecule that are involved in the interaction with integrase were identified using AlphaScreen binding assays, revealing that the integrase interacts with the N-terminal half of TRN-SR2 principally through the HEAT repeats 4, 10, and 11. Combining these results with small-angle X-ray scattering data for the complex of TRN-SR2 with truncated integrase, we propose a molecular model of the complex. We speculate that nuclear import of the PIC may proceed concurrently with the normal nuclear transport.

Inhibitors of the integrase-transportin-SR2 interaction block HIV nuclear import.

BACKGROUND: Combination antiretroviral therapy efficiently suppresses HIV replication in infected patients, transforming HIV/AIDS into a chronic disease. Viral resistance does develop however, especially under suboptimal treatment conditions such as poor adherence. As a consequence, continued exploration of novel targets is paramount to identify novel antivirals that do not suffer from cross-resistance with existing drugs. One new promising class of targets are HIV protein-cofactor interactions. Transportin-SR2 (TRN-SR2) is a ß-karyopherin that was recently identified as an HIV-1 cofactor. It has been implicated in nuclear import of the viral pre-integration complex and was confirmed as a direct binding partner of HIV-1 integrase (IN). Nevertheless, consensus on its mechanism of action is yet to be reached. RESULTS: Here we describe the development and use of an AlphaScreen-based high-throughput screening cascade for small molecule inhibitors of the HIV-1 IN-TRN-SR2 interaction. False positives and nonspecific protein-protein interaction inhibitors were eliminated through different counterscreens. We identified and confirmed 2 active compound series from an initial screen of 25,608 small molecules. These compounds significantly reduced nuclear import of fluorescently labeled HIV particles. CONCLUSIONS: Alphascreen-based high-throughput screening can allow the identification of compounds representing a novel class of HIV inhibitors. These results corroborate the role of the IN-TRN-SR2 interaction in nuclear import. These compounds represent the first in class small molecule inhibitors of HIV-1 nuclear import.