1. Characterization of a novel Tat inhibitor and demonstration of “Block-and-lock” approach

After HIV integration of the proviral DNA into the host genome, the HIV-1 provirus can remain latentor activate transcription. The viral Tat protein is the switch between these two states. Tat binds HIV-1 mRNA (TAR) and efficiently recruits the necessary transcriptional factors to the HIV promoter to initiate exponential viral transcription elongation. Tat has no cellular homolog and is expressed early in the virus life cycle, making it an ideal target for therapeutic intervention. Inhibitors of Tat have been highly sought after; but none is yet in the clinic. We discovered that an analog of a natural product, didehydro-Cortistatin A (dCA), is a very potent inhibitor of HIV-1 transcription (Mousseau et al. Cell Host and Microbe, 2012). dCA binds to the basic domain of Tat and inhibits Tat activated transcription at sub-nanomolar concentrations. The viral promoter activity is directly governed by its chromatin environment. Nucleosome 1 (Nuc-1) downstream from the transcription start site (TSS) directly impede transcription from the HIV-1 promoter. Tat inhibition by dCA results in increased occupancy of Nuc-1, restricting RNAPII recruitment and elongation (Li et al, Epigenetics and Chromatin, 2019). As such, over time dCA prompts the viral promoter into deep transcriptional inhibition, resistant to viral reactivation (Mousseau et al. mBio, 2015).

Illustration: "Block-and-Lock"
Figure – Tat inhibitors are amenable to functional cure approaches, which aim to reduce residual viremia during ART and limit viral rebound during treatment interruption. Using didehydro-Cortistatin A (dCA), Kessing et al. demonstrate the concept in human CD4+ T cells from aviremic individuals and in the bone marrow-liver-thymus mouse model of HIV latency.

Limitations of current primary cell models of HIV-1 latency include reliance on unrepresentative clonal HIV lab strains and use of non-physiologic cytokine cocktails to drive cells into latency. To bypass these limitations, we developed an alternative cellular latency model which uses polyclonally expanded CD4+T cells from HIV-infected patients carrying the autologous HIV reservoir. These cells return to a resting state after 3 weeks in culture. In this model, cells stop producing HIV particles without any manipulation but can be reinduced into HIV production after restimulation (Takata et al, Journal of Virology, 2019). In these human CD4+T cells from aviremic individuals, combining dCA with ART accelerates HIV-1 suppression and prevents viral rebound during treatment interruption. Furthermore, in the bone marrow-liver-thymus (BLT) mouse model of HIV latency and persistence, adding dCA to ART suppressed mice, reduces viral RNA in tissues, and significantly delays and diminishes viral rebound upon treatment interruption (Kessing et al, Cell Reports, 2017). These results are the first in vivo proof-of-principle for a “block-and-lock” approach to a functional cure for HIV.  Specifically, we combined a Tat inhibitor, dCA, with ART to promote a state of sustained latency, halting ongoing viral transcription during ART and blocking reactivation of the latent provirus. The block-an-lock approach is now widely accepted, and we are proud to have pioneered this idea and demonstrated the feasibility of this approach. To date dCA is the most promising Tat inhibitor described. This novel class of inhibitors can prevent viral production from stable reservoirs, reduce residual viremia under ART, limit chronic immune activation observed in suppressed individuals, and treat HIV-associated-neurocognitive disorders (HAND) that are mediated by Tat (Mediouni et al, Current HIV Research, 2015).

Tat is an intrinsically disordered protein, and its conformation changes as it associates with different proteins, to mediate distinct activities. dCA locks Tat in a specific Tat conformer, blocking Tat interaction with several viral and host proteins. Structure activity relationship studies of a large series of dCA derivatives together with molecular docking revealed the importance of dCA’s cycloheptene ring and the isoquinoline nitrogen’s positioning in the interaction with Tat’s basic patch (Lys51, Arg52,Arg55), stabilizing the flexible Tat structure. Our results validate structure-based-design strategies targeting Tat and provide valuable insight into drug development around the dCA pharmacophore (Mediouni et al, mBio, 2019).

A surprising important observation was that resistance to dCA selected for viruses that are Tat-independent, all while highly transcriptional competent. Mutations in Tat/TAR were not identified, consistent with their conservation. Instead, Tat-independent transcription was leveraged thought enhanced basal transcriptional activity that arose from a combination of mutations in the LTR, as well as upregulation of NF-kB activity triggered by mutations in Nef and a Vpr truncation. This high transcriptional fitness correlated with higher susceptibility to CD8+T killing. Highly replicative Tat-independent viruses unable to self-regulate through Tat/TAR may ultimately self-destruct through cytopathic effects and/or immune clearance (Mousseau, mBio, 2019). Study of these unique viruses will provide important insight into the role of Tat in latency.

 HIV-1 and SIV are phylogenetically distinct lentiviruses with independent evolutionary origins and divergent Tat proteins. SIV infection of rhesus macaques (RhMs) is so far, the best characterized model for AIDS research. We demonstrated that dCA specifically binds to the basic domain of SIV Tat, blocking Tat transactivation of the SIV LTR and the recruitment of the RNAP II to the SIV promoter. dCA efficiently inhibits SIV replication in primary rhesus macaques (RhMs) CD4+T cells and potently blocks viral reactivation from CD4+T cells explanted from SIVmac239 viremic rhesus macaques (Mediouni et al, FASEB Journal, 2019). These observations establish the foundation for the study of the impact of dCA on latent infection in RhM.

Ongoing studies:

i) Understanding of the full clinical potential of Tat inhibitors.

Tat inhibitors are unlike any other HIV inhibitor, since the duration of treatment impacts the outcome. This is due to the feedback nature of the Tat-TAR activity, and because epigenetic marks impeding reactivation are deposited at the HIV-1 over time. As such, it is now essential to understand the full clinical potential of Tat inhibitors since many questions still remain. How long does it take for dCA to render residual viral RNA production undetectable? What is the relationship between viral RNA reduction and time to rebound after analytical treatment interruption (ATI)? Can we remove all therapy altogether or should dCA be maintained to keep viral mRNA production undetectable? Does dCA bring benefits if added to front-line therapy? Answering these questions will enable the design of improved treatment schedules and define the potential of this unique type of antiretroviral drugs. We are addressing many of these questions in the more manageable and less expensive BLT mouse model of HIV latency. We expect these will inform the design of ongoing non-human primate and, ultimately, human studies.

ii) Development of novel Tat inhibitors.

It is of course important in drug discovery to have several lead candidates that are down selected by their chemical, pharmacological, pharmacokinetics, safety and toxicity profiles. We are pursuing in parallel classic optimization of the dCA pharmacophore and identify additional clinical candidates that embody equivalent bio-activity to dCA needed in the pre-clinical pipeline.

We developed a high-throughput screen (HTS) and screened 775,000 compounds, using dCA as reference. We have developed appropriated counter screens and have optimized in our laboratory techniques and cell models that will quickly weed out compounds working independently of Tat or TAR.  Although a number of efforts to identify Tat inhibitors were undertaken in the past, especially in the 1990s, we are now in a stronger position to do so.  Specifically, advances in high throughput technologies, the increased size of the small molecule drug libraries, and our increased understanding of Tat and TAR biology and function, make the chance of identifying novel lead compounds much more likely.

iii) Identification and characterization of chromatin regulators of HIV-1 latency.

 HIV-1 transcriptional inhibitors have the unique property of reducing particle production from infected cells. dCA is the proof-of-concept that this novel class of molecules is amenable to block-and-lock functional cure approaches. It is thus important to understand the mechanisms that explain not only dCA’s inhibition of reactivation, but also mechanisms regulating HIV-1 latency in CD4+T memory T cells in general. These efforts will help us improve “block-and-lock” approaches and identify other ways to suppress reactivation.

Human chromatin regulators “write”, “erase”, or “read” chromatin modifications or remodel nucleosome topology. Specificity in gene expression derives from the combinatorial nature of chromatin modifications, and assembly of related chromatin regulator subunits. The rationale for this work is that factors that establish HIV-1 latency are important for viral reactivation, and that by identifying and inhibiting them, a “locked” state of silencing, one strongly resistant to reactivation, can be achieved.

To do so, we combine a comprehensive high-resolution mapping of the nucleosome organization and positioning of chromatin remodeling complexes at the HIV promoter during HIV latency, with a robust pooled shRNAs screening approach to interrogate all chromatin regulatory factors. Primary and secondary screens will be performed in our above mentioned newly developed primary cell system that captures bona fide HIV-1 latency.

Using these approaches, we can correlate high-resolution nucleosome architecture data with their binding to all chromatin remodeling machine families and develop a comprehensive picture of the signals and factors that drive chromatin activity at the latent provirus. These results will generate hypotheses and identify targets that can be subsequently tested. We anticipate that from these candidates, we can infer how HIV-latency is controlled and develop rational therapeutic approaches to modulate HIV latency.

2. Small molecule Inhibitors of HIV-1 capsid dimerization

The HIV-1 capsid plays crucial roles in HIV-1 replication and therefore represents an excellent drug target. We developed a time-resolved fluorescence resonance energy transfer based high-throughput screening assay (HTS-TR-FRET), to identify inhibitors of capsid dimerization, using the C-terminal domain (CTD) of HIV-1 capsid. This assay was used to screen the Library of Pharmacologically Active Compounds, composed of 1,280 in vivo active drugs, and successfully identified Ebselen (2-Phenyl-1,2-benzisoselenazol-3(2H)-one), as an inhibitor of capsid dimerization and HIV-1 replication. This was corroborated by NMR experiments that show perturbation of residues in the dimerization domain of CTD when Ebselen is more than two-fold of the CTD concentration. This small molecule has been shown to act at an early post-entry step prior reverse transcription by stabilizing the viral capsid during HIV-1 infection. Ebselen specifically inhibits diverse retroviruses but has no impact on non-related viruses such as HCV and influenza viruses. The selenium element of Ebselen seems important for the inhibitory activity. This study demonstrated the feasibility of the screening assay to identify small molecule against HIV-1 capsid (Thenin-Houssier et al, Antimicrob Agents Chemother, 2016).

We have now screened a library of 775,000 compounds with excellent statistics, and a hit rate of less than 1%. Secondary screens and mechanistic studies are currently ongoing.

3. Potent suppression HIV-1 replication by natural product extracts

Natural products have always been a valuable resource for the pharmaceutical industry and have been of great benefit in virtually all-clinical therapeutic areas. Kudzu is a climbing vine rich in isoflavones and saponins, and has long been used in traditional Chinese medicine for a variety of purposes. We showed that Kudzu root extract significantly inhibits HIV-1 entry into target cells, cell lines and primary human CD4+T lymphocytes, without detectable cell-associated toxicity. Kudzu inhibits the initial attachment of the viral particle to the surface of the cell, a mechanism that depends on the envelope glycoprotein but is independent from HIV-1 cell receptor CD4 and co-receptors CXCR4/CCR5. This activity seems selective since Kudzu does not interfere with infection by HCV, H1N1, Zika virus Brazil strain, or ADV5 virus.  Importantly, Kudzu acts synergistically or additively depending on the dose with the current cocktails of ARVs, highlighting its value as a supplement to current antiretroviral therapy (Mediouni et al, Retrovirology, 2018). Despite some efforts, we have not been able to identify the chemical entity of Kudzu extract responsible for its antiviral activity, suggesting either that it is a yet-to-be-identified trace component, or that the combinatorial activity of multiple compounds mediates Kudzu’s ability to suppress HIV-1. We will address this question in future studies.