Didehydro-Cortistatin A: a new player in HIV-therapy?

ABSTRACT

Antiretroviral therapy can effectively suppress HIV-1 infection but is ineffective against integrated proviruses. A latent viral reservoir composed of latently infected CD4⁺T cells persists under suppressive therapy, and infected individuals must remain indefinitely on antiretroviral therapy to prevent viral reactivation and propagation. Despite therapy, some degree of low-level ongoing replication is detected and transient viral reactivation may replenish the latent reservoir. An analog of the natural compound, Cortistatin A, blocks HIV-1 transcription by specifically targeting the viral transactivator, Tat. Treatment of latently infected cells with this Tat inhibitor promotes a state of deep-latency from which HIV reactivation capacity is greatly diminished. Here we discuss the use of Tat inhibitors to limit the latent reservoir to achieve a functional cure.

KEYWORDS:

• Tat inhibitor
• didehydro-Cortistatin A
• HIV transcription
• HIV latency
• deep-latency
• latent reservoir
• viral reactivation
• antiretroviral therapy
• functional cure

Antiretroviral therapy shortcomings

Despite the potency of antiretroviral therapy (ART) to eliminate circulating plasma HIV, the virus persists as integrated provirus in resting memory CD4⁺ T cells. As such, patients must remain on ART for life to avoid viral resurgence and progression to AIDS. Moreover, during ART, the latent HIV reservoir is subject to transient reactivation events or small “blips” of viremia that may contribute to the replenishment of the latent reservoir.[1–4] Besides, low-level viremia might still occur in some organs and tissues partly due to inefficient antiretroviral (ARV) distribution.[1,5] Several strategies have been used to eradicate or functionally cure HIV but none has yet demonstrated definitive success.[6]

Mechanisms of transition from active HIV-1 transcription to latency

The viral protein Tat is produced early on in the HIV life cycle and is responsible for exponentially enhancing viral mRNA expression. Tat recruits the P-TEFb complex to the nascent trans-activation response (TAR) element present in all viral mRNAs to promote transcriptional elongation by RNA Polymerase II.[7] The Tat feedback loop is very important to keep an active transcription. Reduction in transcription initiation might decrease the level of Tat below a required threshold inducing the entrance of the provirus into latency.[8] While not completely understood, the entrance and maintenance of latency is characterized by suboptimal levels of Tat, of the positive transcription elongation factor-b (P-TEFb) and of key transcription factors needed for HIV transcription, but also by the presence of repressors of transcription such as YY1 or CBF-1 or by transcriptional interference.[8,9] Consequent to this reduction in viral transcription rates is an accumulation of epigenetic modifications at the two nucleosomes, precisely positioned at the HIV promoter, that characterize a latent provirus.[9]

Cortistatin A, an inhibitor of Tat-dependent HIV transcription

Cortistatin A is a steroidal alkaloid isolated from the marine sponge Corticium simplex found in Southeast Asia, which initial interest sparked out of its anti-proliferative properties.[10] Given Cortistatin A’s scarcity in nature, a synthetic route was devised and the analog didehydro-Cortistatin A (dCA) is now cost effectively produced in gram quantities.[11] In 2012, Mousseau and colleagues reported dCA’s ability to bind to the basic domain of Tat and specifically inhibit Tat-dependent HIV transcriptional amplification in acutely and chronically infected cells at nanomolar EC₅₀ concentrations.[12] In a more recent study by the same group, the long-term activity of dCA was assessed in chronically infected latent cell lines and a multi-log reduction in mRNA levels and viral particle production was observed.[13] Critically, this study demonstrated that discontinuation of dCA treatment did not result in viral rebound above the limit of detection of the assay for up to 4 months. Moreover, reactivation of the virus using several latency-reversing agents (LRAs) was potently inhibited in these cellular models of latency. Using a more relevant primary cellular model that is based on expanded primary CD4⁺ T cells from aviremic-infected individuals under suppressive ART, they confirmed that a long-term treatment (22 days) with a cocktail of ARVs and dCA blocked by 99.9% reactivation initiated with an LRA as compared with ARVs alone. Moreover, the pretreatment with dCA in combination with ARVs limited viral resurgence in the absence of any drugs by 93%.[13]

These results suggest that the inhibition of the viral transactivator Tat by dCA promotes the establishment of epigenetic modifications at the viral promoter that mediate a state of persistent latency or “deep-latency” from which viral reactivation is dramatically restricted. The hypothesis is that epigenetic marks triggered by dCA may be somewhat different from epigenetic marks observed when the virus naturally enters into latency, i.e., just as seen in patients, viral rebound occurs when ARVs are removed from latently infected CD4⁺ T cells, but does not rebound when cells have been previously treated with dCA. One possible explanation is that the occlusion of the basic domain of Tat by dCA blocks Tat’s interaction with proteins associated with the process of transcriptional reactivation from latency such as the histone acetyltransferases p300 and C/EBP [14] and the chromatin remodeling factor SWI/SNF.[15] By interfering with a subset of Tat activities, dCA might trigger a unique set of histone methylations or acetylations in the nucleosomes surrounding the HIV promoter, and/or promote recruitment of repressors of transcription that limit viral reactivation.

Advantages of inclusion of Tat inhibitors in HIV therapy

The use of Tat-specific inhibitors to tackle the latent reservoir has distinctive advantages. Tat is one of the first proteins to be produced early during infection and has no known cellular homologs. Besides, specifically inhibiting Tat blocks the feedback loop that drives exponential production of viral mRNA and viral particles. Tat also plays a determinant role in the entrance and maintenance of latency. As previously mentioned, suboptimal levels of Tat are observed in latently infected memory CD4⁺ T cells.[8] Besides, Tat-deficient viruses are impaired in their ability to reactivate.[16] A direct inhibition of the Tat transcriptional activity might increase the occurrence of proviruses entering latency. Indeed, it has been shown that fewer latent proviruses accumulate in infected T-lymphocytes overexpressing Tat.[17] Interestingly, a recent study demonstrated that the passage from active viral production to latency depends solely on Tat, independently of the state of cellular activation, and the most potent reactivation from latency is the one mediated by Tat.[18] A logical approach to treat HIV would be to prevent Tat activities to limit as much as possible transient viral reactivations causing episodes of clinically detectable viremia or “blips” suspected to participate in reservoir replenishment.[4] Altogether, these studies suggest that a direct inhibition of Tat by molecules such as dCA would block the Tat feedback-loop initiated after low-basal reactivation, and maintain a suboptimal level of available active Tat. A Tat inhibitor added to current ART would promote entrance of the provirus into a prolonged transcriptional silencing, refractory to sporadic viral reactivation and reservoir replenishment. A balance between homeostatic proliferation and death of the long-lived infected memory CD4⁺ T cell could potentially result in a reduction of the size of the viral reservoir over time. Several studies have reported that early ARV treatment, as soon as infection is detected or suspected, correlates with smaller reservoir size and improved virus control, even in the absence of therapy. For example, the Visconti cohort includes 14 HIV-positive individuals referred as post-treatment controllers, which were initially treated with ART for a median of 3 years, and have now an extremely low viral reservoir and have been living ART free for a median of 10 years.[19] In addition, a recent study reported that an 18-year-old French adolescent infected with HIV at birth, treated with ART for the first 6 years of her life, has been in remission for the last 12 years with no detectable viremia.[20] Similarly to these two cases, if a Tat inhibitor such as dCA could decrease the size of the reservoir, one could imagine infected individuals living drug free, eventually heading to a functional cure, defined by the long-term control of virus replication in the absence of ART.[21]

The quest for a specific HIV transcriptional inhibitor

Over the past decades, the quest for small molecule inhibitors of HIV transcription has been the focus of several research groups. Interactions between Tat/TAR, Tat/CyclinT1 and Tat/P-TEFb have been targets of choice for their specific role in HIV gene regulation.[22,23] However, few molecules so far have been found to block Tat either by direct interaction, by modifying its structure or by triggering its degradation.[12,24–26] Tat degradation has been observed with Triptolide, a natural compound isolated from Tripterygium wilfordii, a vine used in traditional Chinese medicine.[26] Triptolide is the only Tat inhibitor that in combination with ART is currently being tested in clinical trials (NCT02219672). This study will assess changes in the size of the HIV reservoir of naïve-ART patients in acute phase of HIV-1 infection. Other targets such as cellular transcription factors (e.g., NF-κB, Sp1, NFAT) or CDK9 (P-TEFb kinase) are still pursued but are less likely to reach the specificity needed to differentiate between cellular and HIV transcription.[22] In sum, despite tremendous efforts by many research groups, molecules specifically blocking HIV transcription have not yet reached the clinic. The elegant dCA study is a proof-of-principle that a Tat inhibitor, or a specific HIV transcriptional inhibitor, combined with ART could block viral reactivation from latently infected CD4⁺ T cells, in the expectation that a prolonged state of latency would decrease the size of viral latent reservoir over time.

Concluding remarks

So far, all intended strategies have failed to reduce the size of the latent reservoir and to cure HIV. The development of Tat-dependent transcriptional inhibitors such as the analog of Cortistatin A aimed at reducing the latent reservoir is an interesting alternative pathway to a functional cure. While promising, studies using dCA have only been performed in vitro in latently infected cell lines and in primary CD4⁺ T cells isolated from infected individuals. Further in vivo studies, using either humanized mice or non-human primates, are needed to understand the impact of combining ART to a Tat inhibitor on reactivation rates and size of the reservoir. If successful, the induction of “deep-latency” by Tat inhibitors would be a significant breakthrough in HIV therapy and for the everyday life of infected individuals. By reducing the latent viral reservoir, this strategy might increase the odds of individuals becoming post-treatment controllers and to achieve the long-awaited long-term remission.

Financial & competing interests disclosure

ST Valente is funded by the NIH-NIAID R01 AI097012 and R01 AI118432-01AI, she owns a patent “A novel inhibitor of HIV replication”-Tat inhibitors U.S. Patent WO021296934 A2; Filed: January 10, 2011; Inventors: Susana Valente and Phil Baran. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.