Our DDX3 RNA Helicase inhibiting Translation Inhibitor class of compounds do not target viral proteins but temporarily inhibit an enzyme of our own RNA cell biology that is of vital importance for the virus during its reproduction cycle. As such the virus finds itself in an impossible situation in which mutating its own viral proteins is no longer helpful in escaping from the drug effect; thus no, or less drug-resistant strains are selected and expanded. Furthermore, the host cells as well as the non-infected cells are not particularly affected by the inhibition of their own enzyme as they can rely on different pathways for their functions. This is confirmed by the almost absent cytotoxic effects of our compounds both in vitro and in vivo. On this basis, a low systemic toxicity is expected even for long term treatments. Rather than mitigating apoptosis in active CD4+ T cells such as provoked by Protease Inhibitors, our Translation Inhibitor class of DDX3 helicase inhibiting compounds does not have such a mitigating mechanism and will even most likely promote cellular apoptosis.
Translation Inhibitors offer unique possibilities uncommon to traditional HIV antivirals, such as significantly reduced resistance development, improved delivery in the body, low toxicity and the possibility of successful co-application in wake-up strategies of dormant reservoir cells.
FIRST HEALTH PHARMACEUTICALS' TIs
WHY DDX3 HELICASE INHIBITION?
Unlike Protease Inhibitors, which actually allow newly formed virions to exit the cell, albeit with dysfunctional viral proteins, Translation Inhibitors effectively block the production of new virions by the inhibition of the DDX3 helicase enzyme, a vital co-factor of the host cell’s protein apparatus, hijacked by the virus, during its translation from proviral host cell DNA. This way, viral components remain trapped within the cell because the synthesis of the essential polyproteins is disrupted. As such, the tentative formation of new viral particles from proviral DNA inevitably leads to the accumulation of unspliced mRNA in the nucleus and spliced mRNA and polyprotein material in the cellular cytoplasm. A part of the protein material will likely be re-metabolised, but the accumulation of cellular waste is generally known to be a pro-apoptotic mechanism. Repeated failed attempts to produce new virions will inevitably accumulate ever more viral waste within the host cell and ultimately promote apoptosis.
A simplified representation of the mechanism that lies behind the action of our DDX3 inhibitor compound
1 – An HIV virion infecting an immune cell
2 – Virus gets into the CD4+ T immune cell. Viral replication begins
3- Transcription and translation of integrated viral genome take place. New virus particles are created
4 – Our small molecule compound inhibits DDX3 blocking the viral mRNA in the nucleus and its translation to proteins. Few or incomplete viral particles are assembled
5 – Cell death by accumulation of cellular waste, thus no newly formed virions and no infected cells remain in the organism
Through the use of HAART we have come very close to deliver HIV a final blow. Viral loads in blood have become virtually undetectable from time to time, but in the end, the virus always comes back! The reason lies in the fact that HIV hides itself in reservoirs such as CNS and the male testis that are separated from the rest of the body by a set of epithelial and endothelial barriers not easily permeable to HAART making them two of the principal reservoirs in the human body. The other anatomical reservoirs where HIV tends to survive are purely functional and characterised by a very high prevalence of the CD4+ type T cells and macrophages in which HIV effectively duplicates. It is known that lymph nodes or gastrointestinal mucosa as well as the female reproductive organs are anatomic locations that function as HIV reservoirs.
THE ROLE OF HIV RESERVOIRS
Virus eradication in patients receiving HAART is not possible if HIV reservoirs are not eliminated. The best picture of a reservoir, without conceiving it as a specific anatomical location, it is rather a pool of resting memory CD4+ T cells carrying latent but replication-competent proviral genomes. It is these dormant memory cells, which are, in fact the true big hurdle left in HIV research and in order to challenge this problem, we carried out new strategies with the aim to wake-up these cells. Resting HIV proviral cells can survive for weeks and even up to decades and their eradication is at present seen as the only solution to the problem. Once the dormant cell has been reactivated it still needs to be killed otherwise it will start producing active virions all over again leading in turn to new resting cells.
Our DDX3 translation inhibiting compounds effectively block the production of new virions by the inhibition of the DDX3 helicase enzyme, a non-essential co-factor of the host cell’s protein apparatus, hijacked by the virus, during its translation from proviral integrated DNA (scheme image – step 3). This way, viral components remain trapped within the cell because the export of viral unspliced mRNAs and the synthesis of the essential polyproteins is strongly inhibited. As such, the tentative formation of new viral particles from proviral DNA inevitably leads to the accumulation of unspliced mRNA in the nucleus and spliced mRNA and polyprotein material in the cellular cytoplasm (scheme image – step 4). Only a part of the protein material will likely be re-metabolised, while the rest will accumulate as intracellular waste, known to be a pro-apoptotic mechanism. Repeated failed attempts to produce new virions will inevitably accumulate ever more viral waste within the host cell and ultimately promote apoptosis (scheme image – step 5).
SHOCK AND KILL
Currently the most promising therapeutic strategy for HIV cure research is the Shock and Kill approach.
SHOCK AND KILL STRATEGY
After decades spent investigating antiretroviral research, seems that HIV therapeutic research is leaning towards an HIV final cure. Even though combinatorial Antiretroviral Therapy (cART) has been the most revolutionary HIV therapeutic approach so far, that alone is not enough to completely eradicate HIV. The virus can hide out in a “latent” form inside certain cells of the immune system such as CD4+ T cells, forming the so-called HIV reservoirs. Because of this latency state the HIV infected cells undergo little or no transcription, thus the immune system cannot detect and eliminate (or attack) them. Once antiretroviral therapy is stopped, these viral-reservoir cells can rapidly fuel HIV rebound.
The Shock and Kill approach is a two-step model which aims at reactivating (Shock) the dormant HIV infected cells – the HIV reservoir – through latency reversal agents (LRAs) and subsequently eradicating (Kill) these cells by means of a combination of antiretroviral drugs, host immune clearance and HIV cytolysis. First Health Pharmaceuticals is currently participating in three multi center public-private collaboration projects on HIV cure strategies mainly focusing on Shock and Kill approach and Reservoirs Reduction. These projects, funded by Health Holland and AIDSfonds, include several high profile research groups from, Amsterdam University Medical Center (AUMC), Erasmus Medical Centre (EMC), Utrecht Medical Centre (UMC), Viiv Healthcare and other industrial partners. Thanks to their potential to contrast HIV reservoir formation and maintenance, the compounds of First Health Pharmaceuticals’ HIV pipeline such as DDX3 RNA Helicase inhibitors, TLR7 agonists and novel generations of antiretroviral agents, will play an important role in the project.
The “Shock” step of the Shock and Kill strategy consists in the reactivation of the provirus hiding in immune CD4+ T cells (the latent HIV infected cells) by means of small pharmacological molecules called Latency Reversal Agents (LRAs).
The target patients are the people living with HIV (PLWH) whose viral load has been suppressed below the level of treatment by effective highly active antiretroviral therapy (HAART) or combined antiretroviral therapy (cART).
Several in vivo and in vitro studies on LRAs have investigated their ability to reactivate and reduce latent reservoirs, alone or in combination with other compounds. Among those, toll-like receptor (TLR) activators have been shown to reactivate latent HIV infected cells.
TOLL-LIKE RECEPTORS AGONISTS
First Health Pharmaceuticals has been investigating potential Shock strategies to be applied in a HIV Shock & Kill approach. Since early 2016, we started studying the potential use of Interleukins and Toll-like Receptor (TLR) agonists, which is a class of compounds that we were already intimately familiar with through our cancer research into TLR7/8.
Toll-like receptors (TLR) class of protein belongs to the family of pattern-recognition receptors (PRR). They are transmembrane receptors line able to recognize conserved molecular structures called pathogen-associated molecular patterns (PAMPs) and promote viral reactivation in CD4+T cells by serving as signaling receptors in the innate immune system. Besides their ability to reactivate latent HIV, TLR agonists also increase immune activation and promote an antiviral response. These combined properties make TLR agonists unique among the different LRAs characterized to date. First Health Pharmaceuticals’ HIV pipeline includes both DDX3 RNA Helicase inhibitors and TLR7 agonists that potentially could contrast HIV reservoir formation and maintenance, and combined can contribute to viral eradication.