4-Deoxyphorbol inhibits HIV-1 infection in synergism with antiretroviral drugs and reactivates viral reservoirs through PKC/MEK activation synergizing with vorinostat
Abstract
The formidable challenge of eradicating human immunodeficiency virus (HIV) infection fundamentally stems from the persistence of latent viral reservoirs. These reservoirs comprise a small, yet critical, population of infected cells, predominantly resting CD4+ T cells, where the viral genetic material lies dormant and is therefore effectively inaccessible to conventional antiretroviral therapies and the host immune system. A highly promising therapeutic strategy to overcome this significant biological barrier involves the pharmacological reactivation of these latently infected T cells, a concept frequently referred to as “shock and kill.” This innovative approach aims to force the quiescent virus to express its genes, thereby rendering the infected cells visible and susceptible to viral clearance mechanisms orchestrated by the immune system or through targeted cell death.
Our current investigations reveal that a novel 4-deoxyphorbol ester derivative, specifically designated 4β-dPE A, which was meticulously isolated from the plant species *Euphorbia amygdaloides subspecies semiperfoliata*, possesses remarkable capabilities in reactivating latent HIV-1. This significant finding strongly suggests that 4β-dPE A could play a pivotal role in the comprehensive reduction of the persistent viral reservoir, thereby offering a promising new avenue in the global pursuit of an HIV cure. Our comprehensive analysis further demonstrates that 4β-dPE A exerts a dual influence on the intricate HIV replication cycle. It exhibits both a discernible inhibitory effect on new viral infection events and a profound capacity for HIV transactivation, which is the crucial process of stimulating dormant viral gene expression. This dual action is remarkably similar to the effects observed with other well-established phorboid compounds known to function as Protein Kinase C (PKC) agonists, such as phorbol myristate acetate (PMA) and prostratin, as well as other diterpene esters like SJ23B, highlighting a shared mechanistic foundation.
A profoundly critical aspect of 4β-dPE A’s therapeutic potential lies in its observed non-tumorigenic profile. This stands in stark contrast to the structurally related compound, PMA, which is widely recognized and documented for its potent tumor-promoting properties, a characteristic that severely limits its clinical utility despite its strong latency-reversing activity. The compelling absence of tumorigenicity in 4β-dPE A is particularly significant given its close structural kinship to PMA. Our accumulated data strongly suggest that this favorable safety profile may be attributed to the specific lack of a long side lipophilic chain in the chemical structure of 4β-dPE A, a molecular feature prominently present in PMA and widely implicated in its adverse tumor-promoting effects. This structural distinction positions 4β-dPE A as a potentially much safer and more viable alternative for clinical application in the context of HIV eradication strategies.
Furthermore, 4β-dPE A demonstrates exceptional potency, impressively activating HIV transcription at remarkably low nanomolar concentrations. This level of activity is considerably lower than the concentrations typically required by many other conventional latency reversing agents (LRAs), such as prostratin, and is notably comparable to that of the highly potent bryostatin. The precise molecular mechanism underlying 4β-dPE A’s potent transcriptional activity and, consequently, its robust anti-latency effect, involves the crucial activation of the Protein Kinase C theta (PKCθ) and Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase (MEK) pathways. These intricate cellular signaling pathways are absolutely essential for transducing signals that ultimately lead to the robust and sustained expression of latent viral genes.
Intriguingly, while PKCθ/MEK activation is unequivocally critical for the transcriptional activity, our detailed investigations reveal that the observed down-regulation of host cell receptors crucial for efficient HIV entry—namely CD4, CXCR4, and CCR5—appears to proceed independently of the PCK/MEK pathway. This fascinating mechanistic dissociation strongly suggests the existence of at least two distinct cellular targets or signaling pathways through which 4β-dPE A exerts its multifaceted effects on HIV. Moreover, consistent with previous findings for other PKC agonists, the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor is profoundly involved in the HIV reactivation induced by 4β-dPE A, underscoring a shared mechanistic underpinning for this important class of compounds.
To further assess its broad therapeutic applicability and potential, we meticulously examined the effects of 4β-dPE A when administered in combination with other established latency reversing agents. When combined with prostratin, another well-known PKC agonist, an unexpected antagonistic effect was observed, suggesting a complex and perhaps competitive interplay between these two compounds at a shared or closely related molecular target. In stark contrast, when 4β-dPE A was combined with vorinostat, a potent histone deacetylase (HDAC) inhibitor, a powerful and highly beneficial synergistic effect was achieved. This synergy was particularly noteworthy as the combination not only synergistically diminished the half maximal effective concentration (EC50) value, thereby indicating a significantly increased potency, but also substantially enhanced the overall efficacy demonstrated by the drugs when administered individually. This compelling finding strongly suggests that combining distinct mechanistic approaches can indeed lead to superior therapeutic outcomes.
Crucially, our studies also extended to combinations of 4β-dPE A with various essential classes of antiretroviral drugs (ART), including CCR5 antagonists, nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs). These comprehensive investigations consistently revealed a robust synergistic effect across all tested ART combinations. This highly promising outcome unequivocally indicates that the concurrent administration of 4β-dPE A with existing antiretroviral therapy would not interfere with the critical efficacy of the established treatment regimen, a supremely important consideration for successful clinical translation and patient management.
Finally, to validate its physiological relevance and potential for real-world application, 4β-dPE A successfully induced latent HIV reactivation in CD4+ T cells isolated directly from infected patients who were undergoing stable antiretroviral therapy. The levels of reactivation achieved in these ex vivo patient samples were strikingly comparable to those observed with the tumorigenic phorbol derivative PMA, unequivocally demonstrating a potent and clinically relevant reactivation effect in a highly representative ex vivo setting.
In summary, our comprehensive study meticulously elucidates the detailed mechanism of action of 4β-dPE A, a novel and remarkably potent deoxyphorbol derivative. Its unique therapeutic profile, characterized by strong and consistent latency reversing activity, a crucial non-tumorigenic nature, exceptional potency at nanomolar concentrations, and demonstrable synergistic effects with both HDAC inhibitors and existing antiretroviral drugs, collectively positions it as an exceptional candidate for strategies aimed at decreasing the persistent and challenging HIV reservoirs, thereby significantly advancing the ultimate and ambitious goal of HIV eradication.
Keywords
HIV-1; Latency reversing agents; Phorbol esters; Protein kinase C; Viral reactivation.
Introduction
The advent of highly effective antiretroviral therapy (ART) has revolutionized the management of human immunodeficiency virus (HIV) infection, leading to a profound decrease in the mortality rates historically attributable to AIDS. This remarkable therapeutic progress has largely been achieved by consistently reducing the circulating viral load in infected individuals to often undetectable levels, thereby significantly delaying disease progression and transforming HIV from a rapidly fatal condition into a manageable chronic illness. Despite these profound successes, a formidable and persistent obstacle to the complete eradication of HIV infection remains: the presence of latent viral reservoirs. These reservoirs are populations of infected cells, predominantly quiescent memory CD4+ T cells, which harbor integrated viral DNA that lies dormant and transcriptionally inactive. This state of latency renders these cells impervious to the direct action of ART, which primarily targets actively replicating virus, and also makes them invisible to the host immune system. Consequently, while these resting T cells do not actively support viral replication, they represent a critical source for viral rebound if ART is interrupted, effectively preventing a functional cure. This phenomenon of viral persistence in a latent state is not unique to HIV, finding parallels in other challenging viral infections such as Ebola virus, which can persist in immune-privileged sites, and hepatitis B virus, human papillomavirus, and most herpesviruses, all of which employ diverse mechanisms to establish and maintain latent infections within their hosts.
The primary mechanism of action for current ART regimens involves interfering with various stages of the active HIV replication cycle. However, since the vast majority of antiretroviral drugs primarily target viral proteins or processes essential for active replication, they are inherently incapable of eliminating these hidden, latent viral reservoirs. This fundamental limitation underscores the continuous and pressing need for the discovery and development of entirely new classes of antiviral agents. Such new therapies are crucial not only for overcoming the challenge of viral latency but also for addressing significant issues associated with long-term ART, including chronic toxicity and the emergence of drug resistance, both of which can compromise the durability and effectiveness of current treatment strategies. In response to this challenge, a pioneering strategy for reservoir elimination, widely known as “shock and kill” or “kick and kill,” has emerged as a central focus of HIV cure research. This innovative approach is predicated on the idea of pharmacologically stimulating viral replication within latently infected CD4+ T cells, effectively “shocking” the dormant virus out of its quiescent state. The goal is to force these cells to produce viral particles and express viral antigens, thereby making them susceptible to either targeted clearance by a robust antiviral immune response or direct cytotoxic effects, while simultaneously preventing the spread of newly produced virus to uninfected cells through the continued administration of conventional antiviral therapy.
The agents responsible for the “shock” component of this strategy are collectively termed latency-reversing agents (LRAs) or anti-latency drugs. This diverse class of compounds encompasses several distinct mechanistic categories, each targeting different cellular pathways involved in maintaining viral latency. Prominent examples include epigenetic modifiers, which alter chromatin structure to promote gene expression; Toll-like receptor (TLR) agonists, which stimulate innate immune pathways; T-cell receptor (TCR) activators, which mimic physiological T-cell activation; modulators of the PI3K/Akt pathway, involved in cell growth and survival; NF-κB agonists, which directly activate a key transcription factor for HIV gene expression; and two of the most extensively studied classes: Protein Kinase C (PKC) agonists and histone deacetylase (HDAC) inhibitors. Among these, PKC agonists such as prostratin and bryostatin, along with HDAC inhibitors like vorinostat, panobinostat, and romidepsin, have garnered significant attention and have progressed to clinical evaluation. Despite considerable research effort and the initiation of several clinical trials involving vorinostat, bryostatin, romidepsin, and TLR7 agonists, the outcomes thus far have not yielded conclusively convincing results regarding a significant reduction in the latent reservoir size. Furthermore, achieving a sustained cure may necessitate an effective “kill” strategy that extends beyond the host’s endogenous immunological response, prompting efforts to integrate immune system modulation into these therapeutic approaches. Examples include studies combining romidepsin (as the “shock”) with a therapeutic vaccine (Vacc-4x) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) for the “kill” phase, which showed only a minor, statistically non-significant reduction in reservoir size. Similarly, the RIVER study, which paired vorinostat as an LRA with two immunomodulatory vaccines, also produced disappointing results. Several potential factors might contribute to the observed limitations of these early clinical interventions. These include methodological challenges in accurately quantifying the reservoir, which can lead to either underestimation or overestimation of its true size; the use of drug doses that may have been too low to achieve optimal efficacy in order to avoid potential toxicity; or simply the intrinsic limited efficacy of the specific drugs selected for these initial trials. Alternative innovative approaches have also been explored, such as the use of CAR T cells engineered to express broadly neutralizing anti-HIV antibodies, which have shown preliminary promise in reducing viral reservoir size in the blood of infected individuals on ART.
Protein Kinase C agonists operate by activating specific isoforms of the protein kinase C enzyme through binding to its regulatory C1 domain, thereby mimicking the action of the physiological ligand diacylglycerol (DAG) and promoting viral reactivation. Prostratin stands out as one of the pioneering PKC agonists thoroughly investigated for its potential as an anti-latency agent. Bryostatin, another PKC agonist and an antineoplastic drug, unfortunately demonstrated a lack of efficacy in reducing the latent reservoir during clinical trials, a outcome likely attributable to the very low doses administered in an attempt to mitigate its significant toxicity. Systemic concentrations of bryostatin were often undetectable at these lower, clinically tolerated doses, suggesting that its toxicity occurs at sub-efficacious levels, which severely restricts its utility as an LRA. If this observed toxicity is directly linked to its on-target mechanism of action, it would pose a substantial limitation for the entire class of PKC agonists. In this context, certain diterpenes and phorboids derived from *Euphorbiaceae* plants have also been identified as LRAs possessing both antiviral and viral reactivation properties. However, even subtle variations in their chemical structures can lead to a complete absence of activity or a distinct preference for specific PKC isoforms, which in turn modulates their efficacy in reactivating the viral reservoir. It is also important to note that the chemical structure of bryostatin is entirely unrelated to that of phorboids, suggesting that its toxic effects *in vivo* could arise from a completely different molecular target, though current data on this aspect are limited. The ongoing discovery and detailed characterization of novel phorboids with diverse PKC binding profiles and, critically, with low toxicity levels, coupled with the identification of other potential molecular targets for these bioactive molecules, would be invaluable in optimizing their efficacy as LRAs while simultaneously minimizing their adverse effects.
Ultimately, similar to the long-established practice in ART, achieving effective and sustained reactivation of the latent reservoir will most likely necessitate the strategic combination of different LRA drugs or potentially antibodies. Such combinatorial approaches are designed to allow for the use of lower doses of individual compounds, thereby reducing cumulative toxicity, while simultaneously achieving a sufficient level of cellular activation to effectively expose and eliminate the viral reservoirs. In this regard, it is paramount to emphasize that any such LRA-based therapy is intended for use in conjunction with ongoing ART. Previous research has indicated that antiretroviral drugs can indeed modulate LRA activity, and conversely, LRAs may influence the antiviral efficacy of ART. For instance, certain protease inhibitors have been shown to diminish the degree of viral reactivation achieved with LRAs, highlighting the complexity of these drug-drug interactions.
We have previously reported on the promising antiviral activity of 4-deoxyphorbol derivative compounds, which were isolated from *Euphorbia amygdaloides subspecies semiperfoliata*. Building upon that foundational work, the present study comprehensively evaluates the detailed mechanism of action and the specific cellular pathways involved in the unique latency-reversal and anti-HIV-1 effects exerted by 4β-dPE A, a particular compound from this class. Our findings confirm that this compound not only demonstrates significant antiviral activity, primarily through its ability to down-regulate host cell receptors critical for viral entry, but also possesses a potent capacity to induce HIV reactivation from latency. This class of compounds, therefore, represents a highly promising avenue for the development of adjuvant therapies aimed at reducing or ultimately eliminating the persistent latent reservoirs of HIV-1, a crucial step towards a complete cure.
Material And Methods
Reagents
The specific 4-deoxyphorbol ester derivative, 4β-dPE A, was rigorously isolated from the botanical source *Euphorbia amygdaloides subspecies semiperfoliata*. This invaluable compound was generously provided by the Centre National de la Recherche Scientifique (CNRS) in France. For experimental use, 4β-dPE A was initially solubilized in dimethyl sulfoxide (DMSO) to achieve a concentrated stock solution of 10 mM, subsequently aliquoted, and stored under ultracold conditions at -80 °C to preserve its integrity and activity. Monoclonal antibodies (mAbs) specifically targeting human CD4, CXCR4, and CCR5 receptors were procured from Becton Dickinson, located in Mountain View, CA, USA, ensuring high specificity for these crucial viral entry molecules. Interleukin-2 (IL-2), a key cytokine for T-cell activation, was obtained from Chiron in Emeryville, CA, USA. Several critical pharmacological inhibitors were employed to dissect specific signaling pathways: rottlerin was sourced from Alexis Co. (Lausanne, Switzerland), while Gö6850 and Gö6976 were purchased from Calbiochem, a division of EMD Biosciences, Inc. (Darmstadt, Germany). Luciferase and Renilla Assay Systems, alongside the CellTiter Glo viability assay kit, essential tools for quantifying gene expression and cell viability, respectively, were supplied by Promega (Madison, WI, USA). Additional reagents included DMSO and the MEK1/2 inhibitor PD184352, both obtained from Sigma-Aldrich (St Quentin-Fallavier, France). A comprehensive panel of antiretroviral drugs and established latency reversing agents were acquired through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH; these included SAHA (vorinostat), raltegravir, lamivudine, tenofovir, emtricitabine, abacavir, efavirenz, and ritonavir. Furthermore, prostratin, phorbol myristate acetate (PMA), and bryostatin-1 were all purchased from Sigma-Aldrich (St Louis, MO), serving as important reference compounds for comparative studies.
Cells
The cellular models employed in this study were meticulously selected to represent various aspects of HIV infection and latency. MT-2 cells (American Type Culture Collection, Ref: CRL-2560) and Jurkat 5.1 LTR-Luc cells (a generous gift from A. Israel, Institut Pasteur, Paris, France) were maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, and a standard antibiotic cocktail comprising penicillin (50 IU/ml) and streptomycin (50 mg/ml), all sourced from Whittaker M.A. Bio-Products (Walkerville, MD, USA). These cultures were routinely split twice weekly to maintain optimal growth. The Jurkat 5.1 LTR-Luc cell line is particularly valuable as it is a stable Jurkat-derived clone engineered to contain a luciferase reporter gene driven by the HIV-1 long terminal repeat (LTR) promoter, allowing for direct measurement of viral transcriptional activity; this cell line was additionally maintained in complete medium supplemented with G418 (200 ug/ml) for selection purposes. Human embryonic kidney 293T cells (American Type Culture Collection, Ref: ATCC CRL-3216) and TZM-bl cells (a cell line stably transfected with an LTR-Luc reporter, obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH, Cat#8129, from Dr. John C. Kappes and Dr. Xiaoyun Wu) were cultured in DMEM medium also containing 10% (v/v) fetal bovine serum, 2 mM L-glutamine, penicillin (50 IU/ml), and streptomycin (50 mg/ml) (Whittaker), and similarly split twice weekly. Peripheral blood mononuclear cells (PBMCs) were freshly isolated from the blood of healthy, anonymous donors via density gradient centrifugation using Ficoll-Hypaque (Pharmacia Corporation, North Peapack, NJ). These cells were subsequently suspended in RPMI 1640 medium enriched with 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics (100 mg/ml streptomycin and 100 U/ml penicillin) (Whittaker M.A. Bio-Products, Walkerville, MD, USA) and cultured at a concentration of 2×10^6 cells/mL. For certain experiments requiring T-cell activation, PBMCs were pre-treated with IL-2 (300 IU/mL) for at least 48 hours. All cell cultures were maintained in a humidified atmosphere at 37 °C with 5% CO2. Rigorous ethical protocols were followed, and proper informed consent was obtained from each blood donor in strict accordance with Spanish legislation on blood donor regulations, ensuring complete confidentiality and privacy.
PBMCs From HIV Infected Patients
For studies directly relevant to the clinical context of HIV infection, PBMCs were acquired from ART-treated HIV-infected patients. These precious samples were kindly provided by Dr. Sonsoles Sanchez Palomino from the Clinic Hospital, Barcelona, Spain. Prior to sample collection, all participating patients provided written informed consent. CD4+ T cells, the primary target cells for HIV, were meticulously purified from these PBMCs using a positive selection method involving human CD4+ T Cell Isolation Kits and MS columns (both from Miltenyi Biotech, Bergisch Gladbach, Germany). The purified CD4+ T cells were then cultured in complete medium at 37 °C in a 5% CO2 humidified atmosphere until they were prepared for experimental use.
Plasmids
A suite of well-characterized plasmids was employed to generate recombinant viruses and reporter systems for evaluating various aspects of HIV replication and transcription. The pNL4.3-luc vector was meticulously constructed by inserting the luciferase reporter gene into the well-established HIV-1 proviral clone pNL4.3, allowing for quantitative assessment of viral replication. Plasmid pNL4.3-Ren was engineered by cloning the *Renilla* luciferase gene into the nef site of pNL4.3, providing an alternative reporter for viral activity. Similarly, the pJR-Ren plasmid was created by cloning the env gene of the HIV-1 JRCSF strain into the pNL4.3-Ren plasmid, enabling studies with an R5-tropic virus. The pNL4.3-Δenv-Luc plasmid, lacking the env gene but containing a luciferase reporter, was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH (pNL4-3.Luc.R-.E- from Dr. Nathaniel Landau). To generate pseudotyped viruses for entry studies, the pcDNA-VSV plasmid, encoding the G protein of vesicular stomatitis virus (VSV), was sourced from Dr. Arenzana-Seisdedos at the Pasteur Institute. For investigating specific transcriptional pathways, the 3-enh-κB-ConA-luc plasmid was utilized, carrying a luciferase gene under the control of three synthetic copies of the κB consensus sequence from the immunoglobulin κ-chain promoter, strategically cloned upstream from the conalbumin transcription start site. The LTR-Luc plasmid, which places the luciferase gene under the direct control of the HIV-1 LTR region, was used for assessing overall viral promoter activity. Lastly, the expression construct pNFAT-LUC, containing three tandem copies of the distal NFAT-binding site from the IL-2 gene promoter coupled to the IL-2 minimal promoter, was a kind gift from Dr. Juan Miguel Redondo (National Center for Cardiovascular Research, Madrid, Spain), allowing for the assessment of NFAT pathway activation. All plasmids were amplified by transforming *E. coli* DH5α cells, which were subsequently cultured in LB medium supplemented with ampicillin at 37 °C for 18 hours. Following bacterial culture, all plasmids were purified using the Qiagen Plasmid Maxi Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, and DNA concentration was precisely determined using a Nanodrop spectrophotometer (Thermoscientific, Waltham, MA, USA).
Viruses
Viral stocks were prepared through transient transfection of producer cell lines with the appropriate plasmids. JR-Ren (an HIV-1 R5-tropic reporter virus) and NL4.3-Ren (an HIV-1 X4-tropic reporter virus) viral supernatants were generated by transfecting 293T cells with pJR-Ren and pNL4.3-Ren plasmids, respectively. For studies on viral entry mechanisms, an HIV-VSV pseudotyped virus (NL4.3-Δenv-VSV-Luc) was produced. This virus contained a luciferase reporter gene in the nef position and expressed the VSV G protein instead of the native HIV-1 env gene, allowing for entry independent of HIV-specific receptors. HIV-VSV was obtained by co-transfection of pNL4.3-Δenv-Luc and pcDNA-VSV plasmids into 293T cells. All viral supernatants were efficiently produced via calcium phosphate transfection of the respective plasmids into 293T cells, ensuring high-titer production.
Anti-HIV-1 Activity Evaluation By Recombinant Virus Assay
The initial assessment of 4β-dPE A’s anti-HIV-1 activity involved a recombinant virus assay. MT-2 cells were seeded into 96-well microplates at a density of 100,000 cells per well in a final volume of 200 µL. These cells were subsequently pre-treated with various concentrations of 4β-dPE A before being infected with an X4-tropic recombinant virus, NL4.3-Ren, obtained from previously transfected 293T cells. After a 48-hour incubation period, cell pellets were harvested, lysed, and the luciferase activity, serving as a direct readout for viral replication, was quantified using a luminometer (Berthold detection systems, Pforzheim, Germany). A similar experimental procedure was followed for infections of IL-2 activated PBMCs, using either X4-tropic HIV (NL4.3-Ren) or R5-tropic HIV (JR-Ren) to assess the compound’s activity across different viral tropisms. To specifically investigate the compound’s effect on HIV-1 entry, MT-2 cells were infected with either HIV-VSV (NL4.3-Δenv-VSV-Luc) or wild-type HIV-1 (NL4.3-Ren) in the presence of varying concentrations of 4β-dPE A. Throughout all experiments, appropriate controls were included, consisting of cells treated solely with the equivalent concentration of DMSO used to dissolve 4β-dPE A. HIV-1 replication inhibition was quantitatively assessed by measuring luminescence activity (relative luminescence units, RLUs), with the untreated, infected cells defining 100% replication. The half-maximal inhibitory concentration (IC50) for each compound was precisely calculated using a non-linear regression formula within GraphPad Prism software.
Toxicity Evaluation
To ensure that observed antiviral or latency-reversing effects were not confounded by cellular toxicity, a rigorous evaluation of cell viability was performed in parallel. Mock-infected cells were treated with the same concentrations of 4β-dPE A or other LRAs (PMA, bryostatin-1, prostratin, and vorinostat) used in the antiviral and anti-latency assays. Additionally, higher concentrations of 4β-dPE A and its combinations with other LRAs or antiretroviral drugs were tested. After a 48-hour incubation period, CellTiter Glo reagent (Promega, Madison, WI, USA) was added to the cultures, and cell viability was quantified by measuring RLUs in a luminometer. Cell viability was expressed as a percentage relative to a non-treated control (receiving only the equivalent DMSO concentration), which was set to 100%. To assess long-term safety, 4β-dPE A toxicity was evaluated over a 14-day period in IL-2 activated PBMCs, at concentrations of 0.1, 1, and 10 µM. Cell viability was determined at multiple time points throughout the treatment course. The half-maximal cytotoxic concentration (CC50) was calculated using a non-linear regression formula in GraphPad Prism software.
Evaluation Of Viral Transcription
Given the extremely low frequency of latently HIV-infected cells *in vivo*, direct purification and comprehensive biochemical analysis of these cells present significant technical challenges. Furthermore, various studies indicate that *in vitro* assays designed to quantify the latent reservoir from PBMCs can yield a wide range of results depending on the specific technique employed. Consequently, to thoroughly evaluate the transcriptional effect of these compounds, a multifaceted approach employing several distinct methods was adopted. Firstly, an *in vitro* model utilizing both MT-2 cells and primary human resting PBMCs was established. These cells were transiently transfected with luciferase reporter plasmids under the control of the entire HIV-1 genome (pNL4.3-Luc). Secondly, transcriptional activation was assessed in non-infected TZM-bl and Jurkat 5.1 LTR-Luc cells. These cell lines are particularly advantageous as they stably contain integrated copies of the luciferase gene driven by the HIV-1 LTR promoter within their genomes, thus obviating the need for *de novo* HIV transfection and allowing for direct and consistent measurement of viral transcriptional activity. In all these experimental systems, 4β-dPE A consistently demonstrated the ability to activate HIV transcription, inducing high levels of HIV-1 gene expression even at nanomolar concentrations.
Furthermore, the half-maximal effective concentrations (EC50 values) for HIV transcription were precisely determined in resting PBMCs. All tested compounds exhibited discernible viral reactivation in these quiescent cells. 4β-dPE A displayed an EC50 value of approximately 2.9 nM as a transcriptional activator, a potency that is notably at least 70-fold lower than that of vorinostat and a remarkable 480-fold lower than prostratin. While 4β-dPE A was less potent than PMA (EC50 of 0.031 nM), it exhibited an EC50 value of 3.4 nM in the same assay, which is almost identical to that of bryostatin. Due to limitations in compound availability, bryostatin concentrations were only tested up to 100 nM, at which point no cell toxicity was observed. Therefore, the CC50 of bryostatin was greater than 100 nM, yielding a specificity index (SI) greater than 21, whereas 4β-dPE A exhibited a CC50 greater than 10,000 nM, indicating a significantly wider therapeutic window. To ensure the observed effects were not influenced by potential cytotoxicity, all compounds were subjected to cell toxicity evaluation in parallel, using the same concentrations as in the anti-latency assays. No toxicity was observed for any of the compounds at the tested concentrations in PBMCs, MT-2, TZM-bl, or Jurkat 5.1 cell lines, confirming that their transcriptional effects were indeed due to specific biological activity rather than cell damage.
4β-dPE A Decreases The Expression Of HIV-1 Entry Receptors
The process of HIV entry into host cells is a meticulously choreographed event involving the crucial interaction of the viral envelope glycoproteins gp120 and gp41 with the host cell’s primary receptor, CD4, and coreceptors, either CXCR4 or CCR5. Previous research has well-established that these critical entry receptors can be down-regulated by various PKC agonists, including prostratin and SJ23B, suggesting a potential mechanism for antiviral activity. To investigate whether 4β-dPE A also modulates these receptors, the expression of cell surface receptors was quantitatively measured using three-color immunophenotyping and flow cytometry on both MT-2 cells and IL-2 activated PBMCs. Cell cultures were stimulated with either PMA, a known PKC agonist, or 4β-dPE A, and receptor expression was assessed at various time points: 2, 24, and 48 hours post-treatment.
Our findings revealed that 4β-dPE A effectively down-regulated the expression of CD4, CXCR4, and/or CCR5 receptors in both MT-2 cells and IL-2 activated PBMCs. This effect was observed to be clearly concentration-dependent. While a concentration of 1 nM 4β-dPE A was not sufficient to induce significant receptor modulation, concentrations of 10 nM and 100 nM were highly effective in achieving substantial receptor down-regulation. The pattern and extent of this down-regulation were remarkably similar to those observed with PMA at a concentration of 100 nM, underscoring a shared mechanistic influence on host cell surface receptor dynamics. This effect suggests that receptor down-regulation contributes to the observed antiviral activity of 4β-dPE A by impeding viral entry.
PKCθ/MEK Pathway Is Required For 4β-dPE A HIV Transcriptional Activity
To rigorously ascertain the dependence of 4β-dPE A-mediated antagonism of HIV latency on the Protein Kinase C (PKC) and Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase (MEK) pathways, a series of targeted inhibition experiments were conducted. Resting PBMCs were pre-treated with specific chemical inhibitors of PKC isoforms: Gö6976 at 1 µM, which primarily inhibits classical PKCs; bisindolylmaleimide at 1 µM (Gö6850), which broadly inhibits both classical and novel PKCs; and rottlerin at 3 µM, a more selective inhibitor of PKCθ. Additionally, the MEK inhibitor PD184352 (IMEK) at 1 µM was utilized to explore the role of the MEK-RAF pathway. Following inhibitor pre-treatment, the HIV transcriptional activity induced by 4β-dPE A was subsequently evaluated. The concentrations for all inhibitors were carefully selected based on previously published studies to ensure maximal target specificity.
Our results demonstrated a clear dependency. The use of a classical PKC inhibitor (Gö6976) did not significantly impair the transcriptional activity induced by 4β-dPE A, suggesting that classical PKC isoforms are not the primary mediators of this effect. However, the broader inhibition of both classical and novel PKCs using bisindolylmaleimide (Gö6850) resulted in a discernible impairment of 4β-dPE A’s transcriptional activity, pointing towards the involvement of novel PKC isoforms. To further pinpoint the specific isoform, we selectively inhibited PKCθ with rottlerin. Crucially, this intervention effectively blocked the transcriptional activity induced by 4β-dPE A, strongly implicating PKCθ as a key mediator in the deoxyphorbol-induced transcriptional response. Furthermore, recognizing that PKC activation can often lead to the activation of the MEK-RAF pathway, which is integral to cellular growth and proliferation, we investigated the effect of MEK inhibition on 4β-dPE A’s transcriptional activity. Our findings confirmed that MEK inhibition significantly decreased the transcriptional activity induced by both 4β-dPE A and PMA (a positive control), providing compelling evidence that the observed effect is indeed dependent on the PKCθ/MEK signaling axis.
PKCθ/MEK Pathway Is Not Involved In 4β-dPE A Activity On Receptors Down-Regulation
To thoroughly investigate whether the same PKC isoforms and downstream signaling pathways involved in transcriptional activation are also responsible for the observed receptor down-regulation, we conducted experiments designed to dissect the specific PKC isoform involved in 4β-dPE A-mediated receptor modulation. IL-2 activated PBMCs were pre-treated with distinct PKC chemical inhibitors, including bisindolylmaleimide (which targets classical and novel PKCs) or rottlerin (a specific PKCθ inhibitor), and the effect of 4β-dPE A on the expression of CD4, CXCR4, and CCR5 receptors was subsequently evaluated. Recognizing that downstream PKC signaling in T cells frequently involves Raf-MEK dependent activation of mitogen-activated protein kinases (MAPKs), the potential role of MEK inhibition in 4β-dPE A’s effects was also explored. Accordingly, PBMCs were additionally treated with the MEK inhibitor PD184352, and the impact on surface cell receptor expression was precisely measured.
Our comprehensive analysis revealed a significant mechanistic dissociation. We found that the inhibition of either PKC or MEK, using the specific inhibitors, did not substantially modify the expression of CD4, CXCR4, or CCR5 receptors, regardless of whether 4β-dPE A treatment was applied or not. This critical finding strongly suggests that the PKC/MEK pathway, despite its crucial role in 4β-dPE A-induced transcriptional activation, is remarkably *not* involved in the profound down-regulation of receptors exerted by 4β-dPE A. Consequently, this indicates that the anti-HIV activity attributed to receptor modulation operates via a distinct, PKC/MEK-independent mechanism. Furthermore, the absence of significant differences in receptor expression between cells treated with PKC inhibitors, MEK inhibitors, and untreated control cells underscores that these inhibitors, by themselves, did not exert any intrinsic influence on receptor expression, thereby validating the specificity of our observations regarding 4β-dPE A’s distinct modes of action.
4β-dPE A Induces A Different Pattern Of Intracellular Distribution And Phosphorylation Of PKCθ
Given that novel PKC isoforms, and particularly PKCθ, have been strongly implicated in the transcriptional response to 4β-dPE A, and knowing that PKCθ is a putative target of PKC agonists in T cells, playing a critical role in the regulation of T-cell activation, proliferation, and differentiation with high expression levels in these cells, we proceeded to analyze its activation status. Specifically, we investigated the phosphorylation of PKCθ at residue T538 after treatment with various LRAs using immunofluorescence microscopy. This approach allowed for a detailed visualization of PKCθ phosphorylation and its subcellular localization.
Our results revealed a compelling and distinct pattern of intracellular distribution of PKCθ when cells were treated with 4β-dPE A, in comparison to treatment with PMA (which served as a pan-PKC agonist control) or an untreated control. While a classic, clear-cut membrane translocation of PKCθ was not overtly observed in the immediate sense, quantitative analysis of the immunofluorescence data unequivocally demonstrated a significant increase in PKCθ phosphorylation. Concurrently, an altered cellular localization of PKCθ was evident, consistent with observations reported by other research groups regarding PKCθ activation. These collective findings strongly suggest that PKCθ is indeed a responsive cellular target within primary human PBMCs to the action of 4β-dPE A, and that its activation, characterized by increased phosphorylation and altered distribution, is a key event contributing to the compound’s biological effects.
NF-κB Transcription Factor Is Involved In 4β-dPE A Transcriptional Activity
To further meticulously unravel the intricate mechanism driving the robust reactivation activity of 4β-dPE A, our investigation delved into its specific influence on key transcriptional factors that are fundamentally crucial for HIV-1 transcription and, importantly, for broader T-cell activation. The ideal therapeutic agent for latency reversal would induce HIV transcription without simultaneously triggering widespread and potentially harmful T-cell activation, which could lead to undesired systemic immune responses. To address this, we employed a sophisticated experimental design utilizing luciferase expression vectors meticulously engineered to be under the control of three distinct promoter elements: three tandem κB consensus repeats (referred to as NF-κB Luc), the NFAT response element (NFAT Luc), or the comprehensive LTR region of HIV (LTR Luc). These vectors were transiently transfected into quiescent, resting PBMCs, which were subsequently treated with 4β-dPE A at three carefully chosen concentrations (1, 10, and 100 nM) or with PMA (at a fixed concentration of 100 nM) as a well-established positive control for T-cell activation and HIV transactivation. After a 48-hour incubation period, the resulting luciferase activity, serving as a direct quantitative measure of transcriptional activation, was meticulously quantified.
Our findings unequivocally demonstrated that 4β-dPE A induced significant activation of both the HIV LTR promoter and the NF-κB transcription factor, with the highest concentration of 100 nM yielding effects comparable to those observed with PMA. This observation strongly supports the notion that 4β-dPE A effectively promotes the transcription of latent HIV by directly engaging the NF-κB pathway, a critical regulator of HIV gene expression. In stark contrast, neither 4β-dPE A nor PMA treatment exerted any discernible influence on the transcriptional activity of NFAT. This stands in sharp distinction to Ionomycin (Io), a known calcium ionophore and potent activator of NFAT, which, as expected, robustly increased its expression. These cumulative data compellingly suggest that the transcriptional activity mediated by PKC agonists, including 4β-dPE A, exhibits a remarkable degree of selectivity, specifically targeting pathways dependent on NF-κB while notably sparing the NFAT pathway. This selectivity is a highly desirable characteristic for LRAs, as it implies a more focused reactivation effect on latent HIV without broadly activating T cells, thereby potentially minimizing unwanted side effects.
4β-dPE A Displays A Synergistic Effect When Combined With Vorinostat But Not With Prostratin
Having firmly established 4β-dPE A as a potent PKC agonist, our subsequent investigative phase focused on exploring its potential combinatorial effects with other classes of latency reversing agents, a strategy often critical for optimizing therapeutic outcomes in complex diseases like HIV. Specifically, we tested its combination with prostratin, another well-characterized PKC agonist, and with vorinostat, a histone deacetylase inhibitor (HDACi), representing distinct mechanistic approaches to latency reversal. Comprehensive combination experiments were performed involving all three drugs: 4β-dPE A in combination with prostratin, 4β-dPE A with vorinostat, and prostratin with vorinostat. For each combination, we meticulously evaluated the impact on both the half-maximal effective concentration (EC50) and the maximal effect (Emax) of viral transcriptional activation. To quantitatively assess the nature of these interactions (synergy, additivity, or antagonism), the Combination Index (CI) was calculated for all combinations, employing the well-established formula developed by Chou and Talalay. According to this widely accepted framework, CI values greater than 1.30 indicate antagonism, values between 1.10 and 1.30 suggest weak antagonism, values between 0.90 and 1.10 denote an additive effect, values between 0.70 and 0.90 signify weak synergy, and critically, CI values below 0.70 are indicative of strong synergy.
Our meticulous analysis revealed distinct and noteworthy patterns of interaction. The combination of 4β-dPE A with prostratin resulted in a pronounced antagonistic effect, with a calculated CI value of 16.65. While this antagonism was primarily reflected in an antagonistic tendency towards lowering the Emax value, it clearly indicates that these two PKC agonists, when combined, counteract each other’s effects rather than enhancing them, suggesting potential competition at a shared or closely related molecular target. In stark contrast, the combination of 4β-dPE A with vorinostat demonstrated a powerful and highly desirable strong synergism, evidenced by a remarkably low CI value of 0.20. Similarly, the combination of prostratin with vorinostat also exhibited strong synergism, with a CI of 0.21. These compelling results underscore a crucial principle: combining 4β-dPE A with another PKC agonist like prostratin paradoxically *increases* the concentrations needed to achieve the same transcriptional activating effect, highlighting a competitive interaction. Conversely, when 4β-dPE A is combined with an HDAC inhibitor such as vorinostat, the profound synergism enables the attainment of the same therapeutic effect at significantly lower concentrations of each individual drug. Furthermore, a particularly exciting finding was that the combination of vorinostat and 4β-dPE A not only reduced the required doses but also dramatically enhanced the overall efficacy of transcriptional activation, increasing it to nearly double the effect observed with either drug administered alone. This suggests a powerful and complementary mechanism of action between these two classes of LRAs, offering a highly promising strategy for future therapeutic interventions aimed at reducing the latent HIV reservoir.
4β-dPE A Displays A Strong Synergistic Antiviral Effect In Combination With NRTIs, NtRTIs, NNRTIs And PIs
Given that latency reversing agent (LRA) treatment is ultimately designed to be co-administered with existing antiretroviral therapy (ART) in HIV-infected individuals, it is paramount to thoroughly evaluate any potential interactions between 4β-dPE A and current ART drugs. Therefore, we meticulously conducted a series of combination experiments involving 4β-dPE A and various classes of antiretroviral drugs, which collectively form the backbone of standard ART regimens. To accomplish this, we combined 4β-dPE A with the entry inhibitor maraviroc (MVC), the nucleoside analogue reverse transcriptase inhibitors (NRTIs) lamivudine, emtricitabine, and abacavir, the nucleotide analogue reverse transcriptase inhibitor (NtRTI) tenofovir, the non-nucleoside analogue reverse transcriptase inhibitor (NNRTI) efavirenz, and the protease inhibitor (PI) ritonavir. These combinations were systematically evaluated using IC50/IC50 ratios to assess their combined inhibitory effect on HIV infection.
Our comprehensive analysis revealed a consistently strong synergistic anti-HIV effect across nearly all tested antiretroviral drug combinations in the presence of 4β-dPE A. This synergy was characterized by Combination Index (CI) values consistently below 0.70 for all tested concentrations, unequivocally indicating that the combined administration significantly diminished the concentrations of each drug required to inhibit HIV infection. This finding is highly advantageous, as it suggests that lower doses of individual drugs could be used, potentially mitigating side effects and improving tolerability. However, it is important to note two specific exceptions: the combination of 4β-dPE A with efavirenz showed only a weak synergism, with a CI of 0.78, indicating a more additive than synergistic interaction. Additionally, the combination with 4β-dPE A and maraviroc exhibited less synergy at higher effective concentrations (e.g., at ED90), suggesting that while beneficial, the magnitude of synergy may vary depending on the specific drug and the desired level of viral inhibition. Overall, these results are highly encouraging, suggesting that 4β-dPE A can be effectively integrated into existing ART regimens to potentially enhance their overall antiviral efficacy.
Antiretroviral Drugs Did Not Modify The Reactivation Capacity Of 4β-dPE A
To thoroughly investigate whether the presence of antiretroviral drugs might interfere with the crucial latency-reversing capacity of 4β-dPE A, a focused single-concentration combination experiment was meticulously designed and executed. The concentrations for this experiment were carefully pre-selected: we utilized the EC90 (the concentration yielding 90% of the maximal effect) derived from our earlier LRA reactivation experiments for both 4β-dPE A and PMA, and the IC90 (the concentration yielding 90% inhibition) obtained from our previous infection experiments for the antiretroviral drugs. It is noteworthy that antiretroviral drugs alone exhibited no discernible viral reactivation effect, confirming their specific mechanism of action targets active replication rather than latency.
Our detailed analysis revealed no statistically significant differences between the rate of viral reactivation achieved by PMA or 4β-dPE A when administered alone, and the rate of reactivation observed when either was combined with an antiretroviral drug. This critical finding indicates that the presence of common antiretroviral medications does not diminish or antagonize the robust latency-reversing activity of 4β-dPE A. While a slight, but statistically non-significant, tendency for abacavir to increase the viral reactivation exerted by both PMA and 4β-dPE A was noted, this observation does not detract from the overall conclusion. Therefore, these results provide strong evidence that the efficacy of 4β-dPE A in reactivating latent HIV *in vivo* should not be adversely affected or altered by the concurrent antiretroviral treatment, a crucial consideration for its potential clinical application in the “shock and kill” strategy.
4β-dPE A Does Not Induce Cell Transformation
A significant and long-standing concern surrounding the clinical application of Protein Kinase C (PKC) agonists, particularly phorbol esters, is their well-documented potential for tumor-promoting activity. However, 4β-dPE A, in contrast to the widely recognized tumor-promoter PMA, notably lacks a hydroxyl group at position 4. Historically, this hydroxyl moiety was thought to be essential for potent PKC activation. More critically, 4β-dPE A also lacks the characteristic long and lipophilic side chains that are widely acknowledged as a defining structural feature of tumorigenic phorbol esters. This structural difference strongly suggests that 4β-dPE A may exert a distinct intracellular activity profile compared to its tumorigenic counterparts. To definitively address the critical question of 4β-dPE A’s potential tumor-promoting activity, we conducted rigorous transforming cell assays using NIH 3T3 fibroblasts, a standard *in vitro* model for assessing cellular transformation.
Our experimental results clearly demonstrated that both 4β-dPE A and prostratin were entirely unable to induce the formation of transforming foci in NIH 3T3 fibroblasts, which are characteristic morphological hallmarks of cellular transformation and oncogenic potential. This stands in stark contrast to our positive control, the oncogene KrasV12 (introduced via transfection), which robustly induced pronounced cellular transformation and extensive foci formation, serving as a clear benchmark for a transforming agent. Furthermore, while PMA treatment did induce foci formation, its effect was markedly less pronounced than that observed with KrasV12, reinforcing its known, albeit often context-dependent, tumor-promoting properties. The unequivocal absence of transforming foci induced by 4β-dPE A in this assay provides compelling evidence that, unlike certain other phorbol esters, 4β-dPE A does not possess tumor-promoting activity, a highly favorable characteristic that greatly enhances its safety profile and potential for clinical development as a latency reversing agent.
4β-dPE A Reactivates HIV-1 In CD4+ T Cells From Infected Patients Receiving ART
While *in vitro* cell lines and transfected PBMCs serve as valuable initial models for evaluating the transcriptional activity of compounds, they do not fully replicate the complex physiological context of latent HIV infection in patients. Although Jurkat 5.1 LTR-Luc and TZM-bl cells contain an integrated copy of the HIV-1 LTR, these are not truly latently infected primary cells. Therefore, to definitively confirm that 4β-dPE A is capable of activating HIV-1 transcription in genuine latently infected T cells, we conducted a critical ex vivo experiment. CD4+ lymphocytes were isolated from three HIV-infected patients who were stable on antiretroviral therapy (ART). These purified CD4+ T cells were then treated with a fixed and effective concentration of 100 nM of 4β-dPE A and compared to treatment with the same concentration of PMA, as well as an untreated control. In this experimental setup, the effect of the compounds is directly assessed within a population of cells that inherently harbor latent virus, as active viral replication is suppressed by the ongoing ART regimen.
Our compelling results demonstrated that 4β-dPE A effectively reactivated HIV from these latently infected CD4+ T cells obtained directly from patients, at a concentration of 100 nM. The levels of viral reactivation achieved by 4β-dPE A were comparable to those induced by PMA, a well-established and potent reactivating agent. This crucial finding strongly suggests that 4β-dPE A possesses the inherent capacity to induce a significant reactivating effect on the viral reservoir *in vivo*, representing a critical step towards validating its therapeutic potential in a clinically relevant setting. The ability of 4β-dPE A to awaken dormant virus in patient-derived cells, even in the presence of ART, underscores its promise as a candidate drug for strategies aimed at reducing or eradicating the persistent HIV reservoir.
Discussion
The ongoing quest for novel therapeutic agents to address challenging diseases like cancer and HIV-1 latency has significantly benefited from the identification of potent natural or synthetic Protein Kinase C (PKC) agonists that crucially lack undesirable tumor-promoting or excessive cellular proliferative activities. Our present study contributes significantly to this endeavor by demonstrating that 4β-dPE A (specifically, 12-O-Tigloyl-13-O-isobutyroyl-20-hydroxyl-4β-deoxyphorbol), a structurally distinct 4-deoxyphorbol ester derivative isolated from the plant species *Euphorbia amygdaloides subspecies semiperfoliata*, exhibits a remarkable capacity to reactivate latent HIV-1 *ex vivo*.
Our comprehensive investigation revealed that 4β-dPE A exerts a dual, impactful influence on the complex HIV replication cycle. Firstly, it robustly inhibits HIV infection, a property previously reported. Secondly, and equally important in the context of latency reversal, it potently induces HIV transcriptional activation. These dual effects are not entirely unique to 4β-dPE A, as similar activities have been well-described for other prominent PKC agonists, including phorbol 12-myristate 13-acetate (PMA), bryostatin, prostratin, and SJ23B. However, a critical distinction lies in their safety profiles. While PMA, a phorbol diester, is a potent inducer of HIV-1 activation, its significant tumor-promoting activity severely restricts its therapeutic applicability. In contrast, prostratin, bryostatin, and SJ23B, despite also being PKC agonists, notably lack the tumorigenic activity characteristic of certain phorbol esters like PMA. Bryostatin, in particular, is chemically distinct from phorbol esters, being a polyacetylated macrolactone initially developed as an anti-tumoral agent. It has been hypothesized that the anti-tumor activity of some PKC agonists might be linked to their capacity to induce sustained, high levels of PKC activation, leading to the eventual degradation of PKC by the proteasome. Conversely, natural ligands such as diacylglycerol (DAG) or phorbol esters possessing long side chains, like PMA, may be inactivated before PKC degradation occurs, thereby promoting prolonged cellular growth and potentially contributing to tumorigenesis. Furthermore, prior research has shown that phorbol esters with extended acyl chains, like PMA, induce a distinct pattern of intracellular PKC translocation, an activity that rapidly diminishes with the shortening of the acyl side chain.
Given this understanding, it is biologically plausible that 4β-dPE A, much like prostratin, and due to its lack of a long side chain, would not exhibit tumor-promoting activity. Our experimental data strongly support this hypothesis. We observed no long-term toxicity after a two-week treatment of PBMCs, and the definitive transformation assay confirmed the complete absence of tumor-promoting activity. Moreover, short-term *in vitro* toxicity experiments further substantiated the low cytotoxicity of 4β-dPE A, as its half-maximal cytotoxic concentration (CC50) was not reached even at concentrations as high as 10 µM in both PBMCs and MT-2 cells. This favorable toxicity profile translates into a remarkably high specificity index for HIV inhibition, exceeding 1500 for MT-2 cells and 30,000 for PBMCs, underscoring its broad therapeutic window.
Operating within these non-toxic concentrations, we meticulously investigated the effect of 4β-dPE A on the HIV-1 replication cycle. Initially, we assessed its HIV-1 inhibitory activity in MT-2 cells and IL-2 activated PBMCs. The IC50 values for 4β-dPE A were found to be remarkably low, ranging between 0.3 and 0.6 nM. While PMA and bryostatin displayed IC50 values within a similar range in MT-2 cells, they were marginally more potent in PBMCs (0.18 nM and 0.17 nM respectively, compared to 0.069 nM and 0.032 nM in MT-2 cells). Crucially, 4β-dPE A proved to be 200–600 times more potent than prostratin as an HIV infection inhibitor (with prostratin’s IC50s at 132.6 nM in MT-2 cells and 204.6 nM in PBMCs), and 100–140 times more potent than vorinostat (with vorinostat’s IC50s at 68.25 nM in MT-2 cells and 43.41 nM in PBMCs).
This potent anti-HIV activity of 4β-dPE A is, at least in part, attributable to its ability to induce the down-regulation of crucial HIV entry receptors: CD4, CXCR4, and CCR5. Our data from VSV-HIV infection experiments and direct receptor down-regulation assays clearly support this mechanism. These findings indicate that 4β-dPE A is at least as effective as SJ23B in inducing receptor down-regulation and significantly more effective than prostratin, highlighting a robust mechanism for inhibiting viral entry.
Among the various PKC agonists explored for HIV anti-latency, only bryostatin has progressed to clinical trials, although the outcomes were largely disappointing. The precise reasons for this clinical failure remain somewhat unclear, but the *in vivo* toxicity of bryostatin is a strong potential factor. Indeed, clinical assays involving bryostatin were prudently designed with the lowest possible doses to circumvent the potential toxicity inherent to PKC agonists. However, these trials ultimately showed no discernible effect on the transcription of latent HIV, likely due to, as the authors themselves suggested, the low plasma concentrations achieved at these minimal doses. Nevertheless, as noted in our introduction, the distinct chemical structure of bryostatin, completely unrelated to phorboids, raises the possibility that its *in vivo* toxicity might stem from interactions with molecular targets unrelated to PKCs, thus not necessarily reflecting a class effect for all PKC agonists.
Consequently, our study thoroughly investigated the effect of 4β-dPE A as a transcriptional reactivator and a bona fide anti-latency drug. Our data unequivocally demonstrate that 4β-dPE A is an exceptionally powerful HIV transcriptional activator, exhibiting *in vitro* activity at remarkably low nanomolar concentrations across a range of cell lines, including MT-2, TZM-bl, Jurkat 5.1 LTR-Luc, and crucially, in resting PBMCs. Specifically, a concentration of 100 nM was consistently sufficient to induce HIV transcription at levels often surpassing those achieved by PMA at the same concentration. In resting PBMCs, 4β-dPE A displayed an EC50 of 2.9 nM, placing it in a similar potency range as bryostatin (3.4 nM) but approximately 100-fold more potent than PMA (0.031 nM).
Furthermore, 4β-dPE A proved to be a significantly more potent transcriptional activator than both vorinostat and prostratin, with EC50 values approximately 250-fold and 1600-fold lower, respectively. This confirms the high potency observed in our earlier infection experiments and reinforces its impressive specificity, as reflected by a specificity index greater than 10,000.
The canonical targets of this class of compounds are PKCs. Classical PKCs (cPKCs) and novel PKCs (nPKCs) possess two tandem C1 domains (C1a and C1b) in their N-terminal region, which exhibit high binding affinities for diacylglycerol (DAG), phorbol esters, and other PKC activators. Intriguingly, while defined phorbol ester binding sites in the PKC C1 domain have been described, often involving a triad of oxygenation including the 4-hydroxyl moiety, our findings with 4β-dPE A demonstrate that the oxygen at position 4 is not strictly required for robust HIV activating effects.
Our results strongly indicate that the viral transcriptional effect of 4β-dPE A is dependent on the activation of the novel PKCθ isoform and the mitogen-activated protein kinase MEK. Previous studies have underscored the critical importance of PKCθ in T lymphocytes, which prompted further investigation into its activation by immunofluorescence. The role of phorbol esters in PKCθ activation and their subsequent anti-HIV latency activity has been extensively documented. While the effect of MEK inhibition on general PKC activity has been described, less is known about its direct involvement in HIV latency reactivation. MEK proteins, a family of intracellular dual kinases, activate ERK/MAPK in response to diverse stimuli, including IL-2 and phorbol esters.
This pathway’s activation is profoundly involved in T-cell proliferation and activation. Therefore, the activation of the PKCθ/MEK pathway is an indispensable requirement for the transcriptional activity, and consequently, the anti-latency property, of 4β-dPE A. Conversely, our data demonstrate that the receptor down-regulation effect, which underpins 4β-dPE A’s anti-HIV activity, appears to be entirely independent of the PKC/MEK pathway. This striking mechanistic dissociation strongly suggests that 4β-dPE A exerts a dual targeting effect in T cells: one involving PKCθ/MEK for transcriptional activation, and another involving a still-unidentified cellular target responsible for inhibiting HIV infection. It is plausible that different chemical moieties within the 4β-dPE A structure contribute to these distinct activities.
We further explored the molecular details of 4β-dPE A’s transcriptional activation mechanism. Our findings reveal that 4β-dPE A potently induces the transactivation of the NF-κB transcription factor. The HIV LTR contains critical binding sites for NF-κB, and its activation is absolutely essential for the initiation of transcription from latent HIV DNA. These effects are mechanistically similar to those exerted by prostratin and SJ23B. However, 4β-dPE A stands out as a highly potent activator of HIV transcription, requiring concentrations similar to bryostatin and notably lower than other phorbol esters in all tested scenarios. In fact, our results indicate that 4β-dPE A is also more powerful than HDAC inhibitors like vorinostat, suggesting its potential utility at lower clinical doses.
Previous research has consistently indicated that anti-latency treatment approaches could be significantly more efficient through the strategic combination of different drugs. While some clinical assays have explored antibody-LRA combinations, drug-drug combinations have been less extensively investigated. Conceptually, combinations of drugs targeting distinct mechanisms or pathways should ideally yield synergistic effects. This theoretical advantage is powerfully exemplified by the combination of PKC agonists, such as prostratin or 4β-dPE A, with an HDAC inhibitor like vorinostat. Our results definitively show that these combinations are strongly synergistic, in stark contrast to the pronounced antagonism observed when two PKC agonists, 4β-dPE A and prostratin, are combined. This critical distinction reinforces the principle that combining LRAs with non-overlapping targets leads to synergy, enabling the use of lower doses of individual compounds while achieving sufficient activation to reduce the size of the latent reservoir. Moreover, the combination of 4β-dPE A and vorinostat not only synergistically lowered the EC50 values but also impressively enhanced their overall transcriptional efficacy, nearly doubling the effect observed with either drug alone. This suggests that such a combination could simultaneously achieve improved potency and greater efficacy in reactivating the viral reservoir.
Considering that latency reversing therapy is envisioned to be co-administered with existing ART in infected patients, the potential implications of LRAs warrant careful consideration. A primary concern revolves around potential toxicity from LRA treatment itself, particularly in patients who already maintain excellent viral control and quality of life on ART. Additionally, the possibility of an antagonistic interaction between LRAs and ART is a significant pharmacological concern that, to our knowledge, has not been thoroughly assessed in many studies.
In this article, we have rigorously analyzed the impact of 4β-dPE A on anti-HIV activity when combined with various families of antiretroviral drugs. Our results consistently demonstrate a strong synergism as HIV infection inhibitors across a broad spectrum of ART classes, including nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs/NtRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), and even with the CCR5 antagonist maraviroc. This synergistic effect is mechanistically plausible for NRTIs, NNRTIs, and PIs, given that 4β-dPE A down-regulates CD4, CXCR4, and CCR5 receptors, thereby inhibiting HIV-1 entry and consequently reducing the rate of T-cell infection.
Although the down-regulation of CCR5 could, in theory, interfere with maraviroc activity, we observed no antagonism between 4β-dPE A and maraviroc in our experiments, effectively ruling out this potential negative interaction. Furthermore, a crucial finding was that antiretroviral drugs did not interfere with the transcriptional activity of the PKC agonists PMA and 4β-dPE A, indicating that the efficacy of 4β-dPE A *in vivo* should not be compromised by concurrent antiretroviral treatment.
Finally, the primary reservoirs of latent HIV are understood to be long-lived resting memory CD4+ T cells. This reservoir forms early in the infection and harbors integrated proviral DNA. The “shock” therapy strategy aims to specifically activate these quiescent cells to render them visible to the host immune system or to a “kill” therapy. To directly assess the reactivating effect of 4β-dPE A in this clinically relevant context, we treated CD4+ T cells isolated from HIV-infected patients undergoing ART with 4β-dPE A. As our findings clearly illustrate, 4β-dPE A effectively reactivated HIV from these patient-derived CD4+ T cells at a concentration of 100 nM. This direct evidence from patient samples strongly suggests that 4β-dPE A has the potential to exert a powerful reactivating effect on the viral reservoir *in vivo*.
In summary, our comprehensive investigation demonstrates that 4β-dPE A, a novel 4-deoxyphorbol derivative, exhibits dual therapeutic potential. It effectively inhibits HIV infection through the down-regulation of CD4, CXCR4, LXS-196, and CCR5 receptors. Simultaneously, it potently reactivates latent HIV in resting PBMCs through the activation of PKCθ/MEK and NF-κB signaling pathways. Crucially, this latter effect of transcriptional activation appears to be independent of receptor down-regulation, as inhibition of PKC/MEK pathways does not modify the receptor expression inhibition induced by 4β-dPE A, suggesting the involvement of distinct cellular targets for these two activities. Moreover, 4β-dPE A displays potent synergism when combined with the HDAC inhibitor vorinostat as an LRA, and also with NRTIs, NNRTIs, and PIs as an HIV inhibitor, underscoring its compatibility and potential to enhance existing ART. Finally, the demonstrated activity of 4β-dPE A in CD4+ T cells from ART-treated infected patients further validates its therapeutic potential in a more realistic clinical scenario. Therefore, 4β-dPE A represents a promising new deoxyphorbol ester derivative with powerful activity as both an anti-latency agent and an HIV infection inhibitor, positioning it as a strong drug candidate for strategies aimed at eradicating HIV reservoirs and ultimately achieving a functional cure.