HE a , Jiménez R a , Bueno P a , Camarero S a , Román L a , Fernández-García De La Torre

Jl

Nothias Lf

Bedoya Lm
email: lmbedoya@ucm.es

4-deoxyphorbol inhibits HIV-1 infection and reactivates viral reservoirs through PKC/MEK activation synergizing with vorinostat and antiretroviral drugs

Keywords: HIV-1, latency reversing agents, proteinkinases C, viral reactivation, phorbol 5 esters HIV-1, latency reversing agents, proteinkinases C, viral reactivation, phorbol 5 esters

Latent HIV reservoirs are the main obstacle to eradicate HIV infection. One strategy 2 proposed to eliminate these viral reservoirs is the pharmacological reactivation of 3 latently infected T cells. We show here that a 4-deoxyphorbol ester derivative isolated 4 from Euphorbia amygdaloides ssp. semiperfoliata, 4β-dPE A, reactivates HIV-1 from 5 latency and could potentially contribute to decrease the viral reservoir. 4β-dPE A shows 6 two effects in the HIV replication cycle, infection inhibition and HIV transactivation, 7 similarly to other phorboids agonists of PKCs such PMA and prostratin and to diterpene 8 esters such SJ23B. 4β-dPE A lacks the long side chain present in PMA, suggesting a 9 non-tumour promoting activity, as our results indicate. 4β-dPE activates HIV 10 transcription at nanomolar concentrations, lower than the concentration needed other 11 latency reversing agents (LRAs) such as prostratin or bryostatin. PKC/MEK activation 12 is required for the transcriptional activity, and thus, anti-latency activity of 4β-dPE A. 13 However, CD4, CXCR4 and CCR5 receptors down-regulation effect seems to be 14 independent of PCK/MEK, suggesting the existence of at least two different targets for 15 4β-dPE A. Further, NF-b transcription factor is involved in 4β-dPE HIV reactivation, as 16 previously shown for other PKCs agonists. We also studied the effects of 4β-dPE A in 17 combination with other LRAs. When 4β-dPE A was combined with another PKC 18 agonists such as prostratin an antagonist effect was achieved, while, when combined 19 with an HDAC inhibitor such as vorinostat, a strong synergistic effect was obtained. 20 Interestingly, the latency reversing effect of the combination was synergistic diminishing 21 the EC 50 value but also increasing the efficacy showed by the drugs alone. Finally, 22 combination of 4β-dPE A with antiretroviral drugs as CCR5 antagonist, NRTIs, NNRTIs and PIs, showed a consistent synergistic effect, suggesting that the combination would 1 not induce undesirable effects on antiretroviral therapy (ART). In summary, we describe 2 here the mechanism of action of a new potent deoxyphorbol derivative as a latency 3 reversing agent candidate to decrease the size of HIV reservoirs.

4

INTRODUCTION

1 Antiretroviral therapy (ART) has significantly decreased mortality attributable to AIDS, 2 reducing the viral load to undetectable levels in infected individuals and delaying 3 disease progression. However, the persistence of latent HIV reservoirs, particularly in 4 resting memory T CD4+ cells, is a major obstacle to HIV eradication. Although resting T 5 cells do not allow viral replication they are a source of HIV when activated [1]. This is 6 also true for other pathogens, such as Ebola virus, found to persist in the so-called sites 7 of immune-privilege [2,3], or hepatitis B virus, human papillomavirus and most 8 herpesvirus, that persist as latent infections using different mechanisms [4].

9

ART acts through interference with different stages of HIV replication cycle but most of 10 antiretrovirals tackle viral proteins so they are unable to eliminate viral reservoirs. Thus, 11 the search for new classes of antiretroviral drugs continues to be indispensable, 12 because of ART long-term toxicity [5] and drug resistances [6]. Treatment with 13 molecules able to induce HIV-1 transcription in resting T cells is a strategy of reservoir 14 elimination named "shock and kill'' or "kick and kill". This strategy aims to tackle the 15 latent reservoir stimulating viral replication in latently infected CD4+ T cells, while 16 blocking the spread of infection with antiviral therapy [7]. 17

The latency-reversing agents (LRAs) or anti-latency drugs will be the "shock" of this 18 strategy. There are several kinds of LRAs including, epigenetic modifiers, TLR agonists,

19

TCR activators, PI3K/Akt pathway modulators, NF-B agonists, PKC agonists and 20 HDAC inhibitors [8]. PKCs agonists (prostratin or bryostatin) and HDAC inhibitors 21 (vorinostat, panobinostat or romidepsin) are the most studied LRAs. Moreover, some clinical studies have been performed with these drugs. Vorinostat [9], bryostatin [10],

1 romidepsin [11] and TLR7 agonists [12] have been tested in clinical trials, but the results 2 obtained were not convincing. Moreover, a cure may require an effective "kill" strategy 3 beyond the immunological response, and some efforts have been made involving 4 immune system modulation [13]. These studies include a vaccine based "kill", as the 5 romidepsin (shock) plus a therapeutic vaccine Vacc-4x and rhGM-CSF (kill) study,

6
showing a slight but non-significant reduction of the reservoir size [14], or the RIVER 7 study, including an LRA as the "shock" (vorinostat) and two immunomodulatory vaccines 8 as the "kill", but results were also disappointing [15]. There could be several causes for 9 this failure: the method of reservoir quantification underestimates or overestimates its 10 size [16], the doses used in the assay were too low to avoid potential toxicity or the low 11 efficacy of the drugs selected. Recently, CAR T cells engineered to bind broadly 12 neutralizing anti-HIV antibodies have been found to reduce the size of the viral 13 reservoirs of the blood of infected individuals on antiretroviral therapy, although this 14 results are too preliminary [17]. 15 PKCs agonist can activate some isoforms of protein kinases C (PKCs) by binding to its 16 regulatory C1 domain, mimicking the action of the physiological ligand diacylglycerol 17 (DAG) and inducing viral reactivation. Prostratin was the first PKC agonist studied as a 18 potential anti-latency agent [18]. Bryostatin, another PKC agonist, showed lack of 19 efficacy in the reduction of the latent reservoir in clinical trials, probably due to the low 20 doses used in the assay to avoid toxicity since systemic concentrations were found 21 undetectable [10,19]. In this sense, some diterpenes and phorboids from 22 Euphorbiaceae have also been described as LRAs with antiviral properties and Arenzana-Seisdedos from Pasteur Institute [27]. The 3-enh-B-ConA-luc plasmid carries 20 a luciferase gene under the control of three synthetic copies of the B consensus of the 21 immunoglobulin k-chain promoter cloned into the BamHI site located upstream from the 22 conalbumin transcription start site [28]. The LTR-Luc plasmid carries a luciferase gene 23 under the control of HIV-1-LTR region [29]. The expression construct pNFAT-LUC 1 containing three tandem copies of the distal NFAT-binding site of the IL-2 gene promoter 2 coupled to the IL-2 minimal promoter was kindly provided by Dr. Juan Miguel Redondo 3 (National Center for Cardiovascular Research, Madrid, Spain) [30]. receptors by PKCs agonists such as prostratin [32] and SJ23B [20]. Therefore, As expected, 4β-dPE A down-regulates CD4, CXCR4 and/or CCR5 expression in MT-2 2 cells and IL-2 activated PBMCs, in a concentration-dependent manner. 4β-dPE A 1nM 3 was not effective, but 10 and 100 nM concentrations were enough to down-regulate 4 receptors with a pattern of down-regulation similar to that as PMA at 100 nM (Figure 2). The very low number of latently HIV-infected cells in vivo makes purification and 7 biochemical analysis of these cells hard to achieve. Moreover, several studies show that 8 in vitro assays used to quantify the reservoir from PBMCs display a large range 9 depending on the technique used [33]. In this work, we have used a model based on HIV-1 reactivation with low concentrations (Figure 3). transcriptional activity of 4β-dPE A was also evaluated (Figure 4). is not related to the down-regulation of receptors exerted by 4β-dPE A, and thus, to the 22 anti-HIV activity. Further, the differences of receptors expression between PKC inhibitor 23 treated cells, MEK inhibitor treated cells and cell treated with no inhibitor were not 1 significant, suggesting that this inhibitors did not affect the receptor expression by 2 themselves (Figure 5).

Antiretroviral drugs did not modify the reactivation capacity of 4β-dPE A 22

To test whether antiretroviral drugs could have an effect on the reactivation capacity of Concentrations were selected as follows: we have used the EC 90 obtained from Since the LRA treatment is designed to be administered together with ART, we 13 evaluated the effect of 4β-dPE A in the antiviral effect of antiretroviral therapy. inhibitor (PI) ritonavir were combined with 4β-dPE A using IC 50 /IC 50 ratios (Figure 10). We have found that the anti-HIV effect of all the antiretroviral drugs tested in the 1 presence of 4β-dPE A showed a strong synergism with CIs below 0.70 for all the 2 concentrations tested. However, the combination 4β-dPE A/efavirenz showed only a 3 weak synergism with a CI of 0.78 and the combination with 4β-dPE A/maraviroc was 4 less synergic when concentrations were higher (ED90) (Figure 10). However, 4β-dPE A lacks a key hydroxyl that was thought to be required for PKC 9 activation although nothing is known about its involvement in tumour-promoting activity. We found that both 4β-dPE A and prostratin were unable to elicit transforming foci, while 16 transfection with KrasV12 strongly induced cell transformation. Further, PMA treatment 17 induced foci formation but to a lesser extent than KrasV12 (Fig. 11). reported previously by us [24] and secondly it induces HIV transcriptional activation.

6

These effects have been described for other PKCs agonists such as phorbol 12-7 myristate 13-acetate (PMA), bryostatin [35], prostratin [32] or SJ23B [20]. However, 8 PMA, a phorbol diester, induced activation of HIV-1, but its potent tumor promoting 9 activity turns its therapeutic use unacceptable [36]. Prostratin, bryostatin and SJ23B are 10 PKCs agonists, analogs of diacylglicerol (DAG), but they lack the tumorogenic activity of In this paper, we show that 4β-dPE A is a powerful HIV reactivation agent, with in vitro 14 active concentrations in the low nM range in resting PBMCs. Indeed, 100 nM 15 concentration in both cell lines was enough to induce HIV transcription at higher levels 16 than PMA at the same concentration. In PBMCs resting, just 10 nM was enough to 17 reach the same level of HIV reactivation than PMA at 100nM. Moreover, 4β-dPE A was 18 also at least 20 times more powerful than prostratin as an HIV infection inhibitor [24] and 19 this anti-HIV activity is due to the down-regulation of CD4, CXCR4 and CCR5, as 20 expected for a PKC agonist. These data showed that 4β-dPE A is at least as effective as 21 SJ23B in receptor down-regulation and more effective than prostratin.

On the other hand, 4β-dPE A was also more powerful as transcriptional reactivator than 1 vorinostat and prostratin with EC 50 values 70 and 480 fold lower, respectively, 2 confirming the high potency showed previously in the infectious experiments and its high 3 specificity also as LRA since its specificity index is greater than 1000 (Figure 8). The These pharmacophores included a triad of oxygenation including the 4-hydroxyl moiety 9 [38]. However, we show here that 4β-dPE A does not need the oxygen in position 4 to 10 display a strong PKC activating effect (figure 12). Our results shown that the viral transcriptional effect of 4β-dPE A depends on the 13 activation of the novel PKC and the mitogen-activated protein kinase kinase MEK 14 (figure 4). Previous literature showed the importance of PKC in T lymphocytes [34] and 15 thus, the PKC activation was further confirmed by immunofluorescence (figure 6). The 16 effect of phorbol esters on PKC and its HIV anti-latency activity have been widely 17 described. On the other hand, the effect of MEK inhibition in PKC activity has been 18

previously described [39], but less is known about its involvement in latency reactivation.

22

PKC/MEK activation is required for the transcriptional, and thus, anti-latency activity of 4β-dPE A. On the other hand, receptors down-regulation, and thus, anti-HIV activity, 1 seems to be independent of PCK/MEK (figure 5). This fact suggests a dual target of 4β- and cell lines, and did not allow EC 50 calculation (figure 1).

18

Finally, it has been previously described that anti-latency treatment could be more 19 efficacious with combinations of different drugs. However, in clinical assays only 20 antibody-LRA combinations have been tested [21,22] unlike drug/drug combinations.

21

Combination of drugs with different targets would be, hypothetically, synergistic. This is 22 the case of the combination of PKCs agonists such as prostratin or 4β-dPE A with an 23 HDAC inhibitor as vorinostat. In fact, our results show that these combinations are 1 strongly synergistic, while the combinations of two PKCs agonists such as 4β-dPE A 2 and prostratin showed a strong antagonism. Therefore, the combination of LRAs with 3 different targets would result in a synergism able to diminish the doses used to obtain 4 enough activation to reduce the reservoir size. Moreover, the combination of 4β-dPE A 5 and vorinostat was synergistic not only lowering EC 50 s values but also increasing the 6 Emax (figure 8). Thus, the combination of these two drugs would result in the possibility because of ART long-term toxicity [5] and drug resistances [6]. Treatment with 13 molecules able to induce HIV-1 transcription in resting T cells is a strategy of reservoir 14 elimination named "shock and kill'' or "kick and kill". This strategy aims to tackle the 15 latent reservoir stimulating viral replication in latently infected CD4+ T cells, while 16 blocking the spread of infection with antiviral therapy [7].

17

The latency-reversing agents (LRAs) or anti-latency drugs will be the "shock" of this 18 strategy. There are several kinds of LRAs including, epigenetic modifiers, TLR agonists,

19

TCR activators, PI3K/Akt pathway modulators, NF-B agonists, PKC agonists and 20 HDAC inhibitors [8]. PKCs agonists (prostratin or bryostatin) and HDAC inhibitors 21 (vorinostat, panobinostat or romidepsin) are the most studied LRAs. Moreover, some clinical studies have been performed with these drugs. Vorinostat [9], bryostatin [10],

1 romidepsin [11] and TLR7 agonists [12] have been tested in clinical trials, but the results 2 obtained were not convincing. Moreover, a cure may require an effective "kill" strategy 3 beyond the immunological response, and some efforts have been made involving 4 immune system modulation [13]. These studies include a vaccine based "kill", as the 5 romidepsin (shock) plus a therapeutic vaccine Vacc-4x and rhGM-CSF (kill) study,

6
showing a slight but non-significant reduction of the reservoir size [14], or the RIVER 7 study, including an LRA as the "shock" (vorinostat) and two immunomodulatory vaccines 8 as the "kill", but results were also disappointing [15]. There could be several causes for 9 this failure: the method of reservoir quantification underestimates or overestimates its undetectable [10,19]. In this sense, some diterpenes and phorboids from 22 Euphorbiaceae have also been described as LRAs with antiviral properties and 23 reactivation activity. However, slight differences in their structure lead to lack of activity 1 or a PKC isoform preference, modulating their reservoir reactivating activity [20]. and to achieve cell activation enough to eliminate the reservoirs [21,22]. In this sense, it 9 is important to point out that this therapy is intended to be used in combination with 10 ART. Some previous reports have shown that antiretroviral drugs could modulate the 11 LRAs activity and, conversely, LRAs can affect the antiviral activity of ART. For example, 12 some protease inhibitors diminish the viral reactivation obtained with LRAs [23]. 13 We have reported previously the antiviral activity of 4-deoxyphorbol derivative 14 compounds isolated from Euphorbia amygdaloides ssp. semiperfoliata [24]. In the 15 present study, the mechanism of action and cellular pathways involved in the latency- A strong inhibition of viral replication was observed at nanomolar concentrations, with an 1 IC 50 value of 6.51 nM. Further, when IL-2 activated PBMCs were infected under the 2 same conditions with X4 or R5 tropic HIV, a stronger inhibition of viral replication was 3 obtained, with IC 50 values of 0.31 and 0.24 nM, respectively (Figure 1). Cell death was not observed at the concentrations tested in both, the infection assay 6 and the cytotoxicity assay. Moreover, 4β-dPE A CC 50 was higher than 10,000 nM ( 107 µM) in PBMCs and MT-2 cells (Figure 1). While 4β-dPE A, prostratin and vorinostat 8 showed no cell toxicity in MT-2 cells and PBMCs at 10.000 nM, a similar pattern of cell 9 toxicity were found for romidepsin and panobinostat, although panobinostat only showed 10 PBMCs toxicity at concentrations close to 10.000 nM. Since toxicity is a cause of 11 concern for anti-latency drugs, we further evaluated 4β-dPE A toxicity in a long-term 12 viability assay (14 days) in IL-2 activated PBMCs at concentrations of 0.1, 1 and 10 µM.

13

Cell viability was determined at different times along treatment. As observed in figure 1, 14 4β-dPE A was not toxic to human PBMCs even at 14 days of treatment. receptors by PKCs agonists such as prostratin [32] and SJ23B [20]. Therefore, As expected, 4β-dPE A down-regulates CD4, CXCR4 and/or CCR5 expression in MT-2 2 cells and IL-2 activated PBMCs, in a concentration-dependent manner. 4β-dPE A 1nM 3 was not effective, but 10 and 100 nM concentrations were enough to down-regulate 4 receptors with a pattern of down-regulation similar to that as PMA at 100 nM (Figure 2). The very low number of latently HIV-infected cells in vivo makes purification and 7 biochemical analysis of these cells hard to achieve. Moreover, several studies show that 8 in vitro assays used to quantify the reservoir from PBMCs display a large range 9 depending on the technique used [33]. In this work, we have used a model based on is not related to the down-regulation of receptors exerted by 4β-dPE A, and thus, to the 22 anti-HIV activity. Further, the differences of receptors expression between PKC inhibitor 23 treated cells, MEK inhibitor treated cells and cell treated with no inhibitor were not 1 significant, suggesting that this inhibitors did not affect the receptor expression by 2 themselves (Figure 5).

Antiretroviral drugs did not modify the reactivation capacity of 4β-dPE A 22

To test whether antiretroviral drugs could have an effect on the reactivation capacity of Concentrations were selected as follows: we have used the EC 90 obtained from We have found that the anti-HIV effect of all the antiretroviral drugs tested in the 1 presence of 4β-dPE A showed a strong synergism with CIs below 0.70 for all the 2 concentrations tested. However, the combination 4β-dPE A/efavirenz showed only a 3 weak synergism with a CI of 0.78 and the combination with 4β-dPE A/maraviroc was 4 less synergic when concentrations were higher (ED90) (Figure 10). However, 4β-dPE A lacks a key hydroxyl that was thought to be required for PKC 9 activation although nothing is known about its involvement in tumour-promoting activity. We found that both 4β-dPE A and prostratin were unable to elicit transforming foci, while 16 transfection with KrasV12 strongly induced cell transformation. Further, PMA treatment 17 induced foci formation but to a lesser extent than KrasV12 (Fig. 11). reported previously by us [24] and secondly it induces HIV transcriptional activation.

6

These effects have been described for other PKCs agonists such as phorbol 12-7 myristate 13-acetate (PMA), bryostatin [35], prostratin [32] or SJ23B [20]. However, 8 PMA, a phorbol diester, induced activation of HIV-1, but its potent tumor promoting 9 activity turns its therapeutic use unacceptable [36]. Prostratin, bryostatin and SJ23B are 10 PKCs agonists, analogs of diacylglicerol (DAG), but they lack the tumorogenic activity of In this paper, we show that 4β-dPE A is a powerful HIV reactivation agent, with in vitro 14 active concentrations in the low nM range in resting PBMCs. Indeed, 100 nM 15 concentration in both cell lines was enough to induce HIV transcription at higher levels 16 than PMA at the same concentration. In PBMCs resting, just 10 nM was enough to 17 reach the same level of HIV reactivation than PMA at 100nM. Moreover, 4β-dPE A was 18 also at least 20 times more powerful than prostratin as an HIV infection inhibitor [24] and 19 this anti-HIV activity is due to the down-regulation of CD4, CXCR4 and CCR5, as 20 expected for a PKC agonist. These data showed that 4β-dPE A is at least as effective as 21 SJ23B in receptor down-regulation and more effective than prostratin.

On the other hand, 4β-dPE A was also more powerful as transcriptional reactivator than 1 vorinostat and prostratin with EC 50 values 70 and 480 fold lower, respectively, 2 confirming the high potency showed previously in the infectious experiments and its high 3 specificity also as LRA since its specificity index is greater than 1000 (Figure 8). The These pharmacophores included a triad of oxygenation including the 4-hydroxyl moiety 9 [38]. However, we show here that 4β-dPE A does not need the oxygen in position 4 to 10 display a strong PKC activating effect (figure 12). Our results shown that the viral transcriptional effect of 4β-dPE A depends on the 13 activation of the novel PKC and the mitogen-activated protein kinase kinase MEK 14 (figure 4). Previous literature showed the importance of PKC in T lymphocytes [34] and 15 thus, the PKC activation was further confirmed by immunofluorescence (figure 6). The 16 effect of phorbol esters on PKC and its HIV anti-latency activity have been widely 17 described. On the other hand, the effect of MEK inhibition in PKC activity has been 18

previously described [39], but less is known about its involvement in latency reactivation.

22

PKC/MEK activation is required for the transcriptional, and thus, anti-latency activity of 4β-dPE A. On the other hand, receptors down-regulation, and thus, anti-HIV activity, 1 seems to be independent of PCK/MEK (figure 5). This fact suggests a dual target of 4β- and cell lines, and did not allow EC 50 calculation (figure 1).

18

Finally, it has been previously described that anti-latency treatment could be more 19 efficacious with combinations of different drugs. However, in clinical assays only 20 antibody-LRA combinations have been tested [21,22] unlike drug/drug combinations.

21

Combination of drugs with different targets would be, hypothetically, synergistic. This is 22 the case of the combination of PKCs agonists such as prostratin or 4β-dPE A with an 23 HDAC inhibitor as vorinostat. In fact, our results show that these combinations are 1 strongly synergistic, while the combinations of two PKCs agonists such as 4β-dPE A 2 and prostratin showed a strong antagonism. Therefore, the combination of LRAs with 3 different targets would result in a synergism able to diminish the doses used to obtain 4 enough activation to reduce the reservoir size. Moreover, the combination of 4β-dPE A 5 and vorinostat was synergistic not only lowering EC 50 s values but also increasing the 6 Emax (figure 8). Thus, the combination of these two drugs would result in the possibility

23 the

 23 NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: pNL4-3.Luc.R -.E -from 18 Dr. Nathaniel Landau and pcDNA-VSV encoding the G protein of VSV from Dr.

19

 19

4

  Plasmids were amplified transforming E. coli DH5α cells and cultured in LB 5 supplemented with ampicillin at 37°C for 18 hours. All plasmids were purified using 6 Qiagen Plasmid Maxi Kit (Qiagen, Hilden, Germany) following the manufacturer 7 instructions. DNA concentration was determined in a Nanodrop (Thermoscientific, 8 Waltham, MA, USA).

9 10 JR 6 evaluation 7

 91067 Viruses -Ren (HIV-1 R5 tropic) and NL4.3-Ren (HIV-1 X4 tropic) viral supernatants were 11 obtained from transfection of pJR-Ren and pNL4.3-Ren plasmids. NL4.3-env-VSV-Luc 12 (HIV-1 with a luciferase reporter gene in the position of nef and expressing VSV G 13 protein instead of HIV-1 env gene) was obtained by co-transfection of pNL4.3-env-Luc 14 and pcDNA-VSV encoding the G protein of VSV. Calcium phosphate transfection of 15 plasmids in 293T cells was used to obtain viral supernatants. 16 Anti-HIV-1 activity evaluation by recombinant virus assay 17 The initial evaluation of 4β-dPE A was performed in MT-2 cells infected with NL4.3-Ren 18 obtained previously by transfection in 293T cells. The assay was performed in 96 well 19 microplate seeded with 100,000 cells/well in a final volume of 200 µl. Cell cultures were 20 infected with viral supernatants (100,000 RLUs/w) and after 48 hours cell pellets were 21 lysed and luciferase activity measured in a luminometre (Berthold detection systems, 22 Pforzheim, Germany). X4 (NL4.3-Ren) or R5 (JR-Ren) infections of IL-2 activated 1 PBMCs followed the same procedure. All the experiments were controlled with cells 2 treated with the same DMSO concentration of 4β-dPE A. HIV-1 replication inhibition was 3 evaluated by measuring the luminescence activity or RLUs, with 100% being the 4 infection of non-treated cells. IC 50 was calculated with a non-linear regression formula 5 using GraphPad Prism software. Toxicity Cell toxicity was evaluated by treating mock infected cells with the same concentrations 8 of 4β-dPE A used in the antiviral assays or with higher concentrations of 4β-dPE A and 9 other LRAs (panobinostat, romidepsin and prostratin) or its combination with LRAs or 10 antiretroviral drugs. After 48 hours in culture, CellTiter Glo reagent (Promega, Madison, 11 WI, USA) was added and cell viability measured by RLUs quantification in a 12 luminometre. Cell viability is expressed as percentage of viable cells compared to a non-13 treated (DMSO same concentration as compound) control (100%). Long-term toxicity 14 evaluation at different times was performed with 4β-dPE A in IL-2-activated-PBMCs for 15 14 days. CC 50 was calculated with a non-linear regression formula using GraphPad 16 Prism software.

17

 17

Receptor expression kinetics 18 Single 8 Immunofluorescence Assays 9 For 10 Slide 8 12 ( 18 Evaluation of viral transcription 19 MT- 2 11 MEK 19 ( 21 A

 18891081218192111921 -, double-or three-color immunophenotyping was performed with a FACScalibur 19 flow cytometer using CD4 (clone SK3), CXCR4 (clone 12G5) and CCR5 (clone 20 2D7/CCR5) antibodies conjugated to the appropriate fluorochrome, all obtained from 21 Becton Dickinson (Heidelberg, Germany). Background staining was assessed with the 22 appropriate isotype-and fluorochrome-matched control mAb and subtracted. Activated 1 PBMCs or MT-2 were treated with different concentrations of 4β-dPE A for 48, 24 and 2 2 hours. Cells were then collected and CD4, CXCR4 and/or CCR5 receptors expression 3 were evaluated. The effect of PKCs inhibitors Gö6850 (bisindolylmaleimide), rottlerin 4 and MEK inhibitor PD184352 was also studied in the expression of CD4, CXCR4 and 5 CCR5 receptors in IL-2 activated PBMCs. The analysis was performed using forward-6 versus-side scatter dot plots. Results are shown as percentage of receptor fluorescence 7 mean intensity (%MIF) obtained by FlowJo 8.0 software. immunofluorescence assays, Jurkatt E61 cells and PBMCs were incubated in a µwell ibiTreat (Ibidi GmbH) for 48h, in the presence or absence of PMA or 4β-dPE 11 A (100 nM). Then, they were permeabilized with the cell fixative CytoskelFix Cytoskeleton) for 10 min at room temperature. After washing twice with 0.1% 13 glycine/PBS, cells were incubated with phospho-PKCθ (Thr538) (Cell Signaling 14 Technology, Danvers, MA) and Alexa Fluor 546 goat anti-rabbit IgG (Molecular Probes) 15 antibodies. Washes were performed with 1X PBS-2% BSA-0.05% saponine buffer. 4′,6-16 diamidino-2-phenylindole (Dapi) was used for nuclear staining. Images were obtained 17 with Leica DMI 4000B Inverted Microscope (Leica Microsystems, Wetzlar, Germany). cells or resting PBMCs (in culture for at least 24 hours) were suspended in 350 µL 20 of RPMI without supplements and electroporated using an Easyject plus Electroporator 21 (Equibio, Middlesex, UK) at 260 (MT-2) or 320 (PBMCs) V, 1500 mF and maximum 22 resistance with 1µg/10 6 cells of a luciferase plasmid under the control of the whole 1 genome of HIV-1 (NL4.3-Luc). Afterwards MT-2, resting PBMCs or TZM-bl (non-2 transfected) cells were seeded in 24 well microplates and treated with different 3 concentrations of 4β-dPE A or PMA (0.1 µM), as a reference control of HIV-1 4 transactivation, and left in culture in complete RPMI at 37°C. 48 hours later, cultures 5 were lysed with luciferase buffer and RLUs measured in a luminometer. This procedure 6 was also used for the transfection of luciferase plasmids under the control of 7 transcription factors NFAT (NFAT-Luc) or NF-B (3-enh-B-ConA-luc) or the long 8 terminal repeat of HIV (LTR-Luc) in resting PBMCs to evaluate transcriptional activity.9The transcriptional activity of 4β-dPE A in resting PBMCs was also studied in the 10 presence of PKCs inhibitors Gö6850 (Bisindolylmaleimide), Gö6976 or rottlerin and A was tested as a latency reversing agent alone and in combination with other 14 latency reversing agents as prostratin and vorinostat and a single concentration of 15 antiretrovirals abacavir (ABC 1 µM), efavirenz (EFV 0.01 µM), ritonavir (Rito 0.1 µM), 16 lamivudine (3TC 1 µM), emtricitabine (FTC 1 µM) and tenofovir (TFV 1 µM). The model 17 used to evaluate the anti-latency effect was performed in primary PBMCs isolated from 18 healthy donnors. Briefly, resting PBMCs were transfected with a recombinant HIV-1 NL4.3-Luc) and treated with serial dilutions or single concentrations or the drugs to be 20 tested. After 48 hours, cell culture was lysed and RLUS were obtained in a luminometer. control of the vehicle used for the dilution of the drugs was used for all the 22 concentrations tested (100%). For LRAs, EC 50 and maximum effect (Emax) were 23 calculated for each drug separately and EC 50 /EC 50 ratios were determined. Afterwards, 1 combinations experiments were performed following the same procedure with the drugs 2 alone as compared to their EC 50 /EC 50 combination. For antiretrovirals, a T-Test analysis 3 were performed between the reactivation rates obtained with PMA or 4β-dPE A alone 4 and the combination with the antiretroviral drug.

5 4β- 16 Transformation assays 17 NIH 1 with 2 3 FlowJo 8 .

 516171238 dPE A was also evaluated in combination with antiretrovirals such as the nucleoside 6 analogue reverse transcriptase inhibitors (NRTI) lamivudine, emtricitabine and abacavir, 7 the nucleotide analogue reverse transcriptase inhibitor (NtRTI) tenofovir, the non-8 nucleoside analogue reverse transcriptase inhibitor (NNRTI) efavirenz and the protease 9 inhibitor (PI) ritonavir. Briefly, MT-2 cells were pre-treated with serial dilutions of the 10 drugs to be tested for 30 minutes and infected with a recombinant HIV-1 (NL4.3-Ren).11After 48 hours, cell culture was lysed and RLUS were obtained in a luminometer. A 12 control of the vehicle used for the dilution of the drugs was used for all the 13 concentrations tested (100%). IC 50 was calculated for each drug separately and IC 50 /IC 50 14 ratios were determined. Afterwards, combinations experiments were performed following 15 the same procedure with the drugs alone as compared to their IC 50 /IC 50 combination. 3T3 fibroblasts were maintained in medium DMEM supplemented with 10% calf 18 serum and treated either with SJ23B, prostratin, or PMA. All compounds were used at 19 0.1 mM concentration. As positive control, K-Ras V12 (pCEFL-KZ-HAK-Ras V12) was 20 transfected by the calcium phosphate precipitation technique. As negative control, 21 vector pCEFL-KZ-HA was transfected under similar conditions. Morphologically 22 transformed foci were scored after 2-3 weeks in culture [31]. Cells were washed with 23 PBS 1x, fixed with PFA 4% for 30 min, washed again with deionized water and stained Giemsa staining solution. Transformed cell foci appeared intense blue colored. Statistics 0 software was used to analyze flow cytometry data and results are shown as 4 percentage of receptor fluorescence mean intensity (%MIF) as compared to a non-5 treated control (100%). GraphPad Prism software was used to calculate effective 6 concentration 50%, inhibitory concentration 50%, cytotoxic concentration 50% (EC 50 , 7 IC 50 , CC 50 ) and Emax (top) in all the experiments (Non-linear regression, dose-response 8 stimulation, log[agonist] vs response for anti-latency experiments and dose response 9 inhibition, log[inh] vs response for antiviral experiments) and to represent concentration-10 response curves. Confidence intervals 95% (CI95%) and R 2 are provided for all the 11 calculated concentrations. GraphPad Prism was also used to perform ANOVA or T-test 12 analysis to determine the value of p (* p <0.05, ** p <0.01 *** p <0.001). Finally, the 13 Calcusyn Software was used to evaluate combination index (CI) for each combination. 14 All the experiments were performed at least in triplicates in three independent A inhibits in vitro HIV infection with low citotoxicity 19 4β-dPE A is a phorbol derivative with special chemical characteristics, as the lack of the 20 oxygen at position 4 and thus, we aimed to study its potential antiviral and latency 21 reversing activity. To that end, first we used MT-2 cells infected with an X4 tropic 22 recombinant virus (NL4.3-Ren) in the presence of different concentrations of 4β-dPE A.

23 A

 23 strong inhibition of viral replication was observed at nanomolar concentrations, with an 1 IC 50 value of 6.51 nM. Further, when IL-2 activated PBMCs were infected under the 2 same conditions with X4 or R5 tropic HIV, a stronger inhibition of viral replication was 3 obtained, with IC 50 values of 0.31 and 0.24 nM, respectively (Figure1).

4 5

 4 Cell death was not observed at the concentrations tested in both, the infection assay 6 and the cytotoxicity assay. Moreover, 4β-dPE A CC 50 was higher than 10,000 nM (10 7 µM) in PBMCs and MT-2 cells (Figure1). While 4β-dPE A, prostratin and vorinostat 8 showed no cell toxicity in MT-2 cells and PBMCs at 10.000 nM, a similar pattern of cell 9 toxicity were found for romidepsin and panobinostat, although panobinostat only showed 10 PBMCs toxicity at concentrations close to 10.000 nM. Since toxicity is a cause of 11 concern for anti-latency drugs, we further evaluated 4β-dPE A toxicity in a long-term 12 viability assay (14 days) in IL-2 activated PBMCs at concentrations of 0.1, 1 and 10 µM.13 Cell viability was determined at different times along treatment. As observed in figure 1, 14 4β-dPE A was not toxic to human PBMCs even at 14 days of treatment.15 16 4β-dPE A decreases the expression of CD4, CXCR4 and CCR5 receptors 17 Viral entry involves the interaction of HIV gp120 and gp41 with CD4, CXCR4 and/or 18 CCR5 receptors. It has been described previously the down regulation of these 19

20 expression

 20 of cell surface receptors was measured by flow cytometry in MT-2 cells and 21 IL-2 activated PBMCs. Cell cultures were stimulated with PMA or 4β-dPE A and left in 22 culture for 2, 24 and 48 h (Figure 2).

1

 1

10 MT- 2

 102 cells and primary human resting PBMCs transfected with luciferase plasmids 11 under the control of HIV-1 whole genome (NL4.3-Luc). We have also tested 12 transcriptional activation in non-infected TZMbl cells, since this cell line contains in its 13 genome integrated copies of the luciferase gene under the control of the HIV-A activates HIV transcription in all the scenarios tested, inducing high levels of

17

 17

18 19 PKC

 1819 theta/MEK pathway is required for 4β-dPE A HIV transcriptional activity 20 To assess the PKC/MEK dependence of 4β-dPE A-mediated antagonism of HIV latency, 21 resting PBMCs were treated with PKCs chemical inhibitors Gö6976 1 µM (classical 22 PKCs inhibitor), bisindolylmaleimide 1 µM (Gö6850, classical and novel PKCs inhibitor), 23 rottlerin 3 µM (PKC theta inhibitor) or MEK inhibitor PD184352 (IMEK) 1 µM and HIV 1

2 3 5 (

 25 Surprisingly, novel PKC inhibition did not decreased HIV transcription induced by 4β-4 dPE A in a statistically significant manner. However, a tendency could be detected p=0,1531). However, PKC theta (rottlerin) and MEK inhibition decreased transcriptional 6 reactivation induced by 4β-dPE A and PMA, suggesting a strong PKC theta/MEK 7 dependence.

8 9 11 To

 811 PKC theta/MEK pathway is not involved in 4β-dPE A activity on receptors down-10 regulation study the specific PKC isoform involved in 4β-dPE A receptor down -regulation, we 12 treated IL-2 activated PBMCs with PKCs chemical inhibitors, bisindolylmaleimide or 13 rottlerin, and we evaluated the 4β-dPE A effect on CD4, CXCR4 and CCR5 receptor 14 expression. Downstream PKCs signaling in T cells may involve Raf-Mek dependent 15 activation of MAPKs, and thus MEK inhibition could also play a role in the 4β-dPE A 16 effect. Therefore, PBMCs were treated with MEK inhibitor PD184352 and the effect of

17 4β-

 17 dPE A in the surface cell receptor expression was also measured.

18 19

 18 Surprisingly, inhibition of PKCs or MEK did not modify the expression of CD4, CXCR4 or 20 CCR5 receptors with or without 4β-dPE A treatment, suggesting that PKC/MEK pathway

21

 21

5 Figure 6

 56 Figure6shows the results of PKC translocation in response to PMA or 4β-dPE A

  transcription factor is involved in 4β-dPE A transcriptional activity 14 To further analyze the mechanism of 4β-dPE A reactivation activity, luciferase 15 expression vectors under the control of three tandem B consensus repeats (NF-B 16 Luc), NFAT (NFAT Luc) or LTR region of HIV (LTR Luc) were transfected in resting 17 PBMCs, treated with 4β-dPE A at three different concentrations (1, 10 and 100 nM) or 18 with PMA (100 nM) and luciferase activity was measured after 48 h in culture.

19 20 21

 1920 As observed in figure7, 4β-dPE A induced the activation of the HIV LTR and NF-kB 22 transcription factor with the highest concentration of 100 nM as PMA. However, NFAT was not influenced by 4β-dPE A treatment while PMA 100 nM plus Ionomycine (Io) 1 strongly increased its expression.

5

 5 Since 4β-dPE A is a PKC agonist, we tested its combination with another PKC agonist, 6 prostratin, and with a histone deacetylase inhibitor (HDACi), vorinostat. First, we studied 7 the anti-latency effect of each drug separately. As it can be seen in figure8, all the three 8 compounds showed viral reactivation in resting PBMCs. Combinations were evaluated 9 among all the three drugs, including 4β-dPE A/prostratin, 4β-dPE A/vorinostat and 10 prostratin/vorinostat and the combination effect on EC 50 and Emax was studied.

  A displayed an EC 50 value of around 10 nM as transcriptional reactivator, which 13 is at least 70 and 480 fold lower than the EC 50 of vorinostat and prostratin, respectively, 14 although it was less potent than PMA (data not show, EC 50 0.2 nM). The combination of 15 4β-dPE A with prostratin was antagonistic showing a clear tendency toward lowering of 16 Emax value. Moreover, the combination index (CI) were above 16, showing a strong 17 antagonism. On the other hand, combination of 4β-dPE A with vorinostat showed a 18 strong synergism, with a CI value of 0.20 as well as the combination of prostratin with 19 vorinostat with a CI of 0.21 (Figure 8).

1

  reactivation experiments of 4β-dPE A and PMA and the IC 90 obtained from infection 2 experiments of antiretroviral drugs, since we found no reactivation effect of antiretroviral 3 drugs alone.

4 5

 4 There were no significant differences among the rate of reactivation of PMA or 4β-dPE A 6 alone and the rate of reactivation of the combination with an antiretroviral drug. Only 7 abacavir showed a slight tendency to increase the viral reactivation exerted by PMA and 8 4β-dPE A, but it was not significant.

14

  Therefore, combination experiments with different types of antiretroviral drugs were 15 performed. To that end, the entry inhibitor maraviroc (MVC), the nucleoside analogue 16 reverse transcriptase inhibitors (NRTI) lamivudine, emtricitabine and abacavir, the 17 nucleotide analogue reverse transcriptase inhibitors (NtRTI) tenofovir, the non-18 nucleoside analogue reverse transcriptase inhibitor (NNRTI) efavirenz and the protease 19

20

 20

5 64β-dPE A do not induce cell transformation 7 A

 57 major cause of concern of PKCs agonists is their potential tumour-promoting activity.

8

 8

10 Besides, 4 -

 104 deoxyphorbols lack the long and lipophilic side chain of tumour-promoter 11 PKC agonist PMA, which would suggest a different intracellular activity. To rule out the 12 potential tumour-promoting activity of 4β-dPE A we performed transforming cell assays 13 in NIH 3T3 fibroblasts.

21 1 O 2 from

 2112 The identification of potent natural or synthetic PKC agonists lacking tumor-promoting 22 activity and cellular proliferative activities has opened new research avenues for the treatment of cancer and HIV-1 latency. We show here that 4β-dPE A (12-O-Tigloyl-13--isobutyroyl-20-hydroxyl-4β-deoxyphorbol), a 4-deoxyphorbol ester derivative isolated Euphorbia amygdaloides ssp semiperfoliata, reactivates HIV-1 from latency and 3 could potentially contribute to decrease the viral reservoir size.

4 4β-

 4 dPE A showed two effects in the HIV replication cycle. First, it inhibits HIV infection, 5

11 phorbol

 11 esters as PMA. Bryostatin is chemically unrelated to phorbol esters since it is a 12 polyacetylated macrolactone developed as an anti-tumoral agent. It has been13 hypothesized that the antitumor activity of PKCs agonists in general could be related to 14 a high PKC activation leading to its degradation by the proteasome, while the natural 15 ligand DAG or phorbol esters as PMA, with long side chains, will be inactivated before 16 PKC degradation, promoting cellular growth. Moreover, it has been shown that phorbol esters with long acyl chain(s) such as the PMA, induced a distinct intracellular 18 translocation pattern of PKCs and that this activity rapidly decreased with a shortening 19 of the acyl side chain [37]. 20 Thus, just like prostratin, the 4β-dPE A, lacking the long side chain, should not display 21 this tumor promoting activity. Short and long term toxicity experiments in vitro support 22 this fact. 4β-dPE A CC 50 was not reached at least at 20 µM in PBMCs and 50 µM in MT-2 cells, displaying a high specificity as HIV inhibitors with a specificity index greater than 1 10000 for MT-2 cells and PBMCs. Other LRAs as panobinostat or romidepsin induced 2 cell toxicity at much lower concentrations. Moreover, we did not find long term toxicity 3 after a 2 weeks treatment of PBMCs (figure 1) and transformation assays confirm the 4 lack of tumor-promoting activity (figure 11).

5 Among

 5 PKCs agonists, only prostratin and bryostatin have entered clinical trials as HIV 6 anti-latency agents although results were disappointing. The reasons of this failure are 7 not clear, but prostratin is less powerful than other phorboids [20], including 4β-dPE A, 8 and thus, doses used in vivo should be higher leading to toxic effects more easily. Later, 9 clinical assays with bryostatin were designed with the lowest doses possible to avoid the 10 potential toxicity of PKC agonists. However, clinical assays did not show any effect on 11 the transcription of latent HIV, due probably, as the authors stated, to low plasma 12 concentrations [10].

13

 13

4 classical 5 N

 45 PKCs (cPKCs) and novel PKCs (nPKCs) have two tandem C1 domains in their -terminal domain, the C1a and C1b domains, which show high binding affinities for 6 DAG, phorbol esters and other PKC activators. Interestingly, phorbol esters binding sites 7 in PKC C1 domain have been described and chemical pharmacophores identified.

8

 8

19 MEK

 19 proteins are a family of intracellular dual kinases that activates ERK/MAPK in 20 response to diverse stimuli such as IL-2 and phorbol esters. The activation of this 21 pathway is strongly involved in cell proliferation and activation of T-cells. Therefore,

2 13 (

 13 dPE A in T cells, PKC/MEK as target for transcriptional activation (anti-latency effect) 3 and a still unknown target as an HIV infection inhibitor. Different chemical moieties could 4 be involved in each target.5We have further explored the mechanism of transcriptional activation. 4β-dPE A induces 6 a clear transactivation of NF-B transcription factor (figure7). HIV LTR contains binding 7 sites for NF-B and its activation is crucial for the initiation of transcription of latent HIV 8 DNA. These effects are similar to those exerted by prostratin and SJ23B, the viral 9 transcriptional activation through the NF-B transcription factor[32, 20]. Therefore, 10 phorbol esters act through the activation of HIV-1 gene transcription, although 4β-dPE A 11 seems to be a more powerful activating agent of HIV transcription, since lower 12 concentrations than the other phorbol esters are needed in the three cell lines tested SJ23B EC 50 between 4-8 nM; prostratin EC 50 between 2000-3000 nM, data not shown), 14 and thus, it could be used at lower doses in clinical assays. In fact our results show that 15 4β-dPE A is also more powerful than HDACIs, since vorinostat showed an EC 50 value of 16 80-120 nM. Panobinostat and romidepsin were too toxic in our assays in both PBMCs

17

 17

7 9

 9 of lowering doses and, at the same time, increasing the efficacy as viral reservoir 8 reactivators.Since latency reversing therapy is proposed to be used together with ART in infected 10 patients, the effect of LRAs is a matter of concern if a negative effect results from this 11 treatment in two different aspects: the main one is the potential toxicity due to LRA 12 treatment on its own in patients with excellent viral control and quality of life. Besides, an 13 antagonistic effect of LRA with ART could be envisaged and as far as we know this 14 potential pharmacological interaction has not been assessed. In this article we have 15 analyzed the effect of 4β-dPE A on anti-HIV activity when combined with different 16 families of types of antiretroviral drugs. Our results show a consistent synergism of LRA with nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside 18 reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs) and even with the 19 CCR5 antagonist maraviroc. This synergistic effect would be expected for NRTIs, 20 NNRTIs and PIs, since 4β-dPE A down-regulates CD4, CXCR4 and CCR5 receptors, 21 inhibiting HIV-1 entry and, thus, diminishing the rate of infection of T cells. However, the 22 down-regulation of CCR5 that could theoretically interfere with the maraviroc activity, 23 was not found in our experiments ruling out a potential antagonism between 4β-dPE A 1 and CCR5 antagonists." 2 In summary, we show here that a 4-deoxyphorbol derivative, 4β-dPE A, inhibits HIV 3 infection through CD4, CXCR4 and CCR5 down regulation and reactivates HIV in 4 resting PBMCs through PKC/MEK and NF-B activation. This last effect is independent 5 of receptors downregulation, since inhibition of PKCs/MEK does not modify receptor 6 expression inhibition induced by 4β-dPE A, and, thus, other cellular targets should be 7 involved. Moreover, 4β-dPE A shows a strong synergism with HDAC inhibitor vorinostat 8 as LRA and with NRTIs, NNRTIs and PIs in the antiretroviral therapy. Therefore, 4β-dPE 9 A is a new deoxyphorbol ester derivative with powerful activity as anti-latency agent and 10 HIV infection inhibitor which could be a drug candidate to eradicate HIV reservoirs.

13

 13 This work was supported by Ministry of Education of the Peruvian government 14 (PRONABEC), the Universidad Complutense de Madrid (UCM-Santander PR26/16-15 20281), the Instituto de Salud Carlos III (ISCIII-FIS PI16CIII/00034) and the Spanish 16 AIDS Research Network RD12/0017/0015 that is included in the Spanish I+D+I Plan

1 Conflict of interest 2 There are no conflicts of interest 3 Figure legends 4 Figure 1 .

 12341 Figure legends

Figure 2 . 4 Figure 3 .

 243 Figure 2. Effect of 4β-dPE A on CD4, CXCR4 and CCR5 receptors expression. Upper

Figure 4 .

 4 Figure 4. PKC and MEK dependence of the HIV-1 transcriptional reactivation activity of 11 4β-dPE A. Resting PBMCs were transfected with pNL4.3-Luc (HIV-1) and maintained in cell 12 culture. Cells were then treated with either 4β-dPE A (10 or 100 nM) or PMA (100 nM), in the 13 presence of PKCs inhibitors bisindolylmaleimide 1 µM (pan-PKC inhibitor) or Gö6976 1 µM 14 (novel PKC inhibitor), MEK inhibitor 1 µM (IMEK) and specific PKC theta inhibitor rottlerin 3 µM 15 and cultured at 37°C for 48h. 100% represents the background level of expression without 16 treatment. One way ANOVA analysis (PKCs inhibitors) or t-test (IMEK and rottlerin) was 17 performed to determine the value of p (* p <0.05). All the result are the mean of at least 3 18 different replicates. 19

Figure 5 . 2 Figure 6 . 8 Figure 7 . 15 Figure 8 . 2 Figure 9 . 4 ( 5 ( 10 Figure 10 . 20 Figure 11 . 2 Figure 12 . 6 BIBLIOGRAPHYINTRODUCTION 1

 5268715829451010201121261 Figure 5. Kinetics of expression of CD4, CXCR4 and CCR5 receptors induced by 4β-dPE A

10 size [ 16

 1016 ], the doses used in the assay were too low to avoid potential toxicity or the low 11 efficacy of the drugs selected. Recently, CAR T cells engineered to bind broadly 12 neutralizing anti-HIV antibodies have been found to reduce the size of the viral 13 reservoirs of the blood of infected individuals on antiretroviral therapy, although this 14 results are too preliminary [17]. 15 PKCs agonist can activate some isoforms of protein kinases C (PKCs) by binding to its 16 regulatory C1 domain, mimicking the action of the physiological ligand diacylglycerol 17 (DAG) and inducing viral reactivation. Prostratin was the first PKC agonist studied as a 18 potential anti-latency agent [18]. Bryostatin, another PKC agonist, showed lack of 19 efficacy in the reduction of the latent reservoir in clinical trials, probably due to the low 20 doses used in the assay to avoid toxicity since systemic concentrations were found 21

2 5 cure of infection. 6 Finally

 56 Identification and characterization of new phorboids with different PKCs binding profile, 3 identification of other potential targets of these drugs and selection of non-tumorogenic 4 phorboids with low toxicity could be helpful for the treatment of HIV-1 latency and the , as in ART, reactivation of the latent reservoir could only be achieved by the 7 combination of different drugs or antibodies able to diminish doses, and thus toxicity,

8

 8

16 reversal

 16 and anti-HIV-1 effects of 4β-dPE A, have been evaluated. This compound 17 shows antiviral activity through down-regulation of receptors and is able to induce HIV 18 reactivation. This class of compounds could be used as an adjuvant therapy to eliminate 19 the latent reservoirs of HIV-1.

4 80°C. 6 ( 8 (

 468 A was isolated from Euphorbia amygdaloides ssp. semiperfoliata and provided 2 by CNRS (Centre National de la Recherche Scientifique CNRS, France). The compound 3 was solubilized in DMSO to a final concentration of 10 mM and stored aliquoted at -Monoclonal antibodies (mAbs) to CD4, CXCR4 and CCR5 were supplied by 5 Becton Dickinson (Mountain View, CA, USA). Interleukin-2 (IL-2) was supplied by Chiron Emeryville, CA. USA). The inhibitor rottlerin was obtained from Alexis Co. (Lausanne, 7 Switzerland) and the inhibitors Gö6850 and Gö6976 were obtained from Calbiochem EMD Biosciences, Inc. Darmstadt, Germany). Renilla and Luciferase Assay Systems

9 and 16 Cells 17 MT- 2 12 Plasmids 13 The 3 ( 4

 916172121334 CellTiter Glo viability assay were supplied by Promega (Madison, WI, USA). DMSO 10 and MEK1/2 inhibitor PD184352 were supplied by Sigma-Aldrich (St Quentin-Fallavier, 11 France). The following reagents were obtained through the NIH AIDS Reagent Program, 12 Division of AIDS, NIAID, NIH: SAHA (vorinostat), raltegravir, lamivudine, tenofovir, 13 emtricitabine, abacavir, efavirenz and ritonavir. Romidepsin and panobinostat were 14 purchased from Selleck Chemicals (Houston, TX) and prostratin were purchased from 15 Sigma-Aldrich (St Louis, MO). cells (American Type Culture Collection, Manassas, VA, USA, Ref: CRL-2560) 18 were cultured in RPMI 1640 medium containing 10% (v/v) fetal bovine serum, 2 mM L-19 glutamine, penicillin (50 IU/ml) and streptomycin (50 mg/ml) (all Whittaker M.A. Bio-20 Products, Walkerville, MD, USA) and split twice a week. 293T cells, obtained from the 21 American type culture collection ATCC ref: ATCC® CRL-3216™, and TZM-bl cells (cell 22 line stably transfected with an LTR-Luc), obtained through the NIH AIDS Reagent 23 Program, Division of AIDS, NIAID, NIH: TZM-bl cells (Cat#8129) from Dr. John C. Kappes, and Dr. Xiaoyun Wu, were both cultured in DMEM medium containing 10% 1 (v/v) fetal bovine serum, 2 mM L-glutamine, penicillin (50 IU/ml) and streptomycin (50 2 mg/ml) (Whittaker) and split twice a week. PBMCs (peripheral blood mononuclear cells) 3 were isolated from healthy blood donors by centrifugation through a Ficoll-Hypaque 4 gradient (Pharmacia Corporation, North Peapack, NJ) and were suspended in RPMI 5 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 6 antibiotics (100 mg/ml streptomycin and 100 U/ml penicillin) (all Whittaker M.A. Bio-7 Products, Walkerville, MD, USA) before culture at a concentration of 2x10 6 cells/mL. In 8 some experiments, PBMCs were activated with IL-2 (300 IU/mL) for at least 48 h. Cells 9 were cultured at 37 °C in a 5% CO 2 humidified atmosphere. Proper informed consent 10 was obtained from each subject in accordance with the Spanish legislation on blood 11 donor regulations. Confidentiality and privacy was assured. vector pNL4.3-luc was generated by cloning the luciferase gene in the HIV-1 14 proviral clone pNL4.3 [25]. Plasmid pNL4.3-Ren was generated cloning the renilla gene 15 in the nef site of pNL4.3 and pJR-Ren plasmid was generated cloning the env gene of 16 HIV-1 JR CSF in the pNL4.3-Ren plasmid [26]. pNL4.3-env-Luc was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: pNL4-3.Luc.R -.E -from 18 Dr. Nathaniel Landau and pcDNA-VSV encoding the G protein of VSV from Dr. 19 Arenzana-Seisdedos from Pasteur Institute [27]. The 3-enh-B-ConA-luc plasmid carries 20 a luciferase gene under the control of three synthetic copies of the B consensus of the 21 immunoglobulin k-chain promoter cloned into the BamHI site located upstream from the 22 conalbumin transcription start site [28]. The LTR-Luc plasmid carries a luciferase gene 23 under the control of HIV-1-LTR region [29]. The expression construct pNFAT-LUC 1 containing three tandem copies of the distal NFAT-binding site of the IL-2 gene promoter 2 coupled to the IL-2 minimal promoter was kindly provided by Dr. Juan Miguel Redondo National Center for Cardiovascular Research, Madrid, Spain) [30]. Plasmids were amplified transforming E. coli DH5α cells and cultured in LB 5 supplemented with ampicillin at 37°C for 18 hours. All plasmids were purified using 6 Qiagen Plasmid Maxi Kit (Qiagen, Hilden, Germany) following the manufacturer 7 instructions. DNA concentration was determined in a Nanodrop (Thermoscientific, 8 Waltham, MA, USA).

9 10 JR 16 Anti-HIV-1 activity evaluation by recombinant virus assay 17 The 6 evaluation 7

 910161767 Viruses -Ren (HIV-1 R5 tropic) and NL4.3-Ren (HIV-1 X4 tropic) viral supernatants were 11 obtained from transfection of pJR-Ren and pNL4.3-Ren plasmids. NL4.3-env-VSV-Luc 12 (HIV-1 with a luciferase reporter gene in the position of nef and expressing VSV G 13 protein instead of HIV-1 env gene) was obtained by co-transfection of pNL4.3-env-Luc 14 and pcDNA-VSV encoding the G protein of VSV. Calcium phosphate transfection of 15 plasmids in 293T cells was used to obtain viral supernatants. initial evaluation of 4β-dPE A was performed in MT-2 cells infected with NL4.3-Ren 18 obtained previously by transfection in 293T cells. The assay was performed in 96 well 19 microplate seeded with 100,000 cells/well in a final volume of 200 µl. Cell cultures were 20 infected with viral supernatants (100,000 RLUs/w) and after 48 hours cell pellets were 21 lysed and luciferase activity measured in a luminometre (Berthold detection systems, 22 Pforzheim, Germany). X4 (NL4.3-Ren) or R5 (JR-Ren) infections of IL-2 activated 1 PBMCs followed the same procedure. All the experiments were controlled with cells 2 treated with the same DMSO concentration of 4β-dPE A. HIV-1 replication inhibition was 3 evaluated by measuring the luminescence activity or RLUs, with 100% being the 4 infection of non-treated cells. IC 50 was calculated with a non-linear regression formula 5 using GraphPad Prism software. Toxicity Cell toxicity was evaluated by treating mock infected cells with the same concentrations 8 of 4β-dPE A used in the antiviral assays or with higher concentrations of 4β-dPE A and 9 other LRAs (panobinostat, romidepsin and prostratin) or its combination with LRAs or 10 antiretroviral drugs. After 48 hours in culture, CellTiter Glo reagent (Promega, Madison, 11 WI, USA) was added and cell viability measured by RLUs quantification in a 12 luminometre. Cell viability is expressed as percentage of viable cells compared to a non-13 treated (DMSO same concentration as compound) control (100%). Long-term toxicity 14 evaluation at different times was performed with 4β-dPE A in IL-2-activated-PBMCs for 15 14 days. CC 50 was calculated with a non-linear regression formula using GraphPad 16 Prism software.

17

 17

Receptor expression kinetics 18 Single 8 Immunofluorescence Assays 9 For 10 Slide 8 12 ( 18 Evaluation of viral transcription 19 MT- 2 11 MEK 19 (

 188910812181921119 -, double-or three-color immunophenotyping was performed with a FACScalibur 19 flow cytometer using CD4 (clone SK3), CXCR4 (clone 12G5) and CCR5 (clone 20 2D7/CCR5) antibodies conjugated to the appropriate fluorochrome, all obtained from 21 Becton Dickinson (Heidelberg, Germany). Background staining was assessed with the 22 appropriate isotype-and fluorochrome-matched control mAb and subtracted. Activated 1 PBMCs or MT-2 were treated with different concentrations of 4β-dPE A for 48, 24 and 2 2 hours. Cells were then collected and CD4, CXCR4 and/or CCR5 receptors expression 3 were evaluated. The effect of PKCs inhibitors Gö6850 (bisindolylmaleimide), rottlerin 4 and MEK inhibitor PD184352 was also studied in the expression of CD4, CXCR4 and 5 CCR5 receptors in IL-2 activated PBMCs. The analysis was performed using forward-6 versus-side scatter dot plots. Results are shown as percentage of receptor fluorescence 7 mean intensity (%MIF) obtained by FlowJo 8.0 software. immunofluorescence assays, Jurkatt E61 cells and PBMCs were incubated in a µwell ibiTreat (Ibidi GmbH) for 48h, in the presence or absence of PMA or 4β-dPE 11 A (100 nM). Then, they were permeabilized with the cell fixative CytoskelFix Cytoskeleton) for 10 min at room temperature. After washing twice with 0.1% 13 glycine/PBS, cells were incubated with phospho-PKCθ (Thr538) (Cell Signaling 14 Technology, Danvers, MA) and Alexa Fluor 546 goat anti-rabbit IgG (Molecular Probes) 15 antibodies. Washes were performed with 1X PBS-2% BSA-0.05% saponine buffer. 4′,6-16 diamidino-2-phenylindole (Dapi) was used for nuclear staining. Images were obtained 17 with Leica DMI 4000B Inverted Microscope (Leica Microsystems, Wetzlar, Germany). cells or resting PBMCs (in culture for at least 24 hours) were suspended in 350 µL 20 of RPMI without supplements and electroporated using an Easyject plus Electroporator 21 (Equibio, Middlesex, UK) at 260 (MT-2) or 320 (PBMCs) V, 1500 mF and maximum 22 resistance with 1µg/10 6 cells of a luciferase plasmid under the control of the whole 1 genome of HIV-1 (NL4.3-Luc). Afterwards MT-2, resting PBMCs or TZM-bl (non-2 transfected) cells were seeded in 24 well microplates and treated with different 3 concentrations of 4β-dPE A or PMA (0.1 µM), as a reference control of HIV-1 4 transactivation, and left in culture in complete RPMI at 37°C. 48 hours later, cultures 5 were lysed with luciferase buffer and RLUs measured in a luminometer. This procedure 6 was also used for the transfection of luciferase plasmids under the control of 7 transcription factors NFAT (NFAT-Luc) or NF-B (3-enh-B-ConA-luc) or the long 8 terminal repeat of HIV (LTR-Luc) in resting PBMCs to evaluate transcriptional activity.9The transcriptional activity of 4β-dPE A in resting PBMCs was also studied in the 10 presence of PKCs inhibitors Gö6850 (Bisindolylmaleimide), Gö6976 or rottlerin and A was tested as a latency reversing agent alone and in combination with other 14 latency reversing agents as prostratin and vorinostat and a single concentration of 15 antiretrovirals abacavir (ABC 1 µM), efavirenz (EFV 0.01 µM), ritonavir (Rito 0.1 µM), 16 lamivudine (3TC 1 µM), emtricitabine (FTC 1 µM) and tenofovir (TFV 1 µM). The model 17 used to evaluate the anti-latency effect was performed in primary PBMCs isolated from 18 healthy donnors. Briefly, resting PBMCs were transfected with a recombinant HIV-1 NL4.3-Luc) and treated with serial dilutions or single concentrations or the drugs to be 20 tested. After 48 hours, cell culture was lysed and RLUS were obtained in a luminometer.

21 A

 21 control of the vehicle used for the dilution of the drugs was used for all the 22 concentrations tested (100%). For LRAs, EC 50 and maximum effect (Emax) were 23 calculated for each drug separately and EC 50 /EC 50 ratios were determined. Afterwards, 1 combinations experiments were performed following the same procedure with the drugs 2 alone as compared to their EC 50 /EC 50 combination. For antiretrovirals, a T-Test analysis 3 were performed between the reactivation rates obtained with PMA or 4β-dPE A alone 4 and the combination with the antiretroviral drug.

5 4β-

 5 dPE A was also evaluated in combination with antiretrovirals such as the nucleoside 6 analogue reverse transcriptase inhibitors (NRTI) lamivudine, emtricitabine and abacavir, 7 the nucleotide analogue reverse transcriptase inhibitor (NtRTI) tenofovir, the non-8 nucleoside analogue reverse transcriptase inhibitor (NNRTI) efavirenz and the protease 9 inhibitor (PI) ritonavir. Briefly, MT-2 cells were pre-treated with serial dilutions of the 10 drugs to be tested for 30 minutes and infected with a recombinant HIV-1 (NL4.3-Ren).

11 16 Transformation assays 17 NIH 1 with 2 3 FlowJo 8 .

 16171238 After 48 hours, cell culture was lysed and RLUS were obtained in a luminometer. A 12 control of the vehicle used for the dilution of the drugs was used for all the 13 concentrations tested (100%). IC 50 was calculated for each drug separately and IC 50 /IC 50 14 ratios were determined. Afterwards, combinations experiments were performed following 15 the same procedure with the drugs alone as compared to their IC 50 /IC 50 combination. 3T3 fibroblasts were maintained in medium DMEM supplemented with 10% calf 18 serum and treated either with SJ23B, prostratin, or PMA. All compounds were used at 19 0.1 mM concentration. As positive control, K-Ras V12 (pCEFL-KZ-HAK-Ras V12) was 20 transfected by the calcium phosphate precipitation technique. As negative control, 21 vector pCEFL-KZ-HA was transfected under similar conditions. Morphologically 22 transformed foci were scored after 2-3 weeks in culture [31]. Cells were washed with PBS 1x, fixed with PFA 4% for 30 min, washed again with deionized water and stained Giemsa staining solution. Transformed cell foci appeared intense blue colored. Statistics 0 software was used to analyze flow cytometry data and results are shown as 4 percentage of receptor fluorescence mean intensity (%MIF) as compared to a non-5 treated control (100%). GraphPad Prism software was used to calculate effective 6 concentration 50%, inhibitory concentration 50%, cytotoxic concentration 50% (EC 50 , 7 IC 50 , CC 50 ) and Emax (top) in all the experiments (Non-linear regression, dose-response 8 stimulation, log[agonist] vs response for anti-latency experiments and dose response 9 inhibition, log[inh] vs response for antiviral experiments) and to represent concentration-10 response curves. Confidence intervals 95% (CI95%) and R 2 are provided for all the 11 calculated concentrations. GraphPad Prism was also used to perform ANOVA or T-test 12 analysis to determine the value of p (* p <0.05, ** p <0.01 *** p <0.001). Finally, the 13 Calcusyn Software was used to evaluate combination index (CI) for each combination. 14 All the experiments were performed at least in triplicates in three independent A inhibits in vitro HIV infection with low citotoxicity 19 4β-dPE A is a phorbol derivative with special chemical characteristics, as the lack of the 20 oxygen at position 4 and thus, we aimed to study its potential antiviral and latency 21 reversing activity. To that end, first we used MT-2 cells infected with an X4 tropic 22 recombinant virus (NL4.3-Ren) in the presence of different concentrations of 4β-dPE A.

23

 23

  A decreases the expression of CD4, CXCR4 and CCR5 receptors 17 Viral entry involves the interaction of HIV gp120 and gp41 with CD4, CXCR4 and/or 18 CCR5 receptors. It has been described previously the down regulation of these 19

20 expression

 20 of cell surface receptors was measured by flow cytometry in MT-2 cells and 21 IL-2 activated PBMCs. Cell cultures were stimulated with PMA or 4β-dPE A and left in 22 culture for 2, 24 and 48 h (Figure 2).

1

 1

5 4β-

 5 dPE A induces HIV transcription 6

10 MT- 2

 102 cells and primary human resting PBMCs transfected with luciferase plasmids 11 under the control of HIV-1 whole genome (NL4.3-Luc). We have also tested 12 transcriptional activation in non-infected TZMbl cells, since this cell line contains in its 13 genome integrated copies of the luciferase gene under the control of the HIV-A activates HIV transcription in all the scenarios tested, inducing high levels of 17 HIV-1 reactivation with low concentrations (Figure3).

18 19 PKC

 1819 theta/MEK pathway is required for 4β-dPE A HIV transcriptional activity 20 To assess the PKC/MEK dependence of 4β-dPE A-mediated antagonism of HIV latency, 21 resting PBMCs were treated with PKCs chemical inhibitors Gö6976 1 µM (classical 22 PKCs inhibitor), bisindolylmaleimide 1 µM (Gö6850, classical and novel PKCs inhibitor), 23 rottlerin 3 µM (PKC theta inhibitor) or MEK inhibitor PD184352 (IMEK) 1 µM and HIV 1transcriptional activity of 4β-dPE A was also evaluated (Figure4).

2 3 5 (

 25 Surprisingly, novel PKC inhibition did not decreased HIV transcription induced by 4β-4 dPE A in a statistically significant manner. However, a tendency could be detected p=0,1531). However, PKC theta (rottlerin) and MEK inhibition decreased transcriptional 6 reactivation induced by 4β-dPE A and PMA, suggesting a strong PKC theta/MEK 7 dependence.

8 9 11 To

 811 PKC theta/MEK pathway is not involved in 4β-dPE A activity on receptors down-10 regulation study the specific PKC isoform involved in 4β-dPE A receptor down -regulation, we 12 treated IL-2 activated PBMCs with PKCs chemical inhibitors, bisindolylmaleimide or 13 rottlerin, and we evaluated the 4β-dPE A effect on CD4, CXCR4 and CCR5 receptor 14 expression. Downstream PKCs signaling in T cells may involve Raf-Mek dependent 15 activation of MAPKs, and thus MEK inhibition could also play a role in the 4β-dPE A 16 effect. Therefore, PBMCs were treated with MEK inhibitor PD184352 and the effect of 17 4β-dPE A in the surface cell receptor expression was also measured.

18 19

 18 Surprisingly, inhibition of PKCs or MEK did not modify the expression of CD4, CXCR4 or 20 CCR5 receptors with or without 4β-dPE A treatment, suggesting that PKC/MEK pathway

21

 21

5 Figure 6

 56 Figure6shows the results of PKC translocation in response to PMA or 4β-dPE A

  transcription factor is involved in 4β-dPE A transcriptional activity 14 To further analyze the mechanism of 4β-dPE A reactivation activity, luciferase 15 expression vectors under the control of three tandem B consensus repeats (NF-B 16 Luc), NFAT (NFAT Luc) or LTR region of HIV (LTR Luc) were transfected in resting 17 PBMCs, treated with 4β-dPE A at three different concentrations (1, 10 and 100 nM) or 18 with PMA (100 nM) and luciferase activity was measured after 48 h in culture.

19 20 21

 1920 As observed in figure7, 4β-dPE A induced the activation of the HIV LTR and NF-kB 22 transcription factor with the highest concentration of 100 nM as PMA. However, NFAT was not influenced by 4β-dPE A treatment while PMA 100 nM plus Ionomycine (Io) 1 strongly increased its expression.

5

 5 Since 4β-dPE A is a PKC agonist, we tested its combination with another PKC agonist, 6 prostratin, and with a histone deacetylase inhibitor (HDACi), vorinostat. First, we studied 7 the anti-latency effect of each drug separately. As it can be seen in figure8, all the three 8 compounds showed viral reactivation in resting PBMCs. Combinations were evaluated 9 among all the three drugs, including 4β-dPE A/prostratin, 4β-dPE A/vorinostat and 10 prostratin/vorinostat and the combination effect on EC 50 and Emax was studied.

  A displayed an EC 50 value of around 10 nM as transcriptional reactivator, which 13 is at least 70 and 480 fold lower than the EC 50 of vorinostat and prostratin, respectively, 14 although it was less potent than PMA (data not show, EC 50 0.2 nM). The combination of 15 4β-dPE A with prostratin was antagonistic showing a clear tendency toward lowering of 16 Emax value. Moreover, the combination index (CI) were above 16, showing a strong 17 antagonism. On the other hand, combination of 4β-dPE A with vorinostat showed a 18 strong synergism, with a CI value of 0.20 as well as the combination of prostratin with 19 vorinostat with a CI of 0.21 (Figure 8).

1

  reactivation experiments of 4β-dPE A and PMA and the IC 90 obtained from infection 2 experiments of antiretroviral drugs, since we found no reactivation effect of antiretroviral 3 drugs alone.

4 5

 4 There were no significant differences among the rate of reactivation of PMA or 4β-dPE A 6 alone and the rate of reactivation of the combination with an antiretroviral drug. Only 7 abacavir showed a slight tendency to increase the viral reactivation exerted by PMA and 8 4β-dPE A, but it was not significant.

11 NtRTIs, NNRTIs and PIs 12

 1112 A displays a strong synergistic antiviral effect in combination with NRTIs,Since the LRA treatment is designed to be administered together with ART, we 13 evaluated the effect of 4β-dPE A in the antiviral effect of antiretroviral therapy. 14 Therefore, combination experiments with different types of antiretroviral drugs were 15 performed. To that end, the entry inhibitor maraviroc (MVC), the nucleoside analogue 16 reverse transcriptase inhibitors (NRTI) lamivudine, emtricitabine and abacavir, the 17 nucleotide analogue reverse transcriptase inhibitors (NtRTI) tenofovir, the non-18 nucleoside analogue reverse transcriptase inhibitor (NNRTI) efavirenz and the protease 19inhibitor (PI) ritonavir were combined with 4β-dPE A using IC 50 /IC 50 ratios (Figure10).

20

 20

5 64β-dPE A do not induce cell transformation 7 A

 57 major cause of concern of PKCs agonists is their potential tumour-promoting activity.

8

 8

10 Besides, 4 -

 104 deoxyphorbols lack the long and lipophilic side chain of tumour-promoter 11 PKC agonist PMA, which would suggest a different intracellular activity. To rule out the 12 potential tumour-promoting activity of 4β-dPE A we performed transforming cell assays 13 in NIH 3T3 fibroblasts.

21 1 O 2 from

 2112 The identification of potent natural or synthetic PKC agonists lacking tumor-promoting 22 activity and cellular proliferative activities has opened new research avenues for the treatment of cancer and HIV-1 latency. We show here that 4β-dPE A (12-O-Tigloyl-13--isobutyroyl-20-hydroxyl-4β-deoxyphorbol), a 4-deoxyphorbol ester derivative isolated Euphorbia amygdaloides ssp semiperfoliata, reactivates HIV-1 from latency and 3 could potentially contribute to decrease the viral reservoir size.

4 4β-

 4 dPE A showed two effects in the HIV replication cycle. First, it inhibits HIV infection, 5

11 phorbol

 11 esters as PMA. Bryostatin is chemically unrelated to phorbol esters since it is a 12 polyacetylated macrolactone developed as an anti-tumoral agent. It has been13 hypothesized that the antitumor activity of PKCs agonists in general could be related to 14 a high PKC activation leading to its degradation by the proteasome, while the natural 15 ligand DAG or phorbol esters as PMA, with long side chains, will be inactivated before 16 PKC degradation, promoting cellular growth. Moreover, it has been shown that phorbol esters with long acyl chain(s) such as the PMA, induced a distinct intracellular 18 translocation pattern of PKCs and that this activity rapidly decreased with a shortening 19 of the acyl side chain [37]. 20 Thus, just like prostratin, the 4β-dPE A, lacking the long side chain, should not display 21 this tumor promoting activity. Short and long term toxicity experiments in vitro support 22 this fact. 4β-dPE A CC 50 was not reached at least at 20 µM in PBMCs and 50 µM in MT-2 cells, displaying a high specificity as HIV inhibitors with a specificity index greater than 1 10000 for MT-2 cells and PBMCs. Other LRAs as panobinostat or romidepsin induced 2 cell toxicity at much lower concentrations. Moreover, we did not find long term toxicity 3 after a 2 weeks treatment of PBMCs (figure 1) and transformation assays confirm the 4 lack of tumor-promoting activity (figure 11).

5 Among

 5 PKCs agonists, only prostratin and bryostatin have entered clinical trials as HIV 6 anti-latency agents although results were disappointing. The reasons of this failure are 7 not clear, but prostratin is less powerful than other phorboids [20], including 4β-dPE A, 8 and thus, doses used in vivo should be higher leading to toxic effects more easily. Later, 9 clinical assays with bryostatin were designed with the lowest doses possible to avoid the 10 potential toxicity of PKC agonists. However, clinical assays did not show any effect on 11 the transcription of latent HIV, due probably, as the authors stated, to low plasma 12 concentrations [10].

13

 13

4 classical 5 N

 45 PKCs (cPKCs) and novel PKCs (nPKCs) have two tandem C1 domains in their -terminal domain, the C1a and C1b domains, which show high binding affinities for 6 DAG, phorbol esters and other PKC activators. Interestingly, phorbol esters binding sites 7 in PKC C1 domain have been described and chemical pharmacophores identified.

8

 8

19 MEK

 19 proteins are a family of intracellular dual kinases that activates ERK/MAPK in 20 response to diverse stimuli such as IL-2 and phorbol esters. The activation of this 21 pathway is strongly involved in cell proliferation and activation of T-cells. Therefore,

2 13 (

 13 dPE A in T cells, PKC/MEK as target for transcriptional activation (anti-latency effect) 3 and a still unknown target as an HIV infection inhibitor. Different chemical moieties could 4 be involved in each target.5We have further explored the mechanism of transcriptional activation. 4β-dPE A induces 6 a clear transactivation of NF-B transcription factor (figure7). HIV LTR contains binding 7 sites for NF-B and its activation is crucial for the initiation of transcription of latent HIV 8 DNA. These effects are similar to those exerted by prostratin and SJ23B, the viral 9 transcriptional activation through the NF-B transcription factor[32, 20]. Therefore, 10 phorbol esters act through the activation of HIV-1 gene transcription, although 4β-dPE A 11 seems to be a more powerful activating agent of HIV transcription, since lower 12 concentrations than the other phorbol esters are needed in the three cell lines tested SJ23B EC 50 between 4-8 nM; prostratin EC 50 between 2000-3000 nM, data not shown), 14 and thus, it could be used at lower doses in clinical assays. In fact our results show that 15 4β-dPE A is also more powerful than HDACIs, since vorinostat showed an EC 50 value of 16 80-120 nM. Panobinostat and romidepsin were too toxic in our assays in both PBMCs

17

 17

7 9 2 4 resting

 924 of lowering doses and, at the same time, increasing the efficacy as viral reservoir 8 reactivators.Since latency reversing therapy is proposed to be used together with ART in infected 10 patients, the effect of LRAs is a matter of concern if a negative effect results from this 11 treatment in two different aspects: the main one is the potential toxicity due to LRA 12 treatment on its own in patients with excellent viral control and quality of life. Besides, an 13 antagonistic effect of LRA with ART could be envisaged and as far as we know this 14 potential pharmacological interaction has not been assessed. In this article we have 15 analyzed the effect of 4β-dPE A on anti-HIV activity when combined with different 16 families of types of antiretroviral drugs. Our results show a consistent synergism of LRA with nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside 18 reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs) and even with the 19 CCR5 antagonist maraviroc. This synergistic effect would be expected for NRTIs, 20 NNRTIs and PIs, since 4β-dPE A down-regulates CD4, CXCR4 and CCR5 receptors, 21 inhibiting HIV-1 entry and, thus, diminishing the rate of infection of T cells. However, the 22 down-regulation of CCR5 that could theoretically interfere with the maraviroc activity, was not found in our experiments ruling out a potential antagonism between 4β-dPE A 1 and CCR5 antagonists." In summary, we show here that a 4-deoxyphorbol derivative, 4β-dPE A, inhibits HIV 3 infection through CD4, CXCR4 and CCR5 down regulation and reactivates HIV in PBMCs through PKC/MEK and NF-B activation. This last effect is independent 5 of receptors downregulation, since inhibition of PKCs/MEK does not modify receptor 6 expression inhibition induced by 4β-dPE A, and, thus, other cellular targets should be 7 involved. Moreover, 4β-dPE A shows a strong synergism with HDAC inhibitor vorinostat 8 as LRA and with NRTIs, NNRTIs and PIs in the antiretroviral therapy. Therefore, 4β-dPE 9 A is a new deoxyphorbol ester derivative with powerful activity as anti-latency agent and 10 HIV infection inhibitor which could be a drug candidate to eradicate HIV reservoirs.

13 This

 13 work was supported by Ministry of Education of the Peruvian government 14 (PRONABEC), the Universidad Complutense de Madrid (UCM-Santander PR26/16-15 20281), the Instituto de Salud Carlos III (ISCIII-FIS PI16CIII/00034) and the Spanish 16 AIDS Research Network RD12/0017/0015 that is included in the Spanish I+D+I Plan

17 and 1 Conflict of interest 2 There are no conflicts of interest 3 Figure legends 4 Figure 1 .

 1712341 Figure legends

Figure 2 . 29 ( 4 Figure 3 .

 22943 Figure 2. Effect of 4β-dPE A on CD4, CXCR4 and CCR5 receptors expression. Upper

Figure 4 .

 4 Figure 4. PKC and MEK dependence of the HIV-1 transcriptional reactivation activity of 11 4β-dPE A. Resting PBMCs were transfected with pNL4.3-Luc (HIV-1) and maintained in cell 12 culture. Cells were then treated with either 4β-dPE A (10 or 100 nM) or PMA (100 nM), in the 13 presence of PKCs inhibitors bisindolylmaleimide 1 µM (pan-PKC inhibitor) or Gö6976 1 µM 14 (novel PKC inhibitor), MEK inhibitor 1 µM (IMEK) and specific PKC theta inhibitor rottlerin 3 µM 15 and cultured at 37°C for 48h. 100% represents the background level of expression without 16 treatment. One way ANOVA analysis (PKCs inhibitors) or t-test (IMEK and rottlerin) was 17 performed to determine the value of p (* p <0.05). All the result are the mean of at least 3 18 different replicates. 19

Figure 5 . 2 Figure 6 . 8 Figure 7 . 15 Figure 8 . 2 Figure 9 . 4 ( 5 ( 10 Figure 10 . 20 Figure 11 . 2 Figure 12 .

 52687158294510102011212 Figure 5. Kinetics of expression of CD4, CXCR4 and CCR5 receptors induced by 4β-dPE A

  Table shows the IC 50 , CC 50 and specificity index (SI) calculated at 48 hours of 18 treatment. IC 50 : Inhibitory concentration 50% in infection (HIV) assays. CC 50 : Cytotoxic 19 concentration 50% in viability assays. CI95%: confidence interval 95%. R 2 : R square. All the 20 result are the mean of at least 3 different replicates. 21

  Table shows the IC 50 , CC 50 and specificity index (SI) calculated at 48 hours of 18 treatment. IC 50 : Inhibitory concentration 50% in infection (HIV) assays. CC 50 : Cytotoxic 19 concentration 50% in viability assays. CI95%: confidence interval 95%. R 2 : R square. All the 20 result are the mean of at least 3 different replicates. 21

4β-dPE A, a single concentration combination experiment was performed (figure9). 24

Lower panels: Kinetics of expression of CD4, CXCR4 and CCR5 receptors induced by 4β-dPE A

in IL-2 activated PBMCs. Cells were treated with either 4β-dPE A at 1, 10 or 100 nM or PMA

4β-dPE A at 10 or 100 nM and membrane expression of receptors was measured using specific

monoclonal antibodies at different points along drug treatment (2, 24 and 48 h). Results are

expressed as percentage of expression measured as mean intensity fluorescence (MIF)

Values are shown as % of RLUs compared to a non-treated control (100%). B. Maximum

efficacy (Emax) comparisons (left panels) were performed with the top effects obtained for each

(pCEFL-KZ-HAK-Ras V12) was transfected by the calcium phosphate precipitation technique.

Morphologically transformed foci were detected after 2-3 weeks in culture by giemsa staining of

MEK inhibitor (IMEK) C) Specific PKC theta inhibitor rottlerin (Rot). Cells were treated with either

by Promega (Madison, and MEK1/2 inhibitor PD184352 were supplied by Sigma-Aldrich (St Quentin-Fallavier, 11 France). The following reagents were obtained through the NIH AIDS Reagent Program, 12 Division of AIDS, NIAID, NIH: SAHA AIDS, NIAID, NIH: TZM-bl cells (Cat#8129) from Dr. John C.