Bioactive Compounds from Euphorbia usambarica Pax. with HIV-1 Latency Reversal Activity

Euphorbia usambarica is a traditional medicine used for gynecologic, endocrine, and urogenital illnesses in East Africa; however, its constituents and bioactivities have not been investigated. A variety of compounds isolated from Euphorbia species have been shown to have activity against latent HIV-1, the major source of HIV-1 persistence despite antiretroviral therapy. We performed bioactivity-guided isolation to identify 15 new diterpenoids (1–9, 14–17, 19, and 20) along with 16 known compounds from E. usambarica with HIV-1 latency reversal activity. Euphordraculoate C (1) exhibits a rare 6/6/3-fused ring system with a 2-methyl-2-cyclopentenone moiety. Usambariphanes A (2) and B (3) display an unusual lactone ring constructed between C-17 and C-2 in the jatrophane structure. 4β-Crotignoid K (14) revealed a 250-fold improvement in latency reversal activity compared to crotignoid K (13), identifying that configuration at the C-4 of tigliane diterpenoids is critical to HIV-1 latency reversal activity. The primary mechanism of the active diterpenoids 12–14 and 21 for the HIV-1 latency reversal activity was activation of PKC, while lignans 26 and 27 that did not increase CD69 expression, suggesting a non-PKC mechanism. Accordingly, natural constituents from E. usambarica have the potential to contribute to the development of HIV-1 eradication strategies.


Introduction
Antiretroviral therapy (ART) durably blocks HIV-1 transcription by targeting viral enzymes; however, these drugs do not result in viral eradication due to the presence of replication-competent proviruses that are stably integrated into the genomes of a small population of long-lived memory T cells, known as the latent reservoir [1]. A promising strategy to address HIV-1 persistence is to use small molecules to reactivate latent proviruses in order to expose these cells to immune clearance and/or viral cytopathic effect. Natural products offer much promise regarding the discovery of new latency reversal agents (LRAs) for HIV-1 eradication [2][3][4].

HIV-1 Latency Reversal Activity of Isolated Compounds in Vitro
Jurkat cells with a full-length integrated HIV-1 provirus that have been modified to contain a GFP coding region in place of the env gene (J-lat 10.6 cells) were used for HIV-1 anti-latency activity, cytotoxicity, and cellular activation testing. All 31 compounds were tested at 1, 10, and 100 µM. Through the GFP expression of J-lat 10.6 cells, it was determined that compounds 12, 13, 14, 21, 26, and 27 showed HIV-1 latency reversal activity (Figure 4). These compounds were further tested at additional concentrations to determine dose response and toxicity curves ( Figure 5A-F). Cell viability for all isolated compounds is presented in Figure S106 in the supplementary material.
anti-latency activity, cytotoxicity, and cellular activation testing. All 31 compounds were tested at 1, 10, and 100 μM. Through the GFP expression of J-lat 10.6 cells, it was determined that compounds 12, 13, 14, 21, 26, and 27 showed HIV-1 latency reversal activity (Figure 4). These compounds were further tested at additional concentrations to determine dose response and toxicity curves ( Figure 5A-F). Cell viability for all isolated compounds is presented in Figure S106 in the supplementary material.  4β-Crotignoid K (14) showed high reactivation levels into nM concentrations, ~250fold less than crotignoid K (13), which is a stereoisomer of 14, differing only in the configuration on C-4 ( Figure 6). The striking difference between these compounds isolated from anti-latency activity, cytotoxicity, and cellular activation testing. All 31 compounds were tested at 1, 10, and 100 μM. Through the GFP expression of J-lat 10.6 cells, it was determined that compounds 12, 13, 14, 21, 26, and 27 showed HIV-1 latency reversal activity ( Figure 4). These compounds were further tested at additional concentrations to determine dose response and toxicity curves ( Figure 5A-F). Cell viability for all isolated compounds is presented in Figure S106 in the supplementary material.  4β-Crotignoid K (14) showed high reactivation levels into nM concentrations, ~250fold less than crotignoid K (13), which is a stereoisomer of 14, differing only in the configuration on C-4 ( Figure 6). The striking difference between these compounds isolated from E. usambarica demonstrated a structure-activity relationship (SAR) of an important cellular trigger to induce HIV-1 proviral transcription. A similar SAR has recently been described between protein kinase C (PKC) agonists, 4-deoxyphorbol (4β-dPEA), phorbol myristate acetate (PMA), and prostratin [63]. Euphorbia species have been shown to be enriched for compounds capable of protein kinase C (PKC) activation in human cells [5,9,11,14,15]. In order to determine whether our active compounds were acting through PKC, we evaluated the latency reversal activity of each of these compounds in the presence and absence of a pan-PKC inhibitor, Gö6983 (Figure 7). Compounds 12, 13, 14, and 21 all showed reduced efficacy, indicating likely activation of PKC as their primary mechanism of action. In contrast, compounds 26 and 27 did not show a significant reduction in their activity when PKC was inhibited. In addition, these compounds did not increase CD69 expression (a hallmark of PKC activation), further suggesting an alternative (non-PKC) mechanism of latency reversal.  Euphorbia species have been shown to be enriched for compounds capable of protein kinase C (PKC) activation in human cells [5,9,11,14,15]. In order to determine whether our active compounds were acting through PKC, we evaluated the latency reversal activity of each of these compounds in the presence and absence of a pan-PKC inhibitor, Gö6983 (Figure 7). Compounds 12, 13, 14, and 21 all showed reduced efficacy, indicating likely activation of PKC as their primary mechanism of action. In contrast, compounds 26 and 27 did not show a significant reduction in their activity when PKC was inhibited. In addition, these compounds did not increase CD69 expression (a hallmark of PKC activation), further suggesting an alternative (non-PKC) mechanism of latency reversal. E. usambarica demonstrated a structure-activity relationship (SAR) of an important cellular trigger to induce HIV-1 proviral transcription. A similar SAR has recently been described between protein kinase C (PKC) agonists, 4-deoxyphorbol (4β-dPEA), phorbol myristate acetate (PMA), and prostratin [63]. Euphorbia species have been shown to be enriched for compounds capable of protein kinase C (PKC) activation in human cells [5,9,11,14,15]. In order to determine whether our active compounds were acting through PKC, we evaluated the latency reversal activity of each of these compounds in the presence and absence of a pan-PKC inhibitor, Gö6983 (Figure 7). Compounds 12, 13, 14, and 21 all showed reduced efficacy, indicating likely activation of PKC as their primary mechanism of action. In contrast, compounds 26 and 27 did not show a significant reduction in their activity when PKC was inhibited. In addition, these compounds did not increase CD69 expression (a hallmark of PKC activation), further suggesting an alternative (non-PKC) mechanism of latency reversal.
Furthermore, compounds 12-14, 21, 26, and 27 showed significant HIV-1 latency reversal activity demonstrated by the GFP expression of J-lat 10.6 cells. 4β-Crotignoid K (14) showed the reactivation of HIV-1 latency at a very low concentration of EC 50 about 0.015 µM and a higher CC 50 concentration than 160 µM. The stereoisomer, crotignoid K (13), showed the EC 50 and CC 50 concentrations about 3.75 and 40 µM, respectively, indicating that 4β-crotignoid K (14) was provided with higher safety and efficacy. There is a 250-fold difference in EC 50 and~1000-fold difference in selectivity index (CC 50 /EC 50 ) between these compounds. The structural difference between 13 and 14 is only in the relative configuration on C-4. However, they demonstrated dramatically different biological activity, indicating that the configuration on C-4 of tigliane-type diterpenoids is critical to HIV-1 latency reversal activity and likely reflects improved PKC activation. The primary mechanism of the active compounds 12-14 and 21 for the HIV-1 latency reversal activity was activation of PKC.
Currently, LRAs are still under investigation and have not been approved by the US Food and Drug Administration (US-FDA). Therefore, the intensive study of LRAs is an important topic, especially to discover new candidates from natural sources. For example, a known LRA, ingenol, is isolated originally from Euphorbia peplus [64] and is a US-FDAapproved topical treatment for actinic keratosis (AK) [65], showed a significant effect in the reactivation of HIV-1 latency through the PKC pathway [65]. Both compound 14 and ingenol mebutate are Euphorbia diterpenoids and the potent PKC agonists. In addition, compound 14 presented the lower cytotoxicity, indicating 14 is a promising candidate for the development of an LRA.

Plant Material
Euphorbia usambarica Pax. (Euphorbiaceae) was collected in Taita Taveta county, Kenya in 2019. Identification was performed by Peter Waweru Mwangi (Department of Medical Physiology, School of Medicine, University of Nairobi, Nairobi, Kenya). A voucher specimen (No. EU-001) has been deposited in the Herbarium of the Department of Pharmacognosy, University of Szeged, Szeged, Hungary.

Cell Isolation and Culture
The HIV-1-infected Jurkat T cell line (J-Lat 10.6) was obtained in January 2019 from the NIH/ATCC HIV-1 Reagent Program (www.hivreagentprogram.org) and cultured in RPMIbased media supplemented with 10% fetal calf serum, 1% penicillin, and 1% streptomycin.

Flow Cytometry
After in vitro culture, J-lat cells were washed with phosphate-buffered saline (1× PBS) prior to staining with 0.1 µL fixable viability dye Live/Dead Aqua (Cat L34957, www.thermofisher.com) per 10 5 cells for 30 min at 4 • C. Simultaneously, J-lat cells were also stained with antibodies against CD69 conjugated to an APC fluorophore (APC antihuman CD69 antibody, biolegend.com). Cells were then washed and re-suspended in 1× PBS prior to flow cytometry acquisition evaluating for cellular viability, green fluorescent protein (GFP) expression, and CD69 expression. Flow cytometry was performed with a BD FACSCelesta or FACSCanoto flow cytometer with FACSDiva acquisition software (Becton Dickinson, Mountain View, CA) prior to analysis with FlowJo (TreeStar Inc., Ashland, OR, USA).

Compound Screening
Isolated compounds were resuspended in dimethyl sulfoxide (DMSO; Sigma-Aldrich, Burlington, USA), at a concentration of 10 mM, and diluted with PBS, and tested with J-lat 10.6 cells at concentrations of 100, 10, and 1 µM. J-lat 10.6 cells were tested with compounds at a concentration of 2.5 × 10 5 cells/mL. We performed a minimum of three replicates of each condition for all experiments. Negative controls contained 1% DMSO to account for any effect of DMSO in the highest dilution of compounds. Additional dilutions were tested for those compounds that showed reactivation through increased GFP production in J-lat 10.6 cells.

Statistical Analysis
Statistical significance was analyzed using software from GraphPad Prism Version 7.0c (GraphPad Software, San Diego, CA, USA). The mean values and standard deviations for all replicate J-lat results were calculated and used to create Figures 4-7. Where applicable, Students t-test was used to determine statistical significance of experimental mean results relative to negative controls.

Conclusions
In this study, 4β-crotignoid K (14) revealed a very higher effect improvement compared to crotignoid K (13), indicating that configuration at the C-4 of tigliane diterpenoids is critical to HIV-1 latency reversal activity. (+)-Syringaresinol (26) and dimeric coniferyl acetate (27) showed no exhibition of CD69 expression, suggesting a non-PKC mechanism of latency reversal. Our results provide insights into the stereochemistry importance of bioactive diterpenoids and suggest that isolated compounds from E. usambarica can further research and development into therapeutic strategies for HIV-1 management, particularly as reactivators of latent HIV-1.