Activating PKC-ε induces HIV expression with improved tolerability

Abstract

Despite suppressive antiretroviral therapy (ART), HIV-1 persists in latent reservoirs that seed new HIV infections if ART is interrupted, necessitating lifelong therapy for people with HIV. Activation of latent HIV during ART could improve recognition and elimination of infected cells by the immune system. Protein kinase C (PKC) isozymes increase HIV transcription and hence are potential latency reversal agents. However, the clinical utility of PKCs for this application is limited due to toxicity, which is poorly understood. Our studies showed that PKC activation with multiple classes of agonists leads to widespread platelet activation, consistent with disseminated intravascular coagulation, at concentrations that were similar to those required for T-cell activation. Differential expression analysis indicated that PKC-ε and PKC-η isoforms are expressed at high levels in human CD4+ T cells but not in platelets. Using structure-based drug design, we developed a novel PKC agonist, C-233, with increased selectivity for PKC-ε. C-233 increased both supernatant HIV RNA and p24 expression ex vivo after treatment of CD4+ T cells from ART-suppressed people with HIV. C-233 was 5-fold more potent for T-cell activation relative to platelet activation. Our studies support the use of structure-based drug design to create selective novel PKC agonists for the safe activation of HIV reservoirs and improved tolerability.

Fig 1. PKC agonists and other compounds used in this study.

Fig 2. HIV activation by novel PKC isoforms.

HIV viral RNA (copies/mL) fold change over DMSO (vehicle). Total CD4⁺ T cells from ART-suppressed people with HIV (n = 12) treated with PKC agonists [prostratin (red circles, n = 7)/Comin compound 2 (red triangles, n = 5)] alone, with pan-PKC inhibitor Gӧ-6850 (green circles/green triangles), or with classical PKC inhibitor Gӧ-6976 (blue circles/blue triangles). PMA and ionomycin (black diamonds) were used as positive control. Each symbol represents the geometric mean for a single individual (n = 3 to 8 replicate wells). Horizontal bars indicate the geometric means of all individuals in the indicated condition. Horizontal line on y-axis indicates the normalized value for DMSO. Significant differences were determined by ratio-paired t test.

Fig 3. Crystal structure of DAG-lactone bound to PKC-δ.

(A) Structure of the novel small-molecule PKC agonist, AJH-863 (stick model with green carbon atoms), bound to the PKC-δ C1B domain (ribbon model with zinc atom [purple]). (B) Detailed interactions of AJH-863 (stick model with green carbon atoms) with the C1B domain. Specific hydrogen bonds are displayed as red dashed lines. (C) Overlay of AJH-863 (stick model with green carbon atoms) and phorbol-13-acetate (stick model with yellow carbon atoms) in the C1B binding site. The protein is displayed as a surface and colored by atom type with beige carbons. (D) Sequence alignment of select C1B residues across PKC isoforms.

Fig 4. PKC-θ and HIV expression activation by novel PKC agonist, C-232A.

(A) Confocal microscopy images of A549 cells expressing tGFP-labeled PKC-θ isoform showing translocation of PKC-θ from cytoplasm to cell edge upon treatment with C-232A. Scale bar represents 10 µm. (B) Assay workflow to measure CD69 and HIV activation in CD4⁺ T cells isolated from ART-suppressed people with HIV. CD69 expression on CD4⁺ T cells (n = 4) and HIV RNA in culture supernatants (n = 6) upon treatment with C232A (C) and prostratin (D), respectively. Each dot represents a single donor and data represented is the geometric mean of the values from four replicates per donor. Left y-axis indicates the HIV RNA in cell culture supernatants (blue circles) and right y-axis indicates levels of CD69⁺ CD4⁺ T cells (red triangles). Curve fitting and P values were generated utilizing the 4-parameter-logistics equation in GraphPad Prism.

Fig 5. Pharmacodynamic and safety profile of C-232A in vivo in rats.

Dose-dependent expression of EGR1 (A) and CD69 (B) mRNA levels measured by QuantiGene analysis in rat whole blood (n = 3) at different time points after C-232A dosing. The y-axis indicates mean fold change in post-dose expression levels compared with predose after normalizing to PPIB mRNA levels. Mean values are plotted with error bars indicating standard deviation. Significant increases are seen in EGR1 levels with 0.3 and 10 mg/kg doses by 1 hour postdose compared with predose levels. P values were calculated using paired t test. Rapid (2 hours postdose) dose-dependent changes in circulating platelet levels (C) and mean platelet volume (D) of rats administered C-232A, indicating platelet activation and aggregation. Significant differences between pre-bleed and 2 hours postdose with 3 mg/kg were indicated. P values were calculated using paired t test. Representative H&E-stained histopathology images of lung (E), brain (F), and kidney glomeruli (G) from rats dosed with 3 mg/kg C-232A. Arrows in (E&F) indicate hemorrhage and arrows in (G) indicate thrombi within the tissues. Scale bar in (E&F) represents 250 µm. Scale bar in (G) represents 100 µm.

Fig 6. Pharmacodynamic and safety profile of C-232A in vivo in rhesus macaques.

(A) Dose-dependent expression of CD69 on CD4⁺ T cells as quantified by flow cytometry in rhesus macaque whole blood at different time points after C-232A dosing (n = 1 for 0.03, 0.1 and 1.0 mg/kg doses; n = 3 for 0.3 mg/kg dose and vehicle control). Circulating platelet levels (B) and mean platelet volume (C) upon C-232A dosing are shown. Circulating fibrinogen (D), activated partial thromboplastin time (E), and prothrombin time (F) are shown after administration of C-232A. Statistical significance was calculated using paired t tests where appropriate. Representative H&E-stained histopathology images of lung (G), adrenal gland (H), and kidney glomeruli (I) from a rhesus macaque administered 1 mg/kg C-232A. Arrows in (G&H) indicate hemorrhage and in (I) indicate thrombi within the tissues. Scale bar in (G) represents 250 µm. Scale bar in (H) represents 500 µm. Scale bar in (I) represents 100 µm.

Fig 7. In vitro assay in whole blood predicts PKC agonist platelet toxicity relative to pharmacodynamic potency.

(A) Workflow for an in vitro assay that measures platelet (CD62P⁺) and CD4⁺ T-cell (CD69⁺) activation simultaneously in whole blood treated with PKC agonists using flow cytometry. EC₅₀ values for activation by several classes of PKC agonists were calculated using 4-parameter, non-linear curve fitting method using GraphPad Prism. (B) Correlation data of several classes of PKC agonists. Y-axis represents CD69 on T cells and x-axis represents CD62P on platelets. Each data point indicates mean EC₅₀ value for 2–4 donors for each agonist.

Fig 8. Expression of novel PKC isoforms in human CD4⁺ T cells and platelets.

Similar expression of PKC-θ in both T cells and platelets was observed, whereas PKC-η and ε were abundant in T cells but undetected in platelets. Cell lysates of isolated CD4⁺ T cells and platelets from healthy donors (n = 3) were tested for expression of novel PKC isoforms θ, η, and ε by western blotting utilizing PKC-θ and PKC-η/ε antibodies. Expression of GAPDH was used for normalization to enable accurate interpretation of differences in protein expression.

Fig 9. Model of C-233 binding to PKC-δ.

C-233 (cyan) was overlayed on compound AJH-863 (green). Note how both the pyridine and the para-phenyl moiety offer multiple ways for analoging in directions pointing into the putative cell membrane direction.

Fig 10. C-233 shows improved potency for T cells in vitro and induces HIV activation ex vivo.

(A) Platelet (CD62P⁺) and CD4⁺ T cell activation (CD69⁺) induced by C-233 measured in whole blood from healthy donors (n = 10) by flow cytometry. EC₅₀ values calculated using 4-parameter nonlinear regression curve fit. Y-axis shows the ratio of EC₅₀ values for platelets to CD4⁺ T cells. Each data point represents a single donor and horizontal bars represent geometric mean. P values were determined by Wilcoxon matched-pair signed rank test. (B) Platelet aggregation induced by PKC agonists, prostratin, C-232A, and C-233, measured by light transmittance aggregometry in purified platelet-rich plasma isolated from healthy donors (workflow in S7 Fig). Each data point represents mean values from all four donors measured at the 20-minute stimulation point with respective compounds in a dilution series as shown. Error bars indicate standard deviation. (C) HIV activation induced in CD4⁺ T cells isolated from ART-suppressed people with HIV is shown for C-233, prostratin and PMA with ionomycin. Each data point indicates geometric mean of 8 replicates from each donor and horizontal lines represent the geometric mean of values from all 4 donors. (D) Total CD4⁺ T cells isolated from people with HIV on ART (n = 2) were treated with C-233 in a dilution series. CD4⁺ T cell activation was assessed by measuring cell surface CD69 expression by flow cytometry. Points indicate the mean and error bars indicate standard deviation. (E) Total CD4⁺ T cells isolated from people with HIV on ART (n = 4) were treated with C-233 in a dilution series and HIV-1 viral RNA induction was measured in culture supernatants at 72 hours after stimulation. Data represented is the geometric mean of the values from all 4 donors and error bars indicate standard deviation. Curve fitting and EC₅₀ values were generated utilizing the 4-parameter-logistics equation in GraphPad Prism. (F) p24 levels were measured using an HIV p24 digital immunoassay. Data represented is the mean of the values from all 4 donors (two replicates/donor) and error bars indicate standard deviation. Curve fitting and EC₅₀ values were generated utilizing the 4-parameter-logistics equation in GraphPad Prism.

Fig 11. C-233 shows improved tolerability in vivo in comparison to C-232A.

Expression levels of (A) Egr1 and (B) CD69 mRNA levels measured by QuantiGene analysis in rat whole blood at different time points after C-233 (10 mg/kg) dosing in comparison with C-232A (1 mg/kg). Expression levels were normalized to predose values after normalizing to PPIB mRNA levels (n = 3). Mean and standard deviation are shown. P values were calculated using paired t test. (C, D) Minimal to no changes seen in circulating platelet parameters(C) platelets, and (D) mean platelet volume of rats administered C-233 in comparison with C-232A.