Designed PKC-targeting bryostatin analogs modulate innate immunity and neuroinflammation

Abstract

Neuroinflammation characterizes multiple neurologic diseases, including primary inflammatory conditions such as multiple sclerosis and classical neurodegenerative diseases. Aberrant activation of the innate immune system contributes to disease progression, but drugs modulating innate immunity, particularly within the central nervous system (CNS), are lacking. The CNS-penetrant natural product bryostatin-1 attenuates neuroinflammation by targeting innate myeloid cells. Supplies of natural bryostatin-1 are limited, but a recent scalable good manufacturing practice (GMP) synthesis has enabled access to it and its analogs (bryologs), the latter providing a path to more efficacious, better tolerated, and more accessible agents. Here, we show that multiple synthetically accessible bryologs replicate the anti-inflammatory effects of bryostatin-1 on innate immune cells in vitro, and a lead bryolog attenuates neuroinflammation in vivo, actions mechanistically dependent on protein kinase C (PKC) binding. Our findings identify bryologs as promising drug candidates for targeting innate immunity in neuroinflammation and create a platform for evaluation of synthetic PKC modulators in neuroinflammatory diseases.

Keywords: EAE; PKC; bryolog; bryostatin; innate immunity; microglia; multiple sclerosis; neuroinflammation; prostratin.

Figure 1.. Synthetically Designed Bryologs Replicate the Anti-Inflammatory Effects of Bryo-1 on LPS-Stimulated Dendritic Cells

(A) Structure of bryo-1. Pharmacophoric elements critical for PKC binding are highlighted in red. Nodes for diversification in analog synthesis are highlighted in blue. The C-ring structurally orients the pharmacophoric core, whereas the A- and B-rings regulate PKC interactions with the plasma membrane and downstream functions. (B) Structures of designed bryologs selected for screening in dendritic cells based on PKC binding profiles. See also Data S1/Methods S1 (C) Mouse bone-marrow derived dendritic cells (mBMDCs) were left unstimulated or treated overnight with LPS (100 ng/ml) plus vehicle or the indicated bryologs (50 nM). Concentrations of IL-12 (left) and IL-10 (right) were measured from culture media by ELISA assay. Similar to bryo-1, the selected bryologs inhibited IL-12 and augmented IL-10 production. Data represent mean ± SEM of three biological replicates. **p < 0.01 relative to vehicle control, one-way analysis of variance (ANOVA) with Dunnett multiple-comparisons test

Figure 2.. The Immunologic Actions of Bryo-1 in Dendritic Cells Require PKC Binding and are Replicated by a Structurally Unrelated PKC Modulator

(A) Structures of the naturally-derived PKC modulator prostratin and its higher-affinity analog, SUW014. The altered side chain is highlighted in blue. (B) Structure of SUW275, a bryolog designed to abrogate binding to PKC due to acylation of the pharmacophoric C26 hydroxyl group (highlighted in red). (C) mBMDCs were unstimulated or treated overnight with LPS (100 ng/ml) plus vehicle or the indicated compounds (50 nM), followed by quantification of IL-12 (left) and IL-10 (right) secretion. The prostratin analog SUW014, but not the PKC non-binding bryolog SUW275, replicated the effects of bryo-1. Data represent mean ± SEM of three independent experiments performed in triplicate. *p < 0.05, **p < 0.01, one-way ANOVA with Dunnett multiple-comparisons test

Figure 3.. The Lead Bryolog SUW133 and the Prostratin Analog SUW014, but not the PKC Non-Binding Bryolog SUW275, Replicate Bryo-1 Effects on Macrophage and Microglia Phenotype.

(A) Peritoneal macrophages were unstimulated or treated overnight with LPS (100 ng/ml) plus vehicle or the indicated drugs (50 nM), followed by quantification of IL-12 (left) and IL-10 (right) secretion. Data represent mean ± SEM of three independent experiments performed in triplicate. (B) Mixed glial cultures were stimulated overnight with IL-4 (20 ng/ml) plus vehicle, bryo-1, or SUW133 (50 nM). Expression of arginase-1, a marker of a reparative microglial phenotype, was assessed by immunoblot. Left, Representative immunoblot. Right, Quantification of immunoblots; data represent mean of two independent experiments performed in duplicate. (C) Mixed glial cultures were unstimulated or stimulated overnight with IL-4 (20 ng/ml) plus bryo-1, SUW014, or SUW275 (50 nM), followed by immunoblotting for arginase-1. Left, Representative immunoblot. Right, Quantification of immunoblots; data represent mean of two independent experiments performed in duplicate. **p < 0.01, one-way ANOVA with Dunnett multiple-comparisons test

Figure 4.. The Anti-Inflammatory Effects of Bryo-1 in Macrophages and EAE are Largely Independent of TRIF

(A and B) Peritoneal macrophages derived from TRIF knockout (KO) mice were unstimulated or stimulated overnight with LPS (100 ng/ml) plus vehicle or bryo-1 (50 nM), followed by quantification of IL-12 (A) and IL-10 (B) secretion. Bryo-1 blocked IL-12 production but failed to augment IL-10 production in TRIF KO macrophages. Data represent mean ± SEM of three biological replicates. (C) Bryo-1 abrogated EAE independently of TRIF. EAE was induced in wild-type (WT) and TRIF KO mice, and mice were treated with bryo-1 (35 nmol/kg) or vehicle (n = 5 mice per group) via i.p. injection three days per week, beginning on the day of immunization with MOG_35–55. Data represent mean ± SEM for each time point. (D) Quantification of final clinical scores of TRIF KO mice treated with either vehicle or bryo-1. Data represent median ± interquartile range of 5 mice per group. **p < 0.01, one-way ANOVA with Tukey multiple-comparisons test in (A), Mann-Whitney U-test in (D)

Figure 5.. A lead bryolog (SUW133) attenuates EAE in a PKC-dependent manner

(A) Mice were subjected to EAE and randomized to treatment with vehicle, bryo-1, the lead bryolog SUW133, or the PKC non-binding bryolog SUW275 when they reached a clinical score of 1.0, corresponding to the onset of neurologic deficits. Drug treatment (35 nmol/kg) was given three days per week via i.p. injection. Data represent mean ± SEM of n = 8 (vehicle and SUW133) or n = 9 (bryo-1 and SUW275) mice per group for each time point. (B) Final clinical scores recorded on post-immunization day 30 from the EAE experiment depicted in (A). Data represent median ± interquartile range. *p < 0.05, **p < 0.01, ns = non-significant, Kruskal-Wallis with Dunn multiple-comparisons test.