Modulating Innate and Adaptive Immunity by (R)-Roscovitine: Potential Therapeutic Opportunity in Cystic Fibrosis

Abstract

(R)-Roscovitine, a pharmacological inhibitor of kinases, is currently in phase II clinical trial as a drug candidate for the treatment of cancers, Cushing's disease and rheumatoid arthritis. We here review the data that support the investigation of (R)-roscovitine as a potential therapeutic agent for the treatment of cystic fibrosis (CF). (R)-Roscovitine displays four independent properties that may favorably combine against CF: (1) it partially protects F508del-CFTR from proteolytic degradation and favors its trafficking to the plasma membrane; (2) by increasing membrane targeting of the TRPC6 ion channel, it rescues acidification in phagolysosomes of CF alveolar macrophages (which show abnormally high pH) and consequently restores their bactericidal activity; (3) its effects on neutrophils (induction of apoptosis), eosinophils (inhibition of degranulation/induction of apoptosis) and lymphocytes (modification of the Th17/Treg balance in favor of the differentiation of anti-inflammatory lymphocytes and reduced production of various interleukins, notably IL-17A) contribute to the resolution of inflammation and restoration of innate immunity, and (4) roscovitine displays analgesic properties in animal pain models. The fact that (R)-roscovitine has undergone extensive preclinical safety/pharmacology studies, and phase I and II clinical trials in cancer patients, encourages its repurposing as a CF drug candidate.

Fig. 1

Structure of the two isomers of roscovitine and M3, the major metabolite of (R)-roscovitine. Isomer R of roscovitine has been developed as a cancer drug candidate under the name seliciclib (CYC202).

Fig. 2

Roscovitine corrects the trafficking defect of F508del-CFTR by regulating its proteolytic degradation. Abnormally folded F508del-CFTR is taken up by the ER quality control (ERQC) system. It binds to the calnexin chaperone in a calcium-dependent manner. The complex is then taken up by the ER-associated degradation (ERAD) pathway for proteolytic degradation by the proteasome. Roscovitine depletes ER Ca²⁺ stores, reducing the interaction of F508del-CFTR with calnexin. In addition, roscovitine lowers proteasomal activity in a Ca²⁺-independent manner. Altogether, this favors the stabilization of F508del-CFTR and its trafficking to the plasma membrane.

Fig. 3

Schematic overview of TRPC6 rescue of microbicidal activity in CFTR-deficient AMs through GPCR (G protein-coupled receptor) activation with (R)-roscovitine. Ionic fluxes in alveolar phagosomal membranes are permissive for intraluminal acidification and the development of a microbicidal environment. GPCR stimulation with (R)-roscovitine sets sequential intracellular signaling events in motion, leading to vesicle-mediated TRPC6 translocation and insertion. Calcium-dependent TRPC6 insertion into the plasma membrane and subsequent uptake into phagosomes determines the production of an intraluminal microbicidal environment. a CFTR+/+ AMs. Phagosomal acidification is driven by the flow of protons into the phagosomal lumen through V-ATPase activity, which, if uncompensated, produces charge buildup in the confined intraluminal compartment. Charge compensation is provided by Cl^- influx through CFTR allowing for a decrease in phagosomal membrane potential and enhanced acidification. The phagosomal lumen pH is approximately 5, and the phagosomal membrane potential (Ψ) is low (approx. +28 mV). The acidified phagosomal lumen supports the proteolytic activity of lysosomal enzymes, leading to bacterial killing. b CFTR−/− AMs. The absence of a Cl^- influx pathway reduces the level of acidification, and the phagosomal lumen pH reaches near-neutral levels, leaving a high phagosomal membrane potential. The lack of an acidified phagosomal lumen prevents bacterial lysis and supports bacterial growth. The elevated phagosomal membrane potential reduces proton movement into the phagosomal lumen. c CFTR−/− AMs exposed to roscovitine. Recruitment of the cation channel TRPC6 to the plasma membrane and subsequently to the phagosomal membrane upon particle engulfment provides an alternative charge shunt pathway in the absence of CFTR expression. Activation of TRPC6 in the phagosomal membrane by (R)-roscovitine-generated diacylglycerol (DAG) opens a cation exit pathway from the phagosomal lumen acting as an alternate charge shunt, thereby allowing for pH regulation and acidification. The phagosomal pH is maintained at a level of approximately 5 and the membrane potential is low. These conditions support microbicidal activity [adapted from [79]].

Fig. 4

Roscovitine acidifies the phagolysosomes of F508del-CFTR and cftr−/− macrophages and prevents bacterial growth (b, c). a Intraphagolysosomal pH in cftr+/+, F508del-CFTR or cftr−/− mouse AMs. Mutation or absence of CFTR leads to increased pH. Exposure to roscovitine results in phagolysosomal acidification. Means ± SEM. *** p < 0.001 vs. control (two-way ANOVA). b, c F508del-CFTR (b) or cftr−/− (c) AMs were exposed to DsRed-labeled bacteria, and fluorescence intensity at 607 ± 20 nm was monitored over time following exposure to 20 μM roscovitine, M3 or corresponding amounts of vehicle (control). Bacterial growth is shown as fold increase in DsRed fluorescence. Summary data from at least 3 separate experiments [adapted from [79]]. Mean fluorescence intensities ± SEM. Bacterial growth is prevented by cftr+/+ AM (data not shown), but not by F508del-CFTR or cftr−/− AM. Bacterial growth is prevented by both roscovitine and its metabolite.

Fig. 5

Roscovitine and neutrophils. a The different steps of inflammation: initiation by neutrophil infiltration is followed by neutrophil apoptosis. Macrophage infiltration then allows phagocytosis of apoptotic neutrophils and resolution of inflammation [adapted from [159]]. Neutrophils from CF patients appear to be partially protected from apoptosis. Roscovitine induces apoptosis of neutrophils, improving their elimination by macrophages and thus favoring the resolution of inflammation. b Proposed molecular mechanisms underlying induction of apoptosis by roscovitine [adapted from [36]]. The cell survival/cell death balance is maintained by Mcl-1, a Bcl-2 family member survival factor that binds to and neutralizes proapoptotic proteins such as Noxa. Mcl-1 is a short-lived protein being constantly synthesized (through RNA polymerase 2, itself under the control of CDK7/cyclin H and CDK9/cyclin T) and constantly degraded (through Mcl-1 ubiquitin ligase and proteasome). Roscovitine inhibits CDK7 and CDK9, preventing phosphorylation of RNA polymerase 2 at Ser-2 and Ser-9, respectively. Consequently, mRNA synthesis is transiently inhibited, and short-lived mRNAs and proteins are down-regulated. This is the case for Mcl-1. Reduction in the Mcl-1 protein level results in a transient increase in free Noxa protein, which triggers Bax/Bak-dependent apoptotic cell death.

Fig. 6

Roscovitine and eosinophils. Proposed molecular mechanisms underlying the action of roscovitine on eosinophils. Under resting conditions, Munc-18 binds to syntaxin-4, preventing it from interacting with SNARE proteins. During inflammation, CDK5 catalytic activity is increased, leading to Munc-18 phosphorylation (p) and preventing the binding of syntaxin-4 to Munc-18. Syntaxin-4 is then free to bind SNAREs, allowing the fusion of intracellular granules to the plasma membrane and release of their contents (e.g. EPX, ECP and LTC₄) in the extracellular space. Roscovitine, by inhibiting CDK5, prevents the release of syntaxin-4 from Munc-18 and binding to SNAREs. Consequently, exocytosis is inhibited.

Fig. 7

Roscovitine and lymphocytes. Depending on external stimuli, CD4+ Th0 cells differentiate into Th1 (IL-12), Th2 (IL-4), Th17 (TGF-β, IL-6, IL-1β and IL-23) and iTreg (TGF-β, IL-2 and retinoic acid) lymphocytes. The relative amounts of TGF-β, interleukins, retinoic acid and additional cytokines skew the differentiation of Th0 cells into either highly proinflammatory Th17 (through RORγt) or anti- inflammatory iTreg (through Foxp3) cells. Possibly through inhibition of CDKs and DYRK1A, roscovitine inhibits the differentiation into Th17 cells, lowering the production of proinflammatory interleukins (IL-17A, IL-17F, IL-21 and IL-22). Consequently, the balance of Th17/iTreg shifts towards the anti-inflammatory response.

Fig. 8

Summary of cellular effects of roscovitine which may be beneficial for the treatment of CF. Roscovitine acts independently on epithelial cells, macrophages, neutrophils, eosinophils and lymphocytes. Arrows: induction or enhancement; lines: inhibition or reduction.