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(R)-Roscovitine, a pharmacological inhibitor of kinases, is currently in phase II clinical trial as a drug candidate for the treatment of cancers, Cushing disease and rheumatoid arthritis. We here review the data that support investigation of (R)-roscovitine as a potential therapeutic agent for the treatment of cystic fibrosis (CF). (R)-Roscovitine displays four independent properties that may favourably combine against CF: (1) it partially protects F508del-CFTR from proteolytic degradation and favours 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, (4) roscovitine displays analgesic properties in animal pain models. The fact that (R)-roscovitine has undergone extensive preclinical safety/pharmacology studies, phase I clinical and phase II clinical trials in cancer patients encourage its repurposing as a CF drug candidate.
Cystic fibrosis (CF) is a genetic disease affecting the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) ion channel (7q31.2, 1480 amino acids, 168 kDa), allowing the passage of chloride ions across the apical membrane of epithelial cells. The CFTR channel displays five domains: two hydrophobic membrane-spanning domains (each constituted of six transmembrane helical segments), two hydrophilic nucleotide binding domains (NBD), and a cytoplasmic regulatory domain which is encoded by exon 13 and contains numerous charged residues and most of the potential phosphorylation sites. The most frequent mutation site (F508del) is localized in NBD1. Loss of function of CFTR translates into pulmonary problems including dehydration and overproduction of mucus, respiratory difficulties, chronic infection (Pseudomonas aeruginosa in particular) and inflammation. Good overviews on various aspects of cystic fibrosis can be found in several recent reviews [1–6].
Lung damage secondary to chronic infection is the main cause of death in CF patients. Treatment of lung disease to reduce the impact of dysregulated innate immunity, infections, inflammation and subsequent lung injury is, therefore, of major importance [7–13]. Improved survival, increased mean age of CF patients worldwide is encouraging , but pulmonary infections remain the main problem for CF patients, as mortality in CF directly relates to compromised respiratory function. Despite some progress in the treatment of CF in recent years, transplantation remains the only therapeutic option for subjects reaching the terminal phase of pulmonary disease. Currently, conventional medical treatment has little to offer to these late stage CF patients and effective new agents need to be identified. The current development of new drugs with antimicrobial properties or anti-inflammatory properties, and the recent discovery and use of CFTR correctors and potentiators provide increasing hope for the treatment of CF [15–23].
We here review recent evidence showing that roscovitine, a protein kinase inhibitor developed as a clinical phase II anti-cancer drug candidate, rescues the trafficking defect of the F508del-CFTR protein, positively affects various aspects of the biology of innate immune cells, leading to potentiation of the antimicrobial defense and down-regulation of the inflammatory process and displays analgesic properties. This body of results advocates in favor of the evaluation of roscovitine for the treatment of CF.
The 2,6,9 -trisubstituted purine (R)-roscovitine (referred to as roscovitine above and in the rest of the article) (Figure 1) was discovered in 1997 as a pharmacological inhibitor of ‘cyclin-dependent kinases’ (CDKs) [24, 25; reviews: 26–29], a class of regulators essential for cell division and other major cellular functions [reviews: 29–31]. Its selectivity has been extensively studied: it interacts with various CDKs, casein kinases 1 (CK1s), dual specificity tyrosine phosphorylation regulated kinases (DYRKs) as well as with pyridoxal kinase [32–34]. Roscovitine was co-crystallized with CDK2, CDK5, CDK9 and pyridoxal kinase [24, 33, 35, 36].
Roscovitine (its synthesis and some derivatives) has been patented in the USA, Europe, and Japan for several applications . The ‘Centre National de la Recherche Scientifique’ (CNRS) holds exclusive rights to the patent that applies to cancers, infections and inflammatory diseases as granted to Cyclacel Pharmaceuticals. A second patent covers the use of roscovitine for the treatment of cerebrovascular conditions (e.g. stroke) and was licensed by the CNRS to Neurokin . Finally, a third patent proposing the use of roscovitine for the treatment of CF was purchased from the CNRS and the University of Poitiers by ManRos Therapeutics . The synthesis of roscovitine and related analogs has been largely described and optimized .
The therapeutic potential of roscovitine has been evaluated for numerous medical and veterinary indications. In addition to cancer we can cite stroke , Parkinson’s disease , Alzheimer’s disease (L.H. Tsai, personal communication), cranial trauma , pain signaling (see 3.4.), various viral infections , polycystic kidney disease [45, 46], glomerulonephritis [47–50], glaucoma [51, 52], Lambert-Eaton syndrome [53–55], deafness , Timothy syndrome [57–59], fibrosis , Cushing disease [61, 62] and diabetes . These studies have made it to preclinical trials, with the exception of glaucoma, glomerulonephritis and Cushing disease where roscovitine entered clinical trials. In cancer research, Cyclacel Pharmaceuticals has conducted preclinical, clinical phase I [64–67] and clinical phase II  trials with roscovitine under the name Seliciclib or CYC202. Non-small cell (NSC) lung cancer, breast cancer and nasopharyngeal cancer have been the main indications . Recently roscovitine has entered clinical trials for the treatment of Cushing disease [61, 62, 69] and rheumatoid arthritis . In the animal breeding field, roscovitine has been used as a tool to synchronize nuclei donor cells for the cloning of numerous mammals [see 71, 72, for example].
Kinetic biodistribution analysis in rats revealed that the highest area under the curve (AUC) of roscovitine was observed in lungs . Several mouse models of lung inflammation or injury were efficiently treated with roscovitine by intraperitoneal administration: bleomycin-induced lung injury , lipoteichoic acid and Streptococcus pneumonia -induced lung inflammation , mechanical ventilation -induced lung injury . Furthermore, roscovitine has been evaluated in phase IIa clinical trials against NSC lung cancer, where a substantial increase in overall survival was observed (388 versus 218 days in the placebo arm), despite no difference in progression free survival . Altogether, these data demonstrate that the lung is a viable target for roscovitine.
Roscovitine is orally bioavailable in man [66–68] and rodents [73, 77]. Once in the organism roscovitine is rapidly metabolized by the liver, essentially by oxidation . The main metabolite is the carboxylate product (M3) (Figure 1) [66, 67, 73, 77], which does not inhibit the kinases targeted by roscovitine but may account for other effects of the drug [77–79]. Although the half-life of M3 in humans is similar to that of roscovitine [66, 67], its CF favorable biological activity (see below) could extend that of roscovitine in CF treatment. Alternatively, the M3 compound could feasibly be developed as a drug candidate per se. Indeed, since it is essentially ‘kinase-dead’, the toxic effects of roscovitine associated with its anti-proliferative effects should thus be considerably reduced, permitting chronic administration of M3 over long periods and/or an increase in the administrated dose. As inhibition of CDKs appears to be important in roscovitine’s effects on neutrophils (see below), this could limit the anti-inflammatory action of M3 on CF, thus normalizing the inflammatory response in CF rather than completely abrogating it. It seems, therefore, possible to envisage the development of M3, or one of its analogs, as an alternative CF drug candidate derived from roscovitine.
The described CFTR mutations are grouped into class I (mutations leading to lack of CFTR protein synthesis), class II (mutations leading to anomalies in CFTR processing such as disruption of folding and trafficking to the surface), class III (mutations leading to defective regulation or gating of CFTR), class IV (mutations leading to defective chloride conductance) and class V (mutations leading to alternative splicing and production of insufficient quantity of CFTR polypeptide) [17, 18]. Alteration of CFTR activity in CF, thus, originates from different causes, depending on the type of mutation. Although 2002 mutations have been described in CFTR (http://www.genet.sickkids.on.ca/StatisticsPage.html), deletion of the codon corresponding to phenylalanine 508 (F508del-CFTR) is by far the most frequent, representing almost 70% of all CF cases. Only five other mutations (G542X, G551D, W1282X, N1303K, R553X) represent more than 1% of all CF cases. All other mutations are rare, and even exceptional, often uniquely detected in a single family.
The F508del-CFTR protein is expressed normally but, due to misfolding, is not trafficked to the apical membrane of epithelial cells. The mutation is ‘temperature sensitive’, meaning that physiological activity of F508del-CFTR is partially restored when cells are cultured at a low temperature (27°C). This is probably linked to proper folding, partial restoration of trafficking and correct localization to the plasma membrane. The F508del-CFTR protein is, thus, potentially functional but, at physiological temperatures, deletion of F508 prevents the correct folding and subsequent correct localization of CFTR to the plasma membrane. ‘Correctors’ are usually low molecular weight molecules that allow the localization of F508del-CFTR to the plasma membrane. Correctors are diverse in terms of chemical structure, mechanism of action and potency to rescue the abnormal trafficking and function of F508del-CFTR. However, all of these compounds are only partial correctors [15–19]. We have recently shown that roscovitine also acts as a partial ‘corrector’ of F508del-CFTR . This corrector effect seems to originate both from a negative effect on the recognition of F508del-CFTR by the endoplasmic reticulum conformation-based quality control (ERQC) pathway and from a partial inhibition of F508del-CFTR proteolysis by the endoplasmic reticulum-associated degradation (ERAD) pathway (Figure 2). Depletion of ER Ca2+ stores by roscovitine reduces the Ca2+-dependent interaction of F508del-CFTR with calnexin, preventing F508del-CFTR to be taken up by the ERAD system to proteolysis. In parallel, roscovitine reduces proteolytic degradation of F508del-CFTR by the proteasome in a Ca2+-independent manner. This increases the availability of F508del-CFTR for translocation to the plasma membrane (Figure 2). The resulting ‘corrector’ effect does not require the kinase inhibitory activities of roscovitine as M3, the main hepatic metabolite of roscovitine [73, 77] (Figure 1), also displays ‘corrector’ properties. Furthermore, other roscovitine derivatives which are active on kinases (CR8, olomoucine) do not show a ‘corrector’ activity. Recently a screen to detect potential ‘correctors’ among a chemical library of 231 kinase inhibitors revealed several ‘corrector’ products (active at 10 μM), notably kenpaullone and alsterpaullone, two inhibitors of CDK/GSK-3 . These compounds, which we also identified as ‘correctors’ (unpublished results), were developed during a long-term collaboration between our laboratory in Roscoff and Dr. Conrad KUNICK’s team in Kaiserslautern .
For several years, treatment of CF disease has aimed at correcting the epithelial defect due to CFTR absence or dysfunction. Several lines of evidence are converging to a novel paradigm of a dysregulated innate immunity resulting in the defects in bacterial clearance observed in CF [7–12]. Pivotal to these processes are neutrophils and macrophages .
The intra-phagolysosomal pH of F508del-CFTR macrophages has been shown to be abnormally high (pH 6.5 – 7.2) when compared to the intra-phagolysosomal pH of non-CF macrophages (pH 4.5 – 5.2)  (Figures 3A, 3B). Neither the phagocytic capacity of the macrophages, nor the fusion of phagosomes with lysosomes are affected by the mutant CFTR . However, bacteria, once phagocytosed, are not destroyed in the phagolysosomes [82, 83]. Bacteria are even able to multiply within the phagolysosomes , which sit at neutral pH far from the normal acidic pH optimum for lysosomal lipases and proteases. As a consequence, the inability of F508del-CFTR macrophages to destroy bacteria can aggravate infections that affect CF patients. Elevation of intra-phagolysosomal pH by 2 units is also observed in cftr−/−_macrophages which completely lack ion channel expression.
The pH abnormalities in CF are now being documented in different cellular compartments, cells, and tissues [82–89] despite earlier resistance in certain camps [90–94]. Recent findings from Zhang and colleagues  identify a population of secretory lysosomes that exhibit a higher pH in cftr−/− deficient AMs than in WT lung macrophages. The role of CFTR in bacterial clearance in the lung is underscored in recent studies on CF pigs which develop human-like cystic fibrosis (CF) lung disease [95–97]. Newborn pigs do not exhibit signs of airway inflammation but already display a defect in their ability to eliminate bacteria which leads to the accumulation of bacteria in the lungs (review in ). These studies provide further validation for our data which established that AMs express functional CFTR and cells from CFTR null as well as mutant-mice exhibit defective bactericidal activity [82, 86]. The cause of this deficiency is apparently a failure of lysosomes and phagosomes to acidify properly in the knockout. The severity of the acidification phenotype scales with the mutant genotype with ΔF508 being the most severe . Phagocytosis per se is not affected and it does not appear that CFTR affects phagolysosomal fusion or reactive oxygen species (ROS) production. Interestingly, only AMs showed a dependence of lysosomal acidification upon CFTR expression. Recent published data demonstrate that macrophage tissue source determines dependence of intracellular acidification on CFTR expression . We surmise that other Cl− channels may play a similar role in phagosomal function in other innate immune cells. Mice null for CLC-3 are susceptible to sepsis and Moreland et al.  suggest that CLC-3 is crucial for normal host defense by mechanisms that may involve phagocytic and secretory behavior in neutrophils, observations which are in conflict with those of Painter and colleagues [100–102] who maintain that CFTR mediates halide transport in human neutrophils.
In collaboration with ManRos Therapeutics, the University of Chicago group (VR, AGG, DN) demonstrated that roscovitine reduces the intra-phagolysosomal pH of F508del-CFTR macrophages by more than one unit (Figure 3C). This effect is also observed with the M3 metabolite of roscovitine. We initially thought that roscovitine was acting as a ‘corrector’ in F508del-CFTR alveolar macrophages (AM) with the F508del-CFTR being addressed to the phagolysosome membranes following uptake of the bacterial cargo, and thus correcting the intra-phagolysosomal pH. Unexpectedly, roscovitine also reduced the intra-phagolysosomal pH of cftr−/− macrophages. This demonstrates that the CFTR channel is not involved in the acidifying effect of roscovitine in phagolysosomes. This effect could thus, in principle, take place in any macrophage that shows a neutral intra-phagolysosomal pH linked to a functional inactivation of CFTR, in other words independently of the mutation involved. In terms of therapeutic applications, this signifies that roscovitine could, therefore, have a macrophage phagolysosomal pH correcting effect in many forms of CF, regardless of the mutation affecting the CFTR gene and channel functionality.
The consequence of the intra-phagolysosomal pH rescue, even if partial, is a marked improvement in the ability of cftr−/− and F508del-CFTR macrophages to eliminate P. aeruginosa, the major pathogen in CF (Figure 4). Improvement of the bactericidal properties of these macrophages lacking CFTR or functional CFTR, by treatment with roscovitine, is therefore independent of CFTR and perhaps more importantly of antibiotic resistance profile of bacterial isolates. Roscovitine could therefore have a positive bactericidal effect on most CF forms. Improvement of the bactericidal properties is observed with the M3 metabolite, S-CR8, N6-methyl-roscovitine and O6-benzyl-roscovitine, but is not observed with S-roscovitine, miglustat, olomoucine, finisterine, perharidine or purvalanol A .
Recent results suggest that the effects of roscovitine on the intra-phagolysosomal pH of macrophages could be explained by an action mediated by the Ca2+ permeable channel TRPC6 . TRPC6 belongs to the ‘Transient Receptor Potential’ (TRP) family of ion channels, particularly important in respiratory system diseases. The TRP family comprises 28 members which are grouped into several different classes: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin) and TRPA (ankyrin) (reviewed in [103–108]). The TRPC channels comprise six members, TRPC1, 3, 4, 5, 6, 7. TRPC6 is a channel activated by diacylglycerol derived from the hydrolysis of phospholipids (phosphoinositides) by phospholipases C. TRPC6 is expressed in cells implicated in inflammation and innate immunity, neutrophils [109–111] and macrophages [112, 113]. TRPC6 is highly expressed in the lungs. Its expression is increased in macrophages from patients with chronic obstructive pulmonary disease (COPD) and pulmonary hypertension [112, 113]. Activation of TRPC6 is implicated in pulmonary edema (LIRE, lung ischemia-reperfusion induced) . Deletion of TRPC6 in mice (trpc6−/−) specifically inhibits pulmonary inflammatory reactions of allergic origin . Few antagonists and agonists of TRPC6 have been described; they generally display low efficiency . Hyperforin (from the St. John herb or St. John’s Wort) is an activator of TRPC6 . A series of TRPC6 channel antagonists has been described by Sanofi . Work by Antigny et al. suggests that TRPC6 activity is regulated by the CFTR channel [119, 120]. The physical interaction of the two channels leads to an inhibition of calcium entry through TRPC6. On the other hand, F508del-CFTR is unable to interact with TRPC6 and this would lead to excessive activation of TRPC6 and abnormal entry of calcium. The influx of calcium can be normalized once the trafficking of F508del-CFTR is corrected (miglustat) or by anti-TRPC6 siRNA [119, 120]. Our results show that roscovitine acts as an indirect activator of the TRPC6 channel, independently of CFTR channel expression or mutation. Roscovitine induces the production of diacylglycerol (DAG) which activates the translocation of TRPC6 calcium channels to the plasma membrane. Following phagocytosis, TRPC6 channels are integrated in the phagosomal membrane and contribute to cation depletion inside the phagolysosomes, thus amplifying intra-phagolysosomal acidification due to V-ATPase (which, by hydrolyzing ATP, allows proton entry). This effect is responsible for the intra-phagolysosomal acidification of macrophages (Figure 3C).
Neutrophils represent the first line of defense against microbes but are also powerful pro-inflammatory cells able to injure host tissues (Figure 5A). CF constitutes a representative example of a pathogenic condition in which the deleterious power of neutrophils is at work with uncontrolled activated neutrophils unable to kill invading bacteria . This particular picture has led to a still unresolved neutrophil conundrum in CF which is whether neutrophils could be genetically modified to display such a pro-inflammatory phenotype . Indeed, as in macrophages  and monocytes , CFTR is expressed in neutrophils and regulates bactericidal activity [101, 123–125].
Neutrophil extracellular traps (NETs) contribute to inflammation in a number of diseases such as systemic lupus erythematosus (SLE)  and inflammatory arthritis  and have been described in CF [128–130]. Furthermore, P. aeruginosa can induce NETosis [127, 131–134]. Oxidative burst and NADPH oxidase activity are central to the process of NET formation with myeloperoxidase (MPO) and neutrophil elastase acting as essential co-factors [135, 136]. The importance of NETs in the killing of pathogens is a matter of concerns and has been recently challenged by the report showing that neutrophils from patients with Papillon-Lefèvre syndrome lacking serine proteinases and unable to produce NETs did not show any defect in bacterial killing . Oxidative burst causes downstream activation of peptidyl dearginase 4 (PAD4) which in turn translocates to the nucleus and hypercitrinulates histones, leading to nuclear decondensation . NETosis is entirely distinct from apoptosis  but may involve the activation of autophagic pathways [140, 141]. The clearance of NETs, unlike that of apoptotic neutrophils, is poorly understood with un-degraded NET fragments promoting inflammation in SLE . As such the pro-inflammatory potential of NETs in the CF airway cannot be ignored.
A complex relationship exists between infections and inflammation in the lungs of CF patients [review in 1–7, 143–146]. The persistence of neutrophils in CF lung that failed to clear bacterial infection and are not cleared themselves by macrophages following apoptosis strongly points to the importance of innate immune cells in this process in CF. Furthermore, other forms of neutrophil death such as NETosis may be prevalent in the CF lung and contribute to lung damage and bacterial colonization [147, 148]. Of note is the modulation of the immune response to infections by proteases from neutrophils especially through chemokines .
Through several structural and pharmacological properties, roscovitine targets innate immune cells via different mechanisms. The discovery that CDK inhibitors such as roscovitine could indeed favor the disposal of neutrophils by enhancing both their apoptosis and their phagocytosis by macrophages has opened a promising research field [74, reviews in 150, 151]. Roscovitine resolves the inflammatory response in various animal models [74, 150]. This activity is linked to the pro-apoptotic action of roscovitine on neutrophils (Figure 5A). The molecular mechanism is likely to implicate inhibition of CDK7 and CDK9  which leads to reduced expression of the cell survival and anti-apoptotic factor Mcl-1 [153, 154], an effect we have observed while analyzing the anti-cancer activity of roscovitine and its derivatives  (Figure 5B). Roscovitine also inhibits the production of nitric oxide and inhibits the activation of NFκB in macrophages [156, 157]. Inhibition of the NFκB pathway by CR8, an analogue of roscovitine, was also observed in chronic lymphocytic leukemia cells . The anti-inflammatory effect of roscovitine via enhanced apoptosis of neutrophils was confirmed in a zebrafish inflammatory model [159, 160], a mouse model with pulmonary inflammation induced by Streptococcus pneumoniae and lipoteichoic acid (a pro-inflammatory constituent of gram positive bacteria) , a mouse model of ventilator-induced lung injury  and an experimental mouse model of pneumococcal meningitis . It was also shown that roscovitine, by inhibiting CDK2 and CDK5, blocks endothelial activation and leucocyte/endothelial cell interactions, contributing to the anti-inflammatory effect . One of the specificities of CF is the persistence of an enormous burden of neutrophils in the airways [163, 164, reviewed in 165, 166]. In spite of the overabundance of neutrophils, the deficit in antimicrobial activity results in a chronic P. aeruginosa infection suggesting a defective innate immunity [9, 10, 166–168]. In vitro studies show that the apoptotic death of neutrophils from CF patients is delayed, thus impairing their elimination by macrophages and promoting inflammation [169–171]. The in vitro kinetics of apoptosis is slowed down in neutrophils from CFTR mutation heterozygote parents, as seen with neutrophils of their children (homozygotes) . In vitro, roscovitine restores a normal level of apoptosis in neutrophils isolated from CF patients . Roscovitine-induced apoptosis of neutrophils and their progenitors has been linked to the Noxa-dependent degradation of Mcl-1, which liberates Bim and Puma, two activators of the pro-apoptotic factor Bax , which interestingly has been demonstrated to be deficient in CF . Furthermore, roscovitine inhibits the proliferation of those progenitor neutrophils which managed to escape apoptosis .
The spectrum of biological activities of roscovitine is wide and, importantly, appears to be cell-specific. For instance, the pro-apoptotic effect in neutrophils is extremely effective while no such effect is observed in macrophages. Roscovitine effects should, thus, be studied in each cell type and might also depend on the type of CFTR mutation.
In addition to neutrophils, eosinophils may participate to lung tissue injury in CF [174, 175]. The pathophysiological importance of eosinophils may be specifically relevant in CF patients showing allergic bronchopulmonary aspergillosis .
Upon stimulation eosinophils release the content of their secretory granules which comprise various toxic proteins, such as eosinophil cationic protein (ECP), eosinophil peroxidase (EPX) and produce pro-inflammatory mediators such as leukotriene C4 (LTC4). Although their number remains stable in peripheral blood and lung, eosinophils are activated in CF [177, 178], resulting in enhanced production of ECP and EPX [175–179] and LTC4  compared to healthy controls. Eosinophils isolated from CF patients release higher amounts of ECP than those of control, healthy patients . ECP levels found in the sputum of CF patients reach levels similar to those able to induce pulmonary damage in vitro [179–181] and correlate with ions levels .
Eosinophils express various CDKs, CDK5 in particular [183, 184] and its activating partners p35 and p39 . Upon eosinophil stimulation, CDK5 is phosphorylated on Ser-159 and its catalytic activity is increased . This correlates with phosphorylation of one of its substrates, Munc-18 [184–186], release of syntaxin-4 and its binding to SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). The binding of syntaxin-4 to SNARE proteins allows interaction of vesicular R-SNAREs to plasma membrane Q-SNAREs, subsequent membrane fusion and exocytotic degranulation . Pharmacological inhibitors of CDKs such as roscovitine and AT7519, or CDK5 siRNA reduce EPX release by eosinophils activated by PMA (phorbol 12-myristate 13-acetate) or secretory IgA . These results suggest that roscovitine, by inhibiting CDK5, may reduce degranulation of challenged eosinophils (Figure 6).
Finally, roscovitine also induces apoptosis (assessed by many techniques) of activated human eosinophils in vitro by reducing Mcl-1 expression , possibly by a mechanism involving inhibition of CDK7 and/or CDK9 . Whether roscovitine, other CDK inhibitors or other agents that drive eosinophil apoptosis, enhance the resolution of eosinophilic dominant inflammation in vivo is under intense investigation [189, 190].
Altogether these data suggest that roscovitine may reduce eosinophil numbers and secretory activity, which is expected to be potentially beneficial to CF patients if seen in vivo.
CD4+ T helper cells play a major role in immune responses. Once activated by antigens, these cells differentiate into different cell types, typically Th1 and Th2 lymphocytes, but also Th17 cells and iTregs (induced regulatory T cells) (Figure 7). Pro-inflammatory Th17 cells are characterized by the production of IL-17A and play an important role in auto-immune diseases, cancer and elimination of extracellular bacteria. On the other hand, anti-inflammatory Treg cells play a key role in controlling immunological tolerance and in suppressing excessive immune responses deleterious to the host. There is an intricate link between iTreg and Th17 cell programs of differentiation, which both require TGF-β (transforming growth factor β). Upon activation in the presence of TGF-β, naive CD4+ T cells (Th0) can differentiate into either Th17 or iTreg cells depending on the overall cytokine milieu . Low or intermediate concentrations of TGF-β together with proinflammatory cytokines (IL-6, IL-1β, IL-23) promote the differentiation of Th17 cells, through expression of the nuclear receptor RORγt (retinoic acid -related orphan receptor). Such activation inhibits the expression and function of Foxp3, the transcription factor driving the Treg differentiation program. Conversely, in the absence of proinflammatory cytokines, high levels of TGF-β promote the expression of Foxp3 and differentiation of iTreg cells. This process is further enhanced by IL-2 and retinoic acid, and is associated with inhibition of RORγt expression and function. Th17 and iTregs cells thus reciprocally inhibit their differentiation [192, 193] (Figure 7).
Emerging evidence suggests that imbalance of T cell responses may contribute to CF pathophysiology. A role for the Th17 and Th2 T lymphocytes in chronic pulmonary inflammation in CF patients was recently proposed. Th0 cells from CF patients or mice show a predisposition to differentiate towards the pro-inflammatory Th17 phenotype, while having a normal propensity to differentiate into Th1 and Treg lineages . High peripheral blood Th17 levels are associated with poor lung functions in CF . A specific profile of pro-inflammatory cytokines/chemokines (particularly IL-17A and IL-5) may be a risk factor associated with infections by P. aeruginosa . A link between the inflammatory background mediated by T cells and susceptibility to P. aeruginosa infections remains to be shown. IL-17A plays a major role in the recruitment, activation and migration of neutrophils in CF patients , its expression is increased in CF patients’ sputum  and its overproduction has even been suggested as the cause of chronic lung inflammation in CF patients . Expression of IL-17 could serve as an early biomarker for infection by P. aeruginosa . A robust increase in Th17 lymphocytes (pro-inflammatory) together with enhanced Th2 responses, and a decrease in Treg lymphocytes (anti-inflammatory), observed in cftr−/− mice, were coupled to susceptibility to infections by Aspergillus fumigatus. A reduction in the expression of indoleamine 2,3-dioxygenase (IDO), the first enzyme in the tryptophan degradation pathway, was observed in CF patients and in the cftr−/− mouse model. The imbalance of Th17 vs Treg is linked to the reduction in IDO activity. Inhibition of Th17 activation (IL-17A siRNA) or stimulation of the IDO pathways (kynurenines) restores protection against A. fumigatus . Heightened Th2 responses in CF patients with allergic bronchopulmonary aspergillosis were associated with lower frequencies of Tregs compared with A. fumigatus-colonized CF patients without allergic bronchopulmonary aspergillosis . Previous report suggested significantly lower percentages of circulating Tregs in chidren with CF, and a correlation between decreased frequencies of Tregs and lower FEV1 . A recent study further showed that patients with CF who have chronic P. aeruginosa infection show an age-dependent, quantitative, and qualitative impairment of Tregs. Tregs isolated from CF patients or from cftr−/− mice showed reduced functional suppressive activity compared with Tregs from non-CF controls. Both “extrinsic” P. aeruginosa-induced effects and “intrinsic” CFTR-mediated Treg functional skewing contributed to Treg impairment in CF . Th17 cells, through IL-17 production, may also be involved in CF-related diabetes (CFRD) . The involvement of T lymphocytes in CF is presented in two brief reviews [205, 206].
Several articles have described the effects of roscovitine on T lymphocytes [207–209]. A screen of 256 inhibitors of intracellular signaling pathways has led to the identification of CDK inhibitors, and roscovitine in particular, as suppressors of Th17 differentiation and, thus, as activators of iTregs differentiation . Induction of iTreg cell differentiation by CDK2 inhibition was recently confirmed with kenpaullone, another pharmacological inhibitor of CDKs . Another essential kinase regulating the differentiation of Th17 and regulatory T cells is DYRK1A . Inhibition of DYRK1A enhances Treg differentiation and impairs Th17 differentiation and attenuates inflammation . As roscovitine is also a DYRK1A inhibitor (IC50 in the μM range) [212, 34], its effect on DYRK1A may contribute to its effects on T cell differentiation. In a mouse model, roscovitine suppresses experimental autoimmune encephalomyelitis (EAE), an auto-immune disease mediated by Th17 cells . Roscovitine suppresses CD4+ T helper (Th) cells and has a beneficial effect on a uveitis mouse model, an auto-immune disease . Roscovitine suppresses the production of pro-inflammatory interferon (IFN) and IL-17 , confirming previous results . Roscovitine thus modifies the Th17/Treg balance in a favorable, anti-inflammatory direction (Figure 7). Whether roscovitine displays additional direct effects that may mitigate CFTR-dependent intrinsic functional skewing of Tregs remains to be determined.
Pain is a common event in CF [213, 214]. Among several CDKs, roscovitine inhibits CDK5, a kinase activated by the binding of one of regulatory subunits p35 or p39, and their respective proteolytic fragments, p25 or 29. CDK5 is known to be involved in modulating pain signaling [review in 215]. CDK5 is mostly expressed in the nervous system, namely sensory neurons of dorsal root ganglia (DRG), spinal cord and trigeminal ganglia [216–218] and its expression as well as activity is significantly increased upon pain sensation. Roscovitine exhibits analgesic properties in various animal models of pain. Wang et al.  were the first to carry out behavioral studies based on the antinociceptive properties of roscovitine. Subsequent studies revealed efficacy of roscovitine in attenuating Complete Freund’s Adjuvant (CFA) induced peripheral inflammation. The subcutaneous injection of CFA evokes local inflammation, redness, swelling, hypersensitivity to noxious stimulus (hyperalgesia) that, subsequently, activates protein kinases like CDK5 in primary sensory neurons. Roscovitine treatment significantly reverses heat hyperalgesia induced by intraplantar CFA injection [218, 220–223]. The analgesic effects of roscovitine can occur through inhibition of CDK5 activity, decreased p35 expression  and/or reduced CDK5 phosphorylation at S159 by casein kinase 1, a post-translational modification that promotes CDK5 activity . Roscovitine can also affect CFA induced inflammatory pain by suppressing TrkB (tropomyosin receptor kinase B) levels , reducing synaptophysin expression  and by preventing trafficking to the plasma membrane of transient receptor potential vanniloid 1 (TRPV1), an ion channel known to be involved in the detection of noxious heat [224, 225]. The level of CDK5 activity can also affect heat hyperalgesia from acute inflammation induced by carrageenan  and inhibition of CDK5 by roscovitine in cultured DRG neurons attenuates calcium influx through TRPV1 .
Recent reports have also demonstrated the antinociceptive effects of roscovitine in neuropatic pain models. Significantly increased expression of CDK5 is observed in the dorsal horn of rats following chronic constriction injury of sciatic nerve and intrathecal delivery of roscovitine significantly attenuates mechanical allodynia in these rats . Roscovitine can down-regulate expression of the NR2A receptor, which in turn, can alleviate neuropathic pain caused by chronic DRG compression . Additionally, roscovitine prevented remifentanil-induced postoperative thermal and mechanical hyperalgesia by decreasing expression and activity of CDK5/p35 and phosphorylation of NR2A (S1232), NR2B (Y1472) and mGlur5 (S1167) . Roscovitine can also down-regulate NMDA (N-methyl-D-aspartate) receptors in animal model of cancer pain where roscovitine treatment significantly reduced mechanical allodynia and thermal hyperalgesia, by inhibiting of NR2B receptor . Additional evidence indicates that roscovitine promotes analgesia through of DARPP-32 dephosphorylation (T75) in a formalin-induced model of nociception . Interestingly, CDK5 is found to be involved in cross-organ reflex sensitization and that colon irritation caused an increase in CDK5 expression in spinal cord and DRG, and intrathecal injection of roscovitine attenuates cross-organ sensitivity and colon irritation by decreasing NR2B phosphorylation .
All of these studies indicate promising analgesic effects of roscovitine in different animal models of pain. The antinociceptive properties of roscovitine along with its anti-inflammatory effects will prove helpful in developing effective treatments of pain in CF patients.
Beside its properties in favor of its evaluation as a CF drug candidate, roscovitine has a few weaknesses which can be summarized as follows. First of all, it was not optimized for this specific indication, in particular for its effects on macrophages intra-phagolysosomal pH. One can anticipate that identification of its molecular target(s) in macrophages - different from the usual kinase targets - should allow the optimization of much more potent and selective roscovitine analogs. Secondly, roscovitine was not optimized in terms of action on its anti-inflammatory targets. Analogs much more potent at inhibiting kinases are available, unfortunately with higher toxicity. Thirdly, roscovitine was not tested in animal models of CF. Despite their disputed predictive values, a positive effect would have been encouraging. Fourthly, roscovitine has a short half-life in human plasma. It remains thus to be seen whether sufficient lung biodistribution and exposure can be reached by oral route administration. Possible improved efficacy of roscovitine by administration through inhalation/nebulization has not been but should be evaluated. Despite these weaknesses, and considering its specific favorable properties and the benefits of drug repurposing in general, roscovitine has been favorably evaluated by official institutions to enter a first clinical trial which will both investigate tolerability in P. aeruginosa chronically infected CF patients and possible beneficial effects .
Evidence indicates that roscovitine has diverse biological properties that could potentially converge toward a novel treatment for CF (Figure 8): (1) roscovitine acts as a ‘corrector’ of the F508del-CFTR channel by protecting it from proteolytic degradation, favoring its relocation in the plasma membrane, (2) roscovitine improves the bactericidal properties of macrophages from CF patients, by translocating/activating the TRPC6 calcium channel (independently of the CFTR mutation) and by partially lowering the intra-phagolysosomal pH that is abnormally elevated in CF macrophages, (3) its main hepatic metabolite also shows a F508del-CFTR ‘corrector’ effect and biological activity on macrophages, despite extremely reduced kinase inhibitory effects, (4) roscovitine has an anti-inflammatory effect likely originating from its ability to promote apoptosis in neutrophils and their elimination by macrophages, (5) roscovitine reduces degranulation of eosinophils and promotes their apoptosis, (6) roscovitine suppresses the differentiation of CD4+ T helper cells into Th17 (pro-inflammatory lymphocytes) (thus reducing the production of pro-inflammatory interleukins such as IL-17) and promotes their differentiation into Tregs (anti-inflammatory lymphocytes), (7) roscovitine displays analgesic properties which could contribute to deal with CF-associated pain, (8) roscovitine is an orally available drug which has already undergone preclinical pharmacological and toxicological studies, extensive phase I and phase II clinical trials, in particular against lung cancer. Repurposing this anticancer drug candidate for CF should thus be a therapeutically valid proposal.
Besides direct clinical trials of roscovitine in CF patients we foresee four main developments in this novel approach to CF. Firstly, the effects of roscovitine on CF models (organoïds , ferret [235–237] and pig [95–98, 237, 238] models of CF, P. aeruginosa infection animal models [238, 239]) should be investigated. Secondly, given the host directed antibacterial effects of roscovitine, its action on other pulmonary pathogens associated with CF, besides P. aeruginosa, [240–242], should be investigated. Thirdly, the expected long-term treatments required for CF and the multiple biological consequences of the disease call for serious consideration of combination treatments associating roscovitine with other currently developed treatments and for alternative administration modes. The recent combination therapy Orkambi® composed of the corrector lumacaftor (or VX809) and the potentiator ivacaftor (or VX770) (www.vrtx.com) can now be prescribed to F508del homozygous CF patients . It will thus be important in a future study to compare the combination of roscovitine/ivacaftor with lumacaftor/ivacaftor. Fourthly, the optimization and development of second generation drugs derived from roscovitine, based on its CF relevant molecular and cellular targets, should be envisaged. The chemistry and biology of purines in general [244, 245] and 2,6,9-trisubstituted purines in particular, have been extensively explored, providing a solid starting ground.
This work was supported by the associations ‘Vaincre la Mucoviscidose’, ‘ABCF Mucoviscidose’, the Labex INFLAMEX (ANR-11-IDEX-0005-02), the ‘Chancellerie des Universités de Paris’ (Legs Poix) (VWS).
CONFLICT OF INTEREST STATEMENT
L. Meijer and H. Galons are co-founders of ManRos Therapeutics, L. Meijer is co-inventor on the roscovitine patent and L. Meijer and F. Becq are co-inventors on the “roscovitine & CF” patent.