Kinases as potential targets for treatment of pulmonary hypertension and right ventricular dysfunction

Pulmonary hypertension (PH) is a progressive pulmonary vasculopathy that causes chronic right ventricular pressure overload and often leads to right ventricular failure. Various kinase inhibitors have been studied in the setting of PH and either improved or worsened the disease, highlighting the importance of understanding the specific role of the respective kinases in a spatiotemporal cellular context. In this review, we will summarize the knowledge on the role of kinases in PH and focus on druggable targets for which certain criteria are met: (a) deregulation of the kinase in PH; (b) small‐molecule inhibitors are available (e.g. from the oncology field); (c) preclinical studies have shown their efficacy in PH models; and (d) when available, therapeutic exploitation in human PH has been initiated. Along this line, clinical considerations such as personalized medicine approaches to predict therapy response and adverse side events such as cardiotoxicity together with their clinical management are discussed.


| KINASE SIGNALLING AND INHIBITION IN PULMONARY HYPERTENSION
The pathobiological key finding in pulmonary hypertension (PH) regardless of the different aetiologies is the structural remodelling process within the pulmonary vasculature. This remodelling involves several cell types including pulmonary arterial smooth muscle (PASMCs) and endothelial cells (PAECs) and adventitial fibroblasts as well as cells from the immune system. A complex phenotype of increased proliferation, vasoconstriction, and apoptosis resistance, together with inflammation and in situ thrombosis, can be considered as the common disease origin Schermuly, Ghofrani, Wilkins, & Grimminger, 2011;Tuder et al., 2013). The luminal obstruction leads to an increase in pulmonary artery pressure, right ventricular hypertrophy, and, if untreated, death due to right heart failure. The ability of the right ventricle (RV) to compensate for changes in load has important prognostic value in PAH (Tello et al., 2019;van de Veerdonk et al., 2011) and RV failure due to pressure overload remains the predominant cause of death (Benza et al., 2012). Beside vasoactive mediators like NO, endothelin or prostacyclin and growth factors like PDGF, FGF or EGF are critically involved in the remodelling process. As reviewed elsewhere, growth factors signal via their respective receptors (Alexander, Fabbro, et al., 2017a) and initiate kinase-dependent signalling events ultimately leading to increased proliferation, resistance to apoptosis and metabolic changes. The group of kinases consists of 518 members which phosphorylate their substrates either on tyrosine (TK) or on serine/threonine (serine/threonine kinase) residues. Kinases can also be classified due to their cellular localization, trans-membranous or intracellular. They all serve as master regulators of cellular fate including virtually all signalling pathways from cell regeneration to cell death. In the past, several kinases have been described to be crucial for the altered cellular phenotype and the dysfunction of pathways which not only regulate vascular tone and blood flow in the pulmonary circulation but also control the growth of the pulmonary vascular cells.
For some of them, an aberrant activity has also been shown to be responsible for tumour development. Kinase inhibitors already approved in the oncology field can be of great value for the treatment of PH (reviewed in Woodcock & Chan, 2019).

| EFFICACY AND SAFETY OF KINASE INHIBITOR THERAPY IN PULMONARY HYPERTENSION
The increase in activity of a kinase under disease conditions as compared with control situations is one important parameter that dictates the side-effect profile of a potential drug. This aspect is of great importance with regard to the risk of toxicity observed for cardiac function and mitochondrial dysfunction as this hampers clinical safety of kinase inhibitors. A few inhibitors have been reported in cancer trials to trigger cardiotoxicity with adverse cardiac events and increased heart failure hospitalizations (Chu et al., 2007;Kerkela et al., 2006). While this emphasizes the need for routine cardiac monitoring in cancer trials (Chaar, Kamta, & Ait-Oudhia, 2018), kinase inhibitor cardiotoxicity is not a drug class effect, and the expression level of certain kinases in cardiomyocytes does not necessarily correlate with the toxicity induced by their corresponding inhibitor (Orphanos, Ioannidis, & Ardavanis, 2009). It is the cellular function of the respective kinase that is the major determining factor of whether inhibition of this kinase exerts cardiotoxicity. Therefore, it is crucial to carefully examine the direct effects of kinase inhibitors on the myocardium when re-purposing kinase inhibitors developed as cancer treatments. Along the same line, kinase inhibitors need to be studied under stress conditions as unwanted cardiotoxic effects may be hidden in the healthy myocardium and may be related to ischaemia-related cell death (hidden cardiotoxicity) (Ferdinandy et al., 2019). Further, kinase inhibitors often affect the whole cardio-pulmonary system, and therefore, adverse cardiac events are potentially masked by beneficial anti-remodelling effects in the lung vasculature in preclinical PH settings. These considerations underline the need for an in-depth characterization of a particular kinase in the cardio-pulmonary unit, especially since unexpected adverse cardiac effects are the leading cause of discontinuation of clinical trials and withdrawal of drugs from the market. Therefore, best practices require several experimental PH models in different animal species to concisely describe the role for a particular kinase in the lung and heart (Bonnet et al., 2017).
Direct effects of pharmacological compounds on RV structure and function in the context of PH are best studied in models with an increased but fixed afterload, where direct effects on the RV myocardium are studied without interfering afterload alterations due to lung remodelling. This is, for instance, achieved through surgical banding of the main pulmonary artery (PA) to a defined diameter (Maarman, Lecour, Butrous, Thienemann, & Sliwa, 2013). Depending on the degree of PA stenosis and duration of maintained pressure overload, these animals either develop RV dysfunction with compensated RV hypertrophy or RV failure (Egemnazarov, Crnkovic, Nagy, Olschewski, & Kwapiszewska, 2018), very similar to what is seen in PAH patients (Tello et al., 2019). By utilizing this animal model, direct cardiotoxic effects of kinase inhibitor treatment and their effects hidden in the healthy heart (hidden cardiotoxicity (Ferdinandy et al., 2019) can be unraveled. This is important as those cardiovascular adverse effects are the main factor complicating the use of kinase inhibitors in humans. Cardiovasculature-related adverse effects include hypertension, QT interval prolongation, a potentially life-threatening abnormality of cardiac repolarization, or left ventricular dysfunction (Dong & Chen, 2018). Further, cardiovascular adverse effects can vary from non-symptomatic decrease in ejection fraction or increase in natriuretic peptides to life-threatening heart failure or pressure increase (Lenihan & Kowey, 2013). Cardiovascular adverse effects seem to be drug specific. Therefore, the use of kinase inhibitors only succeeds under careful monitoring for cardiotoxicity signs. Monitoring for cardiotoxicity should include regular echocardiographic evaluation of cardiac function, ECG with special attention to QT interval prolongation and monitoring of cardiac biomarkers such as creatinine kinase, troponin, and natriuretic peptides (Schmidinger et al., 2008). No recommendations exist about the frequency of the monitoring, but for oncological patients, the frequency of monitoring has been increased up to bimonthly. Finally, although there is a high similarity between cancer cells and pulmonary vascular cells with respect to the hyper-proliferative phenotype, it seems to be more complex to simply adapt the same kinase inhibitors to PH. In solid tumours as well as in PH, several cell types interact within their distinct network together to establish, maintain, and to expand the disease associated cellular composition and structure while there are still enough differences between those two diseases: Tumour cells are needed neither for the proper function of the affected organ nor for the survival of the organism. They possess an aberrant phenotype which can be clearly distinguished from the initial status of their healthy counterparts. This is opposite in the case of PH; although diseased cells of the lung or the heart develop an abnormal phenotype with the given characteristic in PH (e.g., hyper-proliferation, resistance to apoptosis, hypertrophy, and fibrosis), they are still embedded in their organ structure and need to be functional in this cellular context unless they can be replaced by normal pulmonary vascular or cardiac cells. For this reason, therapeutic strategies targeting kinase centrally involved in crucial signalling pathways (i.e., survival, apoptosis, hypertrophy, and fibrosis) have to be chosen carefully to restore and maintain functional integrity.

| GROWTH FACTOR RECEPTOR SIGNALLING
One of the first kinases identified to be crucial for development of PH is PDGFRβ (Schermuly et al., 2005). It is primarily expressed on PASMCs and activated by its ligand (PDGF) to induce major signalling pathways like Ras/MAPK, PI3K, and PLCγ, leading to transcriptional programs with an increase in expression of genes responsible for proliferation, resistance to apoptosis and migration.  (Ghofrani, Seeger, & Grimminger, 2005). Imatinib has reduced the severity of symptoms in this patient, including improved haemodynamic parameters and the walking distance. The follow-up proof of concept study has suggested that PAH patients with the highest pulmonary vascular resistance would benefit from the TK inhibitor (TKI) therapy (Ghofrani et al., 2010). Unfortunately, the TKI therapy was associated with the appearance of SAEs (serious adverse effects) such as vomiting, peripheral oedema, and subdural haematomas. The phase III IMPRES study, which also investigated the effects of imatinib on PAH, has demonstrated improvement in walking distance, reduction of pulmonary resistance, and improvement in cardiac output (Hoeper et al., 2013). Although impressive results have been obtained in experimental models of PH (Schermuly et al., 2005) and in the compassionate treatment (Frost et al., 2015;Ghofrani et al., 2005;Speich et al., 2015), the clinical trials were discontinued due to those safety issues of imatinib (Hoeper et al., 2013;Prada, Gavilanes, & Souza, 2015). Furthermore, imatinib inhibits Abl kinase resulting in increased endoplasmic reticulum stress, leading to mitochondrial dysfunction (Rainbolt, Saunders, & Wiseman, 2014).
Nevertheless, it was clearly demonstrated that this type of therapy, that is, targeting the hyper-proliferative phenotype of pulmonary vascular cells in P(A)H, might be exploited especially for patients that had previously be categorized to be incurable with the known standard drugs. For these reasons, testing of inhibitors for other receptor kinases was performed. Here, targeting of EGF receptor (EGFR), fibroblast-derived growth factor receptor (FGFR), vascular endothelial cell-derived growth factor receptor (VEGFR), insulin-like growth factor receptor (IGF-1R), and Src kinases or multi-kinase approaches have been investigated.
In light of the postulated cancer paradigm of severe PAH (Pullamsetti, Savai, Seeger, & Goncharova, 2017), several multitargeted tyrosine-kinase inhibitors approved as promising cancer therapeutics were tested in the setting of PH and RV pressure overload.
Sorafenib, a multi-kinase inhibitor that among others inhibits PDGFRs, VEGFRs, Flt3, c-Kit, c-RAF, and b-RAF as well as sunitinib through inhibition of PDGFRs, VEGFRs, Flt3, KIT, CSF1R, and RET, both improved RV function by inhibiting pro-fibrotic and hypertrophic myocardial remodelling processes when administered over a short 2-week time period in a PA banding rat model with establish RV hypertrophy (Kojonazarov et al., 2013). But an association with clinical cardiac toxicity has been established for both compounds before (Schmidinger et al., 2008 (Chu et al., 2007), yet the exact molecular mechanisms underlying cardiotoxicity still remain unclear. Physicians have learned to manage toxicities such as those of VEGF inhibitors clinically through the development of dosing schedules that improve tolerability without compromising efficacy (two consecutive weeks on sunitinib treatment; 1-week rest; Bracarda et al., 2017) and individualizing dosing adjustments. Preclinical studies need to adjust those dosing schedules when advancing from proof-of-concept to translational relevant studies. Of note, it has been shown that congestive heart failure which develops under TK inhibitor treatment is generally completely reversible but requires treatment interruption-which can be resumed after recovery (Schmidinger, Larkin, & Ravaud, 2012).
After positive animal studies (Kojonazarov et al., 2013), a phase Ib trial was initiated with the aim to evaluate the safety of sorafenib for treatment of PAH (Gomberg-Maitland et al., 2010). Within 16 weeks of observation on 22 study participants, sorafenib has demonstrated a safe profile without the appearance of serious adverse effects but no significant improvement in walking distance and haemodynamics were observed. However, an important factor in interpretation of these data is that severely ill patients with functional class IV were excluded. Median value of mPAP was 51 mm Hg (range 36-64). So PAH patients most likely to benefit from TKI therapy were not involved in the study. Thus, despite TKIs demonstrated efficacy in animal models of PAH, severe adverse effects limited their use for PAH treatment.
Based on promising preclinical results, a phase II investigating nilotinib (Bcr-Abl, c-Kit, PDGFR-α/β inhibitor) for the treatment of PAH was initiated (ClinicalTrials.gov Identifier: NCT01179737). This trial was terminated because the drug was not well tolerated and higher rates of SAE events (such as hepato-biliary disorders, three cases of PAH worsening out of 12 treated patients) were observed in the treatment group. Indeed, nilotinib prolonged QT interval (Lu et al., 2012).
In the context of PH and RV pressure overload, de Raaf and colleagues tested the effects of BIBF1000, a nintedanib analogue that targets VEGFRs, PDGFRs, and FGFRs, in a PA banding rat model and demonstrated that 35 days of treatment with a clinically relevant dosage had no detrimental effects on either RV morphology or function (de Raaf et al., 2016). Unfortunately, limited therapeutic success was observed in clinical settings for nintedanib (Richter et al., 2018) as well as for dasatinib (Bcr-Abl, c-Kit, PDGFR-α/β, and Src inhibitor) Weatherald, Chumais, Savale, et al., 2017) (Hasinoff, Patel, & Wu, 2017). Clinical data from cancer patients further point towards a better safety profile for nintedanib when compared to sunitinib (Eisen et al., 2015), likely because of different affinities of the inhibitors to the targeted TKs such as VEGFRs. This is important as target affinity and "onand off-target" toxicities further define the cellular function of the respective kinases and need to be taken into consideration when re-purposing cancer drugs. But lapatinib appears as to be the safest substance among TKIs in terms of cardiotoxicity as demonstrated by recent meta-analysis of clinical trials (Rahmani, Shahriary, Sheikhi, Ebadi, & Davoodzadeh, 2017).
Along the same line, a key role for signalling through the receptor TK IGF-1R in tumour biology has been postulated and specific inhibitors were generated (King, Aleksic, Haluska, & Macaulay, 2014). Shi et al. tested the effects of GSK1904529A, a specific IGF-1R inhibitor, on RV structure and function in a PA banding mouse model (Shi et al., 2016). Therapeutic administration after RV dysfunction and hypertrophy was established, improved RV function and decreased RV hypertrophy, pointing towards a role for IGF-1R signalling in hypertrophic remodelling of cardiomyocytes.

| MAPK SIGNALLING
Intracellular kinases which are placed downstream of growth factor receptor kinases came into the focus of research on new therapeutic strategies. Some of them are directly involved in mediating the growth stimuli from the previously mentioned growth factor receptor kinases to the level of transcriptional control. p38 MAPK inhibitors were initially developed for the treatment of inflammatory and rheumatoid diseases. Targeting p38-MAPK became an interesting therapeutic option for PH since this would target both remodelling and inflammatory pathways in the pulmonary vasculature. Pharmacological inhibition of the p38-MAPK in adventitial fibroblasts showed impressive effects in vitro and in in vivo models of pulmonary hypertension (Church et al., 2015). Here, the compound PH-797804 was able to prevent and reverse the pulmonary hypertensive phenotype. The authors further identified IL6 as a main pathogenic cytokine negatively affected by PH-797804 which taken together led them to the conclusion that inhibition of p38-MAPKα targets both remodelling and inflammatory pathways in pulmonary vascular disease. Furthermore, PH-797804 led to improved lung function and reduced dyspnoea in COPD patients (MacNee, Allan, Jones, De Salvo, & Tan, 2013). In the study, the patients were randomized to placebo, 0.5, 3, 6, or 10 mg or PH-797804 treatment. Interestingly, the dosage of 6 mg daily was associated with clinical improvement at 6 weeks of treatment, but not the dosage of 10 mg. PH-797804 has demonstrated a good safety profile with the most commonly reported adverse effect being acneform rash. No liver problems were reported (MacNee et al., 2013). The inflammation markers were not measured in the study. Losmapimod, a p38-MAPK inhibitor developed by GlaxoSmithKline, tested for treatment of COPD patients, did not reduce markers of inflammation measured as sputum neutrophil count, circulating levels of IL-6, IL-8, and C-reactive protein (Lomas et al., 2012). p38-MAPK signalling is involved in the pathobiology of atherogenesis, plaque destabilization, and maladaptive remodelling after myocardial infarction. Therefore, losmapimod has been tested for treatment of patients with acute myocardial infarction (O'Donoghue et al., 2016). Losmapimod has reduced the concentration of C-reactive protein and the incidence of liver enzyme abnormalities and of infections were similar by the treatment groups. However, no reduction in the risk of recurrent major adverse cardiovascular events was observed. Therefore, the development of losmapimod for the myocardial infarction treatment was stopped. SCIO-469 (talmapimod), another p38-MAPK inhibitor tested for the treatment of the rheumatoid arthritis, has demonstrated only transient improvement in the markers of the disease activity, additionally it caused unacceptable side effects including liver toxicity and skin reactions (Genovese et al., 2011). Therefore, the clinical development of the substance was discontinued.
Along the same line, an MAPK upstream of the p38-MAPK cascade, Ask1 (apoptosis signal-regulating kinase 1), was targeted with a small molecule inhibitor (GS-444217) to halt disease progression in experimental PH models (Budas et al., 2018). Ask1 belongs to the class of ubiquitously expressed MAP 3K (MAPK kinase kinase) and is activated in response to oxidative stress. Upon activation, Ask1 phosphorylates MAP 2Ks which in turn phosphorylate and activate effector MAPKs, such as p38-MAPK and JNK. In a recent study, GS-444217 blocked p38-MAPK phosphorylation and reduced pulmonary vascular remodelling, decreased pulmonary arterial pressure, and reduced right ventricular hypertrophy in PH models with established disease (Budas et al., 2018). In a PA banding mouse model, selective inhibition of Ask1 with GS-444217 (a selonsertib analogue) as well as p38-MAPK via PH-797804 improved RV function through a robust reduction in RV fibrosis when administered once RV dysfunction and hypertrophy was established (Budas et al., 2018;Kojonazarov et al., 2017). Altogether, these data point towards a pro-fibrotic role for MAPK signalling through p38-MAPK and highlight the anti-fibrotic potential of MAPK inhibitors in the pressure overloaded RV. The role of MAPK signalling in the hypertrophied RV has recently attracted attention in PAH as a phase II clinical trial with selonsertib was conducted (Rosenkranz et al., 2017). Here, selonsertib at the dosages of 2, 6, and 18 mg was tested for PAH treatment (ClinicalTrials.gov Identifier: NCT02234141). Selonsertib did not change the pulmonary vascular resistance (primary endpoint) and 6-min walk distance (Rosenkranz et al., https://erj.ersjournals.com/content/50/suppl_61/ OA1983; Boucherat, Provencher, & Bonnet, 2018). No significant change was observed in the markers of cardiac function: NT-proBNP, cardiac index, the right ventricular cardiac power, or the right ventricular performance index. The numbers of reported SAE was higher in the groups of patients treated with selonsertib 6 mg and 18 mg, being 17 cases in 49 patients (17/49; 34.69%) in the 6-mg treatment group and 16/47 (34.04%) in 18-mg treatment versus 7/37 (18.92%) in the placebo group. SAE included haemorrhagic anaemia (two cases-one case in 6-mg group and one case in 18-mg group), RV failure (four cases in 6-mg and one case in 18-mg groups vs. one case in placebo group), and pneumonia (three cases in 6-mg and two cases in 18-mg groups vs. no cases in placebo group). In a phase II trial testing selonsertib for treatment of nonalcoholic steatohepatitis, no heartrelated SAE were reported (Loomba et al., 2017). This might suggest that compromised RV in PAH could be prone to effects of the inhibitor. Indeed, increased Ask1 activity has been demonstrated in the stressed RV (Budas et al., 2018) and in LV (Nako et al., 2012).

| JAK-STAT SIGNALLING
Jak/STAT is a signalling pathway regulating immunity, cell division, and cell death, whose importance has been recognized in PAH pathobiology (Paulin, Meloche, & Bonnet, 2012). It recently became an attractive drug target as it was shown to be deregulated in a broad range of fibrotic disorders such as myelofibrosis. Therefore, ruxolitinib, a Jak1/2 inhibitor, is approved by the FDA for the treatment of myeloproliferative neoplasms like essential thrombocythemia, polycythemia vera, and myelofibrosis. Jak2 is a major regulator of cytokine and growth factor stimulation leading to the downstream activation of Stat3 signalling and also to the activation of the noncanonical PI3K as well as the MAPK cascade, all of which are involved in the pathogenesis of idiopathic pulmonary fibrosis (IPF) and PH (Paulin et al., 2012). This makes Jak2 an ideal kinase for specific inhibition by compounds like ruxolitinib or JSI-124 (Milara et al., 2018). The authors showed that Jak2 is activated in pulmonary arteries from idiopathic fibrosis (IPF) patients, which is further increased in PH associated to IPF (PH-IPF) and that Jak2 inhibition ameliorates pulmonary artery remodelling, increases pulmonary artery relaxation, and improves the overall symptoms of the disease in this experimental model of bleomycin-induced IPF with PH. Interestingly, ruxolitinib has been reported to improve pulmonary hypertension in patients with myelofibrosis (Tabarroki et al., 2014). It reduced right ventricular pressure (measured by echocardiography), elevated NO, and decreased NT-proBNP. Of note, a case of PAH worsening in a myelofibrosis patient treated with ruxolitinib has been described (Low, Howard, Harrison, & Tulloh, 2015). In a peptide-based kinase-activity screen, Jak2 was identified as highly active in PAH-patient derived PASMCs compared to healthy cells. Jak2 inhibition by ruxolitinib led to an improvement of cardio-pulmonary function in two independent animal models of P(A)H via blockage of the Jak2-Stat3 signalling pathway (Dinesh Yerabolu, Astrid Weiss, Ralph Schermuly, May 15th 2019, personal communication). Jak/STAT is an important signalling pathway involved in homeostasis in many organ systems indicating a high risk for unwanted side effects. Many adverse effects upon Jak/STAT inhibition have been described, such as anaemia, agranulocytosis, thrombocytopenia, gastrointestinal disturbances, and neurotoxicity (Tefferi & Pardanani, 2011). The high rate of the treatment associated adverse effects limits the use of ruxolitinib for myelofibrosis therapy (Verstovsek, 2009). CEP-701 (lestaurtinib), XL019, and TG101348 are other Jak inhibitors in an early clinical phase of development (Verstovsek, 2009).
CDKs together with their corresponding cyclin orchestrate the complex events that drive the cell cycle. Transition to the next phase depends on the activity of CDK1, CDK2, CDK4, and CDK6 all of which phosphorylate and inactivate the Rb (retinoblastoma protein), a tumour suppressor protein that limits the transcription of E2F proliferative target genes. The activity of the CDK-cyclin complexes is often deregulated in cancer cells leading to uncontrolled cell growth due to increased Rb phosphorylation (i.e., its inactivation) and subsequent enhanced transcriptional E2F activity.
Palbociclib, targeting CDK4 and CDK6, has been recently approved for the treatment of advanced breast cancer. The authors could show that CDK4 and CDK6 inhibition by palbociclib reduced proliferation via cell cycle arrest and interference with the downstream CDK-Rb-E2F signalling pathway in pulmonary vascular cells (Weiss et al., 2019). In vivo, palbociclib was able to reverse the pathological disease phenotype by a reduction in right ventricular systolic pressure, reduction in right heart hypertrophy, restoration of the cardiac index, and a reduction pulmonary vascular remodelling.
These results clearly illustrate that CDK inhibition by palbociclib can provide an additional therapeutic option in the treatment of PAH (Weiss et al., 2019). Besides palbociclib, several other CDK inhibitors are currently in the development such as dinaciclib, milciclib, riviciclib, roniciclib, seliciclib, and voruciclib, which differ in their activity and target profile (Galbraith, Bender, & Espinosa, 2019). Their most common adverse effect is myelosuppression causing development of anaemia, infections, and thrombocytopenia (Loibl et al., 2017). Reversible neutropenia was reported as a most common adverse effect of dinaciclib (Mita et al., 2017). For the milciclib, neutropenia and thrombocytopenia were reported as the most common adverse effect (Aspeslagh et al., 2017). No cardiotoxic effects were reported for palbociclib. When administered at the recommended dosing, it did not cause prolonged QT intervals to a clinically relevant level (Durairaj et al., 2018). Roniciclib was tested for the treatment of different kinds of solid tumours Lin, Lin, Hsueh, Chou, & Wong, 2018;Reck et al., 2019). All of the listed drugs are novel substances; thus, more information is awaited from the currently running studies about their efficiency and toxicity.

| TGF RECEPTOR SIGNALLING
In mammals, seven type I (also known as activin receptor-like kinases [ALKs]) and five type II receptors belong to the superfamily of TGF-β receptors (intensively reviewed in Rol, Kurakula, Happe, Bogaard, & Goumans, 2018). Of all receptors belonging to the TGF-β superfamily, the bone morphogenetic protein type 2 receptor (BMPR2) is potentially the most relevant one for PAH.
More than 70% of patients with familial PAH and 20% of patients with idiopathic PAH have heterozygous mutations that interfere with BMPR2 kinase function. These mutations can lead to a lossof-function phenotype associated with a higher disease severity (Evans et al., 2016). Id proteins are transcriptional targets of active BMPR signalling and function as dominant-negative transcriptional regulators. In the absence of ligands (e.g., BMP and GDF), the pathway is maintained in an inactive status by FKBP12 which shields serine and threonine residues of BMPR1 from being phosphorylated by BMPR2. Once activated, the transcriptional targets, Id1, Id2, and Id3, are all expressed by PAECs and PASMCs underlining their critical role in the tight regulation of cellular differentiation and proliferation. In the case of mutated BMPR2, there is a reduction in Id gene transcription which results in a loss of the growth suppressive effects of BMPs. Hence, the therapeutic approaches aim to reconstitute the kinase function of BMPR2 to regain cell cycle control. Recently, tacrolimus (FK506) was successfully tested in a phase IIa trial in PAH patients which showed that the compound is well tolerated and that BMPR2 levels could be increased in a subset of patients (Spiekerkoetter et al., 2017). This further supports the authors' previous encouraging results obtained in two animal models of PH in which a reduction in right ventricular systolic pressure and right ventricular hypertrophy was observed (Spiekerkoetter et al., 2013). In terms of the mode of action, it is still not clear how precisely tacrolimus restores/activates BMPR2 levels. Similarly to BMP-induced pathways, TGF-β signalling is very complex and multifactorial. Here, although TGF-βRII/TGF-βRI is the preferable high affinity signalling complex, in endothelial cells, TGF-β can also signal through TGF-βRII/ALK1/ALK5. In idiopathic PAH, levels of ALK1 and endoglin (membrane glycoprotein) are specifically increased in PAECs, leading to enhanced Smad1/5 phosphorylation when stimulated with TGF-β (Gore et al., 2014). In the context of PAH, until now, only a limited number of kinase inhibitors interfering with TGF-β receptor activation, specifically the ALK5 kinase (TGF-βRI), were applied in animal models of PH. SD-208 (Zaiman et al., 2008) as well as SB525334 (Thomas et al., 2009) significantly attenuated the development of the PH and inhibited right ventricular hypertrophy in a rat model of PAH. SB525334 inhibited the ex vivo TGF-β induced proliferation of the PASMC isolated from PAH patients and reduced vascular remodelling in MCT model (Thomas et al., 2009).
Currently, many substances (including monoclonal antibodies) targeting TGF-β or its receptors are at the preclinical or early clinical stage of development (Haque & Morris, 2017). LY2157299, a small molecule TGF-β inhibitor was tested in a first-in-human dose study and demonstrated a good safety profile with no cardiovascular toxicities and no tumourigenesis (Rodon et al., 2015). without apparent side effects (Abe et al., 2004;Abe et al., 2006;Jiang et al., 2014;Kojonazarov, Myrzaakhmatova, Sooronbaev, Ishizaki, & Aldashev, 2012;Zhang & Wu, 2017). Fasudil is approved in Japan for the treatment of cerebral vasospasm induced by subarachnoid haemorrhage. Its acute vasodilator effects have been shown in the patients with severe IPAH (Fukumoto et al., 2005). In the study, intravenous administration of fasudil caused an acute reduction in PVR and an increase in cardiac output. These observations were confirmed in another study (Ishikura et al., 2006) but only a non-significant decrease in systemic pressure was observed. Selective pulmonary vasodilative effect of fasudil was observed in the Kyrgyz highlanders suffering from hypoxia-induced PAH (Kojonazarov et al., 2012). When compared with the aerosolized ioprost, fasudil was as efficient as iloprost in PVR reduction, but caused more pronounced increase in cardiac output (Jiang et al., 2014). Whether the observed difference is attributed to the direct cardiac effects of fasudil remains open. In fact, recent findings suggest that disturbances in ROCK signalling contribute to heart failure and that ROCK inhibition restores calcium handling in myocardium normalizing cardiac contractility (Olgar et al., 2017). In a model of PAH, Rho kinase has been shown to mediate right ventricular systolic dysfunction (Gosal et al., 2015). The observed beneficial haemodynamic effects of fasudil suggest it to be, potentially, an interesting agent to treat right heart failure in the settings of exaggerated PAH. Indeed, intravenous fasudil treatment improved in-hospital mortality and the 30-day re-hospitalization rate of patients with right heart failure due to severe pulmonary hypertension (Jiang et al., 2015). In the double-blinded placebo-controlled study, 10 PAH patients were treated with fasudil for 3 months, and it did not significantly reduce PVR but improved cardiac index (Fukumoto et al., 2013). In all the cited studies, fasudil was well tolerated. However, its long-term effects on the right heart and the metabolic costs of its possible, cardiac stimulating effects are unknown. A study suggests that it might have beneficial effects on cardiac perfusion by relieving coronary microvascular spasm (Mohri, Shimokawa, Hirakawa, Masumoto, & Takeshita, 2003). Azaindole-1 showed similar effects in hypoxia and MCT-induced PH but also reduced right ventricular hypertrophy . Very recently, a new inhibitor, named compound 3, has been shown to be more potent and highly selective for ROCK-1 and 2 to improve haemodynamic parameters and counteract pulmonary vascular remodelling in experimental PAH (Cantoni et al., 2019). Thus, fasudil and other ROCK inhibitors deserve further investigation with regards to their effects on the right ventricle in PAH.
The sphingosine kinase 1 (SphK1) is another example for an interesting, potentially druggable kinase of the GPCR-induced signalling network. The intracellular SphK1 catalyses the formation of S1P (sphingosine-1-phosphate), a lipid messenger with intracellular and extracellular functions. S1P can be released from pulmonary vascular cells and might act in an autocrine manner via the S1P receptor (GPCR). The SphK1 signalling pathway can regulate expression of Stat3 (increased) and BMPR2 (decreased) thereby building a potential overlap with known PAH disease causing cellular mechanisms. An initial study with SLP7111228, a SphK1 inhibitor, demonstrates effectiveness to improve both cardiac and total pulmonary artery resistance index but without affecting RVSP or RVH in Su/Hox rats (Gairhe et al., 2016). To the contrary, another SphK1 inhibitor, named SKI-II, prevented hypoxia-mediated PH in rats including right ventricular hypertrophy (Chen et al., 2014) which overall shows the discrep-

| OTHER DRUGS FOR THE TREATMENT OF PAH
In contrast to the previously mentioned kinase inhibitors, at least one compound with an unknown set of target structures has been described to exert a therapeutic effect in the treatment of P(A)H.
Although the details of the mode of action for pirfenidone are yet still unclear, this drug showed impressive results in terms of partial reversal of established pulmonary hypertension with a reduction in total pulmonary vascular resistance and remodelling (Poble et al., 2019). In contrast to previously discussed kinase inhibitors, pirfenidone is in clinical use for longer time, and more is known about its efficiency and safety. Currently, it is approved for the treatment of idiopathic pulmonary fibrosis (IPF). In phase III clinical trials assessing pirfenidone in patients with IPF, it reduced lung function decline, slowed disease progression, and reduced mortality . Beside multicentre randomized clinical trials, pirfenidone's efficiency was confirmed in so-called "real-world" studies from centres treating IPF patient (Oltmanns et al., 2014).
The most commonly reported adverse effects of pirfenidone are rash and dysfunction of the gastrointestinal system (Lancaster et al., 2017). Pirfenidone is being tested also for treatment of rheumatic disorders with promising results (Walker & Margolin, 2001 (Pyne & Pyne, 2017). Finally, initial data from the clinical phase I study with an albumin-bound mTOR inhibitor (nabsirolimus, ABI-009) in severe PAH are expected to be published soon (for details see Table 2). Of course, successful application can be limited by ineffectiveness, drug-related but unwanted side effects and/or cardiotoxicity as previously discussed. This demonstrates the necessity for further investigations of existing kinase inhibitors as well as the search for new "druggable" protein kinases. Hopefully, these studies will make a substantial contribution to upcoming therapeutic options in the treatment of PH in addition to other classes of inhibitors (Sitbon et al., 2019).

| FUTURE DIRECTIONS
Targeted kinase inhibition is a reasonable therapeutic approach as demonstrated by the efficacy of distinct compounds in experimental models of PH (in Table 1) or initial clinical studies (Table 2).
However, TK inhibitor induced severe adverse events that limit their use for the treatment of PAH patients. One approach to improve tolerability and to reduce appearance of adverse effects would be to decrease the drug dosages or to establish a treatment regimen with intermittent drug administration similar to oncologic studies. In the best case, the same effective dosages of kinase inhibitors as used for oncologic patients should be applied. In the IMPRES study, the patients received 200-mg imatinib once daily with the subsequent up-titration to 400 mg once daily if tolerated (Frost et al., 2015). Whether the observed therapeutic effect correlated with the achieved dosage is unclear due to low patient numbers. A hint supporting the suggestion to treat PAH patients with lower dosages comes from animal studies. In an experimental model of PAH, imatinib was efficient at the dosages of 50 and 100 mg/kg (Schermuly et al., 2005) and 15 and 50 mg/kg (Leong, Okida, Higuchi, Yamano, & Hikasa, 2018). For comparison, in oncological studies, imatinib was used at the dosages ranging from 100 mg/kg (Beppu, Jaboine, Merchant, Mackall, & Thiele, 2004;Irsan et al., 2007) to 200 mg/kg (Basciani et al., 2005;Beppu et al., 2004).
Altogether, various multi-targeting and highly specific kinase  (Gessler, 2018). Intermittent administration is another opportunity to limit the risk of side effects as stated previously (Bracarda et al., 2017). Here, the underlying assumption is that aberrant pulmonary vascular cells are affected during the time period of drug administration while the healthy cells of the body do not experience any harm and/or only the latter ones can recover from the compounds burden (so called reversal of cardio-toxicity).
It should be kept in mind that the disease-driving force of an increased kinase activity is the result of a multifactorial process.
Epigenetic changes in DNA methylation, histone modifications, and the abundance of non-coding RNAs (like lncRNAs or microRNAs) can account for an over-expression as well as post-transcriptional or post-translational events. Here, a special emphasis lies on protein phosphatases that play an important role on the level of endogenous regulators of kinase activity. They serve as a master switch by removing crucial phosphate residues from their substrate molecules. While protein serine/threonine phosphatases (PPPs) are promiscuous in terms of their substrate selectivity, protein tyrosine phosphatases (PTPs) display a more distinct profile and could be of interest for targeted re-constitution or more easily for inhibition. In  (Shi et al., 2016) Positive effects on lung function and remodeling are reflected by a decrease in mean pulmonary arterial pressure (" Function) and/or a reduction in the amount of structurally remodeled, muscularized pulmonary vessels (# Remodeling). Improved right ventricular performance is demonstrated by a diminished right ventricular systolic pressure or by an elevation in cardiac output (or index) (" Function), reduced ratio of right ventricle to left ventricle plus septum weight (# Hypertrophy) and less collagen deposition or enhanced capillary density (# Fibrosis). In few publications, no significant change was obtained for the selected parameter (!) or it was not determined (nd.).
T A B L E 2 Kinase inhibitors used in clinical trials for the treatment of pulmonary hypertension screening of a compound library and the identification of a lead structure finally resulting in a new chemical molecule with the best performance in activity-based high throughput assays. In PH, many anti-cancer drugs have been analysed for their efficacy to inhibit hyper-proliferation of pulmonary vascular cells and/or to directly interfere with the right ventricular remodelling process. Experimental PH models are employed to demonstrate the kinase inhibitors' mode of action and any signs of systemic and/or cardiac toxicity.
For the latter case, long-term treatment studies in animals could reveal unknown effects (including adverse events) in the lung and the heart. In the final step, clinical studies are conducted to analyse the mode of action, tolerability, and possible signs of (hidden) toxicity in human PH. In phase II and III studies, drug responses will be detectable on the individual basis, and corresponding biomaterial sampling will allow a deeper investigation on the cellular level.
Distinct degrees of responsiveness to a given drug for a patient subgroup (coloured symbols) might be observed, which can be linked to activity or expression profiles, that is, of biomarkers, or even a particular genetic background. As the costs for DNA F I G U R E 1 Targets of kinase inhibitors for the treatment of pulmonary hypertension. Kinases for targeted inhibition are depicted in red while other intracellular signalling proteins are coloured in grey. Membrane receptors are highlighted in beige. Coloured connections reflect activating protein-protein interaction (green) or blockage of protein function (red). Interference with kinases activity in the pulmonary vasculature by the depicted inhibitors leads to a reduction in proliferation, inflammation, and vasoconstriction which subsequently promotes the reversal of the vascular remodelling process. In the right ventricle, anti-hypertrophic and anti-fibrotic effects are observed resulting in improved cardiac function sequencing have been dramatically decreasing, whole-exome sequencing is now affordable and can provide information about possible SNPs or crucial mutations which influence the therapeutic outcome of the individual patients. This hypothesis is a central aspect of a relatively novel field of research named pharmacogenomics which combines pharmacology and genomics to study how individual genes affect a patient's response to a certain drug.
Thus, many critical determinants of successful medication like doses, safety, and efficacy, can be linked to a particular genetic background which allows improved and personalized medicine in a From-bedside-to-bench-and-back fashion. Finally, this approach would offer a comparison of patient characteristics, prognosis, and responses to different classes of medications according to their distinct signatures and ultimately a better choice for the most effective therapy for each patient (Kan, Shumyatcher, & Himes, 2017;Savale, Guignabert, Weatherald, & Humbert, 2018). This idea could be combined with the observation of an individual responsiveness to a particular class of medication across the phenotypically diverse PAH population (Halliday & Hemnes, 2017). But these strategies have just evolved in the recent years, and current limitations need to be addressed such as the small number of datasets, the heterogeneity during sample collection, and missing harmonized protocols. In conclusion, kinase inhibitors have the potential to become an approved treatment option for PH in the near future, if patients can be classified to selective treatments prior drug administration and if (cardio)toxicity can be reduced to a minimum.

| Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY , and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander, Christopoulos, et al., 2017;Alexander, Fabbro, et al., 2017a, 2017b.

ACKNOWLEDGEMENTS
We would like to thank Tatjana Hirschmugl for drawing of Figure 2 and Megan Grether for critical proofreading of our manuscript. This work was funded by the Deutsche Forschungsgemeinschaft (DFG, F I G U R E 2 New concepts in clinical drug development for the therapy of pulmonary hypertension. To characterize and assess the phenotype, including all pathobiological features of PH, data from basic research (research data) as well as from clinical examinations (clinical data) are collected. Despite the heterogeneity amongst all patients suffering from pulmonary hypertension, different subcategories (coloured symbols) can be identified which display different degrees of similarities with results obtained using primary cells or experimental PH models (overlapping areas). Newly available technologies in combination with routine clinical inspections now allow for an in depth phenotyping of all individual patients including the investigations of metabolic status, kinase activity measurements, RNA/DNA sequencing. By this approach, an appropriate kinase can be validated as ideal drug target in vitro including information about its role in PH. This facilitates the selection of compounds of interest (like kinase inhibitors) to be tested in preclinical studies with PH models in vivo. In the final stage, clinical studies are conducted to analyse the mode of action as well as any possible signs of toxicity (systemic and [hidden] cardiotoxicity). Here, individual drug response and prognostic biomarkers might be identified for different sub-categories of PH patients (coloured symbols). This can be achieved by pharmacogenomics, a relatively novel field which combines pharmacology and genomics to study how individual genes affect a patient's response to a certain drug. Through this process many critical determinants of a successful medication like doses, safety, efficacy, can be linked to a person's genetic background which allows improved and personalized medicine. For further details see text