PHARMACOLOGICAL CASPASE INHIBITORS:
RESEARCH TOWARDS THERAPEUTIC PERSPECTIVES

Caspases are key molecules executing cell death during ontogenesis and homeostasis of multicellular organisms (1, 2). They are activated in both main apoptotic pathways: extrinsic, mediated by death receptors, and intrinsic, where mitochondria play a central role. Moreover, caspases are essential for cytokine maturation during inflammation and contribute to biological processes such as proliferation, differentiation and migration.

According to their structure and function, caspases are classified into three groups. Caspases with long prodomains are mainly involved in inflammation (pro-inflammatory caspases) and apical phases of apoptosis (initiators); short prodomain caspases (executioners) act downstream of initiator caspases. Initiator caspases are synthesized as inactive monomers and typically require dimerization. Executioner caspases are synthesized as inactive dimers, they require cleavage to achieve their active conformation (3).

Increased apoptosis and caspase activity are associated with a number of disorders including hepatic and transplant injuries, myocardial infarction and neurodegenerative diseases, while pro-inflammatory caspases are implicated in rheumatoid arthritis or psoriasis (4, 5). For such diseases, pharmacological inhibition of caspases represents a possible therapeutic approach, as proved in animal models and initial clinical trials (6, 7). No caspase inhibitor-based drug has been introduced to the praxis until now and development of pharmaceuticals specifically targeting caspases remains a great challenge and is now the focus of intense biological and clinical interest.

CASPASE INHIBITORS

Hundreds of synthetic caspase inhibitors have been designed with the aim of bringing more insight into the complex caspase network and also to find new pharmaceuticals. Most of the inhibitors are pseudosubstrates (peptide based inhibitors, peptidomimetics and non-peptidic compounds), which block the active site of the enzyme. Allosteric inhibitors seem to be an innovative way to improve target selectivity (8, 9) (Fig. 1).

Fig. 1. Scheme of caspase inhibition. Covalent caspase inhibitors act as pseudosubstrates that block their catalytic site. A llosteric inhibitors form disulphide bonds with an allosteric site of the enzyme in its inactive state thereby preventing the formation of the active conformation of the enzyme and substrate binding to the active site.

The first synthetic caspase inhibitors were peptides based on aspartic acid modified with a reactive electrophilic group (so-called warhead or cysteine trap), forming reversible/irreversible covalent linkage with the nucleophilic active thiol site of the enzyme. A modified aspartate residue per se may serve as an inhibitor (Boc-Asp-FMK), but it is usually a part of a tri/tetrapeptide (P4)-P3-P2, corresponding with the substrate specificity of caspases (Fig. 1).

Inhibitors with different characteristics have been designed by using different electrophilic warheads. Peptide aldehydes, ketones and nitriles are reversible inhibitors with easily hydrolysable linkage (10). In contrast, a-substituted ketones, such as fluormethylketone (FMK) and chlormethylketone (CMK) irreversibly inactivate caspases trough the formation of thiomethylketone with the active site cysteine (10). Moreover, structure-activity relationship (SAR) revealed that 2, 6-dichlorobenzoyl (DCB) and trifluoperazine (TFP) warheads are successful irreversible moieties (11).

FMK inhibitors have a wide range of specificity and efficiency to block caspase activity. Specific tetrapeptide inhibitors Z/AC-DEVD-FMK, AC-YVAD-FMK/CMK, as well as the broad spectrum tripeptide inhibitor Z-VAD-FMK, are the most widely used caspase inhibitors for research purposes (12) and have substantially contributed to the understanding of apoptosis pathologies in animal models. FMK inhibitors show sufficient permeability compared to aldehyde warheads; however, they also affect other cysteine proteases and are metabolized to toxic fluoracetate (13, 14). Thus, next generation broad spectrum caspase inhibitors have been synthesized with an O-phenoxy group added at the C terminus together with a quinolyl group at the N terminus (Q-VD-OPH), which show higher effectiveness, reduced toxicity and enhanced cellular permeability compared to FMK inhibitors and are able to cross the blood-brain barrier (14).

The use of peptide inhibitors is limited for drug development. Despite substantial improvement, their pharmacokinetic properties (poor bioavailability, cell permeability, metabolic stability) are still not sufficient. Therefore, the peptidic nature of caspase inhibitors was reduced by the use of compounds mimicking the peptide backbone, active warheads were modified and chemical libraries were searched for small molecule caspase inhibitors (15). Application of these strategies resulted in the development of several peptidomimetic and non-peptidic caspase inhibitors based on heterocyclic scaffolds (isatin sulphonamides, aza-peptide epoxides, benzyl- and cyclohexyl-amines, pyrazinone mono-amides) with enhanced selectivity, stability and in vivo activity (16-18); some of them even entered clinical testing, such as IDN-6556; VX-765 (Table 1).

Table 1. Prospective clinical applications of caspase inhibitors.

The design of specific caspase inhibitors via active site targeting is very difficult due to a stringent requirement for active site Asp for all caspases. Therefore, researchers focused on the dimerisation interface of the enzymes. This is very variable among caspases and thus offers new unique targets, e.g. BIR3 allosteric inhibition mechanism typical for caspase-9 (19). The newly designed allosteric inhibitors form disulphide bonds with cysteine allosteric sites in the dimerization interface and alter their productive conformation towards one that strongly resembles caspases in their zymogenic form (20, 21) (Fig. 1).

CASPASE INHIBITORS IN ANIMAL MODELS OF VARIOUS DISEASES

Some current applications of caspase inhibitors are in the research field, where they have confirmed their irreplaceable role in understanding mechanisms of cell death in cell cultures and organ explants, as well as in vivo in physiological and mainly pathophysiological situations. In animal models, caspase inhibitors revealed beneficial effects on ischaemia-reperfusion injury, sepsis, hepatitis, fibrosis and neurodegenerative disorders (Table 2).

Table 2. Diseases associated with inappropriate caspase activation and caspase inhibitors used to investigate related disorders.

Liver injury

Beneficial effects of various caspase inhibitors have been shown in several models of liver diseases: the application of caspase inhibitors reduced transaminase elevation and apoptosis and decreased mortality in rodent models of acute liver failure, reduced ischaemia-reperfusion injury in rodent models and reduced hepatic fibrosis in bile duct ligated and NASH (non-alcoholic steatohepatitis) mouse models (22, 23). One of the first reports of the use of caspase inhibitors in vivo was in mouse models of liver injury (24). Since that time, several caspase inhibitors, either broad spectrum (Z-VAD-FMK, MX1122) (25) or caspase-1 specific (YVAD-CMK) (26), have been shown to inhibit hepatic apoptosis and prevent signs of liver damage induced by anti-CD95 antibodies or tumor necrosis factor (TNF) administration and improved recovery from the treatment. VX-166, a pan-caspase inhibitor decreased caspase-3 and fibrosis in liver (23). Based on preclinical data from animal models, several clinical trials have been performed to treat human liver pathologies (Table 1).

Autoimmune disorders

Caspases play also an important role in autoimmune disorders and infections. The inhibition of caspase-1 was effective in models of rheumatoid arthritis, osteoarthritis and psoriasis (27, 28). Z-VAD-FMK was shown to be protective in the asthma model, decreasing the infiltration of airways, mucus hypersecretion, oedema, and Th2 cytokine release (29). Moreover, Z-VAD-FMK effectively blocked pathologic fibrosis, which might result from autoimmune disorders and infections, as shown in the bleomycin-induced lung fibrosis model (30).

Infections and sepsis

Regarding bacterial infections, Z-VAD-FMK accelerated peritoneal bacterial clearance in peritonitis (31) and decreased hippocampal neuronal cell death in pneumococcal meningitis (32) as well as prevented lymphocyte apoptosis and improved survival in the caecal ligation puncture and pneumonia model of sepsis (33-35). Bacterial sepsis is a growing health problem with high mortality rate associated with a substantial loss of lymphocytes. This may be due to dysregulated apoptosis affecting both intrinsic and extrinsic cell death pathways, as shown by the pharmacological as well as genetic ablation of particular apoptotic cascade players (36). Several broad spectrum caspase inhibitors along with caspase-1, caspase-9 and caspase-8 specific inhibitors have been shown to effectively improve sepsis (37-40). Inflammatory caspase-11 and -12 seem to be promising targets for selective anti-caspase therapy, since caspase-11 deficient mice are resistant to LPS-induced shock (41). Caspase-12 polymorphism in humans is related with the vulnerability to sepsis and caspase-12 deficient mice do better in the case of sepsis (42). Previously mentioned pan-caspase inhibitor VX-166 has also potential in treatment for sepsis (43).

Ischaemia-reperfusion injury

Ischaemia-reperfusion injury is a complex phenomenon that comprises oxidative stress, inflammation and apoptosis activation. Several studies in various animal models have revealed the positive effect of caspase inhibitors on the ischaemia-reperfusion injury in the liver (44), lung (45), heart (46), kidney (47), and brain (48, 49).

MMPSI (isatin sulphonamide, reversible caspase-3 and -7 inhibitor) reduced apoptosis in vitro in a hypoxia model of cardiomyocytes (50). The inhibitor Z-VAD-FMK attenuated cardiomyocyte apoptosis in myocardial injury in rats subjected to a coronary occlusion followed by reperfusion (51). Z-LEHD-FMK (a specific caspase-9 inhibitor) was able to protect the rat myocardium during coronary microembolization by inhibiting apoptosis and improving cardiac function (52).

In the case of acute neurologic ischaemia, a neuroprotective effect was achieved using broad spectrum (Z-VAD-FMK, YVAD-CMK, Boc-Asp-FMK, IDN-5370; Q-VD-OPH), caspase-3-specific (DEVD-FMK, M826), caspase-1-specific (VRT-018858) and caspase-9-specific inhibitors (53-57). Moreover, caspase-2 seems to be a promising target for the future treatment of perinatal brain hypoxia-ischaemia. Caspase-2 is highly expressed in the neonatal brain (58) and its inhibition (TRP601) protects the newborn rodent brain against excytotoxicity, hypoxia-ischaemia, and perinatal arterial stroke, with no adverse effects on physiological parameters (59).

Central nervous system traumatic injury

Acute traumatic injury of the brain and spinal cord includes the activation of multiple caspases in neurons and astrocytes, but also in oligodendrocytes and microglia (60, 61). Their general blockade by Z-VAD-FMK was shown to improve the neurological outcome (62). Particularly, caspase-3 and caspase-1 seem to be crucial for neurological dysfunction in a rat model of traumatic brain injury (63-65). Namely broad spectrum caspase inhibitors (Z-VAD-FMK, Q-VD-OPh) (66, 67) and caspase-3 inhibitors (Z-DEVD-FMK, Ac-DMQD-CHO) (68, 69) were effective in the reduction of trauma-induced apoptosis in rodent models of traumatic spinal cord injury as well as the caspase-9 inhibitor, Z-LEHD-FMK (70), which reduced post-traumatic lesion size and improved motor performance. The combination of Z-LEDH-FMK with MgSO4 (N-methyl-D aspartate receptor antagonist) was demonstrated to minimize the effects of secondary injury in spinal cord trauma (71). Additionally, caspases-6 and -8 seem to have a prominent role in the apoptosis of neurons in the central nervous system, as shown using selective caspase inhibitors in the model of injured adult retinal ganglion cells (72). Besides the activation of multiple caspases, neurologic trauma also involves caspase independent, AIF (apoptosis inducing factor) mediated, cell death; thus, superior neuroprotection may be achieved with combined therapeutic strategies directed at multiple pathways of programmed cell death (73).

Neurodegeneration

Caspases are also potential therapeutic targets in neurodegenerative disorders and thus caspases may be involved in retinal degeneration in diseases such as glaucoma, age-related macular degeneration, and diabetic retinopathy (74, 75).

Z-VAD-FMK blocked apoptosis of the hair cells treated with aminoglycoside and streptomycin and thus protected against vestibular function deficits in vivo (76). Furthermore, Z-VAD-FMK could prevent apoptosis resulting from gunshot noise trauma (77). Activation of multiple caspases has been demonstrated in Alzheimer disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD) and amyotrophic lateral sclerosis (ALS) (78). Caspase-3 and caspase-6 have been shown to cleave tau and APP and actively contribute to senile plaque formation in AD models (79). Similarly, caspase-6 cleaves the HD protein into toxic fragments which enter the nucleus of neurons and interfere with gene transcription (80).

Blocking of caspases by Z-VAD-FMK delayed the disease onset and also mortality in ALS, and in mice expressing mutant human SOD1, and it also restored glutamatergic signalling and behavioural dysfunctions in transgenic animals (81). Q-VD-OPH revealed its neuroprotective role in mouse models of AD and HD (82). More recently, peptidomimetic or non-peptide inhibitors demonstrated great potential in neuroprotection. A specific reversible inhibitor of caspase-3, M826, prevented cell death of striatal neurons in a mouse model of HD (83).

Transplantation

Besides apoptosis and inflammation-related disorders, the application of caspase inhibitors might contribute to a better outcome of therapeutic applications, including transplantation and cryopreservation. Caspase inhibitors have been shown to inhibit ischaemia/reperfusion injury and thus improved graft survival after liver, lung, and pancreatic islet transplantation (45, 84-87). Namely, IDN-6556 improved glucose tolerance and pancreatic islet graft survival in mice (87) and even progressed to clinical testing.

Anticancer treatment

Cell death induction is a common approach in treatment based e. g. on cytostatics (88-90). Notably, caspase inhibitors have been shown to potentiate the effect of anticancer treatment. Z-VAD-FMK and M867 sensitized solid tumour cells to irradiation in vitro and in vivo in mouse hind limb tumour model (91, 92). Caspase inhibitors may be a component of differentiation therapy of leukaemia, perhaps in combination with derivatives of vitamin D (93).

Other applications

Recently, caspase inhibitors showed their potential for stem cell-based therapeutic applications. Cultivation of HSPCs in media with Z-VAD-FMK/Z-LLY-FMK and cytokines could be useful to enhance their homing potential (94).

Caspase inhibitors (Z-VAD-FMK) have been reported to improve cryopreservation efficiency when added to cryopreservation and post-thaw culture media. Positive results have been achieved in case of canine spermatozoa, porcine hepatocytes, human embryonic stem cells and amniotic fluid-derived stem cells (95-99). Similarly, the treatment of platelets with caspase-3 inhibitor could increase their functionality and survival in platelet concentrates (100).

CASPASE INHIBITORS IN CLINICAL TRIALS

The first inhibitor which entered clinical trial was VX-740 (Pralnacasan). Pralnacasan is an orally bioavailable pro-drug of the reversible specific inhibitor of caspase-1. The structure is derived from the tetrapeptide sequences YVAD, the preferred recognition sequence of caspase-1. Preclinical studies demonstrated that the drug is able to inhibit type II collagen-induced arthritis in mice and to reduce forepaw inflammation by decreasing disease severity by 70% (27). In addition to these effects Pralnacasan also suppressed the synthesis of IFN-γ in splenocytes (101). In initial Phase I/IIa studies, Pralnacasan was well tolerated in healthy volunteers and patients with rheumatoid arthritis and osteoarthritis. Moreover, the drug displayed good oral bioavailability and safety and significantly reduced joint symptoms and inflammation in patients with rheumatoid arthritis (27). Pralnacasan was withdrawn from clinical trials because of liver toxicity observed after long-term administration in animal studies, despite no adverse side effects occurring in the trial participants, even after long-term exposure (102).

VX-765 (Belnacasan), another specific caspase-1 inhibitor, also progressed into Phase IIa clinical trial in patients with psoriasis. In preclinical studies Belnacasan inhibited cytokine production in animal models of inflammatory, autoimmune joint and skin disease (103, 104). Phase I clinical trials demonstrated a dose-dependent reduction of cytokine levels in plasma and a Phase II trial conducted in patients proved the safety, tolerability and clinical activity. In 2011, a Phase IIb study to evaluate the efficacy and safety of VX-765 was started in subjects with treatment-resistant partial epilepsy (105). The drug was safe but not effective for epilepsy treatment (106).

IDN-6556 (Emricasan) is a broad spectrum irreversible caspase inhibitor that showed a strong effect in the mouse model of liver injury in vivo. In humans, it has been investigated for the treatment of a number of hepatic diseases and diabetes. To date, Emricasan has been administered to six Phase I and four Phase II clinical trials, and has generally been well-tolerated in both healthy volunteers and patients with liver disease. Clinical trials have demonstrated that Emricasan does not inhibit normal levels of caspase activity in healthy individuals (107). Emricasan was well tolerated and significantly improved markers of liver damage including reductions in serum AST and ALT in patients with hepatitis C (108, 109). The use of IDN-6556 has also been expanded to other important indications such as fatty liver disease (NASH), hepatitis B virus, alcoholic hepatitis and chronic liver failure (108). In 2013, dosing of Emricasan was initiated in the Phase II clinical trial in patients with severe alcoholic hepatitis (110), and last year, Phase II clinical trial started to evaluate the pharmacokinetics and pharmacodynamics of Emricasan in patients with acute or chronic liver failure (111). Besides hepatic diseases, IDN-6556 entered clinical trial in islet transplantation as treatment for diabetes (112) in order to minimize early post-transplant apoptosis (7, 87).

GS-9450 (Nivocasan), an irreversible inhibitor of caspases-1, -8, and -9, was applied in the case of hepatic injury. A Phase I trial dosing GS-9450 for 14 days in healthy volunteers proved the compound to be safe and well tolerated. Subsequently, Phase II trials were performed to examine the safety, tolerability, pharmacokinetics and initial activity of GS-9450 in preventing liver damage due to scarring or fibrosis caused by hepatitis C virus infection and non-alcoholic steatohepatitis. In both studies, treatment with GS-9450 resulted in lower ALT-values. Despite the four-week application of GS-9450 showing no adverse side effects in patients with NASH, a larger, 6-month study in hepatitis C subjects, reported episodes of drug-induced liver injury and the trial was terminated (6, 113) (Table 1).

NCX-1000 (2(acetyloxy) benzoic acid-3(nitrooxymethyl) phenyl ester), an antiapoptotic compund (inhibitor caspase-3, -8, -9), entered Phase IIa of clinical trial to prevent portal hypertension. Oral doses were administered for the period of 16 days. NCX-1000 was well tolerated but did not reduce portal hypertension due to the low selectivity (114, 115).

FUTURE OF ANTI-CASPASE TREATMENT

The ongoing clinical trials demonstrate that caspase inhibitors could have significant therapeutic potential; nonetheless, some clinical testing has been discontinued and none of the caspase inhibitors have yet reached Phase III. Thus, there are still key questions and obstacles which prevent drugs based on caspase inhibitors from their routine clinical practice as anti-caspase treatment.

(I) Efficient and specific caspase inhibitors that would minimize adverse effects are still lacking.

(II) Type of cell death, specific caspases activated, possible redundancies and non-apoptotic/non-inflammatory functions within the caspase family that may interfere with the treatment are not entirely clear in numerous clinical states (116-118).

(III) Drug dosing, timing of drug administration, its duration and drug delivery to the site of action are not yet well understood.

Adverse effects interrupted the clinical testing of VX-740 and GS-9450. These may be caused by interference with non-apoptotic or non-inflammatory roles of caspases as well as poor specificity and the selectivity of caspase inhibitors. In particular, medication with irreversible caspase inhibitors may lead to the modulation of non-target caspases or unrelated proteins (119) such as cysteine cathepsins and legumain. Since irreversible inhibitors show better effectivity in vivo, these could be beneficial, despite of their disadvantages, in short-term therapy of acute life threatening diseases. For the long-term therapy of chronic diseases like rheumatoid arthritis or hepatitis C, reversible non-covalent allosteric inhibitors would be the best choice (120). Such inhibitors are much more difficult to synthesize compared to irreversible inhibitors.

Recent advances in combinatorial chemistry, including innovative screening techniques, structural biology, and bioinformatics greatly accelerated the development of new caspase inhibitors. Studies addressing structure and function of particular caspases helped to identify differences between closely related caspases applicable for development of highly specific inhibitors (21, 121). Available bioinformatics tools allow searching for structures effectively inhibiting caspases as well as prediction of their properties such as oral bioavailability, metabolic stability and minimal toxicity. Pharmacophore modelling, docking studies and searches in libraries of potential drug compounds have been performed to find functional caspase inhibitors structures (11, 21, 122-123). These approaches have great potential to identify compounds that could progress into clinically applicable drugs.

Inhibition of the caspase pathway may be detrimental by blocking non-inflammatory apoptotic cell clearance and thereby promoting inflammation. The broad-spectrum caspase inhibitor ZVAD-FMK modulates the three major types of cell death; it blocks apoptotic cell death, sensitizes cells to necrotic cell death, and induces autophagic cell death. Inhibition of caspases by Z-VAD-FMK might induce hyper-acute TNF-induced shock in certain situations; this means exacerbated TNF-α toxicity, and enhanced oxidative stress and mitochondrial damage, resulting in hyper-acute haemodynamic collapse, kidney failure, and death of the patient (124).

Adverse effects of necrosis and inflammation may be minimized by proper timing and dosing of the drugs for every individual pathological state. For ischaemia-reperfusion injury, the therapeutic window of caspase inhibitors can change according to the type of damage, the duration of ischaemia, and also to the other medicines in combination. It has been demonstrated that inhibition of caspases leads to a decrease in apoptosis during early reperfusion, but with prolonged ischaemia it seems to be ineffective or rather detrimental at least in the case of kidney and myocardial ischaemia-reperfusion injury, respectively (117, 124, 125).

In order to lower the risk of toxicity in the long-term use of caspase inhibitors, drugs based on caspase inhibitors must only target the offending cells and not to disturb the natural turnover of normal cells and hypothetically start or extend tumour growth. Therefore, inhibition of specific caspases in selective cell types is the major challenge. For this purpose, the development of specific nontoxic nanocarriers able to cross different body barriers and to safety release caspase inhibitors is of major interest. Especially for neurological disorders, the drug must be able to cross the blood brain barrier; therefore, novel nanoparticulate drug delivery systems like liposomes are hot candidates (126, 127).

We have focused on apoptotic roles of caspases but there are also non-apoptotic functions of caspases (128-131). Caspase inhibitors could therefore potentially block more functions. Despite numerous attempts non-apoptotic functions of caspases are not completely understood yet.

In summary, caspase inhibitors are essential research tools that are useful in various experimental systems to monitor caspase activity, to understand functions of individual caspases in development and everyday tissue homeostasis and in pathological processes. Nevertheless, the path to “anti-caspase” therapy is still very difficult; several studies have been suspended and many important questions are still open regarding side effects or complications such as tumour growth during or after treatment. Despite the mentioned obstacles, there is no doubt about the prospective of caspase inhibitors for clinical praxis (Tables 1 and 2), and intense research in this field with running clinical trials may yield new safe therapeutic strategies towards personalized therapy.

Acknowledgements: Related research is supported by the Grant Agency of the Czech Republic, project P502/12/1285 at the UVPS, 14-37368G at the IAPG ASCR, v.v.i., and 14-28254s at the IAC ASCR, v.v.i. European Regional Development Fund and the State Budget of the Czech Republic (RECAMO, CZ.1.05/2.1.00/03.0101) supports research at the MMCI. E. Matalova is a holder of the L´Oreal UNESCO Fellowship 2015.

Conflict of interests: None declared.

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