L-asparaginase as a critical component to combat Acute Lymphoblastic Leukaemia (ALL): A novel approach to target ALL

European Journal of Pharmacology 771 (2016) 199–210

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Review

L-asparaginase as a critical component to combat Acute Lymphoblastic Leukaemia (ALL): A novel approach to target ALL Usman Ali a, Muhammad Naveed b, Abid Ullah a, Khadija Ali c, Sayed Afzal Shah e, Shah Fahad d, Abdul Samad Mumtaz e,n a

National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China Department of Biochemistry and Molecular Biology, University of Gujrat, Pakistan c Department of Environmental Sciences, International Islamic University, Islamabad, Pakistan d National Key Laboratory of Crop Genetic Improvement, MOA Key Laboratory of Crop Ecophysiology and Farming System in the Middle Reaches of the Yangtze River, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China e Department of Plant Sciences, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan b

art ic l e i nf o

a b s t r a c t

Article history: Received 11 August 2015 Received in revised form 8 December 2015 Accepted 10 December 2015 Available online 15 December 2015

L-asparaginase, an anti-leukaemic drug that has been approved for clinical use for many years in the treatment of childhood Acute Lymphoblastic Leukaemia (ALL), is obtained from bacterial origin (Escherichia coli and Erwinia carotovora). The efficacy of L-asparaginase has been discussed for the past 40 years, and an ideal substitute for the enzyme has not yet been developed. The early clearance from plasma (short half-life) and requirement for multiple administrations and hence frequent physician visits make the overall treatment cost quite high. In addition, a high rate of allergic reactions in patients receiving treatment with the enzyme isolated from bacterial sources make its clinical application challenging. For these reasons, various attempts are being made to overcome these barriers. Therefore, the present article reviews studies focused on seeking substitutes for L-asparaginase through alternative sources including bacteria, fungi, actinomycetes, algae and plants to overcome these limitations. In addition, the role of chemical modifications and protein engineering approaches to enhance the drug's efficacy are also discussed. Moreover, an overview has also been provided in the current review regarding the contradiction among various researchers regarding the significance of the enzyme's glutaminase activity. & 2015 Elsevier B.V. All rights reserved.

Keywords: L-asparaginase Acute Lymphoblastic Leukaemia E. coli Blood plasma Anti-leukaemic drug Algae and plants

Contents 1. 2. 3. 4. 5.

6.

n

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 Historical background of L-asparaginase (L-ASP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 Pharmacological mechanism of L-ASP, especially in treating ALL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 Clinical use of L-asparaginase (L-ASP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Limitations of L asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202 5.1. Complications associated with L-asparaginase therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202 5.2. Immunogenic complications/resistance to the drug . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 Developments in L-asparaginase discovery to overcome limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 6.1. Chemical modifications made to L asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 6.2. Protein engineering approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204 6.3. Searching for novel sources of L-asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 6.3.1. Bacterial L-asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 6.3.2. Fungal L-asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206 6.3.3. Actinomycetes L-asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206 6.3.4. Algal L-asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206

Corresponding author. Tel.: þ 92 51 90643072, þ 92 342 5188685; fax: þ 92 51 9064 3138. E-mail addresses: [email protected], [email protected] (A.S. Mumtaz).

http://dx.doi.org/10.1016/j.ejphar.2015.12.023 0014-2999/& 2015 Elsevier B.V. All rights reserved.

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6.3.5. Plant L-asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206 7. Conclusions and final remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

1. Introduction Acute Lymphoblastic Leukaemia (ALL) is a blood cancer in which enormous numbers of immature white blood cells or lymphocytes are produced in the bone marrow. Globally, 3 in 100,000 individuals may acquire the disease, mostly at ages under six years (Grigoropoulos et al., 2013) with the highest likelihood of developing the disease occurring between the ages of 2 and 4 years. The anti-proliferative properties of L asparaginase on leukaemic cells were first identified and characterised in human clinical trials in 1970s. Since then, the enzyme has become a key component in the treatment of ALL. Biochemically, it catalyses the hydrolysis of asparagine into aspartic acid and ammonia. Leukaemic cells require large amount of asparagine to maintain their malignant growth. To meet this demand, they obtain the non-acidic, hydrophilic amino acid both from blood serum as well as synthesising the amino acid themselves in limited amounts (Narta et al., 2007). L-asparaginase exploits the high demand of leukaemia cells for asparagine by depleting the circulating pool of asparagine from blood serum. The depletion of the circulating pools of asparagine from the blood serum results in the death of the leukaemia cells. Various organisms including animals, bacteria, fungi, actinomycetes, algae and plants have been reported to synthesise L-asparaginase, but the antineoplastic action of the E. coli and Erwinia caratovora forms of L-asparaginase have been studied most extensively (Howard and Carpenter, 1972; Narta et al., 2007; Oza et al., 2011; Paul, 1982; Sarquis et al., 2004; Sudhir et al., 2012) To use L-asparaginase as a key element in the treatment of ALL, it should be devoid of glutaminase activity. However, some investigators such as Parmentier et al. (2015) have provided evidence that the glutaminase activity of L-asparaginase is essential for its cytotoxicity against leukaemic cells. Other key factors for successful use of the enzyme include its high affinity for its substrate asparagine (low Km value), no side effects, no immunogenic complications and delayed clearance from plasma (prolonged halflife) to minimise the required frequency of administration (Nagarethinam et al., 2012; Parmentier et al., 2015). L-asparaginase from bacterial sources contains up to 10% L glutaminase activity. However, the Pyrococcus furiosus L asparaginase demonstrates no glutaminase activity (Bansal et al., 2010; Campbell et al., 1967). The glutaminase activity of L-asparaginase is probably due to the structural similarity of asparagine and glutamine. The structural formulae show a similarity in the amide groups while the difference between these amino acids is the presence of one additional methyl group in glutamine (Ramya et al., 2012). Furthermore, during the biosynthesis of asparagine, transamidation of aspartate occurs in which glutamine serves as the amide group donor (Nagarethinam et al., 2012). The L-asparaginase enzymes from E. coli and Erwinia carotovora, which have Km values of 1.15  10  5 M and 1.8  10  5 M, respectively, have been shown to have value in clinical applications against ALL (Cedar and Schwartz, 1967; Ho et al., 1970a, 1970b). The present article reviews various attributes that are considered to be important for effective ALL therapy, including the Km values, optimal pH, optimal temperature, glutaminase activity and blood plasma clearance rates for enzymes derived from alternative sources including bacteria, fungi, actinomycetes, algae and plants. In addition, strategies to enhance the drug's efficacy including

chemical modification, protein engineering approaches and sitedirected mutagenesis are also discussed. Because the enzymes from alternative sources including plants, fungi and algae have not been extensively characterised, data regarding pre-clinical and clinical trials for blood plasma clearance and glutaminase activity are not included in this article.

2. Historical background of L-asparaginase (L-ASP) Asparaginase activity was first detected by Lang in beef tissues in 1904 (Lang, 1904). In 1922, Clementi provided experimental evidence for Lang’s observation and reported that the amido-hydrolytic activity of the enzyme was exhibited in all tissues of herbivores, only in the livers of omnivorous animals, and was not present in the organs of carnivorous animals, amphibians, or reptiles (Clementi, 1922). The potency of the enzyme as an anticancer drug was reported by Kid in 1953, who observed the antitumor properties of guinea pig serum (Kidd, 1953). Later, Neuman and McCoy in 1956 demonstrated the metabolic differences between normal and malignant cells in vitro in the presence and absence of the amino acid, asparagine (Neuman and McCoy, 1956). Based on insights from these studies, Broom linked the anti-tumour activity of guinea pig serum to asparagine depletion by the enzyme L-asparaginase (Broome, 1963). Despite the acknowledgement of the theory behind the utilisation of the enzyme in malignancies, there were a few challenges in its clinical use because at the time guinea pigs were the main source and the extraction of enzyme from the pig serum in large quantity was difficult. It was only in 1964 that Mashburn and Wriston investigated an alternative source of the enzyme, and L-asparaginase from E. coli became of primary interest. Two asparaginases were isolated from E. coli, i.e., EC-1 (periplasmic) and EC-2 (cytoplasmic) (Mashburn and Wriston, 1964). However, only EC-2 exhibited antitumor activity. These findings provided a practical basis for the production of enzyme in large quantities for pre-clinical and clinical studies. The potential of L-asparaginase as an anticancer drug was first shown by Oettgen et al. in 1967 (Oettgen et al., 1967). Parenteral therapy with foreign proteins in humans is limited by the key issue of the drug's immunogenicity, and hypersensitivity reactions were found to be associated with the enzyme extracted from bacteria. Since then, several attempts have been made to reduce the immunogenicity of the drug while preserving its enzymatic activity. One such attempt involved coupling of the enzyme to polyethylene glycol (PEG) which resulted in reduced immunogenicity without altering the antineoplastic property of the enzyme (Abuchowski et al., 1979). When tested in an animal model, this modified version showed reduced antibody formation compared to native form and a significant prolongation of its half-life (Yoshimoto et al., 1986). Recently, a protein engineering approach resulted in significant stability and higher cytotoxicity of the enzyme against cancer cell lines (Mehta et al., 2014). For these reasons, L-asparaginase has been established as an indispensable component in modern chemotherapeutic procedures. 3. Pharmacological mechanism of L-ASP, especially in treating

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ALL

4. Clinical use of L-asparaginase (L-ASP)

The substrate of L-asparaginase, L-asparagine, is a member of the amide group of amino acids and is a critical nutrient for tumour cells. Due to the enhanced rate of proliferation and metabolic processes in cancerous cells, they require an elevated level of this amino acid (Luhana et al., 2013). The demand for asparagine is met in two ways: (a) from blood serum and (b) through synthesis by the cells themselves. As a drug, L-asparaginase exploits this requirement and hydrolyses L-asparagine into aspartic acid and ammonia, thereby killing the tumour cells (Narta et al., 2007). The resulting low level of asparagine only affects the viability of the cancerous cells without disturbing the normal cells. The transformation of L-asparagine to L-aspartate and ammonia by L-asparaginase involves a double displacement or “Ping Pong” mechanism in which the nucleophilic group of the enzyme attacks the Cγ of the substrate asparagine forming a tetrahedral intermediate that subsequently breaks down to form an acyl-enzyme intermediate. This is followed by the elimination of ammonia. A second nucleophile (normally water), then attacks this intermediate, thus transforming it to aspartic acid and regenerating the free enzyme (Ehrman et al., 1971; Rohm and Van Etten, 1986). Asparagine has important roles in the biosynthesis of protein, DNA and RNA. Cells also need asparagine to proceed through G1 cycle in cell division (Rytting, 2012). The inability of leukaemia cells to synthesise L-asparagine de novo is related to the lack or presence of low levels of asparagine synthetase (Kiriyama et al., 1989; Prager and Bachynsky, 1968). The degradation of L-asparagine by L-asparaginase is shown in Fig. 1. Song et al. (2015) subjected the chronic myeloid leukaemia cell lines, K562 and KU812 cells, to asparaginase. The results indicated that the enzyme induced apoptosis and cytoprotective autophagy in these chronic myeloid leukaemia cells as evidenced by the formation of autophagosomes, microtubule-associated protein light chain 3 (LC3)-positive autophagy-like vacuoles, and the upregulation of LC3-II. Their findings have provided new insight for understanding the mechanism of action of L-asparaginase in treating leukaemia cells (Song et al., 2015).

The data with respect to clinical trials of L-asparaginase from the bacterial source has been plentiful and widely discussed in the last 40 years. However, no such data have been presented for clinical trials of the enzyme from other sources including fungi, yeast, actinomycetes and plants, because it has not been extensively characterised from these sources. As discussed earlier, given the hypersensitivity associated with native preparations, the modified versions of the enzyme appear to have earned expanded significance in clinical applications. During a phase-I dose-escalation study led by Ho et al. thirtyone adult patients were given pegaspargase fortnightly by i.v. administration (dose starting from 500 to 8000 IU/m2) over one h. Only 3 patients developed toxicity symptoms in the form of hypersensitivity reactions. Hyperglycaemia and hepatic dysfunction were additional major toxicities associated with the treatments. The outcome of the study did not demonstrate any correlation between the dose of the drug and the toxicity. Responses were observed in patients with ALL and cancer. This study provided the basis for subsequent trials, which used equivalent doses varying between 2000 and 2500 IU/m2 for the clinical studies (Ho et al., 1986; Keating et al., 1993). On the basis of the clinical trials conducted by Ho and coworkers, Vieira Pinheiro et al were able to maintain serum L-asparaginase activity adequate enough for essential depletion of L-asparagine. They administered 500 U/m2 of PEG-L-asparaginase (Oncaspar TM) in children with relapsed ALL, and the majority of the patients responded well to this dosage (Vieira Pinheiro et al., 2001). In the ALL-2005 protocol trial initiated in 2005, five haematological centres (in Moscow, Saransk, Volgograd, Tambov and Kirov) participated, and a total of 71 adult patients with ALL were treated. Prior to this study, the results of the MB-2002 study with participation of 16 patients aged 16–23 years performed in the State Hematological Research Center (SHRC) were reviewed. The results of ALL-2005 protocol conducted in the Moscow SHRC were good: a complete remission was achieved in 90% patients, early lethality

Fig. 1. Mechanism of action of L-asparaginase.

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was 6%, and resistance was observed in 4%. In the regional centres, the lethality in remission was higher: the 5-year overall survival was 28% (in the SHRC it was 56%), and recurrence-free survival in regional centre was 22% versus 51% in the SHRC. The long-term responses in the ALL-2005 and MB-2002 trials in patients aged 19–23 was the same, but toxicity of the treatment in the ALL-2005 protocol was higher (no lethality and 5, 4% in induction and remission, respectively) (Parovichnikova et al., 2009) The Children's Oncology Group (COG) conducted clinical trials to compare the pharmacokinetics and pharmacodynamics of succinimidyl carbamate-PEG (SC-PEG) to succinimidyl succinate (SSPEG) in patients with newly diagnosed high-risk (HR) B-cell ALL. A total of 165 evaluable patients were randomly allocated at a 2:1 ratio to receive SC-PEG at 2100 IU/m2 (n ¼ 42) or SC-PEG at 2500 IU/m2 versus SS-PEG at 2500 IU/m2 (n ¼54) as part of an otherwise identical chemotherapy regimen. A significantly longer period of asparaginase activity above defined thresholds and asparagine depletion was observed for the SC-PEG2500 compared with SS-PEG2500, and the two forms had comparable toxicity profiles in children with HR B-cell ALL (Angiolillo et al., 2014). Masurekar et al., (2014) analysed thirty-three patients who had previously been treated on the UKALL 2003 protocol in the ALLR3 clinical trial conducted between Jan 2009 and May 2011. Each PEG-ASNase administration was followed by a trough in the PEGASNase activity in the blood samples at 7–14 days. Of the 33 patients, 21 showed ASNase activity, one patient did not show adequate activity while the rest exhibited ASNase activity Z200 IU l  1. Only one of the 33 patients was determined to have antibodies against both PEG-ASNase and native ASNase. This was the same patient who did not have detectable ASNase activity levels. Thus, 1000 IU m  2 of PEG-ASNase given intramuscularly provides adequate therapeutic levels in patients who relapse in a PEG-ASNase frontline protocol (Masurekar et al., 2014). Henriksen et al. (2015) reported on children enroled in Nordic Society of Pediatric Hematology and Oncology (NOPHO) ALL2008 protocol between July 2008 and August 2011. Those who developed an allergy to PEG-asparaginase were identified through the NOPHO ALL2008 toxicity registry. In the NOPHO ALL2008 protocol, the patients were randomised to receive 8 or 15 doses of intramuscular PEG-asparaginase (Oncaspars) 1000 IU/m(2)/dose administered at 2 or 6 weeks intervals during a total period of 30 weeks. Of the 615 evaluable patients, 79 patients developed a clinical PEG-asparaginase allergy (cumulative risk; 13.2%), and therefore PEG-asparaginase therapy was terminated. The PEG-asparaginase allergy occurred after a median of two doses (75% range 2–4, max 14). In 58% of the PEG-asparaginase-hypersensitive patients, the clinical allergic reactions appeared within 2 h after PEG-asparaginase therapy and ranged from mild symptoms to systemic anaphylaxis. Nine patients encountered an anaphylactic reaction within 1 h and 50 min from the asparaginase administration; none were fatal. Four of 68 patients (6%) who subsequently received Erwinase therapy also reacted allergic to Erwinase (Henriksen et al., 2015). Clinical trials were conducted by Kadia et al. (2015) in adult patients with relapsed or refractory ALL in which other regimens, i.e., methotrexate, vincristine or dexamethasone, were combined with PEG-asparaginase. Of 37 patients treated, the complete remission (CR) rate and overall response rate (ORR) were 28% and 39%, respectively. However, side effects including elevations in bilirubin and transaminases, nausea, peripheral neuropathy, and hyperglycaemia were detected, which were rectified through dose adjustments (Kadia et al., 2015). The L-asparaginase used in the clinic is from a bacterial source. However, some limitations or side effects such as early clearance from blood plasma (short half-life) and immunogenic complications are associated with the drug.

5. Limitations of L asparaginase Treatment of ALL with L-asparaginase is associated with side effects such as leucopoenia, neurological seizures, anaphylaxis, coagulation abnormalities, pancreatitis, etc. Another limitation is that the immune system of the patient can react against the drug in many different ways including the depression of the asparagine synthetase gene, production of specific antibodies against the drug, inactivation of caspase 3 or PARP [poly (ADP-ribose) polymerase] and production of glutamine in large quantities by adipocytes. Furthermore, the drug in its native form clears rapidly from the blood serum (short half-life), so the patient needs 2–3 treatments each week, requiring frequent physician visits and therefore making the overall treatment costly. 5.1. Complications associated with L-asparaginase therapy The most common side effects associated with L-asparaginase therapy include imbalances in the formation of clotting factors such as plasminogen, protein C and protein S and a deficiency in antithrombin after prolonged L-asparaginase treatment (Barbui et al., 1983; Homans et al., 1987; Priest et al., 1980; Ranta et al., 2013; Trivedi and Pitchumoni, 2005; Vigano'D'Angelo et al., 1990). The development of acute pancreatitis is also one of the side effects and is a well-documented complication in leukaemia therapy; this resembles drug-induced pancreatitis in most cases, and the symptoms associated with it include vomiting, abdominal or back pain, and anorexia (Imamura et al., 2005). Patients receiving intensive L-Asparaginase therapy were found to be at increased risk for myocardial infarction and also have a chance of developing secondary leukaemia which can be induced as a result of topoisomerase-targeted drugs. Problems associated with immunodeficiency and acute hepatic dysfunctions are the major side effects of L-Asparaginase in leukaemia therapy (Haddy et al., 2006; Hernandez-Espinosa et al., 2006). During therapy, the onset of venous thrombosis in children has been reported (Sahoo and Hart, 2003). Adolescent patients with leukaemia develop cerebral thrombotic complications due to L-asparaginase therapy (Mitchell et al., 1994). Growth hormone deficiency, particularly in children, and an increased risk of thrombosis was observed in patients treated for ALL (Saviola et al., 2004; Omoti and Omoti, 2006). Thromboembolic problems in paediatric patients with ALL were due to the poor regulation of thrombin and prothrombin levels in the blood after the therapy with the enzyme L-asparaginase (Ortega et al., 1977). L-asparaginases were found to be associated with corneal toxicity in patients given combinational chemotherapy. Foreign body sensations, blurred vision, ocular pain, and bilateral conjunctival hyperaemia were observed to be common symptoms (Sutow et al., 1971). Myocardial ischaemia due to L-asparaginase therapy has been reported in patients with ALL. Even symptoms associated with diabetes were observed due to damage of the islet cells of the pancreas and subsequent decreases in the insulin levels (Oettgen et al., 1970; Zollner and Heimstadt, 1971). Simultaneous occurrence of parotitis and abnormalities in lipid metabolism have also been observed during L-asparaginase therapy (Favrot et al., 1984; Meyer et al., 2003; Parsons et al., 1997; Steinherz, 1994). Early reports showed that the levels of serum cholesterol and triglycerides decreased in most patients. Later, hyperlipidaemia followed by hyperlipidaemic were also reported (Land et al., 1972). In patients with T-cell lymphoblastic lymphoma, L-Asparaginaseassociated hyperlipidaemia with hyper-viscosity has been reported (Meyer et al., 2003). Several reports show that hypertriglyceridemia was observed mostly in children and in very few cases in adults undergoing L-asparaginase therapy (Oettgen et al., 1970; Ohnuma et al., 1969). Impairments of CNS functions along

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with agitation, hallucinations, disorientation, convulsions and coma have been observed. Korholz et al. reported that the symptoms of nervous disorders increased after the administration of L-asparaginase. Asparaginases were found to elicit hypersensitivity reactions, and these reactions are due to the production of high titres of IgG3 antibodies, which are associated with a higher risk of anaphylaxis. Many reports clearly indicate that the E. coli L-asparaginase causes more hypersensitivity reactions than that derived from the Erwinia spp (Korholz et al., 1987). 5.2. Immunogenic complications/resistance to the drug Various researchers have presented different opinions regarding resistance to L-asparaginase. Ho et al. (1970a, 1970b) and Worton et al. (1991) both have reported that resistance to the drug is primarily due to suppression of the asparagine synthetase gene (Ho et al., 1970a; Worton et al., 1991). The resistance has also been associated with the production of specific antibodies against the drug (Capizzi, 1993). The L-asparaginase-sensitive cells produce cytokines (Gallagher et al., 1989). These cytokines control the expansion of resistant cells. As soon as the sensitive cells are killed by the L-asparaginase, the resistant cells escape from regulatory control. The cytokines (IL-12 family members) have direct antitumour activity against B-ALL (Cocco and Airoldi, 2011). It has also been reported that the cytokines, G-CSF and GM-CSF, activate quiescent leukaemia cells, stimulate their proliferation and make them sensitive to chemotherapeutic drugs. As evident from the results of Zalewska-Szewczyk et al. (2009), cross reactivity occurs between the antibodies against the native and pegylated forms of the E. coli enzyme, but in this case the Erwinia-derived L-asparaginase is not affected. Thus, in cases of allergy or silent inactivation of PEG-asparaginase, switching to the Erwinia-derived asparaginase may significantly overcome the resistance to the enzyme. However, Kullas et al. (2012) reported that L-asparaginase suppresses the production of cytokines (Kullas et al., 2012). Holleman et al. (2003) associated the resistance to L-asparaginase and prednisolone with inactivation of caspase 3 or PARP [poly(ADPribose) polymerase] (Holleman et al., 2003). Ehsanipour et al. (2013) established a link between obesity and L-asparaginase resistance and demonstrated that adipocytes (fat cells), which produce sufficient quantities of glutamine, cause the leukaemia cells to be resistant to L-asparaginase (Ehsanipour et al., 2013). These findings suggest that obese children have higher risk of leukaemia than their lean counterparts. Because the native preparation of L-asparaginase results in silent inactivation of PEG L-asparaginase, another strategy is to use the PEG L-asparaginase, rather than the native preparation, initially during the induction course. This approach has shown to induce less antibody production, and in the presence of low titres of antibodies, the PEG L-asparaginase still provides adequate activity levels to overcome the resistance (Andrade et al., 2014).

6. Developments in L-asparaginase discovery to overcome limitations To overcome the aforementioned barriers, several strategies have been applied, including chemical modification and protein engineering by site-directed mutagenesis (Mehta et al., 2014; Offman et al., 2011; Yoshimoto et al., 1986). Moreover, an overview of recent developments in L-asparaginase discovery from alternative sources has also been provided in this section. 6.1. Chemical modifications made to L asparaginase Currently three asparaginase preparations, i.e., native E. coli

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asparaginase (Elspars Merck & Co., Inc., West Point, PA, USA), Pegasparagase (Oncaspars Enzon, Inc., Bridgewater, NJ, USA) and Erwinases (Ipsen-Speywood Pharmaceuticals Ltd, UK), are available for ALL therapy in the USA. The native preparations are supplied under different brand names such as Medacs (Kyowa Hakko, Kogyo, Japan), Crasnitins (Bayer AG, Leverkusen, Germany), Leunases (Sanofi-Aventis, Paris, France) Paronals, and Kidrolases etc. in Europe and Asia. Treatment with Medacs resulted in haemorrhagic and thrombotic events, but it exhibited higher biological activity in achieving asparagine depletion (Boos et al., 1996; Ahlke et al., 1997). The available pegasparagase preparations have extended half-lives compared to the native forms of the enzyme. Compare to the E. coli preparations, the Erwinia asparaginase is superior regarding immunogenicity and a reduced induction of coagulation disorders. However, its biological activity for asparagine is significantly lower, i.e., 26 % as evident from the studies reported by Gentili et al. (1996) and Rizzari et al. (2000). Erwinases is approved as second-line treatment for patients exhibiting hypersensitivity to native and pegasparagase in the United Kingdom (Shrivastava et al., 2015). The molecular weight of the enzyme isolated and purified from various strains of E. coli range from 132 to 141 KDa whereas the molecular weight of the Erwinia L-asparaginase turns out to be 138 KDa (Irion and Arens, 1970; Jackson and Handschumacher, 1970; Maita and Matsuda, 1980). The purified enzymes have specific activities that lie between 300 and 400 μmol of substrate/mg protein. The pH range for isoelectric point for the E. coli enzyme is 4.6–5.5 whereas it is 8.7 for the L-asparaginase from Erwinia (Howard and Carpenter, 1972). The approximate Km value for L-asparaginase is 1  10  5 mol/l (Jackson and Handschumacher, 1970; Maita and Matsuda, 1980). These asparaginases contain up to 10% glutaminase activity, which is undesirable. The immunogenic complications, resistance and short half-life (early clearance from plasma) of the native forms from the bacterial sources make its clinical applications challenging. Therefore, in the mid-1970 several groups began to chemically modify L-asparaginase using various methods to identify a form that was less immunogenic, while having a prolonged half-life and retaining good antitumour activity. Among the chemical modifications made to L-asparaginase, PEGylation (conjugation of L-asparaginase to PEG) has been the most reliable method applied to the drug (Abuchowski et al., 1979). The anti-leukaemic properties of this modified enzyme were tested in the L5178Y tumour-bearing BDF mouse model, and the coupling succeeded in abolishing the drug's immunogenicity. The PEGylation resulted in markedly different biochemical properties from that of native form. The apparent molecular weight of the PEGylated form was higher, and its affinity for specific antibodies was very low but increased when subjected to freeze/thaw cycles (Koerholz et al., 1989). This modified form has antineoplastic activities in both animal models and humans and has several advantages over the native forms (Abuchowski et al., 1979; Yoshimoto et al., 1986; Jurgens et al., 1988). The patients treated with the modified form of the enzyme have diminished incidences of hyperglycaemia and pancreatitis and an absence of anaphylaxis (Ettinger et al., 1995). The modified enzyme has a prolonged halflife and requires less frequent administration, i.e., biweekly as opposed to 2–3 doses per week of the native E. coli preparation (Narta et al., 2007). The actual cost of PEG-asparagase is greater than that of native forms, but the less frequent visits to the physician and the reduced incidence of immunogenic complications make the overall treatment cost considerably less than that of native preparations (Narta et al., 2007). A comparison of native and modified L-asparaginases is shown in Table 1. Several other chemical modifications of the enzyme with their outcomes are summarised in Table 2.

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Table 1 Biochemical properties of native versus modified L-asparaginase. Parameters

E. coli

Erwinia (Native)

Native PEGylated Activity(IU)/mg protein Km (μM)-l-asparaginase Km (μM)-l-glutaminase l-Glu/l-Asp(maximal activity) Molecular weight PI

280–400 12 3000 0.03 141,000 5.0

280–400 12 3000 0.03 – 5

650–700 12 1400 0.10 138,000 8.7

Table 2 describes only modifications made to bacterial L-asparaginase. However, Karamitros et al. (2013) encapsulated a poorly characterised yeast asparaginase (ScASNaseI) in two or three layers of poly dextran/poly-L-arginine-based bilayers. This resulted in an increased resistance against proteases such as trypsin and thrombin and against thermal inactivation at 37 °C (Karamitros et al., 2013). This could significantly extend its half-life in vivo during its implementation in ALL. The enzyme from Cladosporium sp. (fungi), has also been subjected to chemical modification with bovine serum albumin, ovalbumin by crosslinking using glutaraldehyde, N-bromosuccinimide, and mono-methoxy polyethylene glycol (Mohan Kumar et al., 2014). Modification with ovalbumin resulted in an improved enzyme activity that was 10fold higher than that of the native enzyme, while modification with bovine serum albumin through glutaraldehyde cross-linking resulted in a high stability of L-asparaginase: 8.5- and 7.62-fold greater than that of the native enzyme at 28 °C and 37 °C, respectively, after 24 h. This modification also markedly prolonged the L-asparaginase half-life and serum stability. The N-bromosuccinimide-modified ASNase demonstrated a greater stability and a prolonged in vitro half-life of 144 h to proteolytic digestion relative to the unmodified enzyme (93 h), but both were far greater than that of the native E. coli form (20 h). However, in vitro trials to evaluate the associated side effects and % glutaminase activity for the modified form in human and animal models are still awaited. 6.2. Protein engineering approaches Several attempts have been made at the protein level to produce L-asparaginase that would be glutaminase-free, possessing no side effects and prolonged half-life to ensure successful implementation for the in vivo treatment of ALL. The purpose of engineering L-asparaginase should be to identify the amino acid residues that play central roles in contributing to enzyme stability, specificity and immunogenicity. presented high-resolution crystal structures of the complexes of Erwinia chrysanthemi L-asparaginase (ErA). By comparing the amino acid sequence and crystal structure of ErA with other bacterial sources, they found that

replacement of glutamine and asparagine amino acids at the active site with Glu63 and Ser254, respectively, may decrease the glutaminase activity of L-Asparaginase (Aghaiypour et al., 2001). Gaofu et al. (2005) constructed a recombinant gene encoding a chimeric protein comprising of asparaginase, a tetanus-toxin peptide (TTP) spacer (831–854 fragment), and the foreign cholesteryl ester transfer protein C-terminal fragment (CETPC). The gene was expressed and directed to the periplasmic region of E. coli, and the purified chimeric enzyme showed approximately 83% of the activity of the native enzyme (Gaofu et al., 2005). Jianhua et al. (2006) applied alanine-scanning mutagenesis to L-asparaginase from E. coli for the purpose of determining the residues that are responsible for the enzyme antigenicity. Four mutant recombinant L-ASPs were constructed and expressed in E. coli and then purified. A change in the alkaline residues at positions 195–197 from RKH to AAA significantly decreased the antigenicity of enzyme assessed using a competitive enzyme-linked immunosorbent assay using polyclonal antibodies raised against the wild-type L-ASP from rabbits (Jianhua et al., 2006). Kotzia and Labrou (2009) used in vitro directed evolution to create a new enzyme variant with better thermal stability. Using the genes encoding the L-asparaginases from Erwinia chrysanthemi and Erwinia carotovora, a library of enzyme variants was generated by a staggered extension process. The parental L-asparaginase amino acid sequences revealed 77% identity, but their half-inactivation temperature (Tm) varied by 10 °C. A thermostable variant of the E. chrysamthemi enzyme was identified that contained a single point mutation (Asp133Val). The Tm of this variant was 55.8 °C, whereas the wild-type enzyme has a Tm of 46.4 °C. At 50 °C, the half-life values for the wild-type and mutant enzymes were 2.7 and 159.7 h, respectively. Analysis of the electrostatic potential of the wild-type enzyme revealed that Asp133 is located at a neutral region on the enzyme surface and makes a significant and unfavourable electrostatic contribution to overall stability. The contribution of position 133 to thermostability of the enzyme was further analysed through site-saturation mutagenesis. A library of random Asp133 mutants was screened, which confirmed the involvement of this position in thermo-stability and showed that the Asp133Leu mutation confers optimal thermo-stability (Kotzia and Labrou, 2009). Offman et al. (2011) successfully engineered an E. coli L-asparaginase that resisted degradation and inactivation by AEP produced by leukaemic blast cells. Two mutants N24A and N24A R195S were designed. The reduced dosage of the former resulted in reduced antigenicity while the latter exhibited reduced glutaminase activity and toxicity. Therefore, these two mutants could be exchanged during cancer therapy according to the needs and observed side effects of the patient (Offman et al., 2011). Mehta et al. (2014) used a rational protein engineering approach and made several mutations in L-asparaginase-II of E. coli to find out the amino acid residues that contribute to enzyme

Table 2 Various Chemical modifications of L-asparaginase. Chemical modification

Outcome

References

PEG L-asparaginase L-asparaginase with Dextran L-asparaginase with poly-dl-alanyl peptides Acylation Palmitoyl L-asparaginase L-asparaginase with polyoxyethylene allyl methyl diether Silk fibroin-L-ASNase bioconjugates

The drug's immunogenicity was abolished Less effective immunogenicity reduction than with PEG Clinical studies have not been reported The enzyme becomes hydrophobic 10 fold prolongation in half life without acute toxicity Immunoreactivity towards anti-asparaginase serum was lost completely

(Yoshimoto et al., 1986) (Davis et al., 1991) (Narta et al., 2007) (Martins et al., 1990) (Jorge et al., 1994) (Kodera et al., 1992)

Lower immunogenicity and antigenicity compared with the native enzyme and has increased thermal stability Achieves a significantly longer period of asparaginase activity above defined thresholds and asparagine depletion

(Zhang et al., 2005; Wang and Cao, 2011) (Angiolillo et al., 2014)

SC-PEG (Conjugation of PEG to Succinimidyl carbamate)

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stability, specificity towards asparagine and glutamine, immunogenicity and the ability to kill leukaemia cells. E. coli L-asparaginase-II variants with the double mutation K288S/Y176F (i.e., replacement of Lys288 with serine and Tyr176 with phenylalanine) were considerably less well recognised by serum antibodies from mice immunised with wild-type L-asparaginase-II and an ALL patient who had been given asparaginase therapy for 45 days. In addition, such variants were also significantly more stable than the wild-type enzyme. Except for mutation Y176S, i.e., replacement of tyrosine (Y) with serine (S), all other mutations at position 176 significantly reduced the glutaminase activity of the enzyme without disturbing its asparaginase activity. Thus, the replacement of tyr176 with phe and the double mutation K288S/Y176F caused a significant reduction in the glutaminase activity. However, these modified enzymes had somewhat higher asparaginase activity and significantly enhanced cytotoxicity than the wild-type E. coli L-asparaginase. This double-mutated variant exhibited reduced antigenicity and was 10-fold less immunogenic than the wild-type enzyme. The variant in which residue Y176 was mutated to S became devoid of glutaminase activity but had significantly reduced cytotoxicity against leukaemia cells compared to the wild-type enzyme. Interestingly, substitutions made at positions 176 (Y176F) and 66 (W66Y) resulted in variants with elevated glutaminase activity but higher cytotoxicity against leukaemia cells. This provided a new insight that in spite of its glutaminase activity, the therapeutic efficiency of the enzyme can be improved through such mutations (Mehta et al., 2014). Thus, L-asparaginase variants possessing lower glutaminase activity but high cytotoxicity may also appear to be effective drugs in the future. Chan et al. (2014) applied saturation mutagenesis to E. coli L-asparaginase followed by enzymatic screening and identified the Q59L (replacement of glutamine with leucine) variant as a promising one that retained asparaginase activity without exhibiting any detectable glutaminase activity (Chan et al., 2014). This observation offers a striking rationale for further consideration of Q59L as a drug candidate. 6.3. Searching for novel sources of L-asparaginase On the basis of amino acid sequence and biochemical properties, the enzymes possessing asparaginase activity can be

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categorised into several families; however, the bacterial and planttype asparaginases are the largest and best characterised families (Borek and Jaskolski, 2001; Oza et al., 2011). The non-immunological adverse effects of L-asparaginase, such as pancreatitis, hyperglycaemia, hepatotoxicity or coagulation disorders, can be eliminated or mitigated by extensive characterisation of novel sources of the enzyme. Here, L-asparaginase from different sources such as bacteria, fungi, actinomycetes, algae and plants, with the associated biochemical properties that are considered important in cancer therapy are discussed. 6.3.1. Bacterial L-asparaginase In bacteria, two types of L-asparaginase, i.e., Type 1 and Type II, have been reported (Campbell et al., 1967). The Type 1 enzymes are expressed in the cytoplasm and hydrolyse both asparagine and glutamine while those of Type II are expressed in the periplasm and require an anaerobic environment for their expression. The Type II L-asparaginases have a high affinity for L-asparagine, which is a major nutrient for tumour cells. The presence of L-asparaginase has been reported for many bacteria such as E. coli, E. carotovora, Bacillus sp, Pseudomonas spp. etc. The biochemical properties of some of the bacterial L-asparaginases are summarised in Table 3. Cappelletti et al. (2008) reported a new L-asparaginase from Helicobacter pylori. The enzyme has a glutaminase activity of 0.01% and requires a pH of 7–10 for optimal asparaginase activity. However, as evident from Table 3, it has a somewhat higher Km value than the E. coli L-asparaginase, and higher cytotoxic effects have also been reported. Recombinant ASNase derived from W. succinogenes also has a very low activity towards Gln, and this trait may result in a prominent reduction in side effects. It also has a low cross-reactivity. The efficacy of the enzyme in treatment of ALL from this source therefore needs further evaluation. The enzyme from Pseudomonas sp. is a GLN-ASNase. Although it is stable and has a prolonged half-life in tumour-bearing hosts, it has low Km value (i.e., 2.2  10  5 M) for glutamine, which is undesirable and hence this character may limit its application in cancer therapy. Angelica et al. (2012) have reported that Rhizobium etli is another novel source of L-asparaginase (Moreno-Enriquez et al., 2012). On the basis of its glutaminase-free activity and the

Table 3 Biochemical properties of some bacterial L-asparaginases. Bacteria

Pseudomonas aeruginosa Proteus Vulgaris Vibrio succinogenes Bacillus coagulans Mycobacterium phlei Erwinia carotovora Erwinia aroideae E.coli Azotobacter vinelandii Pseudomonas fluorescens Pseudomonas stutzeri Acinetobacter calcoaceticus Bacillus subtilis B11-06 Corynebacterium glutamicum Helicobacter pylori Aeromonas spp Pyrococcus furiosus Rhizobium etli Photobacterium sp. strain J15. Bacillus licheniformis Bacillus aryabhattai

Biochemical properties opt pH

Opt. temp (°C)

Km (M) L asparagine

M.W (KDa)

Reference

9 7–8 7.3 8.5–9.5 8.8–9.2 8 7.5 7–8 8.6 8–9 9 8.6 7.5 7 7–10 9 9 9 7 6–10 8.5

37 57 – 55 – 50 – 37 48 – 37 – 40 40 45 50

0.147  10  3 2.6  10  5 4.78  10  5 4.7  10  3 0.7  10  3 1.8  10  5 3  10  3 1  10  5 1.1  10  4 4.1  10  4 1.45  10  4 2  10  3 4.3  10  4 2.5  10  3 2.9  10  4 4.9  10  6 1.2  10  4 8.9  10  3 7.6  10  4 1.4  10  5 2.57  10  4

160 – 146 85 126 125–145 155 141 84 70 34 105 38 each subunit 81 140

(El-Bessoumy et al., 2004) (Tosa et al., 1972) (Distasio et al., 1976) (Law and Wriston, 1971) (Pastuszak and Szymona, 1976) (Cammack et al., 1972; Maladkar et al., 1993) (Peterson and Ciegler, 1969; Tiwari and Dua, 1996) (Jorge et al., 1994; Maita and Matsuda, 1980) (Gaffar and Shethna, 1977) (Nilolaev et al., 1975) (Manna et al., 1995) (Joner et al., 1973) (Jia et al., 2013) (Mesas et al., 1990) (Cappelletti et al., 2008) (Pattnaik et al., 2000) (Bansal et al., 2010) (Moreno-Enriquez et al., 2012) (Yaacob et al., 2014) (Mahajan et al., 2014) (Singh et al., 2013)

37 25 40 40

102 70 134.8 155

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biochemical properties that were determined, the enzyme from this source is considered to be a potential drug for the treatment of ALL. Therefore, further studies are needed to authenticate its use as an anticancer therapeutic drug. 6.3.2. Fungal L-asparaginase L-asparaginase is also efficiently produced by fungi. Compare to bacteria, the fungal L-asparaginase possesses fewer side effects. The production of L-asparaginase by filamentous fungi viz. Aspergillus tamarii and Aspergillus terreus have been reported to be highest in 2% proline medium from A. terreus (Sarquis et al., 2004). Ali et al. (1994) have reported the nontoxic, myelosuppressive and immunosuppressive nature of A. terreus L-asparaginase (Ali et al., 1994). The L-asparaginases from a variety of yeasts have been reported to be currently in use, especially that from Saccharomyces cerevisiae. These enzymes are encoded by the ASP3 gene (Bon et al., 1997). The production of L-asparaginase has also been reported from Pichia polymorpha isolated from Egyptian soils (Foda et al., 1980). The enzyme has also been isolated from the cell culture broth of Candida utilis (Kil et al., 1995). Although the enzyme from Candida utilis has a low Km value, it has a weaker affinity for L-asparagine and clears rapidly from plasma indicating its inferior antitumor potential compared to the enzyme from E. coli (Sakamoto et al., 1977). The reduced antitumor potential of the enzyme from this source is probably due to presence of a mannan moiety in it. Some of the biochemical properties of fungal L-asparaginases are summarised in Table 4. Recently a marine fungus, Beauveria bassiana (SS18/41), has been reported to produce L-asparaginase, but to date, this enzyme has not been characterised with respect to the various parameters considered in ALL treatment (KamalaKumari et al., 2015). The enzyme from Mucorhiemalis possesses desirable traits such as stability at physiological pH and temperature, high substrate specificity and good scavenging activity. Therefore, this enzyme needs further evaluation as an anti-leukaemic agent. The enzyme from fungi has also been sufficiently characterised that it should proceed towards clinical trials. 6.3.3. Actinomycetes L-asparaginase More than 22,000 secondary metabolites are produced by microbes, 70% of which are produced by actinomycetes. Among the actinomycetes, the Streptomyces group are considered to be economically important because out of the approximately more than 10,000 known antibiotics, 50–55% are produced by this genus (Subramani and Aalbersberg, 2012). Much research is being conducted on these organisms because they have gained attention as rich sources of antibiotics, anti-tumour drugs and other bioactive

molecules. Actinomycetes are the least studied organisms among all of the L-asparaginase-producing sources (Sudhir et al., 2012). Among the actinomycetes, several Streptomyces species such as S. karnatakensis, S. venezualae, S. longisporusflavus, S. ginsengisoli and a marine Streptomyces sp. PDK2 have been reported to produce L-asparaginase (Abdel et al 1998; Deshpande et al., 2014; Mostafa, 1979; Mostafa and Salama, 1979). There are also reports of L-asparaginase production from some Streptomyces sp, e.g., Streptomyces gulbargensis and Streptomyces noursei MTCC 10469, which were isolated from the guts of the fish Theraponjarbua and Villoritacyprinoides (Dharmaraj, 2011; Amena et al., 2010). Marine actinomycetes are a potential source of L-asparaginase on the basis of their adaptations to extremes within the marine environment. The L-asparaginase purified and characterised from Streptomyces spp has a Km value of 2.5  10  5 M with an optimum pH of 7.5 for enzyme activity, which is very close to pH of human blood. However, the enzyme shows optimal activity at 50 °C which is far higher than human body temperature (Basha et al., 2009). Han et al have characterised the enzyme isolated from Staphylococcus sp. OJ82 as having optima of temperature and pH and a Km value of 37 °C, 9 and 2.2  10  3 M, respectively (Han et al., 2014). Very little attention is paid to the L-asparaginase activity of marine actinomycetes purely at screening level because of the difficulties in their identification and classification. It would be worthwhile to focus on the properties such as optimal pH, optimal temperature for activity, percent glutaminase activity, toxicity and the Km values of the enzymes for their substrate L-asparagine from actinomycetes sources. 6.3.4. Algal L-asparaginase A micro algal L-asparaginase was purified to near homogeneity for the first time by John H. Paul from marine Chlamydomonas spp. (Paul, 1982). The molecular weight of the purified enzyme is 275 KDa, and it has a Km value for asparagine of 1.34  10  4 M. It shows some degree of thermal stability and possesses optimal activity over a wide pH range (6.8–9.52). The enzyme shows less antitumor activity in an antilymphoma assay in vivo. 6.3.5. Plant L-asparaginase A number of plant species including barley roots, seedlings of Lupinus leutus and Dilichos lab, green chilli, Tamarindus indica, Lupinus angustifolius, soyabean leaves, the leaves, flowers buds and root tips of Lupines arabreus, Arabidopsis thaliana, chickpea seedling cotyledons, Vicia faba and Phaseolus vulgaris seeds, the immature seeds of peas, Lotus japonicas, Withania somnifera, Datura innoxia, Lycpersicum lycopersicum, Vigna unguiculata, Asparagus

Table 4 Biochemical properties of Fungi L-asparaginase. Fungi

Saccharomyces cerevisiae Pichia polymorpha Rhizomucor miehei Penecillium digitatum Candida utilis Penecillium spp Aspergillus aceulatus Fusarium tricinctum Aspergillus terrus Cylindrocarpon obtusisporum Cladosporium sp Aspergillus niger Mucor hiemalis

Biochemical properties Opt pH

Opt Temp (°C)

Km (M)

M.W (KDa)

Reference

6.8 6.7 7 7 – 7 9 7.5–8.7 5–7 7.4 6.3 6 7

– – 45 30 – 37 30 – 40–45 37 30 37 37

3.5  10  4 1.37  10  2 – 1  10  5 7.7  10  5 4  10  3 12.5  10  3 5.2  10  4 5.8  10  4 1  10  3 1  10  1 1.6  10  5 4.3  10  3

800 – 133.7 – 480 66 – 161–170 – 216 121 – 96

(Dunlop et al., 1978) (Foda et al., 1980) (Huang et al., 2014) (Shrivastava et al., 2012) (Sakamoto et al., 1977) (Patro and Gupta, 2012) (Dange and Peshwe., 2011) (Scheetz et al., 1971) (Ali et al., 1994) (Raha et al., 1990) (Mohan Kumar and Manonmani, 2013) (Luhana et al., 2013) (Monica et al., 2013)

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officinalis and Oryza sativa produce L-asparaginase (Bano and Sivaramakrishnan., 1980; El Shora and Ali., 2013; Fyiad et al., 2012; Bruneau et al., 2006; Cho et al., 2007; Dickson et al., 1992; Grover and Chibnall, 1927; Lees and Blakeney, 1970; Lough et al., 1992a, 1992b; Oza et al., 2009; Sodek, 1980). L-asparaginase activity has also been reported in soil of roots of Pinus pinaster and Pinus radiate (Bell and Adams, 2004). A trace amount of the enzyme is also present in Solanum melongena, Arachis hypogea, Glycin wightii, Saraca asoca, Delonix regia, and Casia fistula (Oza et al., 2009). Although L-asparaginase has been reported in many higher plants, little work has been carried out on the purification and characterisation of L-asparaginase from higher plants (Oza et al., 2009). In plants, L-asparagine is the major nitrogen storage and transport compound, and it may also accumulate under stress conditions (Sieciechowicz et al., 1988). Asparaginase liberates from asparagine the ammonia that is necessary for protein synthesis. There are two groups of such proteins, called potassium-dependent and potassium-independent asparaginases. These two types of enzymes have significant levels of sequence similarity. The plant asparaginase amino acid sequences do not have any significant homology with microbial asparaginase but were 23% identical and 66% similar to a human glycosyl asparaginase (Lough et al., 1992a, 1992b). The biochemical properties of L-asparaginase from some of the plant sources are summarised in Table 5. Limited studies are available on anticancer properties of the enzyme from plant sources. Oza et al. (2010) reported on the antitumor activity of the enzyme purified from Withania somnifera on cell cultures (Oza et al., 2010). In the same article, they also reported the superiority of this enzyme over those from bacteria on the basis of having less toxic effects on the cell cultures. The Km value (for asparagine) of the enzyme from this source cloned and expressed in E. coli was determined to be 7.02  10  5 M. The recombinant enzyme showed 2% glutaminase activity, which is comparable to that of the E. coli L-asparaginase. The in vivo use of the enzyme from this source against ALL is still awaited. Therefore, the enzyme from this source may replace bacterial ones following application of chemical modifications or protein engineering approaches. Fyiad et al. (2012) investigated the antitumor activity of the enzymes isolated from Vicia faba and Phaseolus vulgaris on a human hepatocellular carcinoma cell line (Hep-G2). The optimum pH for the enzyme activity at 37 °C from both these sources was 7.5–8.5 for immobilised enzymes and pH 7.0 for the free enzymes. Recently, a total of 51 endophytes (including bacteria, actinomycetes and fungi) have been isolated from rhizomes of some selected medicinal plants of Zingiberaceae family. Of these, 31 endophytes exhibited L-asparaginase activity in the range of 54.17–155.93 U/mL in unoptimised medium (KrishnaPura and Belur, 2015). Future research can lead to the development of a suitable substitute for the current sources of L-asparaginase that can obviate all of the limitations posed by the current asparaginase formulations.

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7. Conclusions and final remarks From the preceding discussion, it can be concluded that L-asparaginase is an indispensable anti-leukaemic drug in the treatment of ALL. The literature suggests that side effects of this enzyme are probably due to its non-target glutaminase activity. However, there are some other requirements for the enzyme which, if not met, could also be unexplored causes for serious side effects. One of the requirements is driven by the prevailing pH environment of human blood. Because human blood has an optimum pH around 7.4, an enzyme with a maximum Km value for asparagine under such pH conditions would be preferred. Another requirement is presented by the human body temperature, which is 37 °C. Therefore, a source of the enzyme with maximum activity at this temperature would be preferred. Because L-asparaginase converts asparagine to aspartic acid and ammonia, its action would definitely increase the concentration of aspartic acid in blood serum. Therefore, due to acidic nature of aspartate, the pH of the blood serum will be decreased. This disturbance in the blood pH may remain a still unexplored cause of the side effects. Thus, further studies need to be undertaken to neutralise the effect of aspartic acid. Several researchers have demonstrated that the glutaminase side activity of L-asparaginase is responsible for its side effects. However, Mehta et al. (2014) reported that when substitutions of amino acids at positions 176 (Y176F) and 66 (W66Y) were made to the enzyme sequence, the enzyme acquired lower glutaminase activity but showed higher cytotoxicity against cancer cell lines. Parmentier et al. (2015) also provided evidence that the glutaminase activity of L-asparaginase is essential for cytotoxicity against leukaemic cells. This means that a form of L-asparaginase possessing lower glutaminase activity but higher cytotoxicity could become an effective drug in the future. Furthermore, comparisons of the toxicity profile of glutaminase-free L-asparaginase versus L-asparaginase having glutaminase side activity may also led to the development of an effective drug for future clinical use that may protect future generations from a leading death-causing disease, i.e., cancer. The only L-asparaginase that has been subjected to extensive mutations is the form of bacterial origin. The enzyme has also been identified in other sources, and we therefore need to make mutations in the enzyme sequence from sources other than bacteria. This may lead to elimination or mitigation of non-immunological adverse effects of the enzyme such as pancreatitis, hyperglycaemia, hepatotoxicity and coagulation disorders, which would therefore be beneficial in a clinically significant manner.

Acknowledgements We are very grateful to Professor Guo Liang, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China who provided funds for language editing of the manuscript.

Table 5 Biochemical properties of Plant L-asparaginase. Plants

Withania somnifera Capsicum annum Pisum sativum Lupinus arabreus Lupinus angustifolius

Biochemical properties Opt pH

Opt temp (°C)

Km (M)

M.W (KDa)

Reference

8.5 8.5 – – –

37 37 – – –

6.1  10  5 3.3  10  3 2.4  10  3 6.6  10  3 7  10  3

72 120 69 75 75

(Oza et al., 2011) (Bano and Sivaramakrishnan, 1980) (Chagas and Sodek, 2001) (Chang and Farnden, 1981) (Chang and Farnden, 1981)

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