glutaminase and Precursor-Cell-Lymphoblastic-Leukemia-Lymphoma

L-asparaginase is an enzyme that catalyzes the degradation of asparagine and successfully used in the treatment of acute lymphoblastic leukemia. L-asparaginase toxicity is either related to hypersensitivity to the foreign protein or to a secondary L-glutaminase activity that causes inhibition of protein synthesis. PEGylated versions have been incorporated into the treatment protocols to reduce immunogenicity and an alternative L-asparaginase derived from Dickeya chrysanthemi is used in patients with anaphylactic reactions to the E. coli L-asparaginase. Alternative approaches commonly explore new sources of the enzyme as well as the use of protein engineering techniques to create less immunogenic, more stable variants with lower L-glutaminase activity. This article reviews the main strategies used to overcome L-asparaginase shortcomings and introduces recent tools that can be used to create therapeutic enzymes with improved features.

Topics: Animals; Antineoplastic Agents; Asparaginase; Glutaminase; Humans; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protein Engineering

The antitumour enzyme L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1, ASNase), which catalyses the deamidation of L-asparagine (Asn) to L-aspartic acid and ammonia, has been used for many years in the treatment of acute lymphoblastic leukaemia. Also NK tumours, subtypes of myeloid leukaemias and T-cell lymphomas respond to ASNase, and ovarian carcinomas and other solid tumours have been proposed as additional targets for ASNase, with a potential role for its glutaminase activity. The increasing attention devoted to the antitumour activity of ASNase prompted us to analyse recent patents specifically concerning this enzyme. Here, we first give an overview of metabolic pathways affected by Asn and Gln depletion and, hence, potential targets of ASNase. We then discuss recent published patents concerning ASNases. In particular, we pay attention to novel ASNases, such as the recently characterised ASNase produced by Helicobacter pylori, and those presenting amino acid substitutions aimed at improving enzymatic activity of the classical Escherichia coli enzyme. We detail modifications, such as natural glycosylation or synthetic conjugation with other molecules, for therapeutic purposes. Finally, we analyse patents concerning biotechnological protocols and strategies applied to production of ASNase as well as to its administration and delivery in organisms.

Topics: Animals; Antineoplastic Agents; Asparaginase; Asparagine; Drug Delivery Systems; Glutaminase; Glutamine; Humans; Metabolic Networks and Pathways; Neoplasms; Precursor Cell Lymphoblastic Leukemia-Lymphoma

Acute lymphoblastic leukaemia (ALL) is one of the leading types of malignant disorder seen in children. Viral infections, genetic factors and exposure to chemical carcinogens are some of the factors responsible for causing ALL. Treatment strategies followed for curing ALL include chemotherapy or radiation therapy, wherein, chemotherapy involves the use of the enzymatic drug L-Asparaginase. The enzyme can be produced from various plants, animals, bacterial and fungal sources but, among them, bacterial sources are widely used for production of this enzyme. The enzyme is non-human in origin having certain bottle necks with L-Asparaginase therapy in the form of side effects such as pancreatitis, thrombosis which are mainly due to glutaminase side activity. Hence, present-day research is mainly focussed on minimizing or completely eliminating the glutaminase activity of the enzyme L-Asparaginase. This review is focussed on the complications associated with glutaminase side activity and use of glutaminase free enzymatic drug L-Asparaginase in treating ALL and the other developments related to the modification of the drug for quality treatment.

Topics: Animals; Asparaginase; Glutaminase; Humans; Precursor Cell Lymphoblastic Leukemia-Lymphoma

Asparagine is a non-essential amino acid since it can either be taken up via the diet or synthesized by asparagine synthetase. Acute lymphoblastic leukemia (ALL) cells do not express asparagine synthetase or express it only minimally, which makes them completely dependent on extracellular asparagine for their growth and survival. This dependency makes ALL cells vulnerable to treatment with L-asparaginase, an enzyme that hydrolyzes asparagine. To date, all clinically approved L-asparaginases have significant L-glutaminase co-activity, associated with non-immune related toxic side effects observed during therapy. Therefore, reduction of L-glutaminase co-activity with concomitant maintenance of its anticancer L-asparaginase effect may effectively improve the tolerability of this unique drug. Previously, we designed a new alternative variant of Erwinia chrysanthemi (ErA; Erwinaze) with decreased L-glutaminase co-activity, while maintaining its L-asparaginase activity, by the introduction of three key mutations around the active site (ErA-TM). However, Erwinaze and our ErA-TM variant have very short half-lives in vivo. Here, we show that the fusion of ErA-TM with an albumin binding domain (ABD)-tag significantly increases its in vivo persistence. In addition, we evaluated the in vivo therapeutic efficacy of ABD-ErA-TM in a B-ALL xenograft model of SUP-B15. Our results show a comparable long-lasting durable antileukemic effect between the standard-of-care pegylated-asparaginase and ABD-ErA-TM L-asparaginase, but with fewer co-glutaminase-related acute side effects. Since the toxic side effects of current L-asparaginases often result in treatment discontinuation in ALL patients, this novel ErA-TM variant with ultra-low L-glutaminase co-activity and long in vivo persistence may have great clinical potential.

Topics: Asparaginase; Asparagine; Aspartate-Ammonia Ligase; Glutaminase; Humans; Leukemia, Myeloid, Acute; Precursor Cell Lymphoblastic Leukemia-Lymphoma

l-Asparaginase is a cornerstone of acute lymphoblastic leukemia (ALL) therapy since lymphoblasts lack asparagine synthetase (ASNS) and rely on extracellular asparagine availability for survival. Resistance mechanisms are associated with increased ASNS expression in ALL. However, the association between ASNS and l-Asparaginase efficacy in solid tumors remains unclear, thus limiting clinical development. Interestingly, l-Asparaginase also has a glutaminase co-activity that is crucial in pancreatic cancer where KRAS mutations activate glutamine metabolism. By developing l-Asparaginase-resistant pancreatic cancer cells and using OMICS approaches, we identified glutamine synthetase (GS) as a marker of resistance to l-Asparaginase. GS is the only enzyme able to synthesize glutamine, and its expression also correlates with l-Asparaginase efficacy in 27 human cell lines from 11 cancer indications. Finally, we further demonstrated that GS inhibition prevents cancer cell adaptation to l-Asparaginase-induced glutamine starvation. These findings could pave the way to the development of promising drug combinations to overcome l-Asparaginase resistance.

Topics: Asparaginase; Glutamate-Ammonia Ligase; Glutaminase; Glutamine; Humans; Pancreatic Neoplasms; Precursor Cell Lymphoblastic Leukemia-Lymphoma

Depletion of circulating asparagine with l-asparaginase (ASNase) is a mainstay of leukemia treatment and is under investigation in many cancers. Expression levels of asparagine synthetase (ASNS), which catalyzes asparagine synthesis, were considered predictive of cancer cell sensitivity to ASNase treatment, a notion recently challenged. Using [U-

Topics: Animals; Antineoplastic Agents; Asparaginase; Asparagine; Aspartate-Ammonia Ligase; Cell Line, Tumor; Glutaminase; Lymphoma, B-Cell; Mice; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Tumor Microenvironment

l-Asparaginase (EC 3.5.1.1) was first used as a component of combination drug therapies to treat acute lymphoblastic leukemia (ALL), a cancer of the blood and bone marrow, almost 50 years ago. Administering this enzyme to reduce asparagine levels in the blood is a cornerstone of modern clinical protocols for ALL; indeed, this remains the only successful example of a therapy targeted against a specific metabolic weakness in any form of cancer. Three problems, however, constrain the clinical use of l-asparaginase. First, a type II bacterial variant of l-asparaginase is administered to patients, the majority of whom are children, which produces an immune response thereby limiting the time over which the enzyme can be tolerated. Second, l-asparaginase is subject to proteolytic degradation in the blood. Third, toxic side effects are observed, which may be correlated with the l-glutaminase activity of the enzyme. This Perspective will outline how asparagine depletion negatively impacts the growth of leukemic blasts, discuss the structure and mechanism of l-asparaginase, and briefly describe the clinical use of chemically modified forms of clinically useful l-asparaginases, such as Asparlas, which was recently given FDA approval for use in children (babies to young adults) as part of multidrug treatments for ALL. Finally, we review ongoing efforts to engineer l-asparaginase variants with improved therapeutic properties and briefly detail emerging, alternate strategies for the treatment of forms of ALL that are resistant to asparagine depletion.

Topics: Adolescent; Asparaginase; Asparagine; Child; Child, Preschool; Glutaminase; Humans; Medical Oncology; Models, Molecular; Polyethylene Glycols; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protein Conformation; Quality Improvement; Young Adult

L-Asparaginase (L-asparagine aminohydrolase, E.C. 3.5.1.1) has been proven to be competent in treating Acute Lymphoblastic Leukaemia (ALL), which is widely observed in paediatric and adult groups. Currently, clinical L-Asparaginase formulations are derived from bacterial sources such as Escherichia coli and Erwinia chrysanthemi. These formulations when administered to ALL patients lead to several immunological and hypersensitive reactions. Hence, additional purification steps are required to remove toxicity induced by the amalgamation of other enzymes like glutaminase and urease. Production of L-Asparaginase that is free of glutaminase and urease is a major area of research. In this paper, we report the screening and isolation of fungal species collected from the soil and mosses in the Schirmacher Hills, Dronning Maud Land, Antarctica, that produce L-Asparaginase free of glutaminase and urease. A total of 55 isolates were obtained from 33 environmental samples that were tested by conventional plate techniques using Phenol red and Bromothymol blue as indicators. Among the isolated fungi, 30 isolates showed L-Asparaginase free of glutaminase and urease. The L-Asparaginase producing strain Trichosporon asahii IBBLA1, which showed the highest zone index, was then optimized with a Taguchi design. Optimum enzyme activity of 20.57 U mL

Topics: Agaricales; Antarctic Regions; Asparaginase; Bryophyta; DNA, Fungal; Glutaminase; Phylogeny; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Sequence Analysis, DNA; Soil Microbiology; Trichosporon; Urease

Type II l‑asparaginase (l‑ASNase) is an FDA approved enzyme drug with extensive applications for treatment of certain blood cancers. However, the therapeutic efficiency of this enzyme is hampered by its undesirable glutaminase activity. Given the pivotal role of this enzyme against cancer, designing engineered mutants with diminished glutaminase activity would be of great therapeutic interest. To this end, N248S mutation was selected as the potential mutation with beneficial effects. Various in silico analyses including MD simulation, molecular docking and QMMM studies were performed to assess the effects of N248S mutation on the activity of the enzyme. Thereafter, this mutation along with N248A, N248V and N248T mutations as controls were exerted in l‑ASNase gene. The results from in silico analyses and experimental efforts indicated that N248S mutation is associated with the suitable l‑ASNase activity, while the glutaminase activity is disturbed due to impaired interactions. It has been shown that glutamine turnover was affected much more strongly than asparagine hydrolysis. The approach of exploiting in silico tools to design mutated enzymes lead to staggering time and cost reduction. Following this strategy, we have designed a mutant l‑ASNase with diminished glutaminase activity, which could be of interest for improved biomedical applications.

Topics: Amino Acid Sequence; Amino Acid Substitution; Asparaginase; Catalytic Domain; Enzyme Stability; Glutaminase; Kinetics; Molecular Docking Simulation; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Mutation; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Quantum Theory

Among the antitumor drugs, bacterial enzyme L-asparaginase has been employed as the most effective chemotherapeutic agent in pediatric oncotherapy especially for acute lymphoblastic leukemia. Glutaminase free L-asparaginase producing actinomycetes were isolated from soil samples collected from Egypt. Among them, a potential culture, strain NEAE-119, was selected and identified on the basis of morphological, cultural, physiological, and biochemical properties together with 16S rRNA sequence as Streptomyces olivaceus NEAE-119 and sequencing product (1509 bp) was deposited in the GenBank database under accession number KJ200342. The optimization of different process parameters for L-asparaginase production by Streptomyces olivaceus NEAE-119 using Plackett-Burman experimental design and response surface methodology was carried out. Fifteen variables (temperature, pH, incubation time, inoculum size, inoculum age, agitation speed, dextrose, starch, L-asparagine, KNO3, yeast extract, K2HPO4, MgSO4·7H2O, NaCl, and FeSO4·7H2O) were screened using Plackett-Burman experimental design. The most positive significant independent variables affecting enzyme production (temperature, inoculum age, and agitation speed) were further optimized by the face-centered central composite design-response surface methodology.

Topics: Asparaginase; Cell Culture Techniques; Glutaminase; Humans; Hypolipidemic Agents; Precursor Cell Lymphoblastic Leukemia-Lymphoma; RNA, Bacterial; RNA, Ribosomal, 16S; Streptomyces

L-Asparaginase (L-ASP) is a key component of therapy for acute lymphoblastic leukemia. Its mechanism of action, however, is still poorly understood, in part because of its dual asparaginase and glutaminase activities. Here, we show that L-ASP's glutaminase activity is not always required for the enzyme's anticancer effect. We first used molecular dynamics simulations of the clinically standard Escherichia coli L-ASP to predict what mutated forms could be engineered to retain activity against asparagine but not glutamine. Dynamic mapping of enzyme substrate contacts identified Q59 as a promising mutagenesis target for that purpose. Saturation mutagenesis followed by enzymatic screening identified Q59L as a variant that retains asparaginase activity but shows undetectable glutaminase activity. Unlike wild-type L-ASP, Q59L is inactive against cancer cells that express measurable asparagine synthetase (ASNS). Q59L is potently active, however, against ASNS-negative cells. Those observations indicate that the glutaminase activity of L-ASP is necessary for anticancer activity against ASNS-positive cell types but not ASNS-negative cell types. Because the clinical toxicity of L-ASP is thought to stem from its glutaminase activity, these findings suggest the hypothesis that glutaminase-negative variants of L-ASP would provide larger therapeutic indices than wild-type L-ASP for ASNS-negative cancers.

Topics: Antineoplastic Agents; Asparaginase; Aspartate-Ammonia Ligase; Drug Resistance, Neoplasm; Escherichia coli; Glutaminase; Humans; K562 Cells; Models, Molecular; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Recombinant Proteins; Tumor Cells, Cultured

Topics: Antineoplastic Agents; Asparaginase; Aspartate-Ammonia Ligase; Glutaminase; Humans; Precursor Cell Lymphoblastic Leukemia-Lymphoma

L-Asparaginase, an enzyme drug used for the treatment of acute lymphoblastic leukemia and its effective usage in clinical arena is complicated owing to the significant Glutaminase side activity. To develop variants of the enzyme with reduced Glutaminase activity, in silico mutagenesis was done by replacing amino acids in the vicinity of the ligand binding site. It was identified that replacement of enzyme's active site amino acid Asp96 with Alanine decreased the Glutaminase activity by 30% and also increased the Asparaginase activity by 40%. Docking studies were carried out by Autodock 4.0 and binding energy for native enzyme when docked with glutamine was found to be -8.08 Kcal/mole, whereas for mutated protein it was found to be -5.97 Kcal/mole. It was also observed that replacement of active site with amino acids other than alanine did not show considerable change in both Asparaginase and Glutaminase activities. The designed enzyme model with reduced Glutaminase side activity may help to develop a variant of enzyme drug through protein engineering by site-directed mutagenesis and thus to produce a drug with reduced side effect for treating acute lymphoblastic leukemia in children.

Topics: Asparaginase; Asparagine; Catalytic Domain; Child; Computer Simulation; Drug Design; Glutaminase; Glutamine; Humans; Ligands; Mutagenesis, Site-Directed; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protein Engineering