Improving the Treatment of Acute Lymphoblastic Leukemia.
ABSTRACT: l-Asparaginase (EC 220.127.116.11) 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.
Project description:An L-asparaginase (EC 18.104.22.168) specific for L-asparagine has been purified from a marine Chlamydomonas species, the first such enzyme to be purified from a microalga. The purified enzyme (mol.wt. 275 000) possessed a Km for asparagine of 1.34 x 10(-4) M and showed limited antitumour activity in an antilymphoma assay in vivo. Properties of the enzyme are contrasted with those of asparaginases from prokaryotic and eukaryotic sources.
Project description:Our long-term goal is the design of a human l-asparaginase (hASNase3) variant, suitable for use in cancer therapy without the immunogenicity problems associated with the currently used bacterial enzymes. Asparaginases catalyze the hydrolysis of the amino acid asparagine to aspartate and ammonia. The key property allowing for the depletion of blood asparagine by bacterial asparaginases is their low micromolar KM value. In contrast, human enzymes have a millimolar KM for asparagine. Toward the goal of engineering an hASNase3 variant with micromolar KM, we conducted a structure/function analysis of the conserved catalytic threonine triad of this human enzyme. As a member of the N-terminal nucleophile family, to become enzymatically active, hASNase3 must undergo autocleavage between residues Gly167 and Thr168. To determine the individual contribution of each of the three conserved active-site threonines (threonine triad Thr168, Thr186, Thr219) for the enzyme-activating autocleavage and asparaginase reactions, we prepared the T168S, T186V and T219A/V mutants. These mutants were tested for their ability to cleave and to catalyze asparagine hydrolysis, in addition to being examined structurally. We also elucidated the first N-terminal nucleophile plant-type asparaginase structure in the covalent intermediate state. Our studies indicate that, while not all triad threonines are required for the cleavage reaction, all are essential for the asparaginase activity. The increased understanding of hASNase3 function resulting from these studies reveals the key regions that govern cleavage and the asparaginase reaction, which may inform the design of variants that attain a low KM for asparagine.
Project description:Asparaginases catalyze the hydrolysis of the amino acid asparagine to aspartate and ammonia. Bacterial asparaginases are used in cancer chemotherapy to deplete asparagine from the blood, because several hematological malignancies depend on extracellular asparagine for growth. To avoid the immune response against the bacterial enzymes, it would be beneficial to replace them with human asparaginases. However, unlike the bacterial asparaginases, the human enzymes have a millimolar K(m) value for asparagine, making them inefficient in depleting the amino acid from blood. To facilitate the development of human variants suitable for therapeutic use, we determined the structure of human l-asparaginase (hASNase3). This asparaginase is an N-terminal nucleophile (Ntn) family member that requires autocleavage between Gly167 and Thr168 to become catalytically competent. For most Ntn hydrolases, this autoproteolytic activation occurs efficiently. In contrast, hASNas3 is relatively stable in its uncleaved state, and this allowed us to observe the structure of the enzyme prior to cleavage. To determine the structure of the cleaved state, we exploited our discovery that the free amino acid glycine promotes complete cleavage of hASNase3. Both enzyme states were elucidated in the absence and presence of the product aspartate. Together, these structures provide insight into the conformational changes required for cleavage and the precise enzyme-substrate interactions. The new understanding of hASNase3 will serve to guide the design of variants that possess a decreased K(m) value for asparagine, making the human enzyme a suitable replacement for the bacterial asparaginases in cancer therapy.
Project description:L-asparaginase is a chemotherapy drug used to treat acute lymphoblastic leukemia (ALL). The main prerequisite for clinical efficacy of L-asparaginases is micromolar KM for asparagine to allow for complete depletion of this amino acid in the blood. Since currently approved L-asparaginases are of bacterial origin, immunogenicity is a challenge, which would be mitigated by a human enzyme. However, all human L-asparaginases have millimolar KM for asparagine. We recently identified the low KM guinea pig L-asparaginase (gpASNase1). Because gpASNase1 and human L-asparaginase 1 (hASNase1) share ~70% amino-acid identity, we decided to humanize gpASNase1 by generating chimeras with hASNase1 through DNA shuffling. To identify low KM chimeras we developed a suitable bacterial selection system (E. coli strain BW5?). Transforming BW5? with the shuffling libraries allowed for the identification of several low KM clones. To further humanize these clones, the C-terminal domain of gpASNase1 was replaced with that of hASNase1. Two of the identified clones, 63N-hC and 65N-hC, share respectively 85.7% and 87.1% identity with the hASNase1 but have a KM similar to gpASNase1. These clones possess 100-140 fold enhanced catalytic efficiency compared to hASNase1. Notably, we also show that these highly human-like L-asparaginases maintain their in vitro ALL killing potential.
Project description:Asparaginases (ASPG, EC 22.214.171.124) catalyze the hydrolysis of the amide group of L-asparagine producing L-aspartate and ammonium. Three ASPG, PpASPG1, PpASPG2, and PpASPG3, have been identified in the transcriptome of maritime pine (<i>Pinus pinaster</i> Ait.) that were transiently expressed in <i>Nicotiana benthamiana</i> by agroinfection. The three recombinant proteins were processed <i>in planta</i> to active enzymes and it was found that all mature forms exhibited double activity asparaginase/isoaspartyl dipeptidase but only PpASPG1 was able to catalyze efficiently L-asparagine hydrolysis. PpASPG1 contains a variable region of 77 amino acids that is critical for proteolytic processing of the precursor and is retained in the mature enzyme. Furthermore, the functional analysis of deletion mutants demonstrated that this protein fragment is required for specific recognition of the substrate and favors enzyme stability. Potassium has a limited effect on the activation of maritime pine ASPG what is consistent with the lack of a critical residue essential for interaction of cation. Taken together, the results presented here highlight the specific features of ASPG from conifers when compared to the enzymes from angiosperms.
Project description:l-Asparaginases of bacterial origin are a mainstay of acute lymphoblastic leukemia treatment. The mechanism of action of these enzyme drugs is associated with their capacity to deplete the amino acid l-asparagine from the blood. However, clinical use of bacterial l-asparaginases is complicated by their dual l-asparaginase and l-glutaminase activities. The latter, even though representing only ?10% of the overall activity, is partially responsible for the observed toxic side effects. Hence, l-asparaginases devoid of l-glutaminase activity hold potential as safer drugs. Understanding the key determinants of l-asparaginase substrate specificity is a prerequisite step toward the development of enzyme variants with reduced toxicity. Here we present crystal structures of the Erwinia chrysanthemi l-asparaginase in complex with l-aspartic acid and with l-glutamic acid. These structures reveal two enzyme conformations-open and closed-corresponding to the inactive and active states, respectively. The binding of ligands induces the positioning of the catalytic Thr15 into its active conformation, which in turn allows for the ordering and closure of the flexible N-terminal loop. Notably, l-aspartic acid is more efficient than l-glutamic acid in inducing the active positioning of Thr15. Structural elements explaining the preference of the enzyme for l-asparagine over l-glutamine are discussed with guidance to the future development of more specific l-asparaginases.
Project description:The L-asparaginases from Escherichia coli and Erwinia chrysanthemi are effective drugs that have been used in the treatment of acute childhood lymphoblastic leukaemia for over 30 years. However, despite their therapeutic potential, they can cause serious side effects as a consequence of their intrinsic glutaminase activity, which leads to L-glutamine depletion in the blood. Consequently, new asparaginases with low glutaminase activity, fewer side effects and high activity towards L-asparagine are highly desirable as better alternatives in cancer therapy. L-Asparaginase from Helicobacter pylori was overexpressed in E. coli and purified for structural studies. The enzyme was crystallized at pH 7.0 in the presence of 16-19%(w/v) PEG 4000 and 0.1 M magnesium formate. Data were collected to 1.6 A resolution at 100 K from a single crystal at a synchrotron-radiation source. The crystals belong to space group I222, with unit-cell parameters a = 63.6, b = 94.9, c = 100.2 A and one molecule of L-asparaginase in the asymmetric unit. Elucidation of the crystal structure will provide insight into the active site of the enzyme and a better understanding of the structure-activity relationship in L-asparaginases.
Project description:We investigated whether an uncharacterized protein from guinea pig could be the enzyme behind Kidd's serendipitous discovery, made over 60 years ago, that guinea pig serum has cell killing ability. It has been long known that an enzyme with l-asparaginase activity is responsible for cell killing, although astonishingly, its identity remains unclear. Bacterial asparaginases with similar cell killing properties have since become a mainstay therapy of certain cancers such as acute lymphoblastic leukemia. By hydrolyzing asparagine to aspartate and ammonia, these drugs deplete the asparagine present in the blood, killing cancer cells that rely on extracellular asparagine uptake for survival. However, bacterial asparaginases can elicit an adverse immune response. We propose that replacement of bacterial enzymes with the guinea pig asparaginase responsible for serum activity, by its virtue of being more closely related to human enzymes, will be less immunogenic. To this goal, we investigated whether an uncharacterized protein from guinea pig with putative asparaginase activity, which we call gpASNase3, could be that enzyme. We examined its self-activation process (gpASNase3 requires autocleavage to become active), kinetically characterized it for asparaginase and ?-aspartyl dipeptidase activity, and elucidated its crystal structure in both the uncleaved and cleaved states. This work reveals that gpASNase3 is not the enzyme responsible for the antitumor effects of guinea pig serum. It exhibits a low affinity for asparagine as measured by a high Michaelis constant, KM, in the millimolar range, in contrast to the low KM (micromolar range) required for asparaginase to be effective as an anticancer agent.
Project description:Asparaginase is widely used in chemotherapeutic regimens for the treatment of acute lymphoblastic leukemia (ALL) and has led to a substantial improvement in cure rates, especially in children. Optimal therapeutic effects depend on a complete and sustained depletion of serum asparagine. However, pronounced interpatient variability, differences in pharmacokinetic properties between asparaginases and the formation of asparaginase antibodies make it difficult to predict the degree of asparagine depletion that will result from a given dose of asparaginase. The pharmacological principles underlying asparaginase therapy in the treatment of ALL are summarized in this article. A better understanding of the many factors that influence asparaginase activity and subsequent asparagine depletion may allow physicians to tailor treatment to the individual, maximizing therapeutic effect and minimizing treatment-related toxicity. Therapeutic drug monitoring provides a means of assessing a patient's current depletion status and can be used to better evaluate the potential benefit of treatment adjustments.