Asparagine synthetase chemotherapy

Abstract

Modern clinical treatments of childhood acute lymphoblastic leukemia (ALL) employ enzyme-based methods for depletion of blood asparagine in combination with standard chemotherapeutic agents. Significant side effects can arise in these protocols and, in many cases, patients develop drug-resistant forms of the disease that may be correlated with up-regulation of the enzyme glutamine-dependent asparagine synthetase (ASNS). Though the precise molecular mechanisms that result in the appearance of drug resistance are the subject of active study, potent ASNS inhibitors may have clinical utility in treating asparaginase-resistant forms of childhood ALL. This review provides an overview of recent developments in our understanding of (a) the structure and catalytic mechanism of ASNS, and (b) the role that ASNS may play in the onset of drug-resistant childhood ALL. In addition, the first successful, mechanism-based efforts to prepare and characterize nanomolar ASNS inhibitors are discussed, together with the implications of these studies for future efforts to develop useful drugs.

Figures

Figure 1

(a) Cartoon representation of the structure of the Cys-1-Ala mutant of Escherichia coli AS-B complexed with glutamine (blue space-filling model) and AMP (green space-filling model) showing the domain organization of the enzyme (35). Helices and β-strands are shown in yellow and red, respectively. The final 40 C-terminal residues are not observed in the crystal structure, presumably due to their disordered conformation in the absence of bound aspartate. (b) Cartoon showing the putative pathway by which ammonia (light blue spheres) travels between the glutaminase (top) and the synthetase (bottom) active sites in AS-B. The side chains of residues defining the ammonia tunnel that are variable and conserved throughout the family of known asparagine synthetases are colored green and red, respectively. Bound glutamine (top) and AMP (bottom) are rendered as gray-white “ball-and-stick” models. Reprinted from (63), Copyright 2003, with permission from Elsevier.

Figure 2

A working model for control of the asparagine synthetase (ASNS) gene by the AAR or UPR pathways (92). Transcription factors shown in color have been localized to the ASNS promoter by chromatin immunoprecipitation analysis. Unidentified or putative components are shown in gray. Transcription from the ASNS gene reaches its highest rate at 1–4 h (phase I) following nutrient stress. ASNS transcription is still elevated relative to the “fed” state between 4–24 h (phase II) following nutrient stress, but the rate is reduced.

Figure 3

Selected metabolic changes that take place in ASNase-resistant MOLT-4 leukemia cells (123). Treatment with ASNase causes a rapid degradation of extracellular asparagine (Asn) and a subsequent depletion of intracellular Asn. Compensatory changes, shown with red arrows, include increases in: transcription from the asparagine synthetase gene, glutamine synthetase activity (post-transcriptional), and active glutamine transport. Conversely, there is a decrease, shown in yellow, in Asn efflux through Na+-independent exchange. There is little or no aspartate uptake by these cells, so synthesis via transamination may play a role in supplying this substrate for the ASNS-catalyzed reaction.

Figure 4

Cartoon representations of the X-ray crystal structures of (a) BLS complexed to its substrate (CPK-colored space-filling model) and AMPCPP (green space-filling model) in the C-terminal domain (52) and (b) Escherichia coli AS-B complexed with glutamine (not shown) and AMP (green space-filling model) (35). The striking structural conservation in both enzymes suggests either a common ancestor or recruitment of AS to provide BLS during the evolution of clavulanic acid biosynthesis. In both structures, helices and β-strands are shown in yellow and red, respectively, whereas water molecules are represented by red spheres.

Figure 5

Views of the working molecular model for the AS-B/βAspAMP/PPi complex. (a) Interactions between PPi and residues defining the ATP pyrophosphatase loop motif. (b) Protein/βAspAMP interactions involving conserved residues Glu-352, Tyr-357, Lys-376, Asp-384, Arg-387, and Lys-449 that illustrate recognition of the α-amino and α-carboxylate groups present in the βAspAMP intermediate 1. (Color coding: C – gray; H – white; O – red; N - blue; P – purple. Light blue lines show locations of putative hydrogen bonds.)

Figure 6

Structures of compounds 3–11 (see text for details).

Figure 7

Computational visualization of transition state mimicry by the adenylated sulfoximine functional group. (a) Semiempirical (PM3) transition state for the attack of ammonia on a computational model of the acyladenylate intermediate formed in the ASNS synthetase site. (b) Graphical representation of the transition state structure showing the isodensity surface color-coded by the electrostatic potential. Note that the hydrogen atoms in ammonia gain substantial positive charge (red). (c) Optimized (PM3) structure for a model phosphorylated sulfoximine. (d) Graphical representation of the phosphorylated sulfoximine in (c) showing the isodensity surface color-coded by the electrostatic potential. Note that the hydrogen atoms of the methyl group have similar steric and electrostatic properties to those on ammonia in the transition state shown in (b). [Color coding in (a) and (c): C – gray; H – white; O – red; N – blue; P – purple; S – yellow. Dotted lines show noncovalent, electrostatic interactions.]

Figure 8

Computational model of the adenylated sulfoximine ASNS inhibitor 7b docked into the synthetase site of AS-B. For ease of comprehension, only selected protein residues are shown, which are all conserved within glutamine-dependent ASNS. Note the distance between the Glu-348 side-chain carboxylate (pink) and the methyl group (cyan) of the ASNS inhibitor. The methyl substituent mimics the location of ammonia in the transition state formed during attack on the βAspAMP intermediate 1. Residues shown in green (Leu-232 and Ser-346) define the C-terminal end of the channel through which ammonia enters the synthetase active site after being released in the N-terminal glutaminase domain. (Color coding: C – gray; H – white; O – red; N – blue; P – purple; S – yellow.)

Scheme 1

Overall transformation catalyzed by glutamine-dependent ASNS, showing the β-aspartyl-AMP intermediate. Ammonia may replace glutamine as a nitrogen source in vitro.

Scheme 2

Hypothetical mechanism for the ASNS-catalyzed formation of βAspAMP 1 and its subsequent reaction with ammonia to form asparagine and AMP.

Scheme 3

A comparison of the chemical reactions catalyzed by BLS and ASNS. (a) Aspartate is activated by adenylation to yield βAspAMP, which undergoes intermolecular attack by ammonia to yield asparagine. (b) CEA, the substrate for BLS, is activated as an acyl-AMP derivative, which then undergoes intramolecular nitrogen attack to give the β-lactam product.