High-resolution crystal structure of human asparagine synthetase enables analysis of inhibitor binding and selectivity

Wen Zhu,Yi Jin,Claudine Bisson,Yuichiro Takagi,David W Rice,Tyzoon K Nomanbhoy,Brian E Nordin,Francisco Martínez-Márquez,Adriana Coricello,Ashish Radadiya,Nigel G J Richards,Patrick Baumann,Alexandria H Berry,Tsuyoshi Imasaki,Svetlana E Sedelnikova,Sabine Wenzel,John W Kozarich

doi:10.1038/s42003-019-0587-z

Abstract

Expression of human asparagine synthetase (ASNS) promotes metastatic progression and tumor cell invasiveness in colorectal and breast cancer, presumably by altering cellular levels of L-asparagine. Human ASNS is therefore emerging as a bona fide drug target for cancer therapy. Here we show that a slow-onset, tight binding inhibitor, which exhibits nanomolar affinity for human ASNS in vitro, exhibits excellent selectivity at 10 μM concentration in HCT-116 cell lysates with almost no off-target binding. The high-resolution (1.85 Å) crystal structure of human ASNS has enabled us to identify a cluster of negatively charged side chains in the synthetase domain that plays a key role in inhibitor binding. Comparing this structure with those of evolutionarily related AMP-forming enzymes provides insights into intermolecular interactions that give rise to the observed binding selectivity. Our findings demonstrate the feasibility of developing second generation human ASNS inhibitors as lead compounds for the discovery of drugs against metastasis.

Highlights

Expression of human asparagine synthetase (ASNS) promotes metastatic progression and tumor cell invasiveness in colorectal and breast cancer, presumably by altering cellular levels of L-asparagine
mass spectrometric (MS)/MS fragmentation and sequence analysis of the tryptic peptides obtained from these reaction mixtures showed that Asparagine synthetase (ASNS) inhibitor 1 suppressed the ability of probe 3 to acylate the side chain of Lys-466 to an extent of 62% when present in the HCT-116 lysate at 10 μM concentration (Fig. 2)
This work establishes the feasibility of obtaining ASNS inhibitors that exhibit considerable selectivity when present at low, or submicromolar concentrations in cells despite the existence of other ATP-utilizing enzymes possessing homologous catalytic domains to ASNS

Summary

Introduction

Expression of human asparagine synthetase (ASNS) promotes metastatic progression and tumor cell invasiveness in colorectal and breast cancer, presumably by altering cellular levels of L-asparagine. MS/MS fragmentation and sequence analysis of the tryptic peptides obtained from these reaction mixtures showed that ASNS inhibitor 1 suppressed the ability of probe 3 to acylate the side chain of Lys-466 (located within the ATP-binding site of human ASNS) to an extent of 62% when present in the HCT-116 lysate at 10 μM concentration (Fig. 2).

Results

Conclusion