Structural insights into human Arginase-1 pH dependence and its inhibition by the small molecule inhibitor CB-1158

Yvonne Grobben; Joost C M Uitdehaag; Nicole Willemsen-Seegers; Werner W A Tabak; Jos de Man; Rogier C Buijsman; Guido J R Zaman

doi:10.1016/j.yjsbx.2019.100014

Structural insights into human Arginase-1 pH dependence and its inhibition by the small molecule inhibitor CB-1158

J Struct Biol X. 2019 Nov 26:4:100014. doi: 10.1016/j.yjsbx.2019.100014. eCollection 2020.

Authors

Yvonne Grobben¹, Joost C M Uitdehaag¹, Nicole Willemsen-Seegers¹, Werner W A Tabak¹, Jos de Man¹, Rogier C Buijsman¹, Guido J R Zaman¹

Affiliation

¹ Netherlands Translational Research Center B.V., Kloosterstraat 9, 5349 AB Oss, The Netherlands.

Abstract

Arginase-1 is a manganese-dependent metalloenzyme that catalyzes the hydrolysis of L-arginine into L-ornithine and urea. Arginase-1 is abundantly expressed by tumor-infiltrating myeloid cells that promote tumor immunosuppression, which is relieved by inhibition of Arginase-1. We have characterized the potencies of the Arginase-1 reference inhibitors (2S)-2-amino-6-boronohexanoic acid (ABH) and N ^ω-hydroxy-nor-L-arginine (nor-NOHA), and studied their pH-dependence and binding kinetics. To gain a better understanding of the structural changes underlying the high pH optimum of Arginase-1 and its pH-dependent inhibition, we determined the crystal structure of the human Arginase-1/ABH complex at pH 7.0 and 9.0. These structures revealed that at increased pH, the manganese cluster assumes a more symmetrical coordination structure, which presumably contributes to its increase in catalytic activity. Furthermore, we show that binding of ABH involves the presence of a sodium ion close to the manganese cluster. We also studied the investigational new drug CB-1158 (INCB001158). This inhibitor has a low-nanomolar potency at pH 7.4 and increases the thermal stability of Arginase-1 more than ABH and nor-NOHA. Moreover, CB-1158 displays slow association and dissociation kinetics at both pH 9.5 and 7.4, as indicated by surface plasmon resonance. The potent character of CB-1158 is presumably due to its increased rigidity compared to ABH as well as the formation of an additional hydrogen-bond network as observed by resolution of the Arginase-1/CB-1158 crystal structure.

Keywords: ABH, (2S)-2-amino-6-boronohexanoic acid; Biochemical inhibition; Cancer immunotherapy; DMSO, dimethyl sulfoxide; IC50, half-maximal inhibitory concentration; ITC, isothermal titration calorimetry; KD, binding affinity; KM, Michaelis constant; Ki, inhibition constant; MQ, MilliQ water; PDB, Protein Data Bank; RMSD, root-mean-square deviation; SD, standard deviation; SPR, surface plasmon resonance; Surface plasmon resonance; Thermal stability; Tm, melting temperature; X-ray crystallography; ka, association rate constant; kcat, catalytic rate constant; kd, dissociation rate constant; nor-NOHA, Nω-hydroxy-nor-L-arginine; ΔTm, melting temperature shift; τ, target residence time.