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 Nω-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.
Highlights
The ability of tumors to modify their microenvironment and thereby evade the immune system of the host is increasingly recognized as an important determinant of cancer progression and patient prognosis (Gooden et al, 2011; Galon et al, 2013)
The role of Arginase-1 in tumor immune suppression and its potential as a drug target for cancer immunotherapy has culminated in the clinical development of CB-1158 (Steggerda et al, 2017)
Given the importance of Arginase-1 inhibitors, we studied the characteristics of (2S)-2-amino-6-boronohexanoic acid (ABH), nor-NOHA and CB-1158 side-by-side in different biochemical and biophysical assays, including surface plasmon resonance (SPR) and protein crystallography
Summary
The ability of tumors to modify their microenvironment and thereby evade the immune system of the host is increasingly recognized as an important determinant of cancer progression and patient prognosis (Gooden et al, 2011; Galon et al, 2013). In the tumor microenvironment, enhanced expression of Arginase-1 by myeloid cells causes the local depletion of the semi-essential amino acid L-arginine. This results in anergy of effector T-cells by inhibition of CD3ζ chain expression (Rodriguez et al, 2004), and induces the suppression of effector T-cell and natural killer cell proliferation (Steggerda et al, 2017; Oberlies et al, 2009). We demonstrate the high potency of the clinical compound CB-1158 (Fig. 1) (fdasis.nlm.nih.gov/srs/auto/cb-1158) (Geiger et al, 2016), display its slow association and dissociation kinetics and reveal a crystal structure of CB-1158 bound in the Arginase-1 active site
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