A Point Mutation That Confers Constitutive Activity to CXCR4 Reveals That T140 Is an Inverse Agonist and That AMD3100 and ALX40-4C Are Weak Partial Agonists

Abstract

CXCR4 is a G protein-coupled receptor for stromalderived factor 1 (SDF-1) that plays a critical role in leukocyte trafficking, metastasis of mammary carcinoma, and human immunodeficiency virus type-1 infection. To elucidate the mechanism for CXCR4 activation, a constitutively active mutant (CAM) was derived by coupling the receptor to the pheromone response pathway in yeast. Conversion of Asn-119 to Ser or Ala, but not Asp or Lys, conferred autonomous CXCR4 signaling in yeast and mammalian cells. SDF-1 induced signaling in variants with substitution of Asn-119 to Ser, Ala, or Asp, but not Lys. These variants had similar cell surface expression and binding affinity for SDF-1. CXCR4-CAMs were constitutively phosphorylated and present in cytosolic inclusions. Analysis of antagonists revealed that exposure to AMD3100 or ALX40-4C induced G protein activation by CXCR4 wild type, which was greater in the CAM, whereas T140 decreased autonomous signaling. The affinity of AMD3100 and ALX40-4C binding to CAMs was less than to wild type, providing evidence of a conformational shift. These results illustrate the importance of transmembrane helix 3 in CXCR4 signaling. Insight into the mechanism for CXCR4 antagonists will allow for the development of a new generation of agents that lack partial agonist activity that may induce toxicities, as observed for AMD3100.

The CXCR4 antagonist 4F-benzoyl-TN14003 stimulates the recovery of the bone marrow after transplantation

“…14 Comparative studies between T-140 and AMD3100 found that each of these agents inhibited CXCR4 through different mechanisms. 11,12,14 Analysis of antagonists revealed that exposure to AMD3100 induced G protein activation by CXCR4, whereas T-140 decreased autonomous signaling. 12 Therefore, AMD3100 was defined to have a weak partial agonist activity, whereas T-140 functions as an inverse agonist.…”

“…11,12,14 Analysis of antagonists revealed that exposure to AMD3100 induced G protein activation by CXCR4, whereas T-140 decreased autonomous signaling. 12 Therefore, AMD3100 was defined to have a weak partial agonist activity, whereas T-140 functions as an inverse agonist. In our current work, we found that SDF-1a at high levels, as well as T140, but not AMD3100, similarly affect the proliferation and differentiation of progenitors, granulocytes and erythroblasts when co-cultured with stromal cells.…”

“…Furthermore, it was suggested that T140 acts as an inverse agonist, and AMD3100 acts as a weak partial agonist when the coupling of CXCR4 to Ga subunits in mammalian cells was assayed by [35S]GTPgS binding. 12 Indeed, blocking the CXCR4 receptor with antagonists, such as T140 or AMD3100, results in the mobilization of HPCs, including neutrophils. 13,14 Moreover, combination of these two antagonists with granulocyte colony-stimulating factor (G-CSF) has an additive effect.…”

CXCR4 antagonists: targeting the microenvironment in leukemia and other cancers

“…71 Also, in a series of experiments to elucidate the mechanism of CXCR4 signaling, it was noticed that T140 decreased autonomous CXCR4 signaling in CXCR4 wild-type or constitutively active CXCR4 mutants, characterizing T140 as an inverse CXCR4 agonist, whereas AMD3100 and ALX40-4C displayed partial agonist activity in this study. 72 Clinical trials will help to determine whether this characteristic of CXCR4 peptide antagonists correlates with a profile of activities and/or side effects that is distinct from AMD3100.…”

Lipid Bilayer Simulations of CXCR4 with Inverse Agonists and Weak Partial Agonists

“…AMD3100 was withdrawn from clinical trials in patients with HIV-1 infections because of cardiac toxicity. 2 The mechanism of antagonist action has been determined using human CXCR4 mutants with constitutive activity derived in yeast strains expressing the receptor functionally coupled to the pheromone response pathway (10). AMD3100 and ALX40-4C demonstrated weak partial agonist activity (10).…”