The G protein-coupled receptor (GPCR) CXCR4 is a co-receptor for HIV and is involved in cancers and autoimmune diseases. We characterized five purine or quinazoline core polyamine pharmacophores used for targeting CXCR4 dysregulation in diseases. All were neutral antagonists for wild-type CXCR4 and two were biased antagonists with effects on beta-arrestin-2 only at high concentrations. These compounds displayed various activities for a constitutively active mutant (CAM). We use the IT1t-CXCR4 crystal structure and molecular dynamics (MD) simulations to develop two hypotheses for the activation of the N119(3.35)A CAM. The N119(3.35)A mutation facilitates increased coupling of TM helices III and VI. IT1t deactivates the CAM by disrupting the coupling between TM helices III and VI, mediated primarily by residue F87(2.53). Mutants of F87(2.53) in N119(3.35)A CXCR4 precluded constitutive signaling and prevented inverse agonism. This work characterizes CXCR4 ligands and provides a mechanism for N119(3.35)A constitutive activation.
