Citation

  • Authors: Lin FY. et al.
  • Year: 2022
  • Journal: Cell 185 3533-3550.e27
  • Applications: in vitro / DNA / FectoPRO
  • Cell type: Expi293F
    Description: Human embryonic kidney Fibroblast
    Known as: Expi 293-F, Expi, HEK-293 Expi

Method

For transfection, 0.5ug plasmid coding aIIb subunit in PD2529 vector (Atum) and 0.5ug plasmid coding b3 or b3_N305T in PD2529 vector were incubated with 1 uL FectoPro (Polyplus) in 100 uL Opti-MEM (Gibco) for 10 min at room temperature were added per ml of Expi293F a5&aV KO cells (3*106/mL) in Expi293 expression medium (Thermo Fisher Scientific). 24 hrs after transfection, valproic acid and glucose were added to the culture to final concentrations of 3mMand 0.4%, respectively. 48 hours post transfection, cells were washed twice with assay medium (Leibovitz’s L-15 medium and 0.1% BSA) containing 5mM EDTA, twice with assay medium alone, and resuspended in assay medium or L15 supplemented with the indicated concentration of serum and 50mMHEPES, pH7.4.

Abstract

Integrins are validated drug targets with six approved therapeutics. However, small-molecule inhibitors to three integrins failed in late-stage clinical trials for chronic indications. Such unfavorable outcomes may in part be caused by partial agonism, i.e., the stabilization of the high-affinity, extended-open integrin conformation. Here, we show that the failed, small-molecule inhibitors of integrins αIIbβ3 and α4β1 stabilize the high-affinity conformation. Furthermore, we discovered a simple chemical feature present in multiple αIIbβ3 antagonists that stabilizes integrins in their bent-closed conformation. Closing inhibitors contain a polar nitrogen atom that stabilizes, via hydrogen bonds, a water molecule that intervenes between a serine residue and the metal in the metal-ion-dependent adhesion site (MIDAS). Expulsion of this water is a requisite for transition to the open conformation. This change in metal coordination is general to integrins, suggesting broad applicability of the drug-design principle to the integrin family, as validated with a distantly related integrin, α4β1.

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