6
A P P L I C A T I O N N O T E
P2X4 – small molecule interaction
Therapeutic opportunities involving the P2X4 receptor are promising, particularly through the de-
velopment of small molecule modulators. High-throughput screening of small molecule binders to
P2X4 offers a powerful strategy to identify compounds that can either inhibit or enhance the recep-
tor's function. The present case study examined the interaction between the P2X4 receptor and the
small molecule antagonist 5-BDBD (5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diaze-
pin-2-one).
As illustrated in Figure 4A, the copolymer nanodisc-stabilized P2X4 had a hydrodynamic radius (rH)
of 10.81 ± 0.45 nm, which aligns with the anticipated size of the nanodisc. The polydispersity index
(PDI) was demonstrated to be 0.24 ± 0.02, indicating that the purified sample was monodisperse
and thus well suited for further biophysical analysis. In addition, DLS analysis of the same sample
in a thermal ramp also shows that the nanodisc-stabilized P2X4 remained thermally stable up to
approximately 55°C.
Results
Following the screening approach outlined in the previous app note, the most suitable co-
polymers were identified and used in the purification of GLP1R, P2X4, NTCP, and L-HBsAg
respectively using the NativeMP
TM
Platform and Prometheus Panta. The quality and stability
of the purified full length membrane proteins was validated through Size Exclusion Chroma-
tography, SDS-PAGE, Western Blot as well as DLS and nanoDSF via the Prometheus Panta
prior to the interaction analysis.
Figure 3: Western blot and SDS PAGE of P2X4 sol-
ubilized with Native MP
TM
. P2X4 with a theoreti-
cal size of 44,6 kDa was detected at an apparent
height of ~ 60 kDa (marked with arrows). Detec-
tion of proteins in SDS Page via specific primary
Rho1D4 antibody and secondary HRP antibody.
