Family-wide Analysis of the Inhibition of Arf Guanine Nucleotide Exchange Factors with Small Molecules: Evidence of Unique Inhibitory Profiles

Arf
GTPases and their guanine nucleotide exchange factors (ArfGEFs)
are major regulators of membrane traffic and organelle structure in
cells. They are associated with a variety of diseases and are thus
attractive therapeutic targets for inhibition by small molecules.
Several inhibitors of unrelated chemical structures have been discovered,
which have shown their potential in dissecting molecular pathways
and blocking disease-related functions. However, their specificity
across the ArfGEF family has remained elusive. Importantly, inhibitory
responses in the context of membranes, which are critical determinants
of Arf and ArfGEF cellular functions, have not been investigated.
Here, we compare the efficiency and specificity of four structurally
distinct ArfGEF inhibitors, Brefeldin A, SecinH3, M-COPA, and NAV-2729,
toward six ArfGEFs (human ARNO, EFA6, BIG1, and BRAG2 and Legionella and Rickettsia RalF). Inhibition was assessed by fluorescence kinetics using pure
proteins, and its modulation by membranes was determined with lipidated
GTPases in the presence of liposomes. Our analysis shows that despite
the intra-ArfGEF family resemblance, each inhibitor has a specific
inhibitory profile. Notably, M-COPA is a potent pan-ArfGEF inhibitor,
and NAV-2729 inhibits all GEFs, the strongest effects being against
BRAG2 and Arf1. Furthermore, the presence of the membrane-binding
domain in Legionella RalF reveals a
strong inhibitory effect of BFA that is not measured on its GEF domain
alone. This study demonstrates the value of family-wide assays with
incorporation of membranes, and it should enable accurate dissection
of Arf pathways by these inhibitors to best guide their use and development
as therapeutic agents.