Investigating Mammalian Formins with SMIFH2 Fifteen Years in: Novel Targets and Unexpected Biology

Abstract

The mammalian formin family comprises fifteen multi-domain proteins that regulate actin dynamics and microtubules in vitro and in cells. Evolutionarily conserved formin homology (FH) 1 and 2 domains allow formins to locally modulate the cell cytoskeleton. Formins are involved in several developmental and homeostatic processes, as well as human diseases. However, functional redundancy has long hampered studies of individual formins with genetic loss-of-function approaches and prevents the rapid inhibition of formin activities in cells. The discovery of small molecule inhibitor of formin homology 2 domains (SMIFH2) in 2009 was a disruptive change that provided a powerful chemical tool to explore formins’ functions across biological scales. Here, I critically discuss the characterization of SMIFH2 as a pan-formin inhibitor, as well as growing evidence of unexpected off-target effects. By collating the literature and information hidden in public repositories, outstanding controversies and fundamental open questions about the substrates and mechanism of action of SMIFH2 emerge. Whenever possible, I propose explanations for these discrepancies and roadmaps to address the paramount open questions. Furthermore, I suggest that SMIFH2 be reclassified as a multi-target inhibitor for its appealing activities on proteins involved in pathological formin-dependent processes. Notwithstanding all drawbacks and limitations, SMIFH2 will continue to prove useful in studying formins in health and disease in the years to come.

Transmembrane formins as active cargoes of membrane trafficking

“…An mDia2-derived peptide outside GBD/FH3 is predicted to bind membranes enriched in PtdIns(4,5)P 2 in molecular dynamics simulations, and in vitro actin nucleation on PtdIns(4,5)P 2 -enriched membranes appears to require formin activity ( Bucki et al , 2019 ). However, the latter observation builds on pharmacological experiments utilizing the broad-spectrum formin inhibitor SMIFH2 (small molecule inhibitor of FH2) that might affect additional targets including myosins ( Nishimura et al , 2021 ; Innocenti, 2023 ). Formin localization at the cytoplasmic face of the plasmalemma is also controlled by membrane phosphoinositide content in crawling Dictyostelium amoebae, where distinct DRF isoforms participate in the formation of actin wave patterns ( Ecke et al , 2020 ).…”

“…Formin-controlled actin rearrangements also drive secretory vesicle fusion in bovine chromaffin cells ( Wen et al , 2016 ). However, since this conclusion is based on experiments utilizing SMIFH2, the above-discussed specificity concerns remain relevant (see Innocenti, 2023 ).…”