Alterations to the broad-spectrum formin inhibitor SMIFH2 modulate potency but not specificity

Abstract

SMIFH2 is a small molecule inhibitor of the formin family of cytoskeletal regulators that was originally identified in a screen for suppression of actin polymerization induced by the mouse formin Diaphanous 1 (mDia1). Despite widespread use of this compound, it is unknown whether SMIFH2 inhibits all human formins. Additionally, the nature of protein/inhibitor interactions remains elusive. We assayed SMIFH2 against human formins representing six of the seven mammalian classes and found inhibitory activity against all formins tested. We synthesized a panel of SMIFH2 derivatives and found that, while many alterations disrupt SMIFH2 activity, substitution of an electron-donating methoxy group in place of the bromine along with halogenation of the furan ring increases potency by approximately five-fold. Similar to SMIFH2, the active derivatives are also pan-inhibitors for the formins tested. This result suggests that while potency can be improved, the goal of distinguishing between highly conserved FH2 domains may not be achievable using the SMIFH2 scaffold.

Cooperative actin filament nucleation by the Arp2/3 complex and formins maintains the homeostatic cortical array inArabidopsisepidermal cells

“…However, the change in filament buckling could be the consequence of different filament growth rates and processive elongation by formins ( Staiger et al 2009 ; Cai et al 2014 ; Cao et al 2016 ). Even though a recent in vitro study reveals that SMIFH2 is a pan-inhibitor for all human formins ( Orman et al 2022 ), we should not exclude the possibility that SMIFH2 can only inhibit a subset of plant FHs. Further genetic and biochemical analyses should be designed to dissect the interaction between SMIFH2 and different FHs to determine the specificity of this chemical inhibitor on plant formins.…”

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

“…The IC50 for formin-mediated elongation of actin filaments was 4.0 μM [28], suggesting that SMIFH2 is more potent on formin- SMIFH2 was also able to fully inhibit profilin-actin assembly [half maximal inhibitory concentration (IC 50 ) 15 µM] and exhibited specificity for formin-mediated actin assembly; indeed, it did not affect spontaneous polymerization of actin monomers or barbed-end elongation, or actin polymerization stimulated by the Arp2/3 complex [28]. This and the fact that it is active on at least six of the seven mammalian formin subfamilies (IC 50 from 6 to 30 µM, see Supplementary Table S2 for details) [29] as well as on formins from evolutionarily distant species (IC 50 from 5 to 15 µM) [28] qualify SMIFH2 as a general inhibitor of actin assembly mediated by formins. The IC 50 for formin-mediated elongation of actin filaments was 4.0 µM [28], suggesting that SMIFH2 is more potent on formininduced actin-filament elongation than nucleation.…”

“…SMIFH2 was also able to fully inhibit profilin-actin assembly [half maximal inhibitory concentration (IC50) 15 μM] and exhibited specificity for formin-mediated actin assembly; indeed, it did not affect spontaneous polymerization of actin monomers or barbed-end elongation, or actin polymerization stimulated by the Arp2/3 complex [28]. This and the fact that it is active on at least six of the seven mammalian formin subfamilies (IC50 from 6 to 30 μM, see Supplementary Table S2 for details) [29] as well as on formins from evolutionarily distant species (IC50 from 5 to 15 μM) [28] qualify SMIFH2 as a general inhibitor of actin assembly mediated by formins. The IC50 for formin-mediated elongation of actin filaments was 4.0 μM [28], suggesting that SMIFH2 is more potent on formin- SMIFH2 was also able to fully inhibit profilin-actin assembly [half maximal inhibitory concentration (IC 50 ) 15 µM] and exhibited specificity for formin-mediated actin assembly; indeed, it did not affect spontaneous polymerization of actin monomers or barbed-end elongation, or actin polymerization stimulated by the Arp2/3 complex [28].…”

“…A recent structure-activity study of SMIFH2 against a panel of human formins that relied on in vitro actin polymerization assays [29] and NMR analyses has addressed this question. The testing of a panel of SMIFH2 derivatives showed that many alterations disrupted SMIFH2 activity, whereas some of them increased potency by up to fivefold, and a few others produced SMIFH2 derivatives with mild selectivity for certain formins [29].…”

Reforming the Barrier: The Role of Formins in Wound Repair

“…As mentioned above, SMIFH2 has been shown to affect myosin proteins in addition to formins, which indicates its off-target activity. Therefore, all data acquired by this inhibitor should be interpreted with caution [ 122 ]. Since formins are heavily involved in wound-repair-related processes, more pharmacological research is required to modulate them in order to investigate their role in wound healing in vitro and in vivo.…”