Phenothiazines inhibit S100A4 function by inducing protein oligomerization

Abstract

S100A4, a member of the S100 family of Ca 2þ -binding proteins, regulates carcinoma cell motility via interactions with myosin-IIA. Numerous studies indicate that S100A4 is not simply a marker for metastatic disease, but rather has a direct role in metastatic progression. These observations suggest that S100A4 is an excellent target for therapeutic intervention. Using a unique biosensorbased assay, trifluoperazine (TFP) was identified as an inhibitor that disrupts the S100A4/myosin-IIA interaction. To examine the interaction of S100A4 with TFP, we determined the 2.3 Å crystal structure of human Ca 2þ -S100A4 bound to TFP. Two TFP molecules bind within the hydrophobic target binding pocket of Ca 2þ -S100A4 with no significant conformational changes observed in the protein upon complex formation. NMR chemical shift perturbations are consistent with the crystal structure and demonstrate that TFP binds to the target binding cleft of S100A4 in solution. Remarkably, TFP binding results in the assembly of five Ca 2þ -S100A4/TFP dimers into a tightly packed pentameric ring. Within each pentamer most of the contacts between S100A4 dimers occurs through the TFP moieties. The Ca 2þ -S100A4/prochlorperazine (PCP) complex exhibits a similar pentameric assembly. Equilibrium sedimentation and cross-linking studies demonstrate the cooperative formation of a similarly sized S100A4/TFP oligomer in solution. Assays examining the ability of TFP to block S100A4-mediated disassembly of myosin-IIA filaments demonstrate that significant inhibition of S100A4 function occurs only at TFP concentrations that promote S100A4 oligomerization. Together these studies support a unique mode of inhibition in which phenothiazines disrupt the S100A4/ myosin-IIA interaction by sequestering S100A4 via small molecule-induced oligomerization.calcium | X-ray crystallography | NMR | small molecule inhibitor | metastasis T he S100 proteins, of which there are more than 20 members, are characterized by their solubility in 100% saturated ammonium sulfate (1 and 2). Each S100 family member contains two Ca 2þ -binding loops; a C-terminal "typical" EF-hand comprised of 12 residues and an N-terminal pseudo EF-hand consisting of 14 residues. The basic organization of the S100 proteins is a symmetric, antiparallel homodimer, in which the N-and C-terminal helices (helices 1 and 4) from each subunit interact to form a stable four helix bundle that serves as the dimer interface. Calcium binding to the C-terminal typical EF-hand significantly alters the angle between helices 3 and 4, which flank the C-terminal Ca 2þ -binding loop, and exposes a hydrophobic cleft that constitutes a binding surface for target proteins (3-5). Thus the S100 proteins operate as calcium-activated switches that bind and regulate the activity of diverse protein targets. S100 proteins are expressed in a tissue and cell specific manner. Elevated expression of individual family members is associated with a number of human pathologies, including cardiomyopathies, cancer, neurodegeneration, and in...

Interference of the complex between NCS-1 and Ric8a with phenothiazines regulates synaptic function and is an approach for fragile X syndrome

“…The substituents in the phenothiazine group promote specificity and drive these molecules to their corresponding targets, thus determining their biological activity (28). In fact, it has been also shown that another derivative, Trifluoperazine, can target the cancer-related Ca 2+ binding protein S100A4, representing a potential inhibitor of metastasis (38).…”

Stabilization of protein–protein interactions by small molecules

“…These inhibitors belong to the renowned phenothiazine chemical class, a family of compounds endowed with antipsychotic, antiallergic and anesthetic properties [76,77], resulting in breakthrough drugs such as chlorpromazine [78]. One of these compounds, trifluoperazine {TFP, 10-[3-(4-methylpiperazin-1-yl)propyl]-2-(trifluoromethyl)-10H-phenothiazine; Table 1}, was later shown in a crystallographic and biochemical study to inhibit S100A4 function by stabilizing an inactive pentamer [79]. In the crystallized pentamer two TFP molecules are sitting adjacent to each other in the interface contributed from each monomer, adding up to four copies of TFP that make extensive mutual contacts and thus stabilize the oligomeric assembly.…”

Crystal structure of the S100A4–nonmuscle myosin IIA tail fragment complex reveals an asymmetric target binding mechanism

“…In two crystal structure of Ca 2þ -bound S100A4 the hydrophobic cleft of each subunit is occupied by the C terminus of an adjacent dimer (16,17), which could explain the formation of S100A4 oligomers that were observed extracellularly (18). These clefts were also shown to interact with the calmodulin antagonist trifluoperazine (19). Interestingly, the known complex structures of the S100 family do not reveal a uniform binding mode as the orientations of the target peptides in each complex are considerably different.…”