br From structural modeling studies the benzyl phenyl
From structural modeling studies, the benzyl-phenyl moiety of Benp fit into the binding pocket through hydrophobic interactions with F225, L243, and F247 in ARPC2 (Fig. 4A). We constructed a putative binding-defective mutant, ARPC2 (F225A), to test this prediction. We conducted co-immunoprecipitation assays from DLD-1 Clozapine N-oxide transfected with ex-pression vector for myc tagged ARPC2 or ARPC2 F225A. As a result, myc-tagged ARPC2 or ARPC2 F225A mutant did not co-im-munoprecipitated with the other Arp2/3 subunits, suggesting the overexpression of ARPC2 and ARPC2 F225A mutant resulted in free ARPC2 (Fig. 4B). Moreover, we performed the DARTS assay and ana-lyzed the amount of pronase-resistant myc-tagged ARPC2 to investigate whether Benp bound to ARPC2 F225A mutant or not. In the presence of Benp, myc-tagged ARPC2 was resistant to proteolysis by pronase in wild-type ARPC2, but was easily degraded in ARPC2 F225A mutant, suggesting that mutating F225A impaired the binding of Benp to ARPC2 (Fig. 4C). As expected, the anti-migratory activity of Benp was pre-vented in ARPC2-overexpressed cells (Fig. 4D). Cell viability of the cells was not affected by Benp-treatment up to 30 μM (data not shown). To validate ARPC2 as a molecular target of Benp, we overexpressed ARPC2 or Arp3 in the cells (Fig. 4E). The inhibitory effect of Benp was sig-nificantly rescued by ARPC2 overexpression but not by Arp3 over-expression (Fig. 4F and G). In addition, an Arp2/3 inhibitor inhibited the migration of DLD-1 cells, and migration was partially rescued by overexpressing Arp3 but not ARPC2 (Fig. 4G). Our data suggest that the Phe 225 residue is crucial for binding of Benp to ARPC2 and confirm that Benp is an inhibitor of ARPC2.
3.4. Validation of ARPC2 as a direct target of Benproperine
Because Arp3 and ARPC2 are subunits of the Arp2/3 complex [13,14], it was necessary to determine which subunit is directly bound to Benp. We synthesized biotin-conjugated Benp (Benp-biotin; Fig. 5A) and then examined an inhibitory effect on cancer cell migration of the compound. Benp-biotin inhibited the migration of DLD-1 cells with an IC50 value of 10 μM (data not shown). We showed the binding specifi-city using a pull-down assay with Benp-biotin in the presence or ab-sence of free Benp as a competitor. Benp bound to ARPC2 but not to Arp3; the binding between ARPC2 and biotin-Benp was efficiently competed with excess free Benp (Fig. 5B). We further confirmed this specific interaction between ARPC2 and Benp using lysates prepared from DLD-1 cells (Fig. 5C). To confirm the direct interactions between ARPC2 and Benp, we applied label-free non-affinity target identifica-tion approaches, including drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA), to further validate ARPC2 as a direct target of Benp [31,32]. As expected, ARPC2 was resistant to proteolysis by various concentrations of pronase in the presence of Benp (Fig. 5D) and Benp also increased the thermal stability of ARPC2 by increasing drug concentration in AsPC-1 cells (Fig. 5E). Taken together, these data show that Benp directly interacts with ARPC2 in cancer cells.
3.5. Inhibition of cancer cell migration by delaying the initiation of actin polymerization
The Arp2/3 complex and its subunits are involved in actin poly-merization . Therefore, we assessed the ability of Benp to regulate actin polymerization using the in vitro pyrene-actin assembly assay . Addition of the Arp2/3 complex and VCA into the actin poly-merization mixture increased the actin polymerization rate; further addition of Benp or CK869 delayed actin polymerization initiation (Fig. 6A). Our data show that Benp attenuates the rate of action poly-merization nucleation by impairing Arp2/3 function.
During cell migration, Arp2/3 complex-mediated branching is
Fig. 5. Direct binding of Benproperine with ARPC2 in cells. (A) Structure of Benp-conjugated biotin (Benp-Biotin). (B) Purified ARPC2-his and Arp3-his proteins or whole cell lysates from DLD-1 cells (C) were incubated with Benp-conjugated biotin and competed with the indicated concentrations of Benp. Representative gel image immunoblotted with the anti-ARPC2 antibody are shown (n = 2). (D) Immunoblotting of ARPC2 (target protein) and GAPDH (non-target protein) in DLD-1 or AsPC-1 cells that were proteolyzed with various concentration of pronase in the presence or absence of Benp (100 μM) (n = 2). (E) CETSA in cell lysates of AsPC-1 cells that were incubated with DMSO or Benp (100 μM) and quantification of the intensity of ARPC2 with increasing drug concentration (n = 2). The data represent means ± s.d.