The small molecule inhibitor NAV-2729 has a complex target profile including multiple ADP-ribosylation factor regulatory proteins

The ADP-ribosylation factor (Arf) GTPases and their regulatory proteins are implicated in cancer progression. NAV-2729 was previously identified as a specific inhibitor of Arf6 that reduced progression of uveal melanoma in an orthotopic xenograft. Here, our goal was to assess the inhibitory effects of NAV-2729 on the proliferation of additional cell types. We found NAV-2729 inhibited proliferation of multiple cell lines, but Arf6 expression did not correlate with NAV-2729 sensitivity, and knockdown of Arf6 affected neither cell viability nor sensitivity to NAV-2729. Furthermore, binding to native Arf6 was not detected; however, we determined that NAV-2729 inhibited both Arf exchange factors and Arf GTPaseactivating proteins. ASAP1, a GTPase-activating protein linked to cancer progression, was further investigated. We demonstrated that NAV-2729 bound to the PH domain of ASAP1 and changed ASAP1 cellular distribution. However, ASAP1 knockdown did not fully recapitulate the cytoskeletal effects of NAV-2729 nor affect cell proliferation. Finally, our screens identified 48 other possible targets of NAV-2729. These results illustrate the complexities of defining targets of small molecules and identify NAV-2729 as a model PH domain- binding inhibitor.

Automated summary

The ADP-ribosylation factor (Arf) GTPases and their regulatory proteins are associated with cancer progression. NAV-2729 is a specific inhibitor of Arf6 and has been shown to reduce the progression of uveal melanoma. In this study, we assessed the inhibitory effects of NAV-2729 on the proliferation of different cell types and found that it inhibited proliferation in multiple cell lines. However, it did not correlate with Arf6 expression and knockdown of Arf6 did not affect cell viability or sensitivity to NAV-2729. We also identified other potential targets of NAV-2729, highlighting the complexity of defining small molecule targets.