Anti-PITPNM3 small molecular compounds reverse breast cancer metastasis by targeting PITPNM3.

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e15005 Background: Recent studies highlight the fundamental roles of PITPNM3 in breast cancer metastasis. PITPNM3 is identified as the functional receptor of CCL18 and promotes breast cancer cell invasion and metastasis by binding with CCL18. Since anti-CCL18 neutralized antibodies shows medium binding affinity which restricts their clinical application, small molecular inhibitors targeting PITPNM3 are needed to be further investigated. Therefore, we identified several first in class small molecular inhibitors potentially targeting PITPNM3 and can inhibit breast cancer metastasis conducted by PITPNM3 activation. Methods: We performed computer-assisted drug design by constructing PITPNM3 homology model, characterizing potential binding pockets and docking preselected high diversity structured small molecule compounds into the static PITPNM3 model. Top 100 small molecules in silico scores were selected and screened through basic experiments. After screening, the anti-metastasis effects of selected compounds were tested through transwell migration and invasion assay. Immunofluorescence and qPCR were applied to confirm the expression of vimentin and CDH1. Western blot were used to clarify the inhibition effects of selected compounds on PITPNM3 signaling pathways. Results: By using homology remodeling, we successfully constructed the PITPNM3(680-920aa) protein model. The PITPNM3(680-920aa) domain is responsible for interacting with PYK2 and phosphorylating PYK2. The phosphorylation of PYK2 conducted by PITPNM3 signaling pathway will lead to metastasis and epithelial-mesenchymal transition (EMT) of breast cancer cells. We then characterized the potential binding pockets of this static model and a druggable site was founded. More than 50K molecules with high diversity were docked into this druggable site and scored through their docking performance. Finally, top 100 scored small molecules were selected. In addition, through 1 rounds of toxicity screening, 1 round of transwell migration assay screening and 1 round of transwell invasion assay screening, 4 small molecules with higher bioactivity is identified and 1 compound with the highest bioactivity as well as docking performance among 50K small molecules is chose. This compound can inhibit CCL18 treatment as well as tumor associated macrophage co-culture mediated migration and invasion. Besides, it can also inhibit the phophorylation of PYK2 and Src without inhibition the expression of PITPNM3. Conclusions: Our findings identify the first-in-class anti-PITPNM3 small molecule inhibitors. These compounds can inhibit PITPNM3 signaling pathway and reverse breast cancer metastasis.