The first attempt in synthesis, identification, and evaluation of SEPT9 inhibitors on human oral squamous carcinomas.

Septin 9 (SEPT9), a GTPase, known as the fourth cytoskeleton, is widely expressed in various cells and tissues. The functions of SEPT9 are partly similar to other cytoskeletons as a structure protein. Further, SEPT9 can interact with other cytoskeletons, participating in actin dynamics and microtubule regulation. SEPT9 is associated with various diseases, such as cancers. Thus, it could be a potential drug target. However, there are no small molecule SEPT9 inhibitors and the only reported septin inhibitor, forchlorfenuron, has no effects on SEPT9 inhibition from our study results. Therefore, the derivatives of forchlorfenuron were synthesized, and their activities were evaluated by a direct SEPT9 inhibition screening platform, followed by localized surface plasmon resonance (LSPR) and cell-based assays. The screening results conveyed that 6b, 8a, and 8b are SEPT9 inhibitors with IC50 values of 91, 99, and 95 μM, respectively. Also, their binding affinities were 4, 18, and 22 μM, respectively, validated through LSPR. Eventually, the SAR concludes that at the para position, small substituents are tolerated, while at the ortho position, a bulky benzene ring substituent can be the best candidate. In cell-based assays, the IC50 of 6a, 8a, and 8b of human oral squamous carcinomas cytotoxicity were 122, 20, and 21 µM, respectively. Additionally, significant suppression of the cell migration and invasion ability was observed with the 8b treatment. The co-localization study revealed that 8b effectively disrupted the structural organization of SEPT9, microtubules, and actins. This is the first article to systematically study SEPT9 inhibitors and their biological properties, hoping to shed some light on septin research.