DISCOVERY OF A POTENT AND SELECTIVE RIOK1 INHIBITOR ACTIVE IN CRC CELL LINES USING STRUCTURE-BASED DRUG DESIGN
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Colorectal cancer (CRC) is the second leading cause of cancer-related death in the United State. CRC has aggressive malignancy characterized by poor prognosis and its metastasis and recurrence is the cancer stem cell driven. Homo sapiens RIOK1 (hsRIOK1 also known as RIOK1) is an atypical protein kinase which is an essential ribosome biogenesis factor, and it supports cancer cellular proliferation via its critical role in ribosome biogenesis and through stimulation of RAS signaling. The Inhibition of RIOK1 activity represents an opportunity to target CRC. So far, several RIOK1 inhibitors have been reported but none of them are active in any cancer cell line. Toyocamycin, 7-cyano-7-deazaadenosine, is the first RIOK1 inhibitor reported with high binding affinity of 40nM. The lead molecule toyocamycin had to be optimized because of its high toxicity to normal cells and low selectivity from targeting three other kinases. Based on two X-ray crystal structures of afRio1-toyocamycin complex and RIOK1(143-494)-ADP-Mg2+ complex having pAsp, toyocamycin analogs with different alkyl groups on C5’ position were designed and virtually screened in the RIOK1 crystal structure. Considering their physicochemical property calculations, a series of toyocamycin and adenosine analogs were synthesized and tested to see their binding affinities to the drug target. In the virtual screening, two major interactions were observed in the hit molecules: pi-pi interaction with PHE328 and hydrogen bond interaction with ASP341. TSA binding assay demonstrated the hydrogen bond might be a critical interaction to increase the RIOK1-drug binding. These small molecule inhibitors were tested in CRC cell/stem cell lines to study their inhibition efficacy and showed their potency with nano molar to single digit micro molar IC50 values in the cancer cell inhibition, reporting the first cell-active RIOK1 inhibitors. These compounds were tested alone or in the presence of MTA to see the effect of a combinatory inhibition against RIOK1 and its binding partner PRMT5 activities on CRC tumorigenesis. As a result, the co-targeting two oncogenic enzymes sensitized CRC cell/stem cell responses to the inhibitors and in the treatment of the compounds, RIOK1 and PRMT5 binding was promoted in their pull-down assay.