LLY-507

LLY-507 potently and selectively inhibits the protein methyltransferase SMYD2 (IC50 <15 nM in an enzyme assay) compared with other pharmacologically important proteins including other HMTs, protein kinases, GPCRs and NHRs. At microM concentrations, the compound can be used in cellular assays (IC50=0.6 uM in U2OS cells for p53 methylation). However, it is preferable to simultaneously use other chemical probes or knock-down technologies for validations studies, because bona fide physiological substrates and functions of SMYD2 are still unknown and enigmatic.

LLY-507 was disclosed as a potent and selective small molecule inhibitor of SMYD2 with cellular target engagement by inhibition of SYMD2 mediated monomethylation of p53 K370. LLY-507 inhibits the proliferation of ESCC, HCC and breast cancer cell lines. LLY-507 was initially shown to be selective (>100-fold) over a panel of representative methyltransferases, kinases, GPCRs and NHRs. However, recent additional screening at the NIMH Psychoactive Drug Screening program has shown LLY-507 to have potent inhibition of several targets (14 targets with Ki <1 uM and 3 targets with Ki <100 nM). These off-target activities of LLY-507 could confound results in cell assays by showing a response not related to inhibition of SMYD2 and potentially have unwanted psychoactive effects in animal models. Hence, it is recommended that an alternative SMYD2 chemical probe is used, such as A-893, AZ-505, or BAY-598; ideally use all three in parallel to ensure a consistent outcome to confidently link the response to inhibition of SMYD2 activity.

LLY-507 is first-in-class inhibitor of SMYD2, where the biology and mechanism of action are poorly understood. It is quite potent (IC50 = 15 nM) in the the scintillation proximity assay; however, in the protein substrate assay, the IC50 = 600 nM. LLY-507 has a high degree of selectivity when screened against other targets like SMYD3 and SUVH420H (other protein or DNA methyltransferases). The inhibitor gives the same phenotype that was seen in the SMYD2 knockdown model. So far, the mechanism of action has been linked to the inhibition of SMYD2-mediated methylation of p53 K370 in cells. A co-crystal structure of LLY-507 and SMYD2 was obtained, and the structure was solved with a resolution near 1.63 angstroms. The structure will prove useful for designing the next generation of inhibitors. Very brief information was given about the stability of LLY-507, indicating stability after storage at -20 C for 2 years. No pharmacokinetic data were presented for animal exposure. The synthetic pathway to this molecule is thoroughly described in the reference with a path forward and few synthesis issues.

LLY-507 shows good potency and specificity for SMYD2 compared with other protein methyltransferases as well as GPCRs and protein kinases in assays.