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SMYD3 is a multifunctional epigenetic enzyme with lysine methyltransferase activity and various interaction partners. It is implicated in the pathophysiology of cancers but with an unclear mechanism. To discover tool compounds for clarifying its biochemistry and potential as a therapeutic target, a set of drug‐like compounds was screened in a biosensor‐based competition assay. Diperodon was identified as an allosteric ligand; its R and S enantiomers were isolated, and their affinities to SMYD3 were determined (K D=42 and 84 μM, respectively). Co‐crystallization revealed that both enantiomers bind to a previously unidentified allosteric site in the C‐terminal protein binding domain, consistent with its weak inhibitory effect. No competition between diperodon and HSP90 (a known SMYD3 interaction partner) was observed although SMYD3–HSP90 binding was confirmed (K D=13 μM). Diperodon clearly represents a novel starting point for the design of tool compounds interacting with a druggable allosteric site, suitable for the exploration of noncatalytic SMYD3 functions and therapeutics with new mechanisms of action., From a distance: SMYD3 lysine methyltransferase is an epigenetic enzyme with multiple cellular functions and the ability to recognize broad range of substrates from histones to cytosolic proteins. An SPR biosensors‐based biophysical strategy for screening SMYD3 ligands was developed and revealed an allosteric binding site. Subsequent crystallographic studies provided a structural description of the novel binding site of SMYD3.

SET and MYND domain-containing protein 3 (SMYD3) is a lysine methyltransferase that plays a central role in a variety of cancer diseases, exerting its pro-oncogenic activity by methylation of key proteins, of both nuclear and cytoplasmic nature. However, the role of SMYD3 in the initiation and progression of cancer is not yet fully understood and further biochemical characterization is required to support the discovery of therapeutics targeting this enzyme. We have therefore developed robust protocols for production, handling, and crystallization of SMYD3 and biophysical and biochemical assays for clarification of SMYD3 biochemistry and identification of useful lead compounds. Specifically, a time-resolved biosensor assay was developed for kinetic characterization of SMYD3 interactions. Functional differences in SMYD3 interactions with its natural small molecule ligands SAM and SAH were revealed, with SAM forming a very stable complex. A variety of peptides mimicking putative substrates of SMYD3 were explored in order to expose structural features important for recognition. The interaction between SMYD3 and some peptides was influenced by SAM. A nonradioactive SMYD3 activity assay using liquid chromatography-mass spectrometry (LC-MS) analysis explored substrate features of importance also for methylation. Methylation was notable only toward MAP kinase kinase kinase 2 (MAP3K2_K-260)-mimicking peptides, although binary and tertiary complexes were detected also with other peptides. The analysis supported a random bi-bi mechanistic model for SMYD3 methyltransferase catalysis. Our work unveiled complexities in SMYD3 biochemistry and resulted in procedures suitable for further studies and identification of novel starting points for design of effective and specific leads for this potential oncology target.

Lysine methyltransferases SMYD2 and SMYD3 are involved in the epigenetic regulation of cell differentiation and functioning. Overexpression and deregulation of these enzymes have been correlated to the insurgence and progression of different tumors, making them promising molecular targets in cancer therapy even if their role in tumors is not yet fully understood. In this light, selective small-molecule inhibitors are required to fully understand and validate these enzymes, as this is a prerequisite for the development of successful targeted therapeutic strategies. The present review gives a systematic overview of the chemical probes developed to selectively target SMYD2 and SMYD3, with particular focus on the structural features important for high inhibitory activity, on the mode of inhibition and on the efficacy in cell-based and in in vivo models.

Summary SMYD3 is frequently overexpressed in a wide variety of cancers. Indeed, its inactivation reduces tumor growth in preclinical in vivo animal models. However, extensive characterization in vitro failed to clarify SMYD3 function in cancer cells, although confirming its importance in carcinogenesis. Taking advantage of a SMYD3 mutant variant identified in a high-risk breast cancer family, here we show that SMYD3 phosphorylation by ATM enables the formation of a multiprotein complex including ATM, SMYD3, CHK2, and BRCA2, which is required for the final loading of RAD51 at DNA double-strand break sites and completion of homologous recombination (HR). Remarkably, SMYD3 pharmacological inhibition sensitizes HR-proficient cancer cells to PARP inhibitors, thereby extending the potential of the synthetic lethality approach in human tumors., Graphical Abstract, Highlights • SMYD3 phosphorylation by ATM favors the formation of HR complexes during DSB response • SMYD3 mediates DSB repair by promoting RAD51 recruitment at DNA damage sites • SMYD3 inhibition triggers a compensatory PARP-dependent DNA damage response • Co-targeting SMYD3/PARP leads to synthetic lethality in HR-proficient cancer cells, Molecular Biology; Cell Biology; Cancer

SMYD3 is a histone lysine methyltransferase that plays an important role in transcriptional activation as a member of an RNA polymerase complex, and its oncogenic role has been described in different cancer types. We studied the expression and activity of SMYD3 in a preclinical model of colorectal cancer (CRC) and found that it is strongly upregulated throughout tumorigenesis both at the mRNA and protein level. Our results also showed that RNAi-mediated SMYD3 ablation impairs CRC cell proliferation indicating that SMYD3 is required for proper cancer cell growth. These data, together with the importance of lysine methyltransferases as a target for drug discovery, prompted us to carry out a virtual screening to identify new SMYD3 inhibitors by testing several candidate small molecules. Here we report that one of these compounds (BCI-121) induces a significant reduction in SMYD3 activity both in vitro and in CRC cells, as suggested by the analysis of global H3K4me2/3 and H4K5me levels. Of note, the extent of cell growth inhibition by BCI-121 was similar to that observed upon SMYD3 genetic ablation. Most of the results described above were obtained in CRC; however, when we extended our observations to tumor cell lines of different origin, we found that SMYD3 inhibitors are also effective in other cancer types, such as lung, pancreatic, prostate, and ovarian. These results represent the proof of principle that SMYD3 is a druggable target and suggest that new compounds capable of inhibiting its activity may prove useful as novel therapeutic agents in cancer treatment.

Accepted Manuscript version. The Published Journal Article is available on theJournal of ChromatographyA, Volume 1363, pages 150-154 (DOI: https://doi.org/10.1016/j.chroma.2014.07.069). SupplementaryMaterial available free of charge on the article webpage. © 2014. This Manuscript version is made available under the CC-BY-NC-ND 4.0 license.https://creativecommons.org/licenses/by-nc-nd/4.0/ ABSTRACT Levamisole, an anthelmintic drug, has been increasingly employed as an adulterant of illicit street cocaine over the last decade; recently, the use of tetramisole, the racemic mixture of levamisole and its enantiomer dexamisole, was also occasionally observed. A new enantioselective high-performance liquid chromatography (HPLC) method, performed on cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phases in normal-phase mode, was validated and applied to determine the enantiomeric composition of tetramisole enantiomers in seized cocaine samples. Furthermore, the hyphenation of the validated HPLC method with a circular dichroism (CD) detection system allowed the direct determination of elution order and a selective monitoring of levamisole and dexamisole in the presence of possible interferences. The method was applied to the identification and quantitation of the two enantiomers of tetramisole in seized street cocaine samples.

Accepted Manuscript version. The Published Journal Article is available on the Journal of Pharmaceutical and Biomedical Analysis, Volume 122, pages 166–172 (DOI: https://doi.org/10.1016/j.jpba.2016.01.051). Supplementary Material available free of charge on the article webpage. © 2016. This Manuscript version is made available under the CC-BY-NC-ND 4.0 license. https://creativecommons.org/licenses/by-nc-nd/4.0/ ABSTRACT Cucurbitacins are a group of tetracyclic triterpenoids, known for centuries for their anti-cancer and anti-inflammatory properties, which are being actively investigated over the past decades in order to elucidate their mechanism of action. In perspective of being used as therapeutic molecules, a pharmacokinetic characterization is crucial to assess the affinity towards blood carrier proteins and extrapolate distribution volumes. Usually, pharmacokinetic data are first collected on animal models and later translated to humans; therefore, an early characterization of the interaction with carrier proteins from different species is highly desirable. In the present study, the interactions of cucurbitacins E and I with human and rat serum albumins (HSA and RSA) were investigated by means of surface plasmon resonance (SPR)-based optical biosensing and circular dichroism (CD) spectroscopy. Active HSA and RSA sensor chip surfaces were prepared through an amine coupling reaction protocol, and the equilibrium dissociation constants (Kd) for the different cucurbitacins-serum albumins complexes were then determined by SPR analysis. Further information on the binding of cucurbitacins to serum albumins was obtained by CD competition experiments with biliverdin, a specific marker binding to subdomain IB of HSA. SPR data unveiled a previously unreported binding event between CucI and HSA; the determined binding affinities of both compounds were slightly higher for RSA with respect to HSA, even though all the compounds can be ranked as high-affinity binders for both carriers. CD analysis showed that the two cucurbitacins modify the binding of biliverdin to serum albumins through opposite allosteric modulation (positive for HSA, negative for RSA), confirming the need for caution in the translation of pharmacokinetic data across species.

SMYD3 is a histone lysine methyltransferase that plays an important role in transcriptional activation as a member of an RNA polymerase complex, and its oncogenic role has been described in different cancer types. We studied the expression and activity of SMYD3 in a preclinical model of colorectal cancer (CRC) and found that it is strongly upregulated throughout tumorigenesis both at the mRNA and protein level. Our results also showed that RNAi-mediated SMYD3 ablation impairs CRC cell proliferation indicating that SMYD3 is required for proper cancer cell growth. These data, together with the importance of lysine methyltransferases as a target for drug discovery, prompted us to carry out a virtual screening to identify new SMYD3 inhibitors by testing several candidate small molecules. Here we report that one of these compounds (BCI-121) induces a significant reduction in SMYD3 activity both in vitro and in CRC cells, as suggested by the analysis of global H3K4me2/3 and H4K5me levels. Of note, the extent of cell growth inhibition by BCI-121 was similar to that observed upon SMYD3 genetic ablation. Most of the results described above were obtained in CRC; however, when we extended our observations to tumor cell lines of different origin, we found that SMYD3 inhibitors are also effective in other cancer types, such as lung, pancreatic, prostate, and ovarian. These results represent the proof of principle that SMYD3 is a druggable target and suggest that new compounds capable of inhibiting its activity may prove useful as novel therapeutic agents in cancer treatment.