Discovery of the 4-aminopiperidine-based compound EM127 for the site-specific covalent inhibition of SMYD3.

Recent findings support the hypothesis that inhibition of SMYD3 methyltransferase may be a therapeutic avenue for some of the deadliest cancer types. Herein, active site-selective covalent SMYD3 inhibitors were designed by introducing an appropriate reactive cysteine trap into reversible first-generation SMYD3 inhibitors. The 4-aminopiperidine derivative EM127 (11C) bearing a 2-chloroethanoyl group as reactive warhead showed selectivity for Cys186, located in the substrate/histone binding pocket. Selectivity towards Cys186 was retained even at high inhibitor/enzyme ratio, as shown by mass spectrometry. The mode of interaction with the SMYD3 substrate/histone binding pocket was revealed by crystallographic studies. In enzymatic assays, 11C showed a stronger SMYD3 inhibitory effect compared to the reference inhibitor EPZ031686. Remarkably, 11C attenuated the proliferation of MDA-MB-231 breast cancer cell line at the same low micromolar range of concentrations that reduced SMYD3 mediated ERK signaling in HCT116 colorectal cancer and MDA-MB-231 breast cancer cells. Furthermore, 11C (5 µM) strongly decreased the steady-state mRNA levels of genes important for tumor biology such as cyclin dependent kinase 2, c-MET, N-cadherin and fibronectin 1, all known to be regulated, at least in part, by SMYD3. Thus, 11C is as a first example of second generation SMYD3 inhibitors; this agent represents a covalent and a site specific SMYD3 binder capable of potent and prolonged attenuation of methyltransferase activity.

Discovery of an Allosteric Ligand Binding Site in SMYD3 Lysine Methyltransferase.

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 (KD =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 (KD =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.

Targeting SMYD3 to Sensitize Homologous Recombination-Proficient Tumors to PARP-Mediated Synthetic Lethality.

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.

Unveiling the Biochemistry of the Epigenetic Regulator 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_K260)-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.

Small-molecule inhibitors of lysine methyltransferases SMYD2 and SMYD3: current trends.

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.

Combination of human acetylcholinesterase and serum albumin sensing surfaces as highly informative analytical tool for inhibitor screening.

In the continuous research for potential drug lead candidates, the availability of highly informative screening methodologies may constitute a decisive element in the selection of best-in-class compounds. In the present study, a surface plasmon resonance (SPR)-based assay was developed and employed to investigate interactions between human recombinant AChE (hAChE) and four known ligands: galantamine, tacrine, donepezil and edrophonium. To this aim, a sensor chip was functionalized with hAChE using mild immobilization conditions to best preserve enzyme integrity. Binding affinities and, for the first time, kinetic rate constants for all drug-hAChE complexes formation/disruption were determined. Inhibitors were classified in two groups: slow-reversible and fast-reversible binders according to respective target residence time. Combining data obtained on drug-target residence time with data obtained on serum albumin binding levels, a good correlation with potency, plasma protein binding in vivo, and administration regimen was found. The outcomes of this work demonstrated that the developed SPR-based assay is suitable for the screening, the binding affinity ranking and the kinetic evaluation of hAChE inhibitors. The method proposed ensures a simpler and cost-effective assay to quantify kinetic rate constants for inhibitor-hAChE interaction as compared with other proposed and published methods. Eventually, the determination of residence time in combination with preliminary ADME studies might constitute a better tool to predict in vivo behaviour, a key information for the research of new potential drug candidates.

Monitoring drug-serum protein interactions for early ADME prediction through Surface Plasmon Resonance technology.

Many molecules fail to reach the market due to poor pharmacokinetic (PK) properties, rendering the potential drug virtually unavailable for the primary target despite efficient administration to the body. PK properties of endogenous and exogenous compounds in mammals are dependent, among other factors, on their ability to interact with serum proteins. The extent of binding can greatly influence their ADME (adsorption, distribution, metabolism and execration) profile. Reliable and cost-effective bioavailability studies, early in the drug discovery process, can lead to an improvement of the success rate for compounds entering clinical trials. Optical biosensors based on surface plasmon resonance (SPR) detection emerged as an efficient approach to obtain large amounts of information about the binding of small molecules to serum proteins. Simple, automated and fast assays provide a good throughput, versatility and highly informative data output, rendering the methodology particularly suited for early screening. The ability to provide basic information on PK can be easily coupled to structure-activity relationship analysis. In this review, features of the technology and its employment for the study of serum protein-small molecule interactions are presented and discussed.

Surface plasmon resonance and isothermal titration calorimetry to monitor the Ni(II)-dependent binding of Helicobacter pylori NikR to DNA.

NikR is a transcription factor that regulates the expression of Ni(II)-dependent enzymes and other proteins involved in nickel trafficking. In the human pathogenic bacterium Helicobacter pylori, NikR (HpNikR) controls, among others, the expression of the Ni(II) enzyme urease by binding the double-strand DNA (dsDNA) operator region of the urease promoter (OP ureA ) in a Ni(II)-dependent mode. This article describes the complementary use of surface plasmon resonance (SPR) spectroscopy and isothermal titration calorimetry (ITC) to carry out a mechanistic characterization of the HpNikR-OP ureA interaction. An active surface was prepared by affinity capture of OP ureA and validated for the recognition process in the SPR experiments. Subsequently, the Ni(II)-dependent affinity of the transcription factor for its operator region was assessed through kinetic evaluation of the binding process at variable Ni(II) concentrations. The kinetic data are consistent with a two-step binding mode involving an initial encounter between the two interactants, followed by a conformational rearrangement of the HpNikR-OP ureA complex, leading to high affinity binding. This conformational change is only observed in the presence of the full set of four Ni(II) ions bound to the protein. The SPR assay developed and validated in this study constitutes a suitable method to screen potential drug lead candidates acting as inhibitors of this protein-dsDNA interaction. Graphical Abstract Pictorial representation of the interaction between HpNikR, flowing in solution, and the OP ureA urease promoter immobilized on the sensor chip surface.

Surface plasmon resonance and circular dichroism characterization of cucurbitacins binding to serum albumins for early pharmacokinetic profiling.

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 toward 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.

Stopped-Flow Enantioselective HPLC-CD Analysis and TD-DFT Stereochemical Characterization of Methyl Trans-3-(3,4-Dimethoxyphenyl)Glycidate.

Caffeic acid-derived polyethers are a class of natural products isolated from the root extracts of comfrey and bugloss, which are endowed with intriguing pharmacological properties as anticancer agents. The synthesis of new polyether derivatives is achieved through ring-opening polymerization of chiral 2,3-disubstituted oxiranes, whose absolute configurations define the overall stereochemistry of the produced polymer. The absolute stereochemistry of one of these building blocks, methyl trans-3-(3,4-dimethoxy-phenyl)glycidate (3), was therefore characterized by the combination of enantioselective high-performance liquid chromatography (HPLC), electronic circular dichroism (ECD) spectroscopy, and time-dependent density functional theory (TD-DFT) calculations. Initial efforts aiming at the isolation of enantiomers by means of a standard preparative HPLC protocol followed by offline ECD analysis failed due to unexpected degradation of the samples after collection. The stopped-flow HPLC-CD approach, by which the ECD spectra of enantiomers are measured online with the HPLC system, was applied to overcome this issue and allowed a fast, reliable, and chemical-saving analysis, while avoiding the risks of sample degradation during the collection and processing of enantiomeric fractions. Subsequent TD-DFT calculations identified ( as the first eluted enantiomeric fraction on the Lux Cellulose-2 column, therefore achieving a full stereochemical characterization of the chiral oxirane under investigation.

A SMYD3 Small-Molecule Inhibitor Impairing Cancer Cell Growth.

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.

Determination of levamisole and tetramisole in seized cocaine samples by enantioselective high-performance liquid chromatography and circular dichroism detection.

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 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.