Identification of small-molecule binding sites of a ubiquitin-conjugating enzyme-UBE2T through fragment-based screening.

UBE2T is an attractive target for drug development due to its linkage with several types of cancers. However, the druggability of ubiquitin-conjugating E2 (UBE2T) is low because of the lack of a deep and hydrophobic pocket capable of forming strong binding interactions with drug-like small molecules. Here, we performed fragment screening using 19 F-nuclear magnetic resonance (NMR) and validated the hits with 1 H-15 N-heteronuclear single quantum coherence (HSQC) experiment and X-ray crystallographic studies. The cocrystal structures obtained revealed the binding modes of the hit fragments and allowed for the characterization of the fragment-binding sites. Further screening of structural analogues resulted in the identification of a compound series with inhibitory effect on UBE2T activity. Our current study has identified two new binding pockets in UBE2T, which will be useful for the development of small molecules to regulate the function of this protein. In addition, the compounds identified in this study can serve as chemical starting points for the development of UBE2T modulators.

Identification and characterization of inhibitors covalently modifying catalytic cysteine of UBE2T and blocking ubiquitin transfer.

UBE2T is an E2 ubiquitin ligase critical for ubiquitination of substrate and plays important roles in many diseases. Despite the important function, UBE2T is considered as an undruggable target due to lack of a pocket for binding to small molecules with satisfied properties for clinical applications. To develop potent and specific UBE2T inhibitors, we adopted a high-throughput screening assay and two compounds-ETC-6152 and ETC-9004 containing a sulfone tetrazole scaffold were identified. Solution NMR study demonstrated the direct interactions between UBE2T and compounds in solution. Further co-crystal structures reveal the binding modes of these compounds. Both compound hydrolysation and formation of a hydrogen bond with the thiol group of the catalytic cysteine were observed. The formation of covalent complex was confirmed with mass spectrometry. As these two compounds inhibit ubiquitin transfer, our study provides a strategy to develop potent inhibitors of UBE2T.

Tethered primary hepatocyte spheroids on polystyrene multi-well plates for high-throughput drug safety testing.

Hepatocyte spheroids are useful models for mimicking liver phenotypes in vitro because of their three-dimensionality. However, the lack of a biomaterial platform which allows the facile manipulation of spheroid cultures on a large scale severely limits their application in automated high-throughput drug safety testing. In addition, there is not yet a robust way of controlling spheroid size, homogeneity and integrity during extended culture. This work addresses these bottlenecks to the automation of hepatocyte spheroid culture by tethering 3D hepatocyte spheroids directly onto surface-modified polystyrene (PS) multi-well plates. However, polystyrene surfaces are inert toward functionalization, and this makes the uniform conjugation of bioactive ligands very challenging. Surface modification of polystyrene well plates is achieved herein using a three-step sequence, resulting in a homogeneous distribution of bioactive RGD and galactose ligands required for spheroid tethering and formation. Importantly, treatment of polystyrene tethered spheroids with vehicle and paradigm hepatotoxicant (chlorpromazine) treatment using an automated liquid handling platform shows low signal deviation, intact 3D spheroidal morphology and Z' values above 0.5, and hence confirming their amenability to high-throughput automation. Functional analyses performance (i.e. urea and albumin production, cytochrome P450 activity and induction studies) of the polystyrene tethered spheroids reveal significant improvements over hepatocytes cultured as collagen monolayers. This is the first demonstration of automated hepatotoxicant treatment on functional 3D hepatocyte spheroids tethered directly on polystyrene multi-well plates, and will serve as an important advancement in the application of 3D tethered spheroid models to high throughput drug screening.

Correction to "Discovery of Irreversible Inhibitors Targeting Histone Methyltransferase, SMYD3".

[This corrects the article DOI: 10.1021/acsmedchemlett.9b00170.].

Discovery of Irreversible Inhibitors Targeting Histone Methyltransferase, SMYD3.

SMYD3 is a histone methyltransferase that regulates gene transcription, and its overexpression is associated with multiple human cancers. A novel class of tetrahydroacridine compounds which inhibit SMYD3 through a covalent mechanism of action is identified. Optimization of these irreversible inhibitors resulted in the discovery of 4-chloroquinolines, a new class of covalent warheads. Tool compound 29 exhibits high potency by inhibiting SMYD3's enzymatic activity and showing antiproliferative activity against HepG2 in 3D cell culture. Our findings suggest that covalent inhibition of SMYD3 may have an impact on SMYD3 biology by affecting expression levels, and this warrants further exploration.

Target mechanism-based whole-cell screening identifies bortezomib as an inhibitor of caseinolytic protease in mycobacteria.

UNLABELLED: A novel type of antibacterial screening method, a target mechanism-based whole-cell screening method, was developed to combine the advantages of target mechanism- and whole-cell-based approaches. A mycobacterial reporter strain with a synthetic phenotype for caseinolytic protease (ClpP1P2) activity was engineered, allowing the detection of inhibitors of this enzyme inside intact bacilli. A high-throughput screening method identified bortezomib, a human 26S proteasome drug, as a potent inhibitor of ClpP1P2 activity and bacterial growth. A battery of secondary assays was employed to demonstrate that bortezomib indeed exerts its antimicrobial activity via inhibition of ClpP1P2: Down- or upmodulation of the intracellular protease level resulted in hyper- or hyposensitivity of the bacteria, the drug showed specific potentiation of translation error-inducing aminoglycosides, ClpP1P2-specific substrate WhiB1 accumulated upon exposure, and growth inhibition potencies of bortezomib derivatives correlated with ClpP1P2 inhibition potencies. Furthermore, molecular modeling showed that the drug can bind to the catalytic sites of ClpP1P2. This work demonstrates the feasibility of target mechanism-based whole-cell screening, provides chemical validation of ClpP1P2 as a target, and identifies a drug in clinical use as a new lead compound for tuberculosis therapy. IMPORTANCE: During the last decade, antibacterial drug discovery relied on biochemical assays, rather than whole-cell approaches, to identify molecules that interact with purified target proteins derived by genomics. This approach failed to deliver antibacterial compounds with whole-cell activity, either because of cell permeability issues that medicinal chemistry cannot easily fix or because genomic data of essentiality insufficiently predicted the vulnerability of the target identified. As a consequence, the field largely moved back to a whole-cell approach whose main limitation is its black-box nature, i.e., that it requires trial-and-error chemistry because the cellular target is unknown. We developed a novel type of antibacterial screening method, target mechanism-based whole-cell screening, to combine the advantages of both approaches. We engineered a mycobacterial reporter strain with a synthetic phenotype allowing us to identify inhibitors of the caseinolytic protease (ClpP1P2) inside the cell. This approach identified bortezomib, an anticancer drug, as a specific inhibitor of ClpP1P2. We further confirmed the specific "on-target" activity of bortezomib by independent approaches including, but not limited to, genetic manipulation of the target level (over- and underexpressing strains) and by establishing a dynamic structure-activity relationship between ClpP1P2 and growth inhibition. Identifying an "on-target" compound is critical to optimize the efficacy of the compound without compromising its specificity. This work demonstrates the feasibility of target mechanism-based whole-cell screening methods, validates ClpP1P2 as a druggable target, and delivers a lead compound for tuberculosis therapy.

Establishment of a novel whole animal HTS technology platform for melioidosis drug discovery.

Melioidosis is a serious emerging endemic infectious disease caused by Burkholderia pseudomallei, a gram-negative pathogen. Septicemic melioidosis has a mortality rate of 50% even with treatment. Like other gram-negative bacteria, B. pseudomallei is resistant to a number of antibiotics and multi-drug resistant B. pseudomallei is beginning to be encountered in hospitals. There is a clear medical need to develop new treatment options to manage this disease. We used Burkholderia thailandensis (a BSL-2 class organism) to infect Caenorhabditis elegans and set up a surrogate whole animal infection model of melioidosis that we could run in a 384 microtitre plate and establish a whole animal HTS assay. We have optimized and validated this assay in a fluorescence-based format that can be run on our automated screening platforms. This assay has now been used to screen over 300,000 compounds from our small molecule library and we are in the process of characterizing the hits obtained and select compounds for further studies. We have thus established a biologically relevant assay technology platform to screen for antibacterial compounds and used this platform to identify new compounds that may find application in treating melioidosis infections.