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.

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

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

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.

Interaction of antimicrobial peptides from Australian amphibians with lipid membranes.

Solid-state NMR and CD spectroscopy were used to study the effect of antimicrobial peptides (aurein 1.2, citropin 1.1, maculatin 1.1 and caerin 1.1) from Australian tree frogs on phospholipid membranes. 31P NMR results revealed some effect on the phospholipid headgroups when the peptides interact with DMPC/DHPC (dimyristoylphosphatidylcholine/dihexanoylphosphatidylcholine) bicelles and aligned DMPC multilayers. 2H NMR showed a small effect of the peptides on the acyl chains of DMPC in bicelles or aligned multilayers, suggesting interaction with the membrane surface for the shorter peptides and partial insertion for the longer peptides. 15N NMR of selectively labelled peptides in aligned membranes and oriented CD spectra indicated an alpha-helical conformation with helix long axis approximately 50 degrees to the bilayer surface at high peptide concentrations. The peptides did not appear to insert deeply into PC membranes, which may explain why these positively charged peptides preferentially lyse bacterial rather than eucaryotic cells.

Cooperative binding interactions of glycopeptide antibiotics.

Glycopeptide antibiotics of the vancomycin group bind to bacterial cell wall analogue precursors, and typically also form dimers. We have studied the interplay between these two sets of noncovalent bonds formed at separate interfaces. Indole-2-carboxylic acid (L) forms a set of hydrogen bonds to the glycopeptide antibiotic chloroeremomycin (CE) that are analogous to those formed by N-Ac-D-Ala. The ligand/CE dimer interactions (in L/CE/CE/L) are shown to occur with positive cooperativity and structural tightening at the dimer interface. From theoretical considerations and from other data, it is inferred, but not proven, that in the exercise of positive cooperativity, the interface that will be tightened to the greatest degree is the one that lies in the shallowest free energy well.