Mycobacterium tuberculosis l,d‐transpeptidases (Ldts), which are involved in cell‐wall biosynthesis, have emerged as promising targets for the treatment of tuberculosis. However, an efficient method for testing inhibition of these enzymes is not currently available. We present a fluorescence‐based assay for Ldt_Mt2, which is suitable for high‐throughput screening. Two fluorogenic probes were identified that release a fluorophore upon reaction with Ldt_Mt2, thus making it possible to assess the availability of the catalytic site in the presence of inhibitors. The assay was applied to a panel of β‐lactam antibiotics and related inhibitors; the results validate observations that the (carba)penem subclass of β‐lactams are more potent Ldt inhibitors than other β‐lactam classes, though unexpected variations in potency were observed. The method will enable systematic structure–activity relationship studies on Ldts, thereby facilitating the identification of new antibiotics active against M. tuberculosis.

Metallo-β-lactamases (MBLs) enable bacterial resistance to almost all classes of β-lactam antibiotics. We report studies on enethiol containing MBL inhibitors, which were prepared by rhodanine hydrolysis. The enethiols inhibit MBLs from different subclasses. Crystallographic analyses reveal that the enethiol sulphur displaces the di-Zn(II) ion bridging 'hydrolytic' water. In some, but not all, cases biophysical analyses provide evidence that rhodanine/enethiol inhibition involves formation of a ternary MBL enethiol rhodanine complex. The results demonstrate how low molecular weight active site Zn(II) chelating compounds can inhibit a range of clinically relevant MBLs and provide additional evidence for the potential of rhodanines to be hydrolysed to potent inhibitors of MBL protein fold and, maybe, other metallo-enzymes, perhaps contributing to the complex biological effects of rhodanines. The results imply that any medicinal chemistry studies employing rhodanines (and related scaffolds) as inhibitors should as a matter of course include testing of their hydrolysis products.

Histone methyltransferase DOT1L catalyzes mono-, di- and trimethylation of histone 3 at lysine residue 79 (H3K79) and hypermethylation of H3K79 has been linked to the development of acute leukemias characterized by the MLL (mixed-lineage leukemia) rearrangements (MLLr cells). The inhibition of H3K79 methylation inhibits MLLr cells proliferation, and an inhibitor specific for DOT1L, pinometostat, was in clinical trials (Phase Ib/II). However, the compound showed poor pharmacological properties. Thus, there is a need to find new potent inhibitors of DOT1L for the treatment of rearranged leukemias. Here we present the design, synthesis, and biological evaluation of a small molecule that inhibits in the nM level the enzymatic activity of hDOT1L, H3K79 methylation in MLLr cells with comparable potency to pinometostat, associated with improved metabolic stability and a characteristic cytostatic effect.

Herein, we disclose the first regio‐, site‐ and chemoselective late‐stage (hetero)aryl C−H bond cyclopropenylation with cyclopropenium cations (CPCs). The process is fast, operationally simple and shows an excellent functional group tolerance in densely‐functionalized drug molecules, natural products, agrochemicals and fluorescent dyes. Moreover, we discovered that the installation of the cyclopropene ring in drug molecules could not only be used to shield against metabolic instability but also as a synthetic tool to reach medicinally‐relevant sp³‐rich scaffolds exploiting the highly‐strained nature of the cyclopropene ring with known transformations.