Hit Recycling: Discovery of a Potent Carbonic Anhydrase Inhibitor by <i>in Silico</i> Target Fishing

<i>In silico</i> target fishing is an emerging tool in drug discovery, which is mostly used for primary target or off-target prediction and drug repositioning. In this work, we developed an <i>in silico</i> target fishing protocol to identify the primary target of GV2–20, a false-positive hit highlighted in a cell-based screen for 14–3–3 modulators. Although GV2–20 does not bind to 14–3–3 proteins, it showed remarkable antiproliferative effects in CML cells, thus raising interest toward the identification of its primary target. Six potential targets of GV2–20 were prioritized <i>in silico</i> and tested <i>in vitro</i>. Our results show that the molecule is a potent inhibitor of carbonic anhydrase 2 (CA2), thus confirming the predictive capability of our protocol. Most notably, GV2–20 experienced a remarkable selectivity for CA2, CA7, CA9, and CA12, and its scaffold was never explored before as a chemotype for CA inhibition, thus becoming an interesting lead candidate for further development

Design, Synthesis, and Biological Evaluation of Pyrazolo[3,4‑<i>d</i>]pyrimidines Active in Vivo on the Bcr-Abl T315I Mutant

Starting from our in-house library of pyrazolo­[3,4-<i>d</i>]­pyrimidines, a cross-docking simulation was conducted on Bcr-Abl T315I mutant. Among the selected compounds (<b>2a</b>–<b>e</b>), the 4-bromo derivative <b>2b</b> showed the best activity against the Bcr-Abl T315I mutant. Deeper computational studies highlighted the importance of the bromine atom in the para position of the N1 side chain phenyl ring for the interaction with the T315I mutant. A series of 4-bromo derivatives was thus synthesized and biologically evaluated. Compound <b>2j</b> showed a good balance of different ADME properties, high activity in cell-free assays, and a submicromolar potency against T315I Bcr-Abl expressing cells. In addition, it was converted into a water-soluble formulation by liposome encapsulation, preserving a good activity on leukemic T315I cells and avoiding the use of DMSO as solubilizing agent. In vivo studies on mice inoculated with 32D-T315I cells and treated with <b>2j</b> showed a more than 50% reduction in tumor volumes