Lipase-Catalyzed Aza-Michael Reaction on Acrylate Derivatives

A methodology has been developed for an efficient and selective lipase-catalyzed aza-Michael reaction of various amines (primary and secondary) with a series of acrylates and alkylacrylates. Reaction parameters were tuned, and under the optimal conditions it was found that <i>Pseudomonas stutzeri</i> lipase and <i>Chromobacterium viscosum</i> lipase showed the highest selectivity for the aza-Michael addition to substituted alkyl acrylates. For the first time also, some CLEAs were examined that showed a comparable or higher selectivity and yield than the free enzymes and other formulations

Identification of the First Highly Subtype-Selective Inhibitor of Human GABA Transporter GAT3

Screening a library of small-molecule compounds using a cell line expressing human GABA transporter 3 (hGAT3) in a [³H]­GABA uptake assay identified isatin derivatives as a new class of hGAT3 inhibitors. A subsequent structure–activity relationship (SAR) study led to the identification of hGAT3-selective inhibitors (i.e., compounds <b>20</b> and <b>34</b>) that were superior to the reference hGAT3 inhibitor, (<i>S</i>)-SNAP-5114, in terms of potency (low micromolar IC₅₀ values) and selectivity (>30-fold selective for hGAT3 over hGAT1/hGAT2/hBGT1). Further pharmacological characterization of compound <b>20</b> (5-(thiophen-2-yl)­indoline-2,3-dione) revealed a noncompetitive mode of inhibition at hGAT3. This suggests that this compound class, which has no structural resemblance to GABA, has a binding site different from the substrate, GABA. This was supported by a molecular modeling study that suggested a unique binding site that matched the observed selectivity, inhibition kinetics, and SAR of the compound series. These compounds are the most potent GAT3 inhibitors reported to date that provide selectivity for GAT3 over other GABA transporter subtypes

Targeting a Subpocket in <i>Trypanosoma brucei</i> Phosphodiesterase B1 (TbrPDEB1) Enables the Structure-Based Discovery of Selective Inhibitors with Trypanocidal Activity

Several trypanosomatid cyclic nucleotide phosphodiesterases (PDEs) possess a unique, parasite-specific cavity near the ligand-binding region that is referred to as the P-pocket. One of these enzymes, <i>Trypanosoma brucei</i> PDE B1 (TbrPDEB1), is considered a drug target for the treatment of African sleeping sickness. Here, we elucidate the molecular determinants of inhibitor binding and reveal that the P-pocket is amenable to directed design. By iterative cycles of design, synthesis, and pharmacological evaluation and by elucidating the structures of inhibitor-bound TbrPDEB1, hPDE4B, and hPDE4D complexes, we have developed 4a,5,8,8a-tetrahydrophthalazinones as the first selective TbrPDEB1 inhibitor series. Two of these, <b>8</b> (NPD-008) and <b>9</b> (NPD-039), were potent (<i>K</i>_i = 100 nM) TbrPDEB1 inhibitors with antitrypanosomal effects (IC₅₀ = 5.5 and 6.7 μM, respectively). Treatment of parasites with <b>8</b> caused an increase in intracellular cyclic adenosine monophosphate (cAMP) levels and severe disruption of <i>T. brucei</i> cellular organization, chemically validating trypanosomal PDEs as therapeutic targets in trypanosomiasis