Open Access

Transcription factors play a crucial role in regulating biological processes such as cell growth, differentiation, organ development and cellular signaling. Within this group, proteins equipped with zinc finger motifs (ZFs) represent the largest family of sequence-specific DNA-binding transcription regulators. Numerous studies have proven the fundamental role of BCL11B for a variety of tissues and organs such as central nervous system, T cells, skin, teeth, and mammary glands. In a previous work we identified a novel atypical zinc finger domain (CCHC-ZF) which serves as a dimerization interface of BCL11B. This domain and formation of the dimer were shown to be critically important for efficient regulation of the BCL11B target genes and could therefore represent a promising target for novel drug therapies. Here, we report the structural basis for BCL11B–BCL11B interaction mediated by the N-terminal ZF domain. By combining structure prediction algorithms, enhanced sampling molecular dynamics and fluorescence resonance energy transfer (FRET) approaches, we identified amino acid residues indispensable for the formation of the single ZF domain and directly involved in forming the dimer interface. These findings not only provide deep insight into how BCL11B acquires its active structure but also represent an important step towards rational design or selection of potential inhibitors.

The potassium channel opening drugs flupirtine and retigabine have been withdrawn from the market due to occasional drug-induced liver injury (DILI) and tissue discoloration, respectively. While the mechanism underlying DILI after prolonged flupirtine use is not entirely understood, evidence indicates that both drugs are metabolized in an initial step to reactive ortho- and/or para-azaquinone diimines or ortho- and/or para-quinone diimines, respectively. Aiming to develop safer alternatives for the treatment of pain and epilepsy, we have attempted to separate activity from toxicity by employing a drug design strategy of avoiding the detrimental oxidation of the central aromatic ring by shifting oxidation toward the formation of benign metabolites. In the present investigation, an alternative retrometabolic design strategy was followed. The nitrogen atom, which could be involved in the formation of both ortho- or para-quinone diimines of the lead structures, was shifted away from the central ring, yielding a substitution pattern with nitrogen substituents in the meta position only. Evaluation of KV7.2/3 opening activity of the 11 new specially designed derivatives revealed surprisingly steep structure–activity relationship data with inactive compounds and an activity cliff that led to the identification of an apparent “magic methyl” effect in the case of N-(4-fluorobenzyl)-6-[(4-fluorobenzyl)amino]-2-methoxy-4-methylnicotinamide. This flupirtine analogue showed potent KV7.2/3 opening activity, being six times as active as flupirtine itself, and by design is devoid of the potential for azaquinone diimine formation.

An in vitro dissolution model, the so-called EyeFlowCell (EFC), was developed to test intravitreal dosage forms, simulating parameters such as the gel-like consistency of the vitreous body. The developed model consists of a stereolithography 3D-printed flow-through cell with a polyacrylamide (PAA) gel as its core. This gel needed to be coated with an agarose sheath because of its low viscosity. Drug release from hydroxypropyl methylcellulose-based implants containing either triamcinolone acetonide or fluorescein sodium was studied in the EFC using a schematic eye movement by the EyeMovementSystem (EyeMoS). For comparison, studies were performed in USP apparatus 4 and USP apparatus 7. Significantly slower drug release was observed in the PAA gel for both model drugs compared with the compendial methods. Drug release from fluorescein sodium-containing model implants was completed after 40 min in USP apparatus 4, whereas drug release in the gel-based EFC lasted 72 h. Drug release from triamcinolone acetonide-containing model implants was completed after 35 min in USP apparatus 4 and after 150 min in USP apparatus 7, whereas this was delayed until 96 h in the EFC. These results suggest that compendial release methods may overestimate the drug release rate in the human vitreous body. Using a gel-based in vitro release system such as the EFC may better predict drug release.