ABSTRACT
Side chain isotope labelling is a powerful tool to study protein structure and interactions by NMR spectroscopy. 1H,13C labelling of side-chain methyl groups in a deuterated background allows studying large molecules, while side-chain aromatic groups are highly sensitive to the interaction with ligands, drugs, and other proteins. In E. coli, side chain labelling is performed by substituting amino acids with isotope-labelled precursors. However, proteins that can only be produced in mammalian cells require expensive isotope-labelled amino acids. Here we provide a simple and cost-effective method to label side chains in mammalian cells, which exploits the reversible reaction catalyzed by endogenous transaminases to convert isotope-labelled α-ketoacid precursors. We show by in-cell and in-lysate NMR spectroscopy that replacing an amino acid in the medium with its cognate precursor is sufficient to achieve selective labelling without scrambling, and how this approach allows monitoring conformational changes such as those arising from ligand binding.
ABSTRACT
Recently we have developed novel oxotriazinoindole inhibitors (OTIs) of aldose reductase (ALR2), characterized by high efficacy and selectivity. Herein we describe novel OTI derivatives design of which is based on implementation of additional intermolecular interactions within an unoccupied pocket of the ALR2 enzyme. Four novel derivatives, OTI-(7-10), of the previously developed N-benzyl(oxotriazinoindole) inhibitor OTI-6 were synthetized and screened. All of them revealed 2 to 6 times higher ALR2 inhibitory efficacy when compared to their non-substituted lead compound OTI-6. Moreover, the most efficient ALR2 inhibitor OTI-7 (IC50 = 76 nM) possesses remarkably high inhibition selectivity (SF ≥ 1300) in relation to structurally related aldehyde reductase (ALR1). Derivatives OTI-(8-10) bearing the substituents -CONH2, -COOH and -CH2OH, possess 2-3 times lower inhibitory efficacy compared to OTI-7, but better than the reference inhibitor OTI-6. Desolvation penalty is suggested as a possible factor responsible for the drop in ALR2 inhibitory efficacy observed for derivatives OTI-(8-10) in comparison to OTI-7.
Subject(s)
Aldehyde Reductase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Indoles/pharmacology , Triazines/pharmacology , Aldehyde Reductase/metabolism , Animals , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Indoles/chemical synthesis , Indoles/chemistry , Lens, Crystalline/enzymology , Male , Molecular Docking Simulation , Molecular Structure , Rats , Rats, Wistar , Structure-Activity Relationship , Triazines/chemical synthesis , Triazines/chemistryABSTRACT
Inhibition of aldose reductase (AR), the first enzyme of the polyol pathway, is a promising approach in treatment of diabetic complications. We proceeded with optimization of the thioxotriazinoindole scaffold of the novel AR inhibitor cemtirestat by replacement of sulfur with oxygen. A series of 2-(3-oxo-2H-[1,2,4]triazino[5,6-b]indol-5(3H)-yl)acetic acid derivatives (OTIs), designed by molecular modeling and docking, were synthesized. More electronegative and less bulky oxygen of OTIs compared to the sulfur of the original thioxotriazinoindole congeners was found to form a stronger H-bond with Leu300 of AR and to render larger rotational flexibility of the carboxymethyl pharmacophore. AR inhibitory activities of the novel compounds were characterized by the IC50 values in a submicromolar range. Markedly enhanced inhibition selectivity relative to the structurally related aldehyde reductase was recorded. To conclude, structure modification of the original carboxymethylated thioxotriazinoindole cemtirestat by isosteric replacement of sulfur with oxygen in combination with variable N(2) simple substituents provided novel analogues with increased AR inhibition efficacy and markedly improved selectivity.