Modifications of diflunisal and meclofenamate carboxyl groups affect their allosteric effects on GABAA receptor ligand binding

Gamma-aminobutyric acid type A receptors (GABAAR) are allosterically modulated by the nonsteroidal anti-inflammatory drugs diflunisal and fenamates. The carboxyl group of these compounds is charged at physiological pH and therefore penetration of the compounds into the brain is low. In the present study we have transformed the carboxyl group of diflunisal and meclofenamate into non-ionizable functional groups and analyzed the effects of the modifications on stimulation of [(3)H]muscimol binding and on potentiation of &gamma;-aminobutyric acid-induced displacement of 4&#39;-ethenyl-4-n-[2,3-(3)H]propylbicycloorthobenzoate. N-Butylamide derivative of diflunisal modulated radioligand binding with equal or higher potency than the parent compound, while diflunisalamide showed reduced allosteric effect as compared to diflunisal. Amide derivative of meclofenamate equally affected radioligand binding parameters, while both diflunisal and meclofenamate methyl esters were less active than the parent compounds. Our study clearly demonstrates that an intact carboxyl group in diflunisal and meclofenamate is not indispensable for their positive GABAAR modulation. Further derivatization of the compound might yield compounds with higher selectivity for GABAARs that could be utilized in drug development.</p

Kemian koe ylioppilastutkinnossa keväällä 2020

Kevään 2020 ylioppilaskokeen tehtävissä etsittiin metsän kätkemiä aarteita, tislattiin, tutkittiin galvaanista kennoa ja penisilliinien toimintaa sekä määritettiin alstoniittimineraalin koostumus. Lopuksi vertailtiin nykyistä ja Mendelejevin järjestämää alkuaineiden jaksollista järjestelmää. Koe koettiin helpoksi, mutta arvosanojen pisterajat pysyivät entisellä tasolla. </p

Candida antarctica Lipase A-Based Enantiorecognition of a Highly Strained 4-Dibenzocyclooctynol (DIBO) Used for PET Imaging

The enantiomers of aromatic 4-dibenzocyclooctynol (DIBO), used for radiolabeling and subsequent conjugation of biomolecules to form radioligands for positron emission tomography (PET), were separated by kinetic resolution using lipase A from Candida antarctica (CAL-A). In optimized conditions, (R)-DIBO [(R)-1, ee 95%] and its acetylated (S)-ester [(S)-2, ee 96%] were isolated. In silico docking results explained the ability of CAL-A to differentiate the enantiomers of DIBO and to accommodate various acyl donors. Anhydrous MgCl2 was used for binding water from the reaction medium and, thus, for obtaining higher conversion by preventing hydrolysis of the product (S)-2 into the starting material. Since the presence of hydrated MgCl2·6H2O also allowed high conversion or effect on enantioselectivity, Mg2+ ion was suspected to interact with the enzyme. Binding site predictions indicated at least two sites of interest; one in the lid domain at the bottom of the acyl binding pocket and another at the interface of the hydrolase and flap domains, just above the active site.</p

Candida antarctica Lipase A-Based Enantiorecognition of a Highly Strained 4-Dibenzocyclooctynol (DIBO) Used for PET Imaging

The enantiomers of aromatic 4-dibenzocyclooctynol (DIBO), used for radiolabeling and subsequent conjugation of biomolecules to form radioligands for positron emission tomography (PET), were separated by kinetic resolution using lipase A from Candida antarctica (CAL-A). In optimized conditions, (R)-DIBO [(R)-1, ee 95%] and its acetylated (S)-ester [(S)-2, ee 96%] were isolated. In silico docking results explained the ability of CAL-A to differentiate the enantiomers of DIBO and to accommodate various acyl donors. Anhydrous MgCl2 was used for binding water from the reaction medium and, thus, for obtaining higher conversion by preventing hydrolysis of the product (S)-2 into the starting material. Since the presence of hydrated MgCl26H2O also allowed high conversion or effect on enantioselectivity, Mg2+ ion was suspected to interact with the enzyme. Binding site predictions indicated at least two sites of interest; one in the lid domain at the bottom of the acyl binding pocket and another at the interface of the hydrolase and flap domains, just above the active site.peerReviewe

Nanomolar Protein Thermal Profiling with Modified Cyanine Dyes.

Protein properties and interactions have been widely investigated by using external labels. However, the micromolar sensitivity of the current dyes limits their applicability due to the high material consumption and assay cost. In response to this challenge, we synthesized a series of cyanine5 (Cy5) dye-based quencher molecules to develop an external dye technique to probe proteins at the nanomolar protein level in a high-throughput one-step assay format. Several families of Cy5 dye-based quenchers with ring and/or side-chain modifications were designed and synthesized by introducing organic small molecules or peptides. Our results showed that steric hindrance and electrostatic interactions are more important than hydrophobicity in the interaction between the luminescent negatively charged europium-chelate-labeled peptide (Eu-probe) and the quencher molecules. The presence of substituents on the quencher indolenine rings reduces their quenching property, whereas the increased positive charge on the indolenine side chain improved the interaction between the quenchers and the luminescent compound. The designed quencher structures entirely altered the dynamics of the Eu-probe (protein-probe) for studying protein stability and interactions, as we were able to reduce the quencher concentration 100-fold. Moreover, the new quencher molecules allowed us to conduct the experiments using neutral buffer conditions, known as the peptide-probe assay. These improvements enabled us to apply the method in a one-step format for nanomolar protein-ligand interaction and protein profiling studies instead of the previously developed two-step protocol. These improvements provide a faster and simpler method with lower material consumption