Animal liver microsomes are a rich source of carboxylesterases with potential for biocatalytic applications. However, their instability and difficulty in reuse limit their practical application. This study investigates the immobilization of animal liver microsomes from four species Mus musculus (house mouse), Sus scrofa (wild boar), Dama dama (fallow deer), and Capreolus capreolus (roe deer) on Perloza MG microparticles for enhanced stability and reusability. Immobilization significantly improved the stability and pH tolerance of the microsomes, particularly those from D. dama, maintaining esterase activity across a broad pH range (5-9) and enabling the reusability over ten consecutive cycles. The immobilized D. dama microsomes were successfully employed in a preparative-scale chemo-enzymatic synthesis of a cyclophilin D inhibitor, achieving a total reaction yield of 68% with 98% final product purity, demonstrating their potential for sustainable organic synthesis.
- Klíčová slova
- Biocatalysis, Carboxylesterases, Chemo-enzymatic synthesis, Immobilization, Magnetic microparticles,
- Publikační typ
- časopisecké články MeSH
The evolution of proteins is primarily driven by the combinatorial assembly of a limited set of pre-existing modules known as protein domains. This modular architecture not only supports the diversity of natural proteins but also provides a robust strategy for protein engineering, enabling the design of artificial proteins with enhanced or novel functions for various industrial applications. Among these functions, oligomerization plays a crucial role in enhancing protein activity, such as by increasing the binding capacity of antibodies. To investigate the potential of engineering oligomerization, we examined the transferability of the sequence domain encoded by exon 5 (Ex5), which was originally responsible for the oligomerization of ameloblastin (AMBN). We designed a two-domain protein composed of Ex5 in combination with a monomeric, globular, and highly stable protein, specifically calmodulin (CaM). CaM represents the opposite protein character to AMBN, which is highly disordered and has a dynamic character. This engineered protein, termed eCaM, successfully acquired an oligomeric function, inducing self-assembly under specific conditions. Biochemical and biophysical analyses revealed that the oligomerization of eCaM is both concentration- and time-dependent, with the process being reversible upon dilution. Furthermore, mutating a key oligomerization residue within Ex5 abolished the self-assembly of eCaM, confirming the essential role of the Ex5 motif in driving oligomerization. Our findings demonstrate that the oligomerization properties encoded by Ex5 can be effectively transferred to a new protein context, though the positioning of Ex5 within the protein structure is critical. This work highlights the potential of enhancing monomeric proteins with oligomeric functions, paving the way for industrial applications and the development of proteins with tailored properties.
- Publikační typ
- časopisecké články MeSH
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by memory loss and behavioral and psychological symptoms of dementia (BPSD). Given that cholinergic neurons are predominantly affected in AD, current treatments primarily aim to enhance cholinergic neurotransmission. However, imbalances in other neurotransmitters, such as γ-aminobutyric acid (GABA), also contribute to AD symptomatology. In the presented research, using a combination of crystallography and computational methods we developed compound 6 as a dual modulator of GABAergic and cholinergic neurotransmission systems. Compound 6 demonstrated inhibition of BuChE (IC50=0.21 μM) and GABA transporter 1 (IC50=10.96 μM) and 3 (IC50=7.76 μM), along with a favorable drug-likeness profile. Subsequent in vivo studies revealed the effectiveness of 6 in enhancing memory retention and alleviating anxiety and depression symptoms in animal models, while also proving safe and bioavailable for oral administration. The innovative multi-target-directed ligand 6 offers a new approach to treating cognitive deficits and BPSD in AD.
- Klíčová slova
- Alzheimer's disease, GABA transporters, butyrylcholinesterase, inhibitors, multitarget drugs,
- MeSH
- acetylcholinesterasa metabolismus MeSH
- Alzheimerova nemoc * farmakoterapie metabolismus MeSH
- butyrylcholinesterasa metabolismus MeSH
- cholinesterasové inhibitory * chemie farmakologie terapeutické užití MeSH
- fluoreny * chemie farmakologie terapeutické užití MeSH
- GABA * metabolismus MeSH
- lidé MeSH
- molekulární struktura MeSH
- myši MeSH
- nervový přenos * účinky léků MeSH
- proteiny přenášející GABA přes plazmatickou membránu metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- acetylcholinesterasa MeSH
- butyrylcholinesterasa MeSH
- cholinesterasové inhibitory * MeSH
- fluoreny * MeSH
- GABA * MeSH
- proteiny přenášející GABA přes plazmatickou membránu MeSH
Cyclophilin D (CypD) is a mitochondrial enzyme widely accepted as a regulator of the mitochondrial permeability transition pore (mPTP). Excessive opening of mPTP is associated with mitochondrial dysfunction and the development of various diseases; thus, suppression of mPTP opening through CypD inhibition presents a promising therapeutic approach. However, only a limited number of selective CypD inhibitors are currently available. In this study, 10 derivatives of 2-(benzyloxy)arylurea similar or identical to previously published CypD/mPTP inhibitors were synthesized. Unlike the original reports that assessed the opening of mPTP at the cellular level, the compounds were tested directly on the purified CypD enzyme to validate their putative mechanism of action. Additionally, the effect of the selected compounds was tested on isolated mitochondria. The obtained results show that the compounds are only weak inhibitors of CypD and mPTP opening, which is in contrast to previous conclusions drawn from the unspecific cellular JC-1 assay.
- Publikační typ
- časopisecké články MeSH
Enzyme handling and utilization bears many challenges such as their limited stability, intolerance of organic solvents, high cost, or inability to reuse. Most of these limitations can be overcome by enzyme immobilization on the surface of solid support. In this work, the recombinant form of human cholinesterases and monoamine oxidases as important drug targets for neurological diseases were immobilized on the surface of magnetic non-porous microparticles by a non-covalent bond utilizing the interaction between a His-tag terminus on the recombinant enzymes and cobalt (Co2+) ions immobilized on the magnetic microparticles. This type of binding led to targeted enzyme orientation, which completely preserved the catalytic activity and allowed high reproducibility of immobilization. In comparison with free enzymes, the immobilized enzymes showed exceptional stability in time and the possibility of repeated use. Relevant Km, Vmax, and IC50 values using known inhibitors were obtained using particular immobilized enzymes. Such immobilized enzymes on magnetic particles could serve as an excellent tool for a sustainable approach in the early stage of drug discovery.
- Klíčová slova
- Cholinesterases, Immobilization, Magnetic microparticles, Monoamine oxidases, Neurological disorders, Sustainability,
- MeSH
- acetylcholinesterasa metabolismus chemie MeSH
- analýza nákladů a výnosů MeSH
- enzymy imobilizované * chemie metabolismus MeSH
- kobalt * chemie MeSH
- lidé MeSH
- monoaminoxidasa metabolismus chemie MeSH
- nemoci nervového systému farmakoterapie enzymologie MeSH
- objevování léků * MeSH
- rekombinantní proteiny chemie metabolismus MeSH
- stabilita enzymů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- acetylcholinesterasa MeSH
- enzymy imobilizované * MeSH
- kobalt * MeSH
- monoaminoxidasa MeSH
- rekombinantní proteiny MeSH
17β-HSD10 is a mitochondrial enzyme that catalyzes the steroidal oxidation of a hydroxy group to a keto group and, thus, is involved in maintaining steroid homeostasis. The druggability of 17β-HSD10 is related to potential treatment for neurodegenerative diseases, for example, Alzheimer's disease or cancer. Herein, steroidal derivatives with an acidic hemiester substituent at position C-3 on the skeleton were designed, synthesized, and evaluated by using pure recombinant 17β-HSD10 converting 17β-estradiol to estrone. Compounds 22 (IC50 = 6.95 ± 0.35 μM) and 23 (IC50 = 5.59 ± 0.25 μM) were identified as the most potent inhibitors from the series. Compound 23 inhibited 17β-HSD10 activity regardless of the substrate. It was found not cytotoxic toward the HEK-293 cell line and able to inhibit 17β-HSD10 activity also in the cellular environment. Together, these findings support steroidal compounds as promising candidates for further development as 17β-HSD10 inhibitors.
- Publikační typ
- časopisecké články MeSH
Mitochondrial enzyme 17β-hydroxysteroid dehydrogenase type 10 (HSD10) is a potential molecular target for treatment of mitochondrial-related disorders such as Alzheimer's disease (AD). Its over-expression in AD brains is one of the critical factors disturbing the homeostasis of neuroprotective steroids and exacerbating amyloid beta (Aβ)-mediated mitochondrial toxicity and neuronal stress. This study was focused on revalidation of the most potent HSD10 inhibitors derived from benzothiazolyl urea scaffold using fluorescent-based enzymatic assay with physiologically relevant substrates of 17β-oestradiol and allopregnanolone. The oestradiol-based assay led to the identification of two nanomolar inhibitors (IC50 70 and 346 nM) differing from HSD10 hits revealed from the formerly used assay. Both identified inhibitors were found to be effective also in allopregnanolone-based assay with non-competitive or uncompetitive mode of action. In addition, both inhibitors were confirmed to penetrate the HEK293 cells and they were able to inhibit the HSD10 enzyme in the cellular environment. Both molecules seem to be potential lead structures for further research and development of HDS10 inhibitors.
- Klíčová slova
- 17β-HSD10, 17β-oestradiol, Alzheimer's disease, CHANA, allopregnanolone, benzothiazole,
- Publikační typ
- časopisecké články MeSH
Cyclophilin D (CypD) is a key regulator of mitochondrial permeability transition pore (mPTP) opening. This pathophysiological phenomenon is associated with the development of several human diseases, including ischemia-reperfusion injury and neurodegeneration. Blocking mPTP opening through CypD inhibition could be a novel and promising therapeutic approach for these conditions. While numerous CypD inhibitors have been discovered to date, none have been introduced into clinical practice, mostly owing to their high toxicity, unfavorable pharmacokinetics, and low selectivity for CypD over other cyclophilins. This review summarizes current knowledge of CypD inhibitors, with a particular focus on small-molecule compounds with regard to their in vitro activity, their selectivity for CypD, and their binding mode within the enzyme's active site. Finally, approaches for improving the molecular design of CypD inhibitors are discussed.
- Klíčová slova
- cyclophilin D, drug discovery, enzyme inhibition, mitochondria, mitochondrial permeability transition pore,
- MeSH
- lidé MeSH
- mitochondriální nemoci * farmakoterapie MeSH
- mitochondrie metabolismus MeSH
- peptidylprolylisomerasa F * antagonisté a inhibitory MeSH
- přechodový pór mitochondriální permeability MeSH
- transportní proteiny mitochondriální membrány * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- peptidylprolylisomerasa F * MeSH
- přechodový pór mitochondriální permeability MeSH
- transportní proteiny mitochondriální membrány * MeSH
Human cyclophilin D is a mitochondrial peptidyl-prolyl isomerase that plays a role in regulating the opening of the mitochondrial permeability transition pore. It is considered a viable and promising molecular target for the treatment of diseases for which disease development is associated with pore opening, e.g., Alzheimer's disease or ischemia/reperfusion injury. Currently available and widely used in vitro methods based on Kofron's assay for determining cyclophilin D activity suffer from serious drawbacks and limitations. In this study, a completely novel approach for an in vitro assay of cyclophilin D activity using RNase T1 refolding is introduced. The method is simple and is more in line with the presumed physiological role of cyclophilin D in protein folding than Kofron's assay, which relies on a peptide substrate. The method is applicable for identifying novel inhibitors of cyclophilin D as potential drugs for the treatment of the diseases mentioned above. Moreover, the description of CypD activity in the in vitro RNase T1 refolding assay reveals new possibilities for investigating the role of cyclophilin D in protein folding in cells and may lead to a better understanding of its pathological and physiological roles.
- MeSH
- Aspergillus oryzae enzymologie MeSH
- guanyloribonukleasa chemie MeSH
- konformace proteinů MeSH
- lidé MeSH
- mitochondrie metabolismus MeSH
- molekulární modely MeSH
- objevování léků * MeSH
- peptidylprolylisomerasa F chemie metabolismus MeSH
- refolding proteinů * MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- guanyloribonukleasa MeSH
- peptidylprolylisomerasa F MeSH
