Electrochemical biosensor for trypsin activity assay based on cleavage of immobilized tyrosine-containing peptide.

In this work, we proposed a biosensor for trypsin proteolytic activity assay using immobilization of model peptides on screen-printed electrodes (SPE) modified with gold nanoparticles (AuNPs) prepared by electrosynthetic method. Sensing of proteolytic activity was based on electrochemical oxidation of tyrosine residues of peptides. We designed peptides containing N-terminal cysteine residue for immobilization on an SPE, modified with gold nanoparticles, trypsin-specific cleavage site and tyrosine residue as a redox label. The peptides were immobilized on SPE by formation of chemical bonds between mercapto groups of the N-terminal cysteine residues and AuNPs. After the incubation with trypsin, time-dependent cleavage of the immobilized peptides was observed by decline in tyrosine electrochemical oxidation signal. The kinetic parameters of trypsin, such as the catalytic constant (kcat), the Michaelis constant (KM) and the catalytic efficiency (kcat/KM), toward the CGGGRYR peptide were determined as 0.33 ± 0.01 min-1, 198 ± 24 nM and 0.0016 min-1 nM-1, respectively. Using the developed biosensor, we demonstrated the possibility of analysis of trypsin specificity toward the peptides with amino acid residues disrupting proteolysis. Further, we designed the peptides with proline or glutamic acid residues after the cleavage site (CGGRPYR and CGGREYR), and trypsin had reduced activity toward both of them according to the existing knowledge of the enzyme specificity. The developed biosensor system allows one to perform a comparative analysis of the protease steady-state kinetic parameters and specificity toward model peptides with different amino acid sequences.

Interactions of galeterone and its 3-keto-Δ4 metabolite (D4G) with one of the key enzymes of corticosteroid biosynthesis - steroid 21-monooxygenase (CYP21A2).

We have investigated interactions of galeterone and its pharmacologically active metabolite - 3-keto-Δ4-galeterone (D4G) - with one of the key enzymes of corticosteroid biosynthesis - steroid 21-monooxygenase (CYP21A2). It was shown by absorption spectroscopy that both compounds induce type I spectral changes of CYP21A2. Spectral dissociation constants (KS ) of complexes of CYP21A2 with galeterone or D4G were calculated as 3.1 ± 0.7 µm and 4.6 ± 0.4 µm, respectively. It was predicted by molecular docking that both ligands similarly bind to the active site of CYP21A2. We have revealed using reconstituted monooxygenase system that galeterone is a competitive inhibitor of CYP21A2 with the inhibition constant (Ki ) value of 12 ± 3 µm, while D4G at the concentrations of 10 and 25 µm does not inhibit the enzyme. Summarizing, based on the in vitro analyses we detected inhibition of CYP21A2 by galeterone and lack of the influence of D4G on this enzyme.

The interactions of a number of steroid-metabolizing cytochromes P450 with abiraterone D4A metabolite: spectral analysis and molecular docking.

The interactions of pharmacologically active 3-keto-Δ4-metabolite of anticancer drug abiraterone (D4A) with steroid-metabolizing cytochromes P450 (CYP51A1, CYP11A1, CYP19A1) was studied by absorption spectroscopy and molecular docking. Both abiraterone and D4A induce type I spectral changes of CYP51A1, one of the enzymes of cholesterol biosynthesis. We have revealed that D4A did not induce spectral changes of CYP11A1, the key enzyme of pregnenolone biosynthesis, unlike abiraterone (type II ligand of CYP11A1). On the contrary, D4A interacts with the active site of CYP19A1, the key enzyme of estrogen biosynthesis, inducing type II spectral changes, while abiraterone does not. Spectral analysis allowed us to calculate spectral dissociation constant (KS) for each complex of cytochrome P450 with respective ligands. The data were supported by molecular docking. The obtained results broaden understanding of interactions of D4A with some of the key steroid-metabolizing cytochromes P450 and allow one to predict possible disproportions of steroid metabolism.

In vitro interactions of abiraterone, erythromycin, and CYP3A4: implications for drug-drug interactions.

Potential drug-drug interactions of the antitumor drug abiraterone and the macrolide antibiotic erythromycin were studied at the stage of cytochrome P450 3A4 (CYP3A4) biotransformation. Using differential spectroscopy, we have shown that abiraterone is a type II ligand of CYP3A4. The dependence of CYP3A4 spectral changes on the concentration of abiraterone is sigmoidal, which indicates cooperative interactions of CYP3A4 with abiraterone; these interactions were confirmed by molecular docking. The dissociation constant (Kd ) and Hill coefficient (h) values for the CYP3A4-abiraterone complex were calculated as 3.8 ± 0.1 µM and 2.3 ± 0.2, respectively. An electrochemical enzymatic system based on CYP3A4 immobilized on a screen-printed electrode was used to show that abiraterone acts as a competitive inhibitor toward erythromycin N-demethylase activity of CYP3A4 (apparent Ki  = 8.1 ± 1.2 µM), while erythromycin and its products of enzymatic metabolism do not affect abiraterone N-oxidation by CYP3A4. In conclusion, the inhibition properties of abiraterone toward CYP3A4-dependent N-demethylation of erythromycin and the biologically inert behavior of erythromycin toward abiraterone hydroxylation were demonstrated.

Comparison of [17(20)E]-21-Norpregnene oxazolinyl and benzoxazolyl derivatives as inhibitors of CYP17A1 activity and prostate carcinoma cells growth.

Four new 4,5-dihydro-1,3-oxazole, and four new benzo-[d]-oxazole derivatives of [17(20)E]-21-norpregnene, differing in the structure of steroid moiety, were synthesized and evaluated for their potency to inhibit 17α-hydroxylase/17,20-lyase (CYP17A1) activity. Among new compounds, the only oxazolinyl derivative comprising 5-oxo-4,5-seco-3-yn- moiety potently inhibited CYP17A1. Binding modes of the oxazolinyl derivatives of [17(20)E]-21-norpregnene were analyzed by molecular dynamics simulations, and model of alternate, water-bridged type II interaction was proposed for these compounds. Eight new compounds, together with two CYP17A1-inhibiting oxazolinyl derivatives synthesized earlier, abiraterone and galeterone were evaluated for their potency to inhibit prostate carcinoma PC-3 and LNCaP cells growth. Oxazolinyl and benzoxazolyl derivatives comprising 3ß-hydroxy-5-ene moieties potently inhibited prostate carcinoma cell growth; inhibitory potencies of 3-oxo-4-en- and 5-oxo-4,5-seco-3-yn- derivatives were significantly lower.

Novel oxazolinyl derivatives of pregna-5,17(20)-diene as 17α-hydroxylase/17,20-lyase (CYP17A1) inhibitors.

New oxazolinyl derivatives of [17(20)E]-pregna-5,17(20)-diene: 2'-{[(E)-3ß-hydroxyandrost-5-en-17-ylidene]methyl}-4',5'-dihydro-1',3'-oxazole 1 and 2'-{[(E)-3ß-hydroxyandrost-5-en-17-ylidene]methyl}-4',4'-dimethyl-4',5'-dihydro-1',3'-oxazole 2 were evaluated as potential CYP17A1 inhibitors in comparison with 17-(pyridin-3-yl)androsta-5,16-dien-3ß-ol 3 (abiraterone). Differential absorption spectra of human recombinant CYP17A1 in the presence of compound 1 (λmax=422 nm, λmin=386 nm) and compound 2 (λmax=416 nm) indicated significant differences in enzyme/inhibitors complexes. CYP17A1 activity was measured using electrochemical methods. Inhibitory activity of compound 1 was comparable with abiraterone 3 (IC50=0.9±0.1 µM, and IC50=1.3±0.1 µM, for compounds 1 and 3, respectively), while compound 2 was found to be weaker inhibitor (IC50=13±1 µM). Docking of aforementioned compounds to CYP17A1 revealed that steroid fragments of compound 1 and abiraterone 3 occupied close positions; oxazoline cycle of compound 1 was coordinated with heme iron similarly to pyridine cycle of abiraterone 3. Configuration of substituents at 17(20) double bond in preferred docked position corresponded to Z-isomers of compounds 1 and 2. Presence of 4'-substituents in oxazoline ring of compound 2 prevents coordination of oxazoline nitrogen with heme iron and worsens its docking score in comparison with compound 1. These data indicate that oxazolinyl derivative of [17(20)E]-pregna-5,17(20)-diene 1 (rather than 4',4'-dimethyl derivative 2) may be considered as potential CYP17A1 inhibitor and template for development of new compounds affecting growth and proliferation of prostate cancer cells.