The present study aims to investigate the substrate (4-methyl catechol and catechol) specificity and inhibition mechanisms (l-ascorbic acid, citric acid, and l-cysteine) of the tyrosinase enzyme (TYR), which is held responsible for browning in foods and hyperpigmentation in the human skin, through kinetic and molecular docking studies. During the experimental studies, the diphenolase activities of TYR were determined, following which the inhibitory effects of the inhibitors upon the diphenolase activities of TYR. The inhibition types were determined as competitively for l-ascorbic acid and citric acid and noncompetitive for l-cysteine. The kinetic results showed that the substrate specificity was better for catechol while l-cysteine showed the best inhibition profile. As for the in silico studies, they also showed that catechol had a better affinity in line with the experimental results of this study, considering the interactions of the substrates with TYR's active site residues and their distance to CuB metal ion, which is an indicator of diphenolase activity. Besides, the inhibitory mechanisms of the inhibitor molecules were explained by the molecular modeling studies, considering the binding number of the inhibitors with the active site amino acid residues of TYR, the number and length of H bonds, negative binding energy values, and their distance to CuB metal ion. Based on our results, we suggest that the novel method used in this study to explain the inhibitory mechanism of l-cysteine may provide an affordable alternative to the expensive methods available for explaining the inhibitory mechanism of TYR and those of other enzymes. Communicated by Ramaswamy H. Sarma Graphical Abstract

The search for an alternative approach of estrus control (induction or suppression) in dogs is an important issue and the use of slow GnRH agonist-releasing implants has been the subject of frequent research in recent years. Studies to date demonstrate that the short- and long-term effects of deslorelin implants applicated at different time points of the prepubertal period are similar to those of adult dogs; however, there are important differences. The age of the prepubertal bitch and the dosage appear to be the main determinants of the response to deslorelin, as well as the individual metabolism of the bitch. Recent studies reported that the deslorelin-mediated long-term delay of puberty does not have negative carry-over effects on subsequent ovarian functionality, serum steroid hormone concentrations, uterine health, and fertility; however, more molecular studies are needed to determine the effects of application time of GnRH agonists on hormone concentrations and peripheral receptor expression. Furthermore, the long-term effects of delay of puberty with deslorelin on joint health, tumor development, the immune system, and social behavior deserve further investigations.

Progesterone (P4) is the only hormone needed to maintain pregnancy in dogs. Therefore, a competitive inhibitor of 3β-hydroxysteroid dehydrogenase (3β-HSD) could be a safe and effective option to terminate pregnancy by inhibiting P4 synthesis. To address this hypothesis, we investigated the efficacy of trilostane (TRL), a competitive inhibitor of 3β-HSD, in terminating pregnancy in dogs. Twenty-one dogs between days 30 and 38 of pregnancy were randomly assigned to one of two treatment groups (trilostane (TRL) and aglepristone (AGL)) and an untreated control (CON) group (n = 7 dogs each). Fetal heart rates (FHRs) (measured at 12 h intervals) and serum P4 concentrations (measured at 6 h intervals) were evaluated. The pregnancy termination rates were 0% and 100% in the TRL and AGL groups, respectively. The decrease in the FHR in the TRL and AGL groups was significantly lower than that observed in the CON group. There was a marked decrease in P4 concentrations in the TRL group 6, 54, and 102 h after the initiation of treatment. The luteal expression of StAR appeared to be weaker in the AGL group than the CON group. In conclusion, although a treatment-induced decrease was observed in plasma P4 concentrations, a seven-day TRL treatment alone was not effective in terminating pregnancies. Further studies are needed on the effects of the prolonged administration of TRL with varying doses and frequencies for the termination of mid-term pregnancy in dogs.