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  • DiscoveryProbe™ Inhibitor Library In conclusion we have prod

    2024-02-22

    In conclusion, we have produced some highly potent inhibitors of P450 in comparison to the standard DiscoveryProbe™ Inhibitor Library KTZ. Also, due to the limited specificity of these compounds against lyase in comparison to the 17α-OHase component, these compounds would be expected to have a major impact on corticosteroid biosynthesis. Furthermore, through the consideration of the modelling of these inhibitors, we have provided an insight into the conformational space available about the area of the active site. Although the compounds have shown high levels of potency, the biochemical evaluation of similar benzyl imidazole-based compounds suggests that the use of these compounds as potential drug substances is limited due to their ability to inhibit other cytochrome P450 enzymes such as cholesterol side chain cleavage or indeed aromatase,, and which therefore suggests a lack of selectivity. Acknowledgements
    Introduction Adrenal and gonadal 17α-hydroxylase/17,20-lyase (P450c17) catalyzes 2 sequential reactions: 17α-hydroxylation of pregnenolone or progesterone and 17,20-bond scission of the 17α-hydroxylated products [1]. The CYP17A1 gene encoding this enzyme is located on chromosome 10q24.3 [2]; and more than 50 mutations in the CYP17A1 gene have been reported to cause combined 17α-hydroxylase/17,20-lyase deficiency or isolated 17,20-lyase deficiency [3], [4], [5], [6]. Adrenal 17α-hydroxylase deficiency is characterized by impaired production of cortisol and compensated hypersecretion of adrenocorticotropin, which stimulates the synthesis of large amount of deoxycorticosterone and corticosterone, leading to hypertension, hypokalemia, and a suppressed renin-angiotensin system. Gonadal 17,20-lyase deficiency prohibits the synthesis of dehydroepiandrosterone, testosterone, and estrogen, resulting in 46,XY disorders of sex development in genetic males and sexual infantilism in both sexes. Combined 17α-hydroxylase/17,20-lyase deficiency is a rare form of congenital adrenal hyperplasia, which accounts for about 1% of cases overall [7]; and isolated 17,20-lyase deficiency is extremely rare [8]. In the present study, we report a Japanese patient with combined 17α-hydroxylase/17,20-lyase deficiency and a novel CYP17A1 mutation.
    Materials and methods
    Results The patient had 2 heterozygous mutations (Fig. 1). One was a deletion of codon 53 or 54 (TCC) encoding Phe in exon 1 (ΔF54). The other was a C to A transversion of the first nucleotide in codon 373 in exon 6, which resulted in a substitution of Asn (AAC) for His (CAC) at codon 373 (H373N). No other mutations were found in the entire exons and the exon-intron boundaries of the patient's CYP17A1 gene. The mother was heterozygous for the ΔF54 mutation, and the father was heterozygous for the H373N mutation (data not shown). Therefore, it was concluded that the patient is a compound heterozygote bearing the ΔF54 mutation on a maternal allele and the H373N mutation on a paternal allele. To determine the functional consequence of the H373N mutation, we transiently expressed the wild-type and H373N mutant P450c17. The COS-1 cells transfected with the wild-type P450c17 cDNA efficiently converted progesterone to 17α-hydroxyprogesterone and androstenedione, whereas those transfected with the empty pRK 5 plasmid failed to convert progesterone to 17α-hydroxyprogesterone and androstenedione (Table 2). The H373N substitution resulted in markedly reduced production of 17α-hydroxyprogesterone (0.2% of the wild-type P450c17) and no production of androstenedione (Table 2).
    Discussion The ΔF54 mutation in exon 1 was first described in a Japanese female patient with partial combined 17α-hydroxylase/17,20-lyase deficiency who presented with hypertension, hypokalemia, and irregular menstruation [10]. Functional expression analyses revealed that the ΔF54 mutation partially reduces the activities of both 17α-hydroxylase and 17,20-lyase [10], [11]. The ΔF54 mutation has been repeatedly identified in Japanese patients with combined 17α-hydroxylase/17,20-lyase deficiency [10], [11], [12], [13], [14], [15], suggesting that this mutation has been derived from a single founder gene.