Characterization of CYP26B1-Selective Inhibitor, DX314, as a Potential Therapeutic for Keratinization Disorders

Abstract

Inhibition of CYP450-mediated retinoic acid (RA) metabolism by RA metabolism blocking agents increases endogenous retinoids and is an alternative to retinoid therapy. Currently available RA metabolism blocking agents (i.e., liarozole and talarozole) tend to have fewer adverse effects than traditional retinoids but lack target specificity. Substrate-based inhibitor DX314 has enhanced selectivity for RA-metabolizing enzyme CYP26B1 and may offer an improved treatment option for keratinization disorders such as congenital ichthyosis and Darier disease. In this study, we used RT-qPCR, RNA sequencing, pathway, upstream regulator, and histological analyses to demonstrate that DX314 can potentiate the effects of all-trans-RA in healthy and diseased reconstructed human epidermis. We unexpectedly discovered that DX314, but not all-trans-RA or previous RA metabolism blocking agents, appears to protect epidermal barrier integrity. In addition, DX314-induced keratinization and epidermal proliferation effects are observed in a rhino mice model. Altogether, the results indicate that DX314 inhibits all-trans-RA metabolism with minimal off-target activity and shows therapeutic similarity to topical retinoids in vitro and in vivo. Findings of a barrier-protecting effect require further mechanistic study but may lead to a unique strategy in barrier-reinforcing therapies. DX314 is a promising candidate compound for further study and development in the context of keratinization disorders.

Preclinical assessment of dual CYP26[A1/B1] inhibitor, DX308, as an improved treatment for keratinization disorders

“…Confluent monolayer KCs were prepared as previously described 24,37 and treated for 20 h with compounds solubilized in growth media (0.1% DMSO vehicle). Reconstructed human epidermis (RHE) was produced as previously described 24,36,38 . A 4‐day treatment was initiated on day 7 of tissue reconstruction.…”

“…Reconstructed human epidermis (RHE) was produced as previously described. 24,36,38 A 4-day treatment was initiated on day 7 of tissue reconstruction. Treatments were stopped on day 11 and RHE was processed for histology or RNA isolation.…”

“…Confluent monolayer KCs were prepared as previously described 24,37 and treated for 20 h with VEIT ET AL.…”

“…RNA samples were sent to the University of Colorado's Genomics and Sequence Core Facility for library preparation and sequencing on the Illumina HiSeq platform as previously described. 24 Bioinformatics analyses were performed using Array Studio (Omicsoft) and R. 41 Sample sequencing quality was assessed using FastQC. 42 Raw reads were aligned to the reference genome Human.B38/Ensembl.R94 using OSA, 43 providing 36.6-62.1 million reads per sample.…”

“…We previously described a CYP26B1-selective RAMBA, DX314, which shows efficacy in vitro and in vivo. 24 This study investigates DX308 (Figure S1), a specific inhibitor with equal affinity (IC 50 = 51 nM) for both CYP26s found in the skin (CYP26A1 and CYP26B1), which is described in US patent US009963439B2 as example 38. 25 In a series of in vitro assays, we characterize DX308's low potential for off-target activity, topical irritation, and genotoxicity at the expected effective dose.…”