Ampyrone is a direct agonist of human tyrosinase and a potential therapeutic for hypopigmentation disorders
Monika B. Dolinska, Yuhong Wang, Nathan P. Coussens, Vijay K. Kalaskar, Zuhal Eraslan, Samuel J. Grondin, Joseph Bonica, Sarah Toay, Matthew D. Hall, Min Shen, Matthew Boxer, Qiuying Chen, Steven S. Gross, Nabeel Attarwala, Yingyos Jittayasothorn, Ramakrishna P. Alur, Dhyanam Shukla, Robin Kee, Charles DeYoung, Cuilee Sha, David R. Adams, Stacie K. Loftus, Tiziana Cogliati, Yuri V. Sergeev, Jonathan H. Zippin, Brian P. Brooks
Monika B. Dolinska, Yuhong Wang, Nathan P. Coussens, Vijay K. Kalaskar, Zuhal Eraslan, Samuel J. Grondin, Joseph Bonica, Sarah Toay, Matthew D. Hall, Min Shen, Matthew Boxer, Qiuying Chen, Steven S. Gross, Nabeel Attarwala, Yingyos Jittayasothorn, Ramakrishna P. Alur, Dhyanam Shukla, Robin Kee, Charles DeYoung, Cuilee Sha, David R. Adams, Stacie K. Loftus, Tiziana Cogliati, Yuri V. Sergeev, Jonathan H. Zippin, Brian P. Brooks
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Research Article Dermatology Ophthalmology

Ampyrone is a direct agonist of human tyrosinase and a potential therapeutic for hypopigmentation disorders

Abstract

Significant loss of pigmentation can increase visual disability, skin cancer risk, and psychosocial stress. Tyrosinase (TYR) catalyzes the first and rate-limiting step of melanin synthesis. Inhibitors of TYR are well established and are currently used in clinical settings; however, there is a dearth of direct activators of TYR. Here, using a human TYR construct, we developed high-throughput screening methods, in cell confirmatory assays employing 13C-tyrosine tracing, and computational analysis techniques, and identified ampyrone (4-aminoantipyrine) as a TYR activator. Ampyrone increased the in vitro catalytic activity of the human recombinant intramelanosomal domain of TYR (hTYR) and its hypomorphic variant, Pro406Leu (P406L), a cause of oculocutaneous albinism type 1B (OCA1B). Moreover, ampyrone induced melanin synthesis in both WT and OCA1B human melanocytes, mouse OCA2 melanocytes, as well as 3-dimensional (3D) human skin cultures. Computational studies provided additional insight into the effects of direct TYR agonists on enzyme activity. Our results identify ampyrone as a lead candidate for TYR activation, potentially supporting the development of therapies for patients with genetic and acquired diseases of hypopigmentation.

Authors

Monika B. Dolinska, Yuhong Wang, Nathan P. Coussens, Vijay K. Kalaskar, Zuhal Eraslan, Samuel J. Grondin, Joseph Bonica, Sarah Toay, Matthew D. Hall, Min Shen, Matthew Boxer, Qiuying Chen, Steven S. Gross, Nabeel Attarwala, Yingyos Jittayasothorn, Ramakrishna P. Alur, Dhyanam Shukla, Robin Kee, Charles DeYoung, Cuilee Sha, David R. Adams, Stacie K. Loftus, Tiziana Cogliati, Yuri V. Sergeev, Jonathan H. Zippin, Brian P. Brooks

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Figure 1

Validation and performance of a miniaturized HTS assay for simultaneously identifying both activators and inhibitors of hTYRWT diphenol oxidase activity in vitro.

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Validation and performance of a miniaturized HTS assay for simultaneousl...
(A) The optimized diphenol oxidase activity assay was performed with hTYRWT (0.75 μM) in the absence and presence of increasing concentrations of the vehicle. No reduction in enzymatic activity was observed at concentrations up to 1.73%. All data show the mean ± SD (n = 3 technical replicates). (B) Kinetics of the optimized assay controls including the high activity control (triangles, 3-fold hTYRWT, 4.5 μM), baseline control (squares, hTYRWT, 1.5 μM), and low activity control (diamonds, no enzyme). Together, the controls enabled the assessment of assay performance to screen for activators (Z’ = 0.8) and inhibitors (Z’ = 0.83). All data show the mean ± SD (n = 3 technical replicates). (C) Endpoint percentage activity of the controls within a 1536-well microplate including the high activity control (n = 32), baseline control (n = 32), and low activity control (n = 32). (D) Signal/background values from the controls to identify activators (filled circles) and inhibitors (open circles) among the 26 microplates of the primary HTS. The average signal/background for the low activity control to identify inhibitors (no enzyme, open circles) was 11.75 (dotted line) and 2.0 for the high activity control to identify activators (3-fold hTYRWT, filled circles). (E) The average coefficient of variation from the DMSO-treated baseline control for 26 microplates was 4.1% (dotted line), which demonstrates low variability. (F) The Z’-factor was calculated from the controls of each microplate to serve as an acceptance criteria and to assess assay performance, where a Z’-factor value ≥ 0.5 (dashed line) is considered to be excellent. For the low activity control (open circles), Z’-factor values ranged between 0.61 and 0.85, with an average of 0.79 (dotted line) among 26 microplates. For the high activity control (filled circles), Z’-factor values ranged between 0.54 and 0.87, with an average of 0.74 (dotted line) among 26 microplates.