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. 2025 Mar 29;26(7):3181.
doi: 10.3390/ijms26073181.

A Comprehensive In Vitro and In Silico Approach for Targeting 4-Hydroxyphenyl Pyruvate Dioxygenase: Towards New Therapeutics for Alkaptonuria

Giulia Bernardini  1 , Alfonso Trezza  1 , Elena Petricci  1 , Giulia Romagnoli  1 , Demetra Zambardino  1 , Fabrizio Manetti  1 , Daniela Braconi  1 , Michela Geminiani  1 , Annalisa Santucci  1   2
Affiliations

A Comprehensive In Vitro and In Silico Approach for Targeting 4-Hydroxyphenyl Pyruvate Dioxygenase: Towards New Therapeutics for Alkaptonuria

Giulia Bernardini et al. Int J Mol Sci. .

Abstract

Alkaptonuria (AKU) is an ultra-rare genetic disorder caused by mutations in the homogentisate 1,2-dioxygenase (HGD) gene, leading to the accumulation of homogentisic acid (HGA). Current treatment options are limited, with Nitisinone (Orfadin or NTBC) being the only approved drug. However, its long-term use raises concerns due to significant adverse effects, highlighting the urgent need for safer alternatives. AKU manifests with progressive and often painful symptoms, severely impacting patients' quality of life. Identifying new therapeutic approaches to inhibit 4-hydroxyphenyl pyruvate dioxygenase (4-HPPD) is critical to improving outcomes for AKU patients. In this study, we present a novel integrated in vitro and in silico strategy to assess the residence time of 4-HPPD inhibitors. In particular, we evaluated several features of a set of triketone compounds including their inhibitory efficacy, residence time, and ochronotic pigment accumulation. By means of our integrated approach, we investigated the pharmacokinetic and pharmacodynamics properties of novel 4-HPPD inhibitors and provided a promising foundation for the development of safer and more effective treatments for AKU.

Keywords: 4-HPPD; AKU; IC50; IC90; NTBC; docking simulation; inhibitors; molecular dynamics simulation; molecular modeling; residence time; steered molecular dynamics (SMD).

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Inhibition curves obtained by testing different concentrations of the 4-HPPD inhibitors with corresponding IC50 and IC90 values.
Figure 2
Figure 2
4-HPPD/compound residence time evaluation. (A) Enzyme activity evaluated in presence of each compound. (B) Details of the tested compounds with their corresponding residence time (τ) and half-life values. Magenta: NTBC; dark green: c1; turquoise: c2; purple: c3; ocher: c4; light green: c5; light blue: c6; black: c7.
Figure 3
Figure 3
SMD simulations. Force profiles of compounds pulled out of the 4-HPPD binding pocket along the unbinding pathway.
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