RARE DISEASE
Alkaptonuria
Alkaptonuria
Alkaptonuria
Synonyms: Hereditary ochronosis, Homogentisic acid oxidase deficiency
Synonyms: Hereditary ochronosis, Homogentisic acid oxidase deficiency
Synonyms: Hereditary ochronosis, Homogentisic acid oxidase deficiency
Drug discovery
4
drugs
With orphan designations
Overview
Alkaptonuria (AKU) is a rare autosomal recessive disorder caused by HGD gene mutations, resulting in deficient homogentisate 1,2-dioxygenase. This leads to systemic accumulation of homogentisic acid (HGA), causing ochronosis (blue-black connective tissue pigmentation) and early-onset osteoarthritis. Complications include cardiac valve calcification, renal/prostatic stones, and osteoporosis [1][2][7]. Diagnosis involves urinary HGA quantification, genetic testing, and imaging for arthropathy. First-line therapy with nitisinone reduces HGA production, supplemented by symptomatic management (analgesics, joint replacement) [3][6][8].
Burden
Severe quality-of-life impact: Chronic pain, mobility loss, and frequent surgeries (50% require joint replacement by age 55) [4][8].
Increased risks: Cardiovascular disease (22% aortic stenosis), renal stones, and Parkinson’s disease (20× higher prevalence) [9][16][18].
Economic burden: High costs from repeated surgeries and lifelong multidisciplinary care [4][20].
Categories: rare genetic diseases, rare inborn errors of metabolism, rare ophthalmic disorders, rare skin diseases
Research Papers
272 drug discovery papers about Alkaptonuria, with 3 first-in-class and 3 next-in-class emerging drug candidates forecasted to outperform the average preclinical success rate. Recent publications:
272 drug discovery papers about Alkaptonuria, with 3 first-in-class and 3 next-in-class emerging drug candidates forecasted to outperform the average preclinical success rate. Recent publications:
categories:
Small molecules
gene therapies
2025-11-11 | Inborn Errors of Amino Acid Metabolism Revisited: Clinical Implications and Insights into Current Therapies
Inborn errors of amino acid metabolism (IEAAMs) are a heterogeneous group of genetic disorders caused by defects in enzymes, cofactors, or transporters of amino acid catabolism, biosynthesis, or transport. These defects result in toxic metabolite accumulation and/or deficiency of essential metabolites. This review aims to provide an updated overview of diagnosis, clinical implications, management, and evolving therapeutic approaches across major IEAAMs. A narrative review of recent literature was undertaken, focusing on established and novel therapeutic strategies for key IEAAMs, including phenylketonuria, alkaptonuria, tyrosinemia, homocystinuria, and maple syrup urine disease. Key management strategies include amino acid-restricted diets/restriction of natural protein with restriction of dietary precursors, dietary supplementations, including disease-specific amino acid supplements, medications to reduce formation of offending metabolites, pharmacotherapies, enzyme/cofactor replacement or pharmacological chaperones, enhancing residual enzyme activity and promoting alternative path-ways/accessory pathways. Emergency therapy is essential in severe types and focuses on promoting anabolism, limiting catabolism, reducing formation, and enhancing clearance of toxic metabolites. Other treatment options include organ transplantation, and new emerging modalities, such as mRNA therapies and gene therapies/in vivo gene editing offer potential for definitive interventions. Despite advancements in therapy and close monitoring, many IEAAMs remain associated with significant comorbidities. Future research is essential to optimise current treatment standards, particularly neuroprotective and metabolic regulatory features. While an in-depth discussion of innovative person-alised therapies is beyond the scope of this article, we believe that collective experiences will thrust future research in this field and expand access to innovative personalised therapies.
2025-04-17 | A Comprehensive In Vitro and In Silico Approach for Targeting 4-Hydroxyphenyl Pyruvate Dioxygenase: Towards New Therapeutics for Alkaptonuria.
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.
2020-06-01 | Conditional targeting in mice reveals that hepatic homogentisate 1,2-dioxygenase activity is essential in reducing circulating homogentisic acid and for effective therapy in the genetic disease alkaptonuria.
Alkaptonuria is an inherited disease caused by homogentisate 1,2-dioxygenase (HGD) deficiency. Circulating homogentisic acid (HGA) is elevated and deposits in connective tissues as ochronotic pigment. In this study, we aimed to define developmental and adult HGD tissue expression and determine the location and amount of gene activity required to lower circulating HGA and rescue the alkaptonuria phenotype. We generated an alkaptonuria mouse model using a knockout-first design for the disruption of the HGD gene. Hgd tm1a -/- mice showed elevated HGA and ochronosis in adulthood. LacZ staining driven by the endogenous HGD promoter was localised to only liver parenchymal cells and kidney proximal tubules in adulthood, commencing at E12.5 and E15.5 respectively. Following removal of the gene trap cassette to obtain a normal mouse with a floxed 6th HGD exon, a double transgenic was then created with Mx1-Cre which conditionally deleted HGD in liver in a dose dependent manner. 20% of HGD mRNA remaining in liver did not rescue the disease, suggesting that we need more than 20% of liver HGD to correct the disease in gene therapy. Kidney HGD activity which remained intact reduced urinary HGA, most likely by increased absorption, but did not reduce plasma HGA nor did it prevent ochronosis. In addition, downstream metabolites of exogenous 13C6-HGA, were detected in heterozygous plasma, revealing that hepatocytes take up and metabolise HGA. This novel alkaptonuria mouse model demonstrated the importance of targeting liver for therapeutic intervention, supported by our observation that hepatocytes take up and metabolise HGA.
2019-05-29 | Control of Alkaptonuria with Nitisinone and Gene Therapy: A Systematic Review
Alkaptonuria (AKU) is a genetic disorder inherited in accordance with Mendel first law. Mutations in the HGA gene result in the AKU disorder. Three major features of this disorder: arthritis, ochronosis, and the presence of Homogentisic Acid (HGA) in the urine. The author searched the PubMed Databases at National Center for Biotechnology Information (NCBI) for articles on AKU published between 2014 and 2019. All articles were open access and in English. In this systematic review, the author included one’s own references and other relevant publications. Search results showed that detection tools for people with AKU can include x-rays and genetic tests. No adequate treatment is available for AKU at present. However, counselors of genetic counseling may help patients with AKU and give counseling to them and their families. Candidate drugs of AKU are nitisinone and genetic manipulation techniques. Research results on the use of nitisinone on AKU have shown remarkable improvements. In the future, genetic manipulation techniques may be beneficial for treating AKU. These techniques are such as modified CRISPR/Cas9 (FokI-dCas9), End-Joining Homology Techniques (EJHTs) and induced Pluripotent Stem Cells (iPSCs).
2016-09-22 | Identification of Homogentisate Dioxygenase as a Target for Vitamin E Biofortification in Oilseeds
Soybean (Glycine max) is a major plant source of protein and oil and produces important secondary metabolites beneficial for human health. As a tool for gene function discovery and improvement of this important crop, a mutant population was generated using fast neutron irradiation. Visual screening of mutagenized seeds identified a mutant line, designated MO12, which produced brown seeds as opposed to the yellow seeds produced by the unmodified Williams 82 parental cultivar. Using forward genetic methods combined with comparative genome hybridization analysis, we were able to establish that deletion of the GmHGO1 gene is the genetic basis of the brown seeded phenotype exhibited by the MO12 mutant line. GmHGO1 encodes a homogentisate dioxygenase (HGO), which catalyzes the committed enzymatic step in homogentisate catabolism. This report describes to our knowledge the first functional characterization of a plant HGO gene, defects of which are linked to the human genetic disease alkaptonuria. We show that reduced homogentisate catabolism in a soybean HGO mutant is an effective strategy for enhancing the production of lipid-soluble antioxidants such as vitamin E, as well as tolerance to herbicides that target pathways associated with homogentisate metabolism. Furthermore, this work demonstrates the utility of fast neutron mutagenesis in identifying novel genes that contribute to soybean agronomic traits.
proteins
2026-06-02 | Liver-directed AAV gene therapy metabolically corrects AKU in Hgd deficient mice
Abstract Background Alkaptonuria (AKU) is a rare autosomal recessive metabolic disorder caused by deficiency of homogentisate 1,2-dioxygenase (HGD), resulting in systemic accumulation of homogentisic acid (HGA), ochronosis, and progressive multisystem disease. Although nitisinone (NTBC) lowers HGA levels, it does not correct the underlying genetic defect and induces hypertyrosinemia, highlighting the need for curative treatment approaches. We evaluated liver-directed adeno-associated virus (AAV)-mediated HGD gene therapy as a potential treatment for AKU. Methods Hgd-deficient ( Hgd -/- ) mice received liver-directed AAV2/8 vectors expressing codon-optimized human HGD under a liver-specific promoter. Reporter vectors were first used to assess hepatic biodistribution and transduction efficiency. Therapeutic efficacy was subsequently evaluated following AAV2/8-HGD administration (1 x 10 12 vg/mouse). HGD expression was assessed by DNAscope, Western blotting, and RT-qPCR. Metabolic correction was determined using targeted LC-MS/MS and untargeted LC-HRMS metabolomics and compared with NTBC-treated Hgd -/- mice. Results Reporter studies demonstrated liver-predominant transduction, with dose-dependent hepatocyte transduction reaching 89-93% at the highest dose. AAV2/8-HGD treatment produced robust hepatic HGD expression, with codon-optimized human HGD transcript levels approximately 33-fold higher than endogenous murine Hgd expression. Twelve weeks after treatment, plasma and urinary HGA levels were significantly reduced, with plasma HGA restored to near wild-type concentrations. Untargeted metabolomics further demonstrated marked reductions in HGA-derived phase I and II metabolites and revealed significant modulation of tricarboxylic acid cycle metabolism, consistent with partial restoration of metabolic homeostasis. Compared with NTBC-treated mice, AAV2/8-HGD achieved comparable plasma HGA reduction without elevation of upstream tyrosine pathway metabolites. Conclusions Liver-directed AAV2/8-HGD gene therapy achieved substantial biochemical correction in Hgd -/- mice and restored metabolic flux without inducing hypertyrosinemia. These findings provide proof-of-concept supporting AAV-mediated HGD replacement as a promising long-term therapeutic strategy for AKU.
2025-07-07 | Competitions for tyrosine breakdown: In synthetic microbial communities and between a gut microbial pathway and a human pathway
ABSTRACT Tyrosine, a versatile amino acid that undergoes diverse transformations to produce both beneficial and detrimental metabolites. The modulation of these metabolites results from direct competition among different metabolic pathways responsible for the breakdown of tyrosine whether it be the competition between distinct microbes or the rivalry between a microbe and its host. The fight between microbes for the available tyrosine might drive potential changes to the communities present in various environments. In contrast, if the similar contest for tyrosine is presented between a gut microbial pathway and a human pathway, it can hold potential to affect the human health. In this work, we present various metabolic outcomes of tyrosine within synthetic microbial communities which are prominently driven by specific enzyme activities of tyrosine breakdown pathways. Additionally, we developed a metabolic diversion of the human tyrosine breakdown pathway facilitated by a gut microbial enzyme. This approach holds promise as a novel strategy to develop potential therapeutic interventions in future for addressing metabolic disorders like tyrosinemias (I, II, III), hawkinsinuria, and alkaptonuria, associated with the human tyrosine breakdown pathway.
2025-01-08 | Exploring the Osteoinductive Potential of Bacterial Pyomelanin Derived from Pseudomonas aeruginosa in a Human Osteoblast Model.
Alkaptonuria (AKU) is a genetically determined disease associated with disorders of tyrosine metabolism. In AKU, the deposition of homogentisic acid polymers contributes to the pathological ossification of cartilage tissue. The controlled use of biomimetics similar to deposits observed in cartilage during AKU potentially may serve the development of new bone regeneration therapy based on the activation of osteoblasts. The proposed biomimetic is pyomelanin (PyoM), a polymeric biomacromolecule synthesized by Pseudomonas aeruginosa. This work presents comprehensive data on the osteoinductive, pro-regenerative, and antibacterial properties, as well as the cytocompatibility, of water-soluble (PyoMsol) or water-insoluble (PyoMinsol) PyoM. Both variants of PyoM support osteoinductive processes as well as the maturation of osteoblasts in cell cultures in vitro due to the upregulation of bone-formation markers, osteocalcin (OC), and alkaline phosphatase (ALP). Furthermore, the cytokines involved in these processes were elevated in cell cultures of osteoblasts exposed to PyoM: tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10. The PyoM variants are cytocompatible in a wide concentration range and limit the doxorubicin-induced apoptosis of osteoblasts. This cytoprotective PyoM activity is correlated with an increased migration of osteoblasts. Moreover, PyoMsol and PyoMinsol exhibit antibacterial activity against staphylococci isolated from infected bones. The osteoinductive, pro-regenerative, and antiapoptotic effects achieved through PyoM stimulation prompt the development of new biocomposites modified with this bacterial biopolymer for medical use.
2024-09-18 | Evaluation of a casein glycomacropeptide-based protein substitute, in the dietary management of NTBC-induced tyrosinaemia in patients with alkaptonuria: A prospective open-label study.
2-(2-Nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) treatment of alkaptonuria (AKU) leads to increased blood tyrosine levels, causing skin issues and potentially sight-threatening corneal keratopathy. Adherence to dietary management of NTBC-induced tyrosinemia, a low-protein diet with or without protein substitutes, can be difficult for patients. This 28-day interventional study evaluated a low tyrosine casein glycomacropeptide (cGMP) protein substitute (TYR sphere)®, a 20 g protein equivalent, cGMP-based protein substitute, in terms of adherence, palatability, usability, comparison to amino acid (AA)-based protein substitutes, gastrointestinal tolerance and metabolic control in adults with NTBC-induced tyrosinaemia. Four adults (mean 61.1 years, range 53.3-69.3 years) with AKU and NTBC-induced tyrosinaemia were recruited from the United Kingdom National Alkaptonuria Centre (NAC). The cGMP protein substitute was prescribed based on individual nutritional requirements, replacing ≥1 AA-based protein substitute. Participants recorded product-related data in study diaries, using five-point Likert scales and daily and weekly logs. To determine metabolic control, prestudy blood tyrosine levels were compared to weekly blood spot tests during the study. Median cGMP protein substitute adherence was 98%. Most participants rated palatability and usability positively, and preferred cGMP protein substitute to AA-based products. There were no notable gastrointestinal changes, and metabolic control was maintained. cGMP protein substitute is a palatable and well-tolerated option in the dietary management of AKU patients with NTBC-induced tyrosinaemia.
2024-03-01 | Abstract 1891 Exploring Tyrosine Metabolism: Examining the implications of competition within a microbial community and the health consequences arising from the rivalry between a gut microbial pathway and the human pathway
Tyrosine, a versatile amino acid crucial for protein synthesis, undergoes diverse conversions leading to both beneficial and detrimental metabolites. The modulation of these metabolite levels results from direct competition among different metabolic pathways responsible for tyrosine breakdown, whether it be the competition between distinct microbes or the rivalry between a microbe and its host. This study aims to understand the breakdown of tyrosine through two distinct scenarios: 1) within a microbial community, and 2) during a competition between a microbe and a host. Our objectives include understanding the mechanisms of these competitions, discerning the metabolic outcomes, and identifying the efficiency of different pathways. To achieve this, we constructed a synthetic microbial community comprising various E. coli strains, each differing solely by the presence of a specific enzyme from a distinct tyrosine metabolic pathway. To assess metabolic outcomes in these communities, we developed a tool employing a phenotypic assay. Our findings reveal that breakdown product levels vary based on the type and combination of metabolic pathways within the community, with the metabolic outcome directly proportional to strain ratios. The methodologies employed in this study include SDS-PAGE analysis, genetic manipulation, colorimetric assays, phenotypic assays, and HPLC. Moreover, the inability to metabolize tyrosine resulting from a flawed tyrosine breakdown pathway in humans contributed to diverse metabolic disorders such as tyrosinemias (I, II, III), hawkinsinuria, and alkaptonuria. Utilizing the tool derived from our experiments on tyrosine breakdown within a microbial community, we illustrate a potential metabolic diversion of the human tyrosine breakdown pathway facilitated by a gut microbial enzyme. This approach holds promise as a novel therapeutic intervention for addressing metabolic disorders associated with the human tyrosine breakdown pathway. We thank the New College of Interdisciplinary Arts and Sciences (NCIAS) at Arizona State University for the funding support.
cell therapies
2025-02-16 | Ochronotic tendinopathy of Achilles tendon-A rare case report
Alkaptonuria, a rare autosomal recessive genetic disorder, resulting in blackish discoloration of urine, connective tissue, sclera, heart valves, blood vessels and skin.We report, a 57 yrs old / Female presented to us with complain of chronic right ankle pain since 9-months. Patient had difficulty in weight bearing and walking. Patient had a history of twisting injury of right ankle 1 month ago, with popping sound.On Examination, palpable gap was felt on Right Tendo Achilles region. O’Brien needle test, Simmonds-Thompson test and Single heel raise test were found to be positive. Intraoperatively Blackish discoloration of Tendo Achilles was seen.Open Trans osseous Tendo Achilles repair with modified Krakow suture and Plantaris reinforcement procedure was done.Postoperatively, laboratory investigations showed an increase in homogentisic acid levels in urine (1025%; normal value
2024-10-01 | An in vitro cell model for exploring inflammatory and amyloidogenic events in alkaptonuria.
Alkaptonuria (AKU) is a progressive systemic inherited metabolic disorder primarily affecting the osteoarticular system, characterized by the degeneration of cartilage induced by ochronosis, ultimately leading to early osteoarthritis (OA). However, investigating AKU pathology in human chondrocytes, which is crucial for understanding the disease, encounters challenges due to limited availability and donor variability. To overcome this obstacle, an in vitro model has been established using homogentisic acid (HGA) to simulate AKU conditions. This model employed immortalized C20/A4 human chondrocytes and serves as a dependable platform for studying AKU pathogenesis. Significantly, the model demonstrates the accumulation of ochronotic pigment in HGA-treated cells, consistent with findings from previous studies. Furthermore, investigations into inflammatory processes during HGA exposure revealed notable oxidative stress, as indicated by elevated levels of reactive oxygen species and lipid peroxidation. Additionally, the model demonstrated HGA-induced inflammatory responses, evidenced by increased production of nitric oxide, overexpression of inducible nitric oxide synthase, and cyclooxygenase-2. These findings underscore the model's utility in studying inflammation associated with AKU. Moreover, analysis of serum amyloid A and serum amyloid P proteins revealed a potential interaction, corroborating evidence of amyloid fibril formation. This hypothesis was further supported by Congo red staining, which showed fibril formation exclusively in HGA-treated cells. Overall, the C20/A4 cell model provided valuable insights into AKU pathogenesis, emphasizing its potential for facilitating drug development and therapeutic interventions.
2005-12-01 | Ochronotic arthropathy: disappearance of alkaptonuria after liver transplantation for hepatitis B-related cirrhosis.
In Brief The deficiency of homogentisic acid oxidase, an enzyme that is mainly found in hepatocytes, is associated with alkaptonuria and ochronosis. We report a patient with clinical and radiologic findings of ochronotic arthropathy in whom alkaptonuria disappeared and the progressive course of the disease stopped after liver transplantation for hepatitis B-related cirrhosis. This patient with classic ochronosis had reversal of the alkaptonuria after liver transplantation, which appears to have replaced the missing homogenetic acid oxidase.
2005-06-28 | A genetically engineered strain of Pseudomonas putida as a useful tool for identifying new therapeutic herbicides.
A genetically engineered strain of Pseudomonas putida U designed for the identification of new therapeutic herbicides has been obtained. In this bacterium, deletion of the homogentisate gene cluster (hmgRABC) confers upon this mutant huge biotechnological possibilities since it can be used: (i) as a target for testing new specific herbicides (p-hydroxy-phenylpyruvate dioxygenase inhibitors); (ii) to identify new therapeutic drugs-effective in the treatment of alkaptonuria and other related tyrosinemia – and (iii) as a source of homogentisic acid in a plant–bacterium association.
2004-07-01 | Bilateral Achilles Tendon Ruptures in a Patient With Ochronosis
We report the case of a 67-year-old man with ochronosis who had bilateral Achilles tendon ruptures. We reconstructed the Achilles tendon using pull-out wiring for the right side and an anchoring system for the left side, and reinforced the repair site using the peroneus brevis tendon for both sides. He could walk without a cane at 3 months postoperatively. Tendon ruptures in patients with ochronosis should be treated as pathologic ruptures because histologic examination reveals that both ends of the ruptured tendon and the insertion site at the calcaneus have extensive black pigment depositions where homogentisic acid and its metabolites have accumulated, and there are no normal collagen bundles present. Even if an Achilles tendon rupture is clinically diagnosed as an acute injury, the ruptured Achilles tendon should be primarily repaired and reinforced with autologous tissue because there are a few viable cells at the ruptured site, and because the tendon ruptures mainly at the insertion site of the calcaneus. Although this is a preliminary report, the short-term result is good and the reconstructed sites have showed no rerupture.
small molecules
2026-06-30 | SMILES-based degree molecular descriptors and machine learning for QSPR modeling of anti-alkaptonuria drugs.
Quantitative Structure-Property Relationship (QSPR) modelling provides an efficient computational framework for predicting physicochemical properties of drug molecules when experimental data are limited. In this study, we investigate the predictive capability of degree-based topological indices (TIs) derived from SMILES (Simplified Molecular Input Line Entry System) representations for modelling physicochemical properties of anti-alkaptonuria drugs. Nine representative compounds, including Nitisinone, Ascorbic Acid, Ibuprofen, Naproxen, Paracetamol, Tramadol, Methotrexate, Sulfasalazine, and Glucosamine, were analysed using several molecular descriptors such as molecular weight, logP, hydrogen bond donors and acceptors, rotatable bonds, and polar surface area. A total of 58 regression models were developed using Linear Regression (LR) and two machine learning algorithms, Random Forest (RF) and Extreme Gradient Boosting (XGBoost, abbreviated XGB). Model performance was evaluated using Mean Absolute Error (MAE), Mean Squared Error (MSE), Root Mean Squared Error (RMSE), and the coefficient of determination R 2 . The results demonstrate that machine learning models significantly outperform classical regression, with XGB achieving the most accurate and stable predictions for the investigated physicochemical properties. This study introduces a machine learning-driven QSPR framework that integrates SMILES-derived degree-based topological indices with ensemble learning techniques for predicting physicochemical properties of anti-alkaptonuria drugs. The proposed approach demonstrates improved predictive performance on small datasets and highlights the effectiveness of combining graph-theoretic molecular descriptors with advanced machine learning methods.
2026-06-12 | Architecture and dynamics of a supramolecular oxygen transport system in human homogentisate 1,2-Dioxygenase.
Alkaptonuria (AKU) is an ultra-rare inherited metabolic disorder caused by impaired activity of homogentisate 1,2-dioxygenase (HGD), a Fe(II)-dependent enzyme that catalyzes the oxidative cleavage of homogentisic acid in the tyrosine degradation pathway. Although high-resolution structures of human HGD have been solved, a fundamental mechanistic question has remained unresolved: how molecular oxygen reaches the deeply buried catalytic iron required for catalysis. Here, we identify a previously unreported AKU-associated HGD variant, c.925G>A (p.G309R), and use it as a mechanistic perturbation reference to dissect the structural determinants of oxygen access. By integrating replicated classical and steered molecular dynamics simulations with transient pocket detection, tunnel mapping, O2 spatial-occupancy analysis, residue-level tunnel composition, and PCA/tICA-based dynamic validation, we identify a structurally accessible and dynamically supported O2-translocation architecture connecting the central pore of the hexameric enzyme to the non-heme Fe(II) active sites. This pathway is not intrinsic to a single subunit but emerges from a cooperative arrangement of residues contributed by three protomers, generating six symmetry-related O2-access routes per hexamer. The G309R substitution perturbs the architecture and continuity of this tunnel system, providing a mechanistic explanation for enzyme dysfunction without evidence of active-site structural perturbation or global destabilization. Together, our findings support oxygen-tunnel integrity as a previously unrecognized mechanistic requirement for human HGD activity and introduce disruption of oxygen trafficking as an additional pathogenic mode in AKU, with implications for structure-guided variant interpretation and precision-medicine strategies.
2026-06-03 | Deciphering and Improving Human Homogentisate 1,2-Dioxygenase Function Through Knowledge Gaining Directed Evolution: Implications for Alkaptonuria
Abstract Human homogentisate 1,2-dioxygenase (HGD) catalyses the oxidative cleavage of homogentisic acid (HGA) to maleylacetoacetate (MAA), a key step in tyrosine degradation. Loss of HGD activity causes alkaptonuria (AKU), a rare inherited metabolic disorder characterized by toxic HGA accumulation. Current therapy with nitisinone lowers HGA levels but does not restore HGD function, motivating further investigation of HGD structure-function relationships. In this study, we applied the Knowledge Gaining Directed Evolution (KnowVolution) strategy to investigate how amino acid substitutions influence catalytic activity and structural integrity of human HGD. Catalytic activity was evaluated in Escherichia coli using an assay quantifying MAA formation over time. Across four KnowVolution phases, multiple substitutions were identified that modulated catalytic activity while preserving enzyme function. Notably, none of the influential substitutions were located within the catalytic pocket; instead, they occurred predominantly at surface-exposed or structural positions. Structural mapping, interface analysis, and computational stability predictions indicated that some substitutions contribute to hexamer stabilization, whereas others likely alter activity through indirect, non-catalytic mechanisms involving pocket remodelling. Combined substitutions showed non-additive effects that were either cooperative or antagonistic, demonstrating that their impact could not be predicted from individual contributions. Tunnel and pocket analyses showed that N31S, S54D and D86H produced a more compact hexamer, whereas a Q354P+P359E double mutant reduced catalytic pocket solvent accessibility and volume, supporting the observed activity differences. Overall, these findings demonstrate that HGD activity can be modulated by substitutions outside the catalytic pocket, providing new insight into HGD function and genotype-phenotype relationships underlying AKU.
2026-03-01 | 1466: AGGRESSIVE MULTIMODAL MANAGEMENT OF ALKAPTONURIA-INDUCED HEMOLYSIS AND METHEMOGLOBINEMIA
Introduction: Alkaptonuria (AKU) is a rare disorder of the tyrosine metabolic pathway that results in significant accumulation of homogentisic acid (HGA). A fatal complication of AKU is the development of hemolysis and methemoglobinemia. The pathophysiology is thought to be secondary to HGA buildup alongside its oxidative metabolite benzoquinone acetic acid that causes the oxidization of hemoglobin to methemoglobin. This rare and fatal phenomenon has only been described six other times in literature. Description: The patient is a 66 y/o male with PMHx of HFpEF, COPD, AKU who initially presented with a CHF exacerbation. He developed an AKI during diuresis requiring HD. Seven days into his admission he developed hypoxia. An ABG was drawn that showed dark chocolate-colored blood with PO2 of 346. Methemoglobin level was too high to be calculated. LDH was 1,093, haptoglobin undetectably low, and potassium was 6.5, consistent with hemolysis. The patient was started on CVVH. Peripheral smear showed anisopoikilocytosis with bite and blister cells consistent with oxidative hemolysis. The patient was given methylene blue with improvement in his methemoglobin level to 45.4%. The patient was given N-acetylcysteine and high dose ascorbic acid for antioxidant affects. Both plasma exchange and RBC exchange transfusion were utilized to treat the hemolytic anemia. Methemoglobin levels normalized and the patient’s condition stabilized. The orphan drug Nitisinone was emergently obtained. The patient developed a severe ischemic hepatocellular injury 36 hours after presentation resulting in overwhelming vasoplegic shock. He was compassionately extubated and passed shortly after. Discussion: Previous patients have been treated with a combination of antioxidants, methylene blue, and transfusions. Our patient’s condition was rapidly identified and aggressively treated with methylene blue, CVVH, ascorbic acid, NAC, plasma exchange, and exchange transfusion with significant improvement in his condition. Nitisinone, an inhibitor of an upstream enzyme that reduces the production of HGA, was also obtained and started within 24 hours. This combination of therapies has not been reported in literature. We suspect that the combination of our treatments was successful, and it was the initial ischemic injury that led to his demise.
2026-01-27 | Effect of Nitisinone on Aortic Stenosis Disease Progression in Patients With Alkaptonuria: An Analysis of the Suitability of Nitisinone in Alkaptonuria (SONIA) 2 Study.
Background and aim Alkaptonuria (AKU) is a rare metabolic disorder characterised by the accumulation of homogentisic acid (HGA). Deposition of HGA in the aortic valve leading to progressive aortic stenosis is a serious complication. Nitisinone has been shown to improve morbidity and slow disease progression in AKU, but the effects of this treatment on aortic stenosis progression have not yet been described. The objective of this study was to evaluate whether treatment with nitisinone attenuated the progression of aortic stenosis, as assessed by peak trans-aortic valve pressure (Pmax), in patients with AKU. This post-hoc analysis used longitudinal echocardiographic data from the Suitability of Nitisinone in Alkaptonuria (SONIA) 2, a four-year multicenter randomised controlled trial, to examine aortic stenosis disease progression. Methods Data were obtained from echocardiograms performed on 138 patients over 48 months of follow-up. A linear mixed-effects regression model was used to ascertain the difference in the maximal trans-aortic valve pressure gradient (Pmax) at baseline and 48 months between the treatment and control groups, adjusting for baseline Pmax and other covariates. Results At baseline, 18/138 patients (13.0%) had aortic stenosis of varying degrees of severity, and 25/138 (18.1%) had aortic sclerosis. The difference in Pmax between the control (N=69) and treatment (N=69) groups at baseline was 0.063 mmHg [95% CI: -0.054 mmHg to 0.18 mmHg) and did not reach statistical significance (p=0.23). At the end of the four-year treatment period, the difference in Pmax was 0.10 mmHg (95% CI: -0.0007 mmHg to 0.20 mmHg) (p = 0.05), representing a modest but statistically significant between-group treatment effect. Conclusion Nitisinone may attenuate the progression of aortic stenosis in patients with AKU. Given the small absolute effect size and post-hoc nature of the analysis, these findings should be interpreted as exploratory and hypothesis-generating rather than clinically definitive. Additional research is needed to determine whether nitisinone produces clinically meaningful outcomes for aortic stenosis in this population.
gene therapies
2025-11-11 | Inborn Errors of Amino Acid Metabolism Revisited: Clinical Implications and Insights into Current Therapies
Inborn errors of amino acid metabolism (IEAAMs) are a heterogeneous group of genetic disorders caused by defects in enzymes, cofactors, or transporters of amino acid catabolism, biosynthesis, or transport. These defects result in toxic metabolite accumulation and/or deficiency of essential metabolites. This review aims to provide an updated overview of diagnosis, clinical implications, management, and evolving therapeutic approaches across major IEAAMs. A narrative review of recent literature was undertaken, focusing on established and novel therapeutic strategies for key IEAAMs, including phenylketonuria, alkaptonuria, tyrosinemia, homocystinuria, and maple syrup urine disease. Key management strategies include amino acid-restricted diets/restriction of natural protein with restriction of dietary precursors, dietary supplementations, including disease-specific amino acid supplements, medications to reduce formation of offending metabolites, pharmacotherapies, enzyme/cofactor replacement or pharmacological chaperones, enhancing residual enzyme activity and promoting alternative path-ways/accessory pathways. Emergency therapy is essential in severe types and focuses on promoting anabolism, limiting catabolism, reducing formation, and enhancing clearance of toxic metabolites. Other treatment options include organ transplantation, and new emerging modalities, such as mRNA therapies and gene therapies/in vivo gene editing offer potential for definitive interventions. Despite advancements in therapy and close monitoring, many IEAAMs remain associated with significant comorbidities. Future research is essential to optimise current treatment standards, particularly neuroprotective and metabolic regulatory features. While an in-depth discussion of innovative person-alised therapies is beyond the scope of this article, we believe that collective experiences will thrust future research in this field and expand access to innovative personalised therapies.
2025-04-17 | A Comprehensive In Vitro and In Silico Approach for Targeting 4-Hydroxyphenyl Pyruvate Dioxygenase: Towards New Therapeutics for Alkaptonuria.
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.
2020-06-01 | Conditional targeting in mice reveals that hepatic homogentisate 1,2-dioxygenase activity is essential in reducing circulating homogentisic acid and for effective therapy in the genetic disease alkaptonuria.
Alkaptonuria is an inherited disease caused by homogentisate 1,2-dioxygenase (HGD) deficiency. Circulating homogentisic acid (HGA) is elevated and deposits in connective tissues as ochronotic pigment. In this study, we aimed to define developmental and adult HGD tissue expression and determine the location and amount of gene activity required to lower circulating HGA and rescue the alkaptonuria phenotype. We generated an alkaptonuria mouse model using a knockout-first design for the disruption of the HGD gene. Hgd tm1a -/- mice showed elevated HGA and ochronosis in adulthood. LacZ staining driven by the endogenous HGD promoter was localised to only liver parenchymal cells and kidney proximal tubules in adulthood, commencing at E12.5 and E15.5 respectively. Following removal of the gene trap cassette to obtain a normal mouse with a floxed 6th HGD exon, a double transgenic was then created with Mx1-Cre which conditionally deleted HGD in liver in a dose dependent manner. 20% of HGD mRNA remaining in liver did not rescue the disease, suggesting that we need more than 20% of liver HGD to correct the disease in gene therapy. Kidney HGD activity which remained intact reduced urinary HGA, most likely by increased absorption, but did not reduce plasma HGA nor did it prevent ochronosis. In addition, downstream metabolites of exogenous 13C6-HGA, were detected in heterozygous plasma, revealing that hepatocytes take up and metabolise HGA. This novel alkaptonuria mouse model demonstrated the importance of targeting liver for therapeutic intervention, supported by our observation that hepatocytes take up and metabolise HGA.
2019-05-29 | Control of Alkaptonuria with Nitisinone and Gene Therapy: A Systematic Review
Alkaptonuria (AKU) is a genetic disorder inherited in accordance with Mendel first law. Mutations in the HGA gene result in the AKU disorder. Three major features of this disorder: arthritis, ochronosis, and the presence of Homogentisic Acid (HGA) in the urine. The author searched the PubMed Databases at National Center for Biotechnology Information (NCBI) for articles on AKU published between 2014 and 2019. All articles were open access and in English. In this systematic review, the author included one’s own references and other relevant publications. Search results showed that detection tools for people with AKU can include x-rays and genetic tests. No adequate treatment is available for AKU at present. However, counselors of genetic counseling may help patients with AKU and give counseling to them and their families. Candidate drugs of AKU are nitisinone and genetic manipulation techniques. Research results on the use of nitisinone on AKU have shown remarkable improvements. In the future, genetic manipulation techniques may be beneficial for treating AKU. These techniques are such as modified CRISPR/Cas9 (FokI-dCas9), End-Joining Homology Techniques (EJHTs) and induced Pluripotent Stem Cells (iPSCs).
2016-09-22 | Identification of Homogentisate Dioxygenase as a Target for Vitamin E Biofortification in Oilseeds
Soybean (Glycine max) is a major plant source of protein and oil and produces important secondary metabolites beneficial for human health. As a tool for gene function discovery and improvement of this important crop, a mutant population was generated using fast neutron irradiation. Visual screening of mutagenized seeds identified a mutant line, designated MO12, which produced brown seeds as opposed to the yellow seeds produced by the unmodified Williams 82 parental cultivar. Using forward genetic methods combined with comparative genome hybridization analysis, we were able to establish that deletion of the GmHGO1 gene is the genetic basis of the brown seeded phenotype exhibited by the MO12 mutant line. GmHGO1 encodes a homogentisate dioxygenase (HGO), which catalyzes the committed enzymatic step in homogentisate catabolism. This report describes to our knowledge the first functional characterization of a plant HGO gene, defects of which are linked to the human genetic disease alkaptonuria. We show that reduced homogentisate catabolism in a soybean HGO mutant is an effective strategy for enhancing the production of lipid-soluble antioxidants such as vitamin E, as well as tolerance to herbicides that target pathways associated with homogentisate metabolism. Furthermore, this work demonstrates the utility of fast neutron mutagenesis in identifying novel genes that contribute to soybean agronomic traits.
proteins
2026-06-02 | Liver-directed AAV gene therapy metabolically corrects AKU in Hgd deficient mice
Abstract Background Alkaptonuria (AKU) is a rare autosomal recessive metabolic disorder caused by deficiency of homogentisate 1,2-dioxygenase (HGD), resulting in systemic accumulation of homogentisic acid (HGA), ochronosis, and progressive multisystem disease. Although nitisinone (NTBC) lowers HGA levels, it does not correct the underlying genetic defect and induces hypertyrosinemia, highlighting the need for curative treatment approaches. We evaluated liver-directed adeno-associated virus (AAV)-mediated HGD gene therapy as a potential treatment for AKU. Methods Hgd-deficient ( Hgd -/- ) mice received liver-directed AAV2/8 vectors expressing codon-optimized human HGD under a liver-specific promoter. Reporter vectors were first used to assess hepatic biodistribution and transduction efficiency. Therapeutic efficacy was subsequently evaluated following AAV2/8-HGD administration (1 x 10 12 vg/mouse). HGD expression was assessed by DNAscope, Western blotting, and RT-qPCR. Metabolic correction was determined using targeted LC-MS/MS and untargeted LC-HRMS metabolomics and compared with NTBC-treated Hgd -/- mice. Results Reporter studies demonstrated liver-predominant transduction, with dose-dependent hepatocyte transduction reaching 89-93% at the highest dose. AAV2/8-HGD treatment produced robust hepatic HGD expression, with codon-optimized human HGD transcript levels approximately 33-fold higher than endogenous murine Hgd expression. Twelve weeks after treatment, plasma and urinary HGA levels were significantly reduced, with plasma HGA restored to near wild-type concentrations. Untargeted metabolomics further demonstrated marked reductions in HGA-derived phase I and II metabolites and revealed significant modulation of tricarboxylic acid cycle metabolism, consistent with partial restoration of metabolic homeostasis. Compared with NTBC-treated mice, AAV2/8-HGD achieved comparable plasma HGA reduction without elevation of upstream tyrosine pathway metabolites. Conclusions Liver-directed AAV2/8-HGD gene therapy achieved substantial biochemical correction in Hgd -/- mice and restored metabolic flux without inducing hypertyrosinemia. These findings provide proof-of-concept supporting AAV-mediated HGD replacement as a promising long-term therapeutic strategy for AKU.
2025-07-07 | Competitions for tyrosine breakdown: In synthetic microbial communities and between a gut microbial pathway and a human pathway
ABSTRACT Tyrosine, a versatile amino acid that undergoes diverse transformations to produce both beneficial and detrimental metabolites. The modulation of these metabolites results from direct competition among different metabolic pathways responsible for the breakdown of tyrosine whether it be the competition between distinct microbes or the rivalry between a microbe and its host. The fight between microbes for the available tyrosine might drive potential changes to the communities present in various environments. In contrast, if the similar contest for tyrosine is presented between a gut microbial pathway and a human pathway, it can hold potential to affect the human health. In this work, we present various metabolic outcomes of tyrosine within synthetic microbial communities which are prominently driven by specific enzyme activities of tyrosine breakdown pathways. Additionally, we developed a metabolic diversion of the human tyrosine breakdown pathway facilitated by a gut microbial enzyme. This approach holds promise as a novel strategy to develop potential therapeutic interventions in future for addressing metabolic disorders like tyrosinemias (I, II, III), hawkinsinuria, and alkaptonuria, associated with the human tyrosine breakdown pathway.
2025-01-08 | Exploring the Osteoinductive Potential of Bacterial Pyomelanin Derived from Pseudomonas aeruginosa in a Human Osteoblast Model.
Alkaptonuria (AKU) is a genetically determined disease associated with disorders of tyrosine metabolism. In AKU, the deposition of homogentisic acid polymers contributes to the pathological ossification of cartilage tissue. The controlled use of biomimetics similar to deposits observed in cartilage during AKU potentially may serve the development of new bone regeneration therapy based on the activation of osteoblasts. The proposed biomimetic is pyomelanin (PyoM), a polymeric biomacromolecule synthesized by Pseudomonas aeruginosa. This work presents comprehensive data on the osteoinductive, pro-regenerative, and antibacterial properties, as well as the cytocompatibility, of water-soluble (PyoMsol) or water-insoluble (PyoMinsol) PyoM. Both variants of PyoM support osteoinductive processes as well as the maturation of osteoblasts in cell cultures in vitro due to the upregulation of bone-formation markers, osteocalcin (OC), and alkaline phosphatase (ALP). Furthermore, the cytokines involved in these processes were elevated in cell cultures of osteoblasts exposed to PyoM: tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10. The PyoM variants are cytocompatible in a wide concentration range and limit the doxorubicin-induced apoptosis of osteoblasts. This cytoprotective PyoM activity is correlated with an increased migration of osteoblasts. Moreover, PyoMsol and PyoMinsol exhibit antibacterial activity against staphylococci isolated from infected bones. The osteoinductive, pro-regenerative, and antiapoptotic effects achieved through PyoM stimulation prompt the development of new biocomposites modified with this bacterial biopolymer for medical use.
2024-09-18 | Evaluation of a casein glycomacropeptide-based protein substitute, in the dietary management of NTBC-induced tyrosinaemia in patients with alkaptonuria: A prospective open-label study.
2-(2-Nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) treatment of alkaptonuria (AKU) leads to increased blood tyrosine levels, causing skin issues and potentially sight-threatening corneal keratopathy. Adherence to dietary management of NTBC-induced tyrosinemia, a low-protein diet with or without protein substitutes, can be difficult for patients. This 28-day interventional study evaluated a low tyrosine casein glycomacropeptide (cGMP) protein substitute (TYR sphere)®, a 20 g protein equivalent, cGMP-based protein substitute, in terms of adherence, palatability, usability, comparison to amino acid (AA)-based protein substitutes, gastrointestinal tolerance and metabolic control in adults with NTBC-induced tyrosinaemia. Four adults (mean 61.1 years, range 53.3-69.3 years) with AKU and NTBC-induced tyrosinaemia were recruited from the United Kingdom National Alkaptonuria Centre (NAC). The cGMP protein substitute was prescribed based on individual nutritional requirements, replacing ≥1 AA-based protein substitute. Participants recorded product-related data in study diaries, using five-point Likert scales and daily and weekly logs. To determine metabolic control, prestudy blood tyrosine levels were compared to weekly blood spot tests during the study. Median cGMP protein substitute adherence was 98%. Most participants rated palatability and usability positively, and preferred cGMP protein substitute to AA-based products. There were no notable gastrointestinal changes, and metabolic control was maintained. cGMP protein substitute is a palatable and well-tolerated option in the dietary management of AKU patients with NTBC-induced tyrosinaemia.
2024-03-01 | Abstract 1891 Exploring Tyrosine Metabolism: Examining the implications of competition within a microbial community and the health consequences arising from the rivalry between a gut microbial pathway and the human pathway
Tyrosine, a versatile amino acid crucial for protein synthesis, undergoes diverse conversions leading to both beneficial and detrimental metabolites. The modulation of these metabolite levels results from direct competition among different metabolic pathways responsible for tyrosine breakdown, whether it be the competition between distinct microbes or the rivalry between a microbe and its host. This study aims to understand the breakdown of tyrosine through two distinct scenarios: 1) within a microbial community, and 2) during a competition between a microbe and a host. Our objectives include understanding the mechanisms of these competitions, discerning the metabolic outcomes, and identifying the efficiency of different pathways. To achieve this, we constructed a synthetic microbial community comprising various E. coli strains, each differing solely by the presence of a specific enzyme from a distinct tyrosine metabolic pathway. To assess metabolic outcomes in these communities, we developed a tool employing a phenotypic assay. Our findings reveal that breakdown product levels vary based on the type and combination of metabolic pathways within the community, with the metabolic outcome directly proportional to strain ratios. The methodologies employed in this study include SDS-PAGE analysis, genetic manipulation, colorimetric assays, phenotypic assays, and HPLC. Moreover, the inability to metabolize tyrosine resulting from a flawed tyrosine breakdown pathway in humans contributed to diverse metabolic disorders such as tyrosinemias (I, II, III), hawkinsinuria, and alkaptonuria. Utilizing the tool derived from our experiments on tyrosine breakdown within a microbial community, we illustrate a potential metabolic diversion of the human tyrosine breakdown pathway facilitated by a gut microbial enzyme. This approach holds promise as a novel therapeutic intervention for addressing metabolic disorders associated with the human tyrosine breakdown pathway. We thank the New College of Interdisciplinary Arts and Sciences (NCIAS) at Arizona State University for the funding support.
cell therapies
2025-02-16 | Ochronotic tendinopathy of Achilles tendon-A rare case report
Alkaptonuria, a rare autosomal recessive genetic disorder, resulting in blackish discoloration of urine, connective tissue, sclera, heart valves, blood vessels and skin.We report, a 57 yrs old / Female presented to us with complain of chronic right ankle pain since 9-months. Patient had difficulty in weight bearing and walking. Patient had a history of twisting injury of right ankle 1 month ago, with popping sound.On Examination, palpable gap was felt on Right Tendo Achilles region. O’Brien needle test, Simmonds-Thompson test and Single heel raise test were found to be positive. Intraoperatively Blackish discoloration of Tendo Achilles was seen.Open Trans osseous Tendo Achilles repair with modified Krakow suture and Plantaris reinforcement procedure was done.Postoperatively, laboratory investigations showed an increase in homogentisic acid levels in urine (1025%; normal value
2024-10-01 | An in vitro cell model for exploring inflammatory and amyloidogenic events in alkaptonuria.
Alkaptonuria (AKU) is a progressive systemic inherited metabolic disorder primarily affecting the osteoarticular system, characterized by the degeneration of cartilage induced by ochronosis, ultimately leading to early osteoarthritis (OA). However, investigating AKU pathology in human chondrocytes, which is crucial for understanding the disease, encounters challenges due to limited availability and donor variability. To overcome this obstacle, an in vitro model has been established using homogentisic acid (HGA) to simulate AKU conditions. This model employed immortalized C20/A4 human chondrocytes and serves as a dependable platform for studying AKU pathogenesis. Significantly, the model demonstrates the accumulation of ochronotic pigment in HGA-treated cells, consistent with findings from previous studies. Furthermore, investigations into inflammatory processes during HGA exposure revealed notable oxidative stress, as indicated by elevated levels of reactive oxygen species and lipid peroxidation. Additionally, the model demonstrated HGA-induced inflammatory responses, evidenced by increased production of nitric oxide, overexpression of inducible nitric oxide synthase, and cyclooxygenase-2. These findings underscore the model's utility in studying inflammation associated with AKU. Moreover, analysis of serum amyloid A and serum amyloid P proteins revealed a potential interaction, corroborating evidence of amyloid fibril formation. This hypothesis was further supported by Congo red staining, which showed fibril formation exclusively in HGA-treated cells. Overall, the C20/A4 cell model provided valuable insights into AKU pathogenesis, emphasizing its potential for facilitating drug development and therapeutic interventions.
2005-12-01 | Ochronotic arthropathy: disappearance of alkaptonuria after liver transplantation for hepatitis B-related cirrhosis.
In Brief The deficiency of homogentisic acid oxidase, an enzyme that is mainly found in hepatocytes, is associated with alkaptonuria and ochronosis. We report a patient with clinical and radiologic findings of ochronotic arthropathy in whom alkaptonuria disappeared and the progressive course of the disease stopped after liver transplantation for hepatitis B-related cirrhosis. This patient with classic ochronosis had reversal of the alkaptonuria after liver transplantation, which appears to have replaced the missing homogenetic acid oxidase.
2005-06-28 | A genetically engineered strain of Pseudomonas putida as a useful tool for identifying new therapeutic herbicides.
A genetically engineered strain of Pseudomonas putida U designed for the identification of new therapeutic herbicides has been obtained. In this bacterium, deletion of the homogentisate gene cluster (hmgRABC) confers upon this mutant huge biotechnological possibilities since it can be used: (i) as a target for testing new specific herbicides (p-hydroxy-phenylpyruvate dioxygenase inhibitors); (ii) to identify new therapeutic drugs-effective in the treatment of alkaptonuria and other related tyrosinemia – and (iii) as a source of homogentisic acid in a plant–bacterium association.
2004-07-01 | Bilateral Achilles Tendon Ruptures in a Patient With Ochronosis
We report the case of a 67-year-old man with ochronosis who had bilateral Achilles tendon ruptures. We reconstructed the Achilles tendon using pull-out wiring for the right side and an anchoring system for the left side, and reinforced the repair site using the peroneus brevis tendon for both sides. He could walk without a cane at 3 months postoperatively. Tendon ruptures in patients with ochronosis should be treated as pathologic ruptures because histologic examination reveals that both ends of the ruptured tendon and the insertion site at the calcaneus have extensive black pigment depositions where homogentisic acid and its metabolites have accumulated, and there are no normal collagen bundles present. Even if an Achilles tendon rupture is clinically diagnosed as an acute injury, the ruptured Achilles tendon should be primarily repaired and reinforced with autologous tissue because there are a few viable cells at the ruptured site, and because the tendon ruptures mainly at the insertion site of the calcaneus. Although this is a preliminary report, the short-term result is good and the reconstructed sites have showed no rerupture.
small molecules
2026-06-30 | SMILES-based degree molecular descriptors and machine learning for QSPR modeling of anti-alkaptonuria drugs.
Quantitative Structure-Property Relationship (QSPR) modelling provides an efficient computational framework for predicting physicochemical properties of drug molecules when experimental data are limited. In this study, we investigate the predictive capability of degree-based topological indices (TIs) derived from SMILES (Simplified Molecular Input Line Entry System) representations for modelling physicochemical properties of anti-alkaptonuria drugs. Nine representative compounds, including Nitisinone, Ascorbic Acid, Ibuprofen, Naproxen, Paracetamol, Tramadol, Methotrexate, Sulfasalazine, and Glucosamine, were analysed using several molecular descriptors such as molecular weight, logP, hydrogen bond donors and acceptors, rotatable bonds, and polar surface area. A total of 58 regression models were developed using Linear Regression (LR) and two machine learning algorithms, Random Forest (RF) and Extreme Gradient Boosting (XGBoost, abbreviated XGB). Model performance was evaluated using Mean Absolute Error (MAE), Mean Squared Error (MSE), Root Mean Squared Error (RMSE), and the coefficient of determination R 2 . The results demonstrate that machine learning models significantly outperform classical regression, with XGB achieving the most accurate and stable predictions for the investigated physicochemical properties. This study introduces a machine learning-driven QSPR framework that integrates SMILES-derived degree-based topological indices with ensemble learning techniques for predicting physicochemical properties of anti-alkaptonuria drugs. The proposed approach demonstrates improved predictive performance on small datasets and highlights the effectiveness of combining graph-theoretic molecular descriptors with advanced machine learning methods.
2026-06-12 | Architecture and dynamics of a supramolecular oxygen transport system in human homogentisate 1,2-Dioxygenase.
Alkaptonuria (AKU) is an ultra-rare inherited metabolic disorder caused by impaired activity of homogentisate 1,2-dioxygenase (HGD), a Fe(II)-dependent enzyme that catalyzes the oxidative cleavage of homogentisic acid in the tyrosine degradation pathway. Although high-resolution structures of human HGD have been solved, a fundamental mechanistic question has remained unresolved: how molecular oxygen reaches the deeply buried catalytic iron required for catalysis. Here, we identify a previously unreported AKU-associated HGD variant, c.925G>A (p.G309R), and use it as a mechanistic perturbation reference to dissect the structural determinants of oxygen access. By integrating replicated classical and steered molecular dynamics simulations with transient pocket detection, tunnel mapping, O2 spatial-occupancy analysis, residue-level tunnel composition, and PCA/tICA-based dynamic validation, we identify a structurally accessible and dynamically supported O2-translocation architecture connecting the central pore of the hexameric enzyme to the non-heme Fe(II) active sites. This pathway is not intrinsic to a single subunit but emerges from a cooperative arrangement of residues contributed by three protomers, generating six symmetry-related O2-access routes per hexamer. The G309R substitution perturbs the architecture and continuity of this tunnel system, providing a mechanistic explanation for enzyme dysfunction without evidence of active-site structural perturbation or global destabilization. Together, our findings support oxygen-tunnel integrity as a previously unrecognized mechanistic requirement for human HGD activity and introduce disruption of oxygen trafficking as an additional pathogenic mode in AKU, with implications for structure-guided variant interpretation and precision-medicine strategies.
2026-06-03 | Deciphering and Improving Human Homogentisate 1,2-Dioxygenase Function Through Knowledge Gaining Directed Evolution: Implications for Alkaptonuria
Abstract Human homogentisate 1,2-dioxygenase (HGD) catalyses the oxidative cleavage of homogentisic acid (HGA) to maleylacetoacetate (MAA), a key step in tyrosine degradation. Loss of HGD activity causes alkaptonuria (AKU), a rare inherited metabolic disorder characterized by toxic HGA accumulation. Current therapy with nitisinone lowers HGA levels but does not restore HGD function, motivating further investigation of HGD structure-function relationships. In this study, we applied the Knowledge Gaining Directed Evolution (KnowVolution) strategy to investigate how amino acid substitutions influence catalytic activity and structural integrity of human HGD. Catalytic activity was evaluated in Escherichia coli using an assay quantifying MAA formation over time. Across four KnowVolution phases, multiple substitutions were identified that modulated catalytic activity while preserving enzyme function. Notably, none of the influential substitutions were located within the catalytic pocket; instead, they occurred predominantly at surface-exposed or structural positions. Structural mapping, interface analysis, and computational stability predictions indicated that some substitutions contribute to hexamer stabilization, whereas others likely alter activity through indirect, non-catalytic mechanisms involving pocket remodelling. Combined substitutions showed non-additive effects that were either cooperative or antagonistic, demonstrating that their impact could not be predicted from individual contributions. Tunnel and pocket analyses showed that N31S, S54D and D86H produced a more compact hexamer, whereas a Q354P+P359E double mutant reduced catalytic pocket solvent accessibility and volume, supporting the observed activity differences. Overall, these findings demonstrate that HGD activity can be modulated by substitutions outside the catalytic pocket, providing new insight into HGD function and genotype-phenotype relationships underlying AKU.
2026-03-01 | 1466: AGGRESSIVE MULTIMODAL MANAGEMENT OF ALKAPTONURIA-INDUCED HEMOLYSIS AND METHEMOGLOBINEMIA
Introduction: Alkaptonuria (AKU) is a rare disorder of the tyrosine metabolic pathway that results in significant accumulation of homogentisic acid (HGA). A fatal complication of AKU is the development of hemolysis and methemoglobinemia. The pathophysiology is thought to be secondary to HGA buildup alongside its oxidative metabolite benzoquinone acetic acid that causes the oxidization of hemoglobin to methemoglobin. This rare and fatal phenomenon has only been described six other times in literature. Description: The patient is a 66 y/o male with PMHx of HFpEF, COPD, AKU who initially presented with a CHF exacerbation. He developed an AKI during diuresis requiring HD. Seven days into his admission he developed hypoxia. An ABG was drawn that showed dark chocolate-colored blood with PO2 of 346. Methemoglobin level was too high to be calculated. LDH was 1,093, haptoglobin undetectably low, and potassium was 6.5, consistent with hemolysis. The patient was started on CVVH. Peripheral smear showed anisopoikilocytosis with bite and blister cells consistent with oxidative hemolysis. The patient was given methylene blue with improvement in his methemoglobin level to 45.4%. The patient was given N-acetylcysteine and high dose ascorbic acid for antioxidant affects. Both plasma exchange and RBC exchange transfusion were utilized to treat the hemolytic anemia. Methemoglobin levels normalized and the patient’s condition stabilized. The orphan drug Nitisinone was emergently obtained. The patient developed a severe ischemic hepatocellular injury 36 hours after presentation resulting in overwhelming vasoplegic shock. He was compassionately extubated and passed shortly after. Discussion: Previous patients have been treated with a combination of antioxidants, methylene blue, and transfusions. Our patient’s condition was rapidly identified and aggressively treated with methylene blue, CVVH, ascorbic acid, NAC, plasma exchange, and exchange transfusion with significant improvement in his condition. Nitisinone, an inhibitor of an upstream enzyme that reduces the production of HGA, was also obtained and started within 24 hours. This combination of therapies has not been reported in literature. We suspect that the combination of our treatments was successful, and it was the initial ischemic injury that led to his demise.
2026-01-27 | Effect of Nitisinone on Aortic Stenosis Disease Progression in Patients With Alkaptonuria: An Analysis of the Suitability of Nitisinone in Alkaptonuria (SONIA) 2 Study.
Background and aim Alkaptonuria (AKU) is a rare metabolic disorder characterised by the accumulation of homogentisic acid (HGA). Deposition of HGA in the aortic valve leading to progressive aortic stenosis is a serious complication. Nitisinone has been shown to improve morbidity and slow disease progression in AKU, but the effects of this treatment on aortic stenosis progression have not yet been described. The objective of this study was to evaluate whether treatment with nitisinone attenuated the progression of aortic stenosis, as assessed by peak trans-aortic valve pressure (Pmax), in patients with AKU. This post-hoc analysis used longitudinal echocardiographic data from the Suitability of Nitisinone in Alkaptonuria (SONIA) 2, a four-year multicenter randomised controlled trial, to examine aortic stenosis disease progression. Methods Data were obtained from echocardiograms performed on 138 patients over 48 months of follow-up. A linear mixed-effects regression model was used to ascertain the difference in the maximal trans-aortic valve pressure gradient (Pmax) at baseline and 48 months between the treatment and control groups, adjusting for baseline Pmax and other covariates. Results At baseline, 18/138 patients (13.0%) had aortic stenosis of varying degrees of severity, and 25/138 (18.1%) had aortic sclerosis. The difference in Pmax between the control (N=69) and treatment (N=69) groups at baseline was 0.063 mmHg [95% CI: -0.054 mmHg to 0.18 mmHg) and did not reach statistical significance (p=0.23). At the end of the four-year treatment period, the difference in Pmax was 0.10 mmHg (95% CI: -0.0007 mmHg to 0.20 mmHg) (p = 0.05), representing a modest but statistically significant between-group treatment effect. Conclusion Nitisinone may attenuate the progression of aortic stenosis in patients with AKU. Given the small absolute effect size and post-hoc nature of the analysis, these findings should be interpreted as exploratory and hypothesis-generating rather than clinically definitive. Additional research is needed to determine whether nitisinone produces clinically meaningful outcomes for aortic stenosis in this population.
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Drug Discovery Landscape
4 orphan drug designations for Alkaptonuria, including 1 approved therapy.
4 orphan drug designations for Alkaptonuria, including 1 approved therapy.
Drug | Therapy type | Regulator | Orphan designation | Approval | Sponsor |
|---|---|---|---|---|---|
nitisinone [Harliku] | small molecules | FDA | 2023-06-12 | 2025-06-10 | Cycle Pharmaceuticals Ltd. |
Methotrexate | small molecules | EMA | 2016-08-29 | — | aimAKU (Associazione Italiana Malati di Alcaptonuria) |
Nitisinone | small molecules | EMA | 2002-03-13 | — | Swedish Orphan Biovitrum AB (publ) |
nitisinone | small molecules | FDA | 2001-10-19 | — | Swedish Orphan Biovitrum AB (publ) |
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