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Primary hyperoxaluria type I (PH1) is a rare kidney disease due to the deficit of alanine:glyoxylate aminotransferase (AGT), a pyridoxal-5'-phosphate-dependent enzyme responsible for liver glyoxylate detoxification, which in turn prevents oxalate formation and precipitation as kidney stones. Many PH1-associated missense mutations cause AGT misfoldi...
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... RESULTS AND DISCUSSION Selection of Commercially Available AGT Ligands. In the search for a specific AGT ligand effective as PC, we started from a known hit compound (compound 1, Figure 1). 23 The scaffold of compound 1 was used to launch a small-size screening campaign of commercially available molecules that were tested in silico for their putative ability to bind at the AGT active site. ...
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... The scaffold of compound 1 was used to launch a small-size screening campaign of commercially available molecules that were tested in silico for their putative ability to bind at the AGT active site. Based on their predicted binding affinity for the AGT active site, we selected four analogues of compound 1 (compounds 2, 3, 4, and 5) (Figure 1). ...
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... evaluated the interaction of compounds 1, 2, 3, 4, and 5 with AGTwt and the folding-defective G41R variant 25 by absorbance spectroscopy. As already observed for AOA, 23 the interaction with each ligand caused the disappearance of the bands at 423 and 340 nm, attributed to the ketoenamine and enolimine tautomers of the internal aldimine, respectively, 3 and the concomitant appearance of a peak at 374 nm ( Figure S1A). Upon excitation of the enzyme−inhibitor complexes at 374 nm, a fluorescence emission spectrum with maximum at ∼450 nm was observed ( Figure S1C). ...
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... already observed for AOA, 23 the interaction with each ligand caused the disappearance of the bands at 423 and 340 nm, attributed to the ketoenamine and enolimine tautomers of the internal aldimine, respectively, 3 and the concomitant appearance of a peak at 374 nm ( Figure S1A). Upon excitation of the enzyme−inhibitor complexes at 374 nm, a fluorescence emission spectrum with maximum at ∼450 nm was observed ( Figure S1C). Similar spectral changes occurred for the G41R variant, although in the latter case two absorbance bands centered at 370 and at 334 nm were observed in the presence of each ligand ( Figure S1B,D) due to subtle active site differences caused by the mutation of Gly41 that affect the tautomeric equilibrium. ...
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... excitation of the enzyme−inhibitor complexes at 374 nm, a fluorescence emission spectrum with maximum at ∼450 nm was observed ( Figure S1C). Similar spectral changes occurred for the G41R variant, although in the latter case two absorbance bands centered at 370 and at 334 nm were observed in the presence of each ligand ( Figure S1B,D) due to subtle active site differences caused by the mutation of Gly41 that affect the tautomeric equilibrium. 25 Overall, these results indicate that all compounds are able to bind the AGT active site through the formation of an oxime between the carbonyl group of PLP and the amino group of the ligand. ...
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... the other hand, the viability of CHO-GO-G41R cells increased in the presence of compound 10d and compounds 29u and 25o without PN or in the presence of compounds 25o and 29u and PN, while it slightly decreased in the presence of compound 20l with or without PN. Overall, by pairing the data obtained on AGT expression and activity with those on cell viability, we could observe that (i) the five selected compounds (10d, 20l, 25o, 29s, 29u) increase the specific activity of G41R, a model of a misfolded variant, although with different efficiency; (ii) compounds 20l and 25o lead to improved activity and to improved or unaltered expression in the absence and in the presence of PN in both AGTwt and G41R variant, and they do not affect cell viability. Since the effects of compound 25o are higher than those of compound 20l, we chose compound 25o as the best hit for subsequent studies. ...
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... changes of AGT wt and the G41R variant in the presence of compound 1 and analogues ( Figure S1); effect of compound 1 and analogues on AGTwt and the G41R variant expressed in mammalian cells in the presence of PN ( Figure S2); binding studies of synthetized compounds on AGT and G41R variant in the purified form ( Figure S3); effects of synthetic compound treatment on AGTwt and the G41R variant expressed in mammalian cells in the presence of PN ( Figure S4); cytotoxic effect of selected hit compounds ( Figure S5); ranking of five best synthetized compounds (Table S1); and LC−MS analytical method for assessing purity of key target compounds (Annex) (PDF) ...
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Background:
Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disease stemming from a deficiency in liver-specific alanine-glyoxylate aminotransferase, resulting in increased endogenous oxalate deposition and end-stage renal disease. Organ transplantation is the only effective treatment. However, its approach and timing remain contr...
Introduction:
Combined or sequential liver and kidney transplantation (CLKT/SLKT) restores kidney function and corrects the underlying metabolic defect in children with end-stage kidney disease in primary hyperoxaluria type 1 (PH1). However, data on long-term outcome, especially in children with infantile PH1, are rare.
Methods:
All pediatric PH...
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Background
Primary hyperoxaluria type 1 is characterized by hepatic oxalate overproduction, leading to nephrocalcinosis, kidney stones, kidney failure and systemic oxalosis, including oxalate osteopathy. Combined liver-kidney transplantation (CLKT) and kidney after liver transplantation (KALT) were established therapeutic options to stop the devast...
Background
Primary hyperoxaluria type 1 (PH1) is a rare disease with autosomal recessive transmission, characterized by increased urinary excretion of oxalate, resulting in chronic kidney disease secondary to recurrent urolithiasis, nephrocalcinosis, and accumulation of oxalate in various organs and tissues (systemic oxalosis). Since 2020, an innov...
Citations
... The role of misfolding in PH1 pathogenesis has also prompted the identification of small AGT ligands working as pharmacological chaperones for the holo-form, that is, competitive inhibitors that stabilize the native structure and dissociate in the presence of the substrate [5]. A recent study has identified a hit compound as putative drug able to partly rescue the effect of the most common pathogenic mutations in a cellular model of PH1 [43,44]. ...
Purpose of review
Primary hyperoxalurias (PHs) are rare disorders caused by the deficit of liver enzymes involved in glyoxylate metabolism. Their main hallmark is the increased excretion of oxalate leading to the deposition of calcium oxalate stones in the urinary tract. This review describes the molecular aspects of PHs and their relevance for the clinical management of patients.
Recent findings
Recently, the study of PHs pathogenesis has received great attention. The development of novel in vitro and in vivo models has allowed to elucidate how inherited mutations lead to enzyme deficit, as well as to confirm the pathogenicity of newly-identified mutations. In addition, a better knowledge of the metabolic consequences in disorders of liver glyoxylate detoxification has been crucial to identify the key players in liver oxalate production, thus leading to the identification and validation of new drug targets.
Summary
The research on PHs at basic, translational and clinical level has improved our knowledge on the critical factors that modulate disease severity and the response to the available treatments, leading to the development of new drugs, either in preclinical stage or, very recently, approved for patient treatment.
... In our HDX-MS studies, we certainly observed destabilizing effects of P11L and G170R but of local nature and not very large (Fig. 5), indicating that structural destabilization of the native state plays a role (but maybe not the main role) in PH1 pathogenesis. Consistent with this view, development of very high affinity ligands (with apparent dissociation constants even in the low nM range) only leads to a modest correction in an aggregating PH1 variant in model cells of PH1 [47] and an AGT variant with extremely high kinetic stability do not show higher expression levels in transfected eukaryotic cells [46]. A plausible alternative for a main mechanism leading to protein misfolding in PH1 is a reduced folding efficiency in vivo (e.g., as observed as steady-state enhanced interaction with molecular chaperones in PH1 variants; [28,29]). ...
Primary hyperoxaluria type I (PH1) is caused by deficient alanine:glyoxylate aminotransferase (AGT) activity. PH1‐causing mutations in AGT lead to protein mistargeting and aggregation. Here, we use hydrogen‐deuterium exchange (HDX) to characterize the wild‐type (WT), the LM (a polymorphism frequent in PH1 patients) and the LM G170R (the most common mutation in PH1) variants of AGT. We provide the first experimental analysis of AGT structural dynamics, showing that stability is heterogeneous in the native state and providing a blueprint for frustrated regions with potentially functional relevance. The LM and LM G170R variants only show local destabilization. Enzymatic transamination of the pyridoxal 5‐phosphate cofactor bound to AGT hardly affects stability. Our study, thus, supports that AGT misfolding is not caused by dramatic effects on structural dynamics.
... Dindo and coworkers describe in this Special Issue the importance of the dimerization of human alanine:glyoxylate amino transferase (AGT) for the proper folding of the enzyme in cells and its import to peroxisomes, where the enzyme is metabolically useful, as well as the chaperone role of the protein cofactor pyridoxal 5 -phosphate (PLP) for dimerization and function [24]. More recently, the same group have described the successful development of pharmacological chaperones that partially restore the normal AGT activity of PH1-causing mutations [25]. Moya-Garzón and coworkers present in this Special Issue an alternative for the treatment of PH1 based on inhibitors of oxalate formation that is currently under further development by using tools from medicinal chemistry [22,26]. ...
Advances in DNA sequencing technologies are revealing a vast genetic heterogeneity in human population, which may predispose to metabolic alterations if the activity of metabolic enzymes is affected [...]
Primary Hyperoxaluria Type 1 (PH1) is a rare autosomal disease caused by mutations in AGXT that lead to the deficiency of alanine:glyoxylate aminotransferase (AGT). AGT is a liver pyridoxal 5'-phosphate (PLP)-dependent enzyme that detoxifies glyoxylate inside peroxisomes. The lack of AGT activity results in a build-up of glyoxylate that is oxidized to oxalate, then culminating in hyperoxaluria often leading to kidney failure. Most pathogenic mutations reduce AGT specific activity because of catalytic defects, improper folding, mistargeting to mitochondria, reduced intracellular stability, dimerization, and/or aggregation. Administration of pyridoxine (PN), a precursor of PLP, is a therapeutic option available for PH1 patients carrying responsive genotypes through the ability of the coenzyme to behave as a chaperone. Here, we report the clinical and biochemical characterization of the novel mutation c.1093G > T (p.Gly365Cys) identified in a Japanese patient. In silico studies predict that the p.Gly365Cys mutation causes a steric clash resulting in a local rearrangement of the region surrounding the active site, thus possibly affecting PLP binding and catalysis. Indeed, the purified p.Gly365Cys mutant displays proper folding but shows an extensive decrease of catalytic efficiency due to an altered PLP-binding. When expressed in AGXT1-KO HepG2 cells the variant shows reduced specific activity and protein levels in comparison with wild type AGT that cannot be rescued by PN treatment. Overall, our data indicate that the mutation of Gly365 induces a conformational change at the AGT active site translating into a functional and structural defect and allow to predict that the patients will not be responsive to vitamin B6, thus supporting the usefulness of preclinical studies to guide therapeutic decisions in the era of precision medicine.
Primary hyperoxaluria type 1 (PHT1) treatment is mainly focused on inhibiting the enzyme glycolate oxidase, which plays a pivotal role in the production of glyoxylate, which undergoes oxidation to produce oxalate. When the renal secretion capacity exceeds, calcium oxalate forms stones that accumulate in the kidneys. In this respect, detailed QSAR analysis, molecular docking, and dynamics simulations of a series of inhibitors containing glycolic, glyoxylic, and salicylic acid groups have been performed employing different regression machine learning techniques. Three robust models with less than 9 descriptors—based on a tenfold cross (Q2CV) and external (Q2EXT) validation—were found i.e., MLR1 (Q2CV = 0.893, Q2EXT = 0.897), RF1 (Q2CV = 0.889, Q2EXT = 0.907), and IBK1 (Q2CV = 0.891, Q2EXT = 0.907). An ensemble model was built by averaging the predicted pIC50 of the three models, obtaining a Q2EXT = 0.933. Physicochemical properties such as charge, electronegativity, hardness, softness, van der Waals volume, and polarizability were considered as attributes to build the models. To get more insight into the potential biological activity of the compouds studied herein, docking and dynamic analysis were carried out, finding the hydrophobic and polar residues show important interactions with the ligands. A screening of the DrugBank database V.5.1.7 was performed, leading to the proposal of seven commercial drugs within the applicability domain of the models, that can be suggested as possible PHT1 treatment.
