CYP24A1

C H A P T E R

6 CYP24A1: Structure, Function, and Physiological Role René St-Arnaud1, Glenville Jones2 1Shriners

Hospitals for Children – Canada, Montreal, QC, Canada; 2Queen’s University, Kingston, ON, Canada

O U T L I N E Introduction81

Putative CYP24A1 Involvement in Other Systems

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CYP24A1-Catalyzed Pathways

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CYP24A1 in Chronic Kidney Disease

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C24-Oxidation Pathway

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Role of 24,25(OH)2D in Chondrocyte Maturation

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C23-Hydroxylation Pathway

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24,25(OH)2D and Fracture Repair

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CYP24A1, a Multifunctional Enzyme

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Biological Relevance of the C24-Oxidation Pathway

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Structure–Function Relationships

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Perspectives90 Idiopathic Infantile Hypercalcemia Diagnosis and Treatment90 24,25(OH)2D Supplementation for Fracture Repair 90 CYP24A1 Inhibitors 90

Mutations of CYP24A1 and Idiopathic Infantile Hypercalcemia85

References91

Preclinical Models of Idiopathic Infantile Hypercalcemia 87

chain (Fig. 6.1). CYP24A1 is a mitochondrial inner-membrane cytochrome P450 enzyme that utilizes the soluble iron–sulfur protein adrenodoxin and the flavoenzyme, adrenodoxin reductase for NADPH-derived electron transfer into its heme center. The enzyme exhibits multifunctionality and the pathways catalyzed by the protein have been mapped out. The physiological relevance of these pathways has been confirmed in mice deficient for the Cyp24a1 gene. Recent work has focused on structure–function analysis and several homology models were described before the crystal structure of the rat CYP24A1 protein was reported. This chapter will review the enzymatic pathways, the structure– function relationships, and the role of CYP24A1 in the catabolism of 1,25(OH)2D. In addition, other putative biological roles of the enzyme and one of its main catalytic products will be presented, together with the perspectives offered by pharmacological modulation of its activity.

List of Abbreviations CKD  Chronic kidney disease IIH  Idiopathic infantile hypercalcemia

INTRODUCTION In a classic endocrine negative-feedback loop, the vitamin D hormone, 1,25(OH)2D, induces in target tissues the expression of the main effector of its catabolic breakdown, the CYP24A1 enzyme. This vitamin D receptor (VDR)–mediated transcriptional response insures attenuation of the 1,25(OH)2D biological signal inside target cells and helps regulate vitamin D homeostasis. Catabolism of 1,25(OH)2D occurs through CYP24A1mediated modification of the secosteroid’s aliphatic side

Vitamin D, Volume 1: Biochemistry, Physiology and Diagnostics, Fourth Edition http://dx.doi.org/10.1016/B978-0-12-809965-0.00006-9

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© 2018 Elsevier Inc. All rights reserved.

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6.  CYP24A1: STRUCTURE, FUNCTION, AND PHYSIOLOGICAL ROLE

FIGURE 6.1  Enzymatic pathways catalyzed by CYP24A1. The C24-oxidation pathway products are shown on the left; the C23-hydroxylation products are depicted at the right. Only the products of 1,25(OH)2D are represented; refer to the text for the 25(OH)D products.

CYP24A1-CATALYZED PATHWAYS The main substrates for the hydroxylation reactions catalyzed by CYP24A1 are 25(OH)D and 1,25(OH)2D. In the first case, the reaction leads to the formation of 24,25(OH)2D, a metabolite that circulates in the bloodstream. In the second instance, the initial, short-lived enzymatic product is 1,24,25(OH)3D. For CYP24A1-dependent hydroxylation, electrons are derived from NADPH and transferred in sequence through NADPH-adrenodoxin reductase and adrenodoxin to the heme molecule in the enzyme’s active center where molecular oxygen is bound, then split into a reactive oxyferryl center that is directed to the targeted substrate and subsequently reduced to a hydroxyl group. The enzyme is termed a mixedfunction oxidase because the other oxygen atom is reduced to H2O [1]. The specificity of this catalytic reaction is determined by substrate orientation within the enzyme’s active site, where the target carbon atom must be in close proximity to the hemeoxyferryl center. Interestingly, the CYP24A1 enzyme is able

to hydroxylate both the C23 or the C24 side-chain carbons of 25(OH)D or 1,25(OH)2D [2]. The relative degree of C23- and C24-hydroxylase activity appears species-specific, and the structural basis of this altered specificity has been examined using sequence alignment and site-directed mutagenesis and will be described in ’Structure-Function Relationships’. C24-hydroxylation leads to side-chain cleavage and oxidation to a carboxylic acid (C24-oxidation pathway), whereas hydroxylation at carbon 23 results in lactone formation (C23hydroxylation pathway).

C24-OXIDATION PATHWAY In the mid- to late-seventies, the C24-hydroxylation of 1,25(OH)2D was shown to be induced by 1,25(OH)2D itself [3,4]. Since the product of that reaction, 1,24,25(OH)3D was 10 times less active than the 1,25(OH)2D substrate [3], investigators began to reason that the C24-hydroxylation reaction

I.  HISTORY, CHEMISTRY METABOLISM, CIRCULATION & REGULATION

CYP24A1, a Multifunctional Enzyme

was perhaps the first step in an inactivation process. It was also discovered that the C24-hydroxylated metabolites, 24,25(OH)2D and 1,24,25(OH)3D, could be further converted to different metabolic products sporting C24-oxo and/or C23-hydroxy groups [5–7]. Studies using perfused rat kidney then led to the identification of additional metabolites: a C23-alcohol [8] and a C23-acid, calcitroic acid [9,10]. Those metabolites had not been previously identified in vitro. Calcitroic acid was shown to be the main biliary excretory form of 1,25(OH)2D [11]. Each metabolite was identified by a combination of high-performance liquid chromatography (HPLC), UV spectrophotometric, and mass spectrometric techniques. With most metabolites identified, investigators deduced that C24-hydroxylation initiates the C24-oxidation pathway that leads to 1,25(OH)2D degradation [9,10]. This pathway comprises five enzymatic steps (Fig. 6.1): it begins with 24-hydroxylation of 1,25(OH)2D to yield 1,24,25(OH)3D. This metabolite is ketonized to 24-oxo-1,25(OH)2D. Carbon 23 is then hydroxylated to generate 24-oxo-1,23,25(OH)3D, and this compound is metabolized by oxidative cleavage of the carbon– carbon bond between C23 and C24 to produce 24,25,26,27-tetranor-1,23(OH)2D. This C23 alcohol converts to calcitroic acid, the excretory product of 1,25(OH)2D in bile. When the initial substrate is 25(OH)D, the metabolic intermediates are 24,25(OH)2D, 24-oxo-25(OH)D, 24-oxo23,25(OH)2D, 24,25,26,27-tetranor-23(OH)D, and finally calcitroic acid. The 1,25(OH)2D-inducible 24-hydroxylation and calcitroic acid production were observed in several cell lines from kidney, bone, intestine, skin, and breast [12–14], demonstrating that the C24-oxidation catabolic pathway can be induced in a number of vitamin D target cells.

C23-HYDROXYLATION PATHWAY The discovery of a C23-oxidative pathway for 25(OH)D emerged from the identification of 25(OH)D-26,23-lactone, 1,25(OH)2D-26,23-lactone, and their respective metabolic precursors [15–20]. Using kidney mitochondria isolated from a variety of species, the 24-hydroxylase and 23-hydroxylase activities were found to copurify [21]. Interestingly, some species use both pathways, such as in humans [2,22–24], whereas others preferentially 23-hydroxylate (guinea pig, opossum) [25,26] or primarily 24-hydroxylate (rat) [21]. Classical biochemical studies hinted that the two enzymatic activities might have different kinetic parameters [21], but for quite some time it was unclear whether the reactions were catalyzed by a single enzyme or by distinct proteins. The cloning and expression of recombinant CYP24A1 from different species resolved the discrepancy and demonstrated that a single polypeptide chain was capable of both C23- and C24-hydroxylation activities. The structural basis of the C23- versus C24-hydroxylation regioselectivity has been examined using site-specific mutagenesis [27] and will be detailed in ’Structure-Function Relationships’ below. The human CYP24A1 was expressed in bacteria and used in a reconstituted system consisting of the bacterial membrane fraction, adrenodoxin, and adrenodoxin reductase.

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This system was used to study the metabolism of 25(OH)D or 1,25(OH)2D by HPLC and mass spectrometric analysis [2]. All steps of the C23-hydroxylation pathway were catalyzed by the recombinant human CYP24A1 enzyme: from 25(OH)D to 23,25(OH)2D, then 23,25,26(OH)3D, followed by conversion to 25(OH)D-26,23-lactol, and finally 25(OH)D-26,23-lactone [2]. When the initial substrate is 1,25(OH)2D, the metabolic intermediates are 1,23,25(OH)3D, 1,23,25,26(OH)4D, 1,25(OH)2D-26,23-lactol, and finally 1,25(OH)2D-26,23-lactone (Fig. 6.1). The biological activity of the C23-hydroxylation metabolites is unclear, but there have been claims that the terminal 1,25(OH)2D-derived product, 1,25(OH)2D-26,23-lactone, could act as a VDR antagonist [28,29]. It appears surprising that CYP24A1 would catalyze two independent pathways from the 25-hydroxylated forms of vitamin D. The finding that 1,25(OH)2D-26,23-lactone acts as a VDR antagonist [28,29] suggests that the lactone pathway provides a redundant or fail-safe mechanism to more efficiently and rapidly dampen the vitamin D signal [27].

CYP24A1, A MULTIFUNCTIONAL ENZYME The CYP24A1 enzyme has been purified to homogeneity from rat kidney mitochondria [30], and the purified protein was used to raise antibodies [31] that permitted cloning of the cDNA [32]. This then allowed production of the recombinant protein in parallel with the cloning of the gene from various species [33–35]. The recombinant CYP24A1 protein, when associated with its electron-transport cofactors, NADPH-adrenodoxin reductase and adrenodoxin, has been shown to perform multiple steps of the C24-oxidation pathway. This includes 23-hydroxylation, dehydrogenation of the 24-hydroxyl group, and side-chain cleavage [22,23]. CYP24A1 is thus a bona fide multicatalytic enzyme. The recombinant human CYP24A1 protein exhibited release of the product at each step of the C23- and C24-hydroxylation pathways [2]. Thus multiple intermediate metabolites, such as 24,25(OH)2D, can be released from the CYP24A1 substrate-binding pocket. When metabolism of 25(OH)D was studied using kidney homogenates from vitamin D-treated or vitamin D-deficient chicks, conversion from 25(OH)D to 25(OH)D-26,23-lactol to 25(OH)D-26,23-lactone was measured in samples from both D-replete and D-deficient animals [36]. Because Cyp24a1 is a 1,25(OH)2D-induced gene, the conversion to 25(OH)D-26,23lactone measured in vitamin D-deficient chicks hints that it can be catalyzed even in the absence of CYP24A1, although it is clear that the recombinant CYP24A1 protein has the ability to perform this conversion. It has been suggested that an unknown aldehyde oxidase could catalyze this reaction in vitamin D-deficient chicks [36]. Conversion rates from 1,25(OH)2D-26,23-lactol to 1,25(OH)2D-26,23-lactone in tissue homogenates from 1,25(OH)2D-deficient chicken range from 55% to 61% of those measured in 1,25(OH)2D-supplemented chicken [37]. These measured differences may represent the relative contributions of the putative aldehyde oxidase and CYP24A1 for the conversion to the lactone under physiological conditions.

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BIOLOGICAL RELEVANCE OF THE C24OXIDATION PATHWAY The hypothesis that the main role of the C24-oxidation pathway is attenuation of the 1,25(OH)2D biological signal inside target cells was tested in vitro using cytochrome P450 inhibitors. Blocking P450 activity by treatment of cells with the antifungal imidazole derivative, ketoconazole, inhibits catabolism and results in 1,25(OH)2D accumulation and extended hormone action [38]. This hypothesis was also tested and confirmed in vivo by engineering Cyp24a1-deficient mice to examine the role of the CYP24A1 enzyme in vitamin D homeostasis [39]. Fifty percent of Cyp24a1−/− mice die before 3 weeks of age [39,40]. Analysis of macrophage function ruled out impaired responses to infection as the cause for postnatal death. The perinatal lethality is most likely a consequence of hypercalcemia secondary to hypervitaminosis D because the inactivation of the Cyp24a1 gene in mice impaired the ability of the animals to clear 1,25(OH)2D. Bolus and chronic 1,25(OH)2D administration resulted in a marked elevation in serum 1,25(OH)2D levels in the mutant animals [39]. Chronic 1,25(OH)2D administration in Cyp24a1−/− mutants resulted in histological changes consistent with hypervitaminosis D in the kidney: cortical tubular dilation, necrotic debris, and mineralization (nephrocalcinosis). The inability to regulate 1,25(OH)2D and calcium homeostasis presumably leads to fatal hypercalcemia. Indeed, extremely high levels of circulating 1,25(OH)2D and calcium were measured in runted animals that died before weaning [39]. Because half of the mutant progeny appear unaffected by the Cyp24a1 deficiency, these surviving animals most likely use alternate means of regulating vitamin D homeostasis. The pharmacokinetics of labeled 1,25(OH)2D were measured in Cyp24a1−/− survivors and heterozygote controls. These experiments have shown that Cyp24a1-null mice have impaired clearance of 1,25(OH)2D. Surprisingly, Cyp24a1−/− mice demonstrate total absence of both 24-hydroxylated metabolites and 1,25(OH)2D-26,23-lactone [41]. Similar results were obtained in VDR-knockout mice [41,42], demonstrating that the VDRdependent induction of Cyp24a1 expression is required for production of calcitroic acid and 1,25(OH)2D-26,23-lactone in vivo. These data raise doubts about any physiological contribution of an unknown aldehyde oxidase for the conversion to the lactone [36], at least in mice. Furthermore, these findings suggest that the Cyp24a1-null survivors adapt to the impaired vitamin D catabolism not by using an alternative catabolic route but by limiting the synthesis of the active compound [41].

been steady pharmacological interest in the development of CYP24A1 inhibitors to prolong the bioactivity of 1,25(OH)2D or its relevant analogs [44–47]. Information on tertiary structure of the substrate-binding pocket was perceived as relevant to develop specific inhibitors. A seminal finding within the cytochrome P450 field was obtained when the crystal structure of class I P450s (bacterial/ mitochondrial, receiving their electrons from a two-protein redox chain) was aligned with that of class II enzymes (microsomal, receiving electrons from a single reductase). This led to the observation that despite extensive differences in amino acid sequence, all P450 molecules possess similar tertiary structure [48]. This feature was used extensively to generate CYP24A1 homology models and test the role of multiple residues in substrate binding and catalysis using site-directed mutagenesis [27,49–53]. These modeling studies successfully predicted multiple residues in the CYP24A1-binding pocket as confirmed by the crystal structure. Recombinant CYP24A1 crystals were obtained following adrenodoxin-Sepharose affinity chromatography purification of bacterially expressed rat Cyp24a1 sequence deleted of its mitochondrial import signal (Δ2–32) [49] and mutated at residue 57 (S57D) to stabilize the recombinant protein [43,54]. The CYP24A1 crystal structure displays the canonical P450 fold, including the 12 α-helices (A–L) and 4 β-sheet systems (β1– β4). Five additional short helices (A′, B′, G′, K′, and K″) were also identified [43]. The substrate-binding cavity is defined by the β1 and β4 sheets, the B–C loop, and helices E, F, G, I, and K surrounding the heme. Alignment of helices A′ and G′ with other mitochondrial P450 sequences identifies them as membrane insertion sequences, and computational modeling suggests that residues in the hydrophobic surfaces of helices A′ and G′ can penetrate ∼7 Å into the mitochondrial inner membrane to serve as anchors flanking the substrate access channel (Fig. 6.2).

STRUCTURE–FUNCTION RELATIONSHIPS There has been significant interest in performing structure– function analyses and generating homology models of the CYP24A1 structure while awaiting crystal structure resolution, which fortunately was recently reported [43]. Efforts stemmed from two different perspectives: on the one hand, investigators were intrigued by the species-specific bias for C23- or C24-hydroxylation [2,21–24]. On the other hand, there has

FIGURE 6.2  Structure of the rat CYP24A1 protein inserted in the inner mitochondrial membrane. The ribbon structure was obtained from the atomic coordinates deposited in the Protein Data Bank, code 3K9V (http://www.pdb.org). The arrow defines the substrate access channel and points to the heme. MIS, membrane insertion sequence.

I.  HISTORY, CHEMISTRY METABOLISM, CIRCULATION & REGULATION

Mutations of CYP24A1 and Idiopathic Infantile Hypercalcemia

The crystal structure identified 19 residues (from nine regions of the folded sequence) that surround the active-site cavity [43]. These are Leu129 and Ile131 (from the B–B′ region); Trp134 (within the B′-helix); Met148 (B′–C region); Met245, Met246, and Phe249 (from the F-helix); His271 and Trp275 (within the G-helix); Leu325, Ala326, Glu329, and Thr330 (four residues of the I-helix); Val391 (K-helix/β1-4 loop); Phe393, Thr394, and Thr395 (part of the β1-4 sheet); and Gly499 and Ile500 (from the β4-1/β4-2 turn). Of these 19 residues, 13 were correctly predicted as relevant for substrate binding and catalysis in homology modeling studies: Ile131, Trp134, Met148, Met245, Met246, Phe249, Ala326, Glu329, Thr330, Val391, Thr394, Gly499, and Ile500 [50,51,53]. The P450 structural motif–based method of 3D-homology modeling used by Masuda et al. [53] appears particularly effective as it allowed the authors to make strong predictions concerning the positioning and role of six residues (Ile131, Trp134, Met148, Met246, Ala326, and Gly499) within the CYP24A1 active site. These predictions were born out by the solved crystal structure. As mentioned previously, CYP24A1 is a multifunctional enzyme that, amongst other catalyzed reactions, can either 23-hydroxylate or 24-hydroxylate the 1,25(OH)2D substrate. The degree to which CYP24A1 performs each reaction is species-dependent [2,21–24], and it was reasoned that speciesspecific sequence differences could explain the behavior of the enzyme. A systematic site-directed mutagenesis effort to convert the putative substrate-binding residues of the rat sequence to those of the human sequence identified the residues at positions 416 (Thr in rat and Met in human) and 500 (Ile in rat and Thr in human) as important for C23-hydroxylation. However, the changes in C24-/C23-hydroxylation ratio measured in that study were quite modest [52]. Furthermore, the amino acid differences between the human and rat sequences at residues 416 and 500 are not consistently different in nonhuman species including opossum and guinea pig, which predominantly 23-hydroxylate vitamin D substrates. This observation hinted that it was unlikely that residues 416 and 500 were the primary determinants of the species-specific differences in regioselectivity. Alignment of the I-helix (residues 312–345) from 19 species identified residue 326 (Ala in human and Gly in opossum) as a potential key determinant [27]. When alanine 326 in the human CYP24A1 is changed to a glycine, as it is in opossum and guinea pig, the catalytic pattern is dramatically altered to favor 23-hydroxylation. No other mutation produced a comparable radical change in hydroxylation pattern [27]. The side chain of Ala326 appears as a major determinant of the depth of substrate penetration within the enzyme’s binding pocket and of the alignment of the hydroxylation site above the heme iron atom. Helix I was confirmed as a structural element defining the substrate-binding cavity in the crystal structure [43]. When substrate docking simulations with 1,25(OH)2D were performed on the open CYP24A1 crystal structure [43], the calculations yielded proper nanomolar affinity (Ki = 2.65 nM) and a reproducible conformation in accord with predictions made by homology modeling [50,51,53]. In the crystal structure–based simulations, the 1,25(OH)2D molecule was stabilized by two hydrogen bonds and multiple hydrophobic

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interactions among key conserved residues identified in the homology models: Ile131, Trp134, Met246, Phe249, Ala326, Val391, Thr394, Thr395, Gly499, and Ile500. The two hydrogen bonds were computed between the C3-hydroxyl group of the secosteroid A ring and the B–B′ loop at amino acid Leu129 and between the C25-hydroxyl group of the side chain and residue Leu325 within the I-helix [43]. In the calculated configuration using the open conformation of CYP24A1 [43], the C21-methyl group is positioned over the heme iron, and thus it is unlikely that the open structure computation represents the substrate’s terminal binding configuration relevant for catalysis (which requires the C23 or C24 carbons to be aligned with the heme). However, the ability of the 1,25(OH)2D substrate to bind deep within the active site and interact with the heme group should accommodate the cavity collapse to a closed state required to exclude solvent for catalysis and proper substrate positioning. Improper substrates bound in a shallow conformation would not be able to stabilize the closed state, and this may contribute to enzymatic specificity [43]. Site-directed mutagenesis of a related vitamin D metabolic enzyme, CYP27B1, had identified the invariant arginine pair within helix L (Arg465 and Arg466) as critical for protein folding and/or heme binding and more clearly linked Arg466 to the electron and oxygen transfer steps required for catalytic function [55]. The crystal structure of CYP24A1 reveals that these basic residues’ side chains protrude below the heme and that Arg466 is positioned to promote electron transfer between adrenodoxin and the heme iron. The side chains of Arg465 and Arg466 are also within 8–10 Å of the conserved K-helix residues (Lys378 and Lys382) involved in adrenodoxin recognition and electron transfer in CYP27B1 [55]. Overall, the CYP24A1 crystal structure identified conserved residues from helices K, K″, and L that face an adjacent lysine-rich loop to provide the interface for binding of the redox protein [43]. The CYP24A1 crystal structure thus provides a template for understanding membrane insertion, substrate binding, and electron chain partner interactions. This molecular understanding of the structure–function relationships of the CYP24A1 protein will greatly facilitate biorational drug development.

MUTATIONS OF CYP24A1 AND IDIOPATHIC INFANTILE HYPERCALCEMIA Based on numerous reports in recent years [56–80], it has become evident that loss-of-function mutations of CYP24A1 are one of the principal genetic causes of hypercalcemia. Schlingmann et al. [74] reported several families with hypercalcemia and an assortment of homozygous recessive and compound heterozygous mutations causing defective 25(OH) D3-24-hydroxylase enzyme activity, elevated serum 1,25(OH)2D3, and hypercalcemia that ultimately result in hypercalciuria, nephrolithiasis, and nephrocalcinosis. This was followed by various publications reporting the same consequences in adults [60,77] including one in a 70-year-old male [65]. Thus the name, idiopathic infantile hypercalcemia (IIH), is a misnomer because the cause of the disease is no longer unknown and is

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not confined to childhood [78]. In fact, there are at least three known diseases under the constellation of IIH:   

1. l oss-of-function mutations of CYP24A1 at cytogenetic location 20q13.2 (sometimes known as Lightwood syndrome [68]); 2. Williams–Beuren syndrome (WBS) caused by a large deletion on chromosome 7 (cytogenetic location 7q11.23) of 28 genes including the elastin gene (Online Mendelian Inheritance in Man, OMIM, #194050); and 3. loss-of-function mutations of the Na-Pi cotransporter at cytogenetic location 5q35.3 (also known as SLC34A1) [81];   

There is likely to be more genetic bases for hypercalcemia to be described in the future. There are currently 21 missense and deletion mutations of CYP24A1 described in the literature and the position of these are illustrated in Fig. 6.3. These mutations must be distinguished from numerous polymorphisms of the CYP24A1 gene. Lossof-function mutations can be predicted by various computer programs, but comprehensive confirmation of their effects on enzyme activity only comes from expression of the defective gene. Schlingmann et al. [74] used site-directed mutagenesis of the wild-type human CYP24A1 gene, followed by transient or stable expression of the mutant in V-79 Chinese hamster lung fibroblasts, and incubation of the cells with [1β-3H]1,25-(OH)2D3 to demonstrate loss of enzyme activity. Others (Nesterova et al. [72]) have used cultured fibroblasts to show loss of CYP24A1 enzyme function. More recently, Molin et al. [71] used liquid chromatography and tandem mass spectrometry (LC-MS/MS) analysis of the 1,24,25-(OH)3D3 product, instead of use of a radioactive substrate, to demonstrate loss of CYP24A1 enzyme activity. Although in IIH it is critical to show that there are two genetically mutated CYP24A1 alleles, and that these are lossof-function mutations and not polymorphisms, the value of a reliable rapid screening test for IIH has also been a principal research focus. Kaufmann et al. [82] described such an LC-MS/MS method for measuring 24,25-(OH)2D3 in 100 μL of serum or less based on derivatization with DMEQ-TAD {4-[2-(6,7-dimethoxy-4-ethyl-3,4-dihydroquinoxalinyl-)

ethyl]-1,2,4-triazoline-3,5-dione}, a dienophile which couples to all vitamin D metabolites in the blood. 24,25-(OH)2D3 is the main product of CYP24A1 found in the blood, is indicative of the enzyme activity of the kidney enzyme, and is roughly proportional to the level of the substrate 25(OH)D3. The absolute concentration of 24,25-(OH)2D3 is a fairly reliable measure of CYP24A1 enzyme activity in vivo, and the metabolite is low in most IIH patients with CYP24A1 mutations, even though most untreated IIH patients have elevated serum 25(OH)D3 values [71]. However, a more reliable indicator of IIH due to CYP24A1 mutations is the ratio of the metabolite to its precursor 25(OH) D3, namely serum 25(OH)D3:24,25-(OH)2D3 [58,71,82,83]. We have found that this ratio is even more conclusive than serum 24,25-(OH)2D3 alone because it eliminates the possibility that the patient might have a low serum 24,25-(OH)2D3 level due to vitamin D deficiency. Our experience of this analytical test using the serum 25(OH)D3:24,25-(OH)2D3 ratio in over 100 IIH patients has convinced us that it does not produce false negative results [71,82]. The other known causes of IIH, namely WBS mutations (OMIM #194050) and Na-Pi cotransporter defects [81], have normal serum 25(OH)D3:24,25-(OH)2D3 ratios and are thus distinguished from CYP24A1 defects by this rapid test (Kaufmann et al., submitted). One question about IIH due to CYP24A1 mutations is why patients experience a history of hypercalcemia/hypercalciuria in early childhood, and then often become normocalcemic but suffer occasional hypercalcemic episodes throughout life? Part of the answer comes from studies of the CYP24A1-knockout mouse, which shows a 50% lethality at weaning with survivors exhibiting a partial downregulation of expression of CYP27B1, thereby reducing their serum 1,25-(OH)2D3 concentrations [41]. We presume that lethality stems from an inability to regulate CYP27B1 to counter the reduced rate of catabolism. Aside from the pressures of vitamin D intake in IIH patients, which may underlie hypercalcemic episodes, there have now been several reports documenting increased risks of hypercalcemia during pregnancy in IIH patients [61,67,75,80]. Dinour et al. [61] and Shah et al. [75] both reported multiple episodes of hypercalcemia in patients with proven CYP24A1 mutations

FIGURE 6.3  CYP24A1 mutations. Reported mutations in idiopathic infantile hypercalcemia patients are listed above the primary protein structure of CYP24A1. Primary structure features (α-helices and β-strands) are numbered/labeled and indicated in different colors.

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CYP24A1 in Chronic Kidney Disease

coinciding with successive pregnancies. It is postulated that the well-documented, increased 1,25-(OH)2D3 production by the placenta during pregnancy [84,85] upsets calcium homeostasis in these individuals and normocalcemia returns after pregnancy. Although the heterozygotic newborn has been reported to be normal in such pregnancies, the mother can suffer from the harmful effects of the hypercalcemia, namely renal stones, pancreatitis, and calcification of the placenta [61,67,75,80]. Interestingly, the gestating female Cyp24a1-knockout mouse fails to carry to term and does not survive pregnancy (authors unpublished observations). From studies thus far, it would appear that all IIH patients with CYP24A1 mutations should be placed on vitamin D-restricted intakes and female patients should be counseled on their increased risk of hypercalcemia during pregnancy. Successful treatments for acute hypercalcemic episodes in IIH include use of general cytochrome P450 inhibitors such as ketoconazole and fluconazole, which block many endogenous drug and steroidal hydroxylases as well as CYP27B1 [72,73]. As described in the preceding chapter (Chapter 4), the development of specific CYP27B1 inhibitors has been initiated but has thus far not advanced to testing in clinical trials.

PRECLINICAL MODELS OF IDIOPATHIC INFANTILE HYPERCALCEMIA The currently available Cyp24a1-knockout strain [39] exhibits hypercalcemia, hypercalciuria, and nephrocalcinosis and could represent a model of IIH. Supporting this view, it was shown that surviving Cyp24a1-null animals possess a much reduced ability to clear a bolus dose of [1β-3H]1α,25(OH)2D3 compared with normal wild-type littermates [41]. However, it should be noted that the Cyp24a1-deficient mouse strain was generated with a selection cassette replacing the heme-binding exon and does not express any Cyp24a1 message [39]. This contrasts with the situation in patients, which in many cases express the mutated CYP24A1 message and protein (authors’ unpublished observations). Moreover, some of the CYP24A1 mutations identified in IIH patients are hypomorphic mutations, demonstrating residual or altered activity [74]. There is thus a need to generate improved preclinical animal models of IIH based on human mutations to better understand the effects of dysfunctional vitamin D metabolism on IIH patients. This work is currently in progress in the authors’ laboratories. Two types of mutations were selected to expand our comprehension of the impact of a modified CYP24A1 sequence on the pathophysiology of IIH: a missense loss-of-function mutation (R396W) [74] and two missense mutations with altered activity that are well-described from in vitro studies (A326G and V391L) [27,86]. The R396W loss-of-function mutation is from a patient with IIH [74]. The A326G and V391L variants have been studied in vitro as part of an exhaustive structure–function analysis [27,86]. Although they have not yet been identified in IIH patients, these mutations should be extremely informative about the role of vitamin D catabolism

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in hypercalcemia. The single amino acid A326G change alters the regioselectivity of the native wild-type enzyme from a 24-hydroxylation phenotype to a 23-hydroxylating phenotype [27]. The V391L change is also interesting because it gives the catabolic CYP24A1 some anabolic properties, namely the addition of 25-hydroxylase activity [86]. Mutagenesis studies have proven invaluable to understand the structure–activity relationships of CYP24A1. Furthering these observations in the context of global animal physiology (i.e., in vivo structure– function analysis) through knock-in mutagenesis will provide key information on vitamin D metabolism and mineral ion homeostasis.

PUTATIVE CYP24A1 INVOLVEMENT IN OTHER SYSTEMS The prevalent view implicates CYP24A1 mainly in the attenuation of the calcemic 1,25(OH)2D biological activity. But the enzyme is inducible in all 1,25(OH)2D target tissues and evidence has emerged that CYP24A1 expression might be relevant for vitamin D functions distinct from mineral homeostasis. Furthermore, intermediate metabolites can be released from the CYP24A1 substrate-binding pocket. One such metabolite, 24,25(OH)2D, is the most abundant dihydroxylated metabolite in the circulation and it has been proposed that it may exert biological effects.

CYP24A1 IN CHRONIC KIDNEY DISEASE Chronic kidney disease (CKD) shows steadily increasing worldwide incidence due to an aging population and augmenting obesity with its associated complications of hypertension and adult-onset diabetes [87]. Stages 3 and 4 CKD (moderate) are characterized by progressively decreasing kidney function as assessed by glomerular filtration rate. Severe CKD (stage 5) is associated with minimal or altogether absent kidney function and patients require regular dialysis or kidney transplantation for survival [88]. With declining renal function, kidney failure patients experience declining 1,25(OH)2D levels, which causes decreased systemic calcium and increased phosphate levels that lead to elevations of two hormones: parathyroid hormone (PTH) [89–91] and fibroblast growth factor 23 (FGF23) [92]. Elevated serum levels of PTH, defined as secondary hyperparathyroidism (SHPT), leads to the development of renal osteodystrophy, characterized by disorganized bone remodeling and accompanied by loss of bone strength and integrity, with associated morbidity [93–95]. Untreated, SHPT of renal failure has also been associated with increased mortality through increased cardiovascular calcification and associated ischemic events [96–98]. Treatment with vitamin D analogs are advocated for the clinical care of renal patients [99,100]. These compounds act to lower PTH levels close to the normal range and help to maintain normocalcemia, bone health, and cardiovascular integrity [101–104].

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Some in vitro and animal studies have suggested that FGF23 not only suppresses the expression of CYP27B1 but also leads to elevated expression of CYP24A1 [105–108], particularly at the mRNA level. This effect of FGF23 may be exacerbated by hormonal replacement therapeutic regimen with current vitamin D analogs. As CYP24A1 inactivates prohormonal, hormonal, and analog forms of vitamin D, the aberrant elevation in CYP24A1 expression may contribute to vitamin D insufficiency and exacerbate SHPT in CKD patients. These novel findings suggest that treatment of CKD patients might be another clinical management situation that could benefit from the availability of specific inhibitors of CYP24A1 [107,109]. Consequently, in CKD where FGF23 is high, one might expect that CYP24 expression and serum 24,25-(OH)2D3 levels would be elevated [110]. Somewhat perplexing is the finding that serum 24,25-(OH)2D3 levels are not elevated, but are subnormal, in CKD patients on dialysis [111,112]. Based on our current knowledge, it is difficult to reconcile these apparently conflicting effects of FGF23 on CYP24A1 mRNA expression and CYP24A1 enzyme activity in CKD patients. However, there is no doubt that the situation in dialysis patients is complicated by the loss of functional kidney cells and the fact that PTH and FGF23 are both highly elevated simultaneously. The recent demonstration [113] in the VDR-knockout mouse that a high-calcium, high-phosphate rescue diet suppresses PTH to normal and restores basal expression of CYP24A1 suggests that high PTH not only has effects on CYP27B1 expression but also on CYP24A1 expression. Thus in dialysis patients, we may be observing a dominant effect of PTH over FGF23 when it comes to CYP24A1 enzyme activity, as measured by 24,25(OH)2D3 levels.

ROLE OF 24,25(OH)2D IN CHONDROCYTE MATURATION An extensive literature demonstrates that Cyp24a1 is expressed in growth plate chondrocytes and that cells from the growth plate respond to 24,25(OH)2D in a cell maturation– dependent manner (reviewed in Refs. [114,115]). Most of these studies were performed using the in vitro rat costochondral primary culture system. Dissection of the tissue allows isolation of cells from different regions of the growth plate. Each region represents a different maturation stage along the chondrocytic differentiation pathway. In this model system, the less differentiated cells of the resting zone, also called the reserve zone, respond to 24,25(OH)2D. The more mature cells of the growth zone, including the prehypertrophic and hypertrophic compartments, respond primarily to 1,25(OH)2D. The effects of 24,25(OH)2D were also recently observed in the well-characterized prechondrocytic cell line ATDC5 [116,117]. In resting zone cells, 24,25(OH)2D decreases cell proliferation but stimulates differentiation and maturation: the metabolite stimulates extracellular matrix production through stimulation of the synthesis of sulfated glycosaminoglycans [118]. It also causes the cells to produce extracellular

matrix vesicles that contain neutral metalloproteinases [119] but reduces total matrix vesicle metalloproteinase activity [120,121]. 24,25(OH)2D was shown to act on resting zone chondrocytes via phospholipase D [122,123]. The proposed mechanism invokes that 24,25(OH)2D-mediated stimulation of phospholipase D promotes the conversion of phosphatidylcholine to phosphatidic acid, leading to lysophosphatidic acid (LPA) production. LPA in turn stimulates increases in alkaline phosphatase activity and sulfation, while protecting resting zone cells from apoptotic cell death [124]. The antiapoptotic effect is thought to involve LPA signaling through LPA receptor-1 or -3, which decreases p53 abundance and increases the Bcl-2/Bax ratio [125]. Interestingly, treatment of resting zone chondrocytes with 24,25(OH)2D induces a change in maturation state, resulting in downregulation of responsiveness to 24,25(OH)2D and upregulation of responsiveness to 1,25(OH)2D [126]. These observations support the hypothesis that 24,25(OH)2D plays a role in cartilage development. It is worth mentioning that growth plates from Cyp24a1−/− mice do not show major defects [39,127]. These observations suggest that the absence of CYP24A1 activity does not affect growth plate development and that 24,25(OH)2D is not required for chondrocyte maturation in vivo [39]. It remains possible, however, that a redundant endocrine system is able to compensate for the function of 24,25(OH)2D in animals.

24,25(OH)2D AND FRACTURE REPAIR It has been proposed that 24,25(OH)2D, the enzymatic product of the CYP24A1 activity on the 25(OH)D substrate, might also play a role in fracture repair, but there is limited information available on this putative function of the metabolite. The healing of fractures is a unique postnatal biological repair process resulting in the restoration of injured skeletal tissue to a state of normal structure and function. Fracture repair involves a complex multistep process that involves response to injury, intramembranous bone formation, chondrogenesis, endochondral bone formation, and bone remodeling [128,129]. Several studies have described a complex pattern of gene expression that occurs during the course of these events [130–133]. Taken together, results from gene expression monitoring during bone repair suggest that the molecular regulation of fracture healing is complex but recapitulates some aspects of embryonic skeletal formation [134,135]. A role for 24,25(OH)2D in fracture repair is supported by the observation that the circulating levels of 24,25(OH)2D increase during fracture repair in chickens because of an increase in CYP24A1 activity [136] (Fig. 6.4, top panel). When the effect of various vitamin D metabolites on the mechanical properties of healed bones was tested, treatment with 1,25(OH)2D alone resulted in poor healing [137]. However, the strength of healed bones in animals fed with 24,25(OH)2D in combination with 1,25(OH)2D was equivalent to that measured in a control

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24,25(OH)2D AND FRACTURE REPAIR

FIGURE 6.4  Cyp24a1 expression during fracture healing in chicks and mice. Top panel: the changes in CYP24A1 activity and circulating 24,25(OH)2D concentrations are listed besides the temporal sequence of fracture healing. ↑, increase; N.D., not determined. The putative expression of a receptor/binding protein for 24,25(OH)2D at day 10 postfracture is expressed by a question mark. Bottom panel: quantitative reverse-transcription polymerase chain reaction on mRNA extracted from the callus of the fractured right tibia (Fracture) and a diaphysial section of the left nonfractured tibia (Contralateral) of wild-type mice at 14 days postfracture. The expression of Cyp24a1 was significantly increased in the fractured bone, confirming the observations previously obtained in chicks. *, P < .05. Top panel: Based on the data described in references Seo EG, Norman AW. Three-fold induction of renal 25-hydroxyvitamin D3-24hydroxylase activity and increased serum 24,25-dihydroxyvitamin D3 levels are correlated with the healing process after chick tibial fracture. J Bone Miner Res 1997;12(4):598–606; Seo EG, Einhorn TA, Norman AW. 24R,25-dihydroxyvitamin D3: an essential vitamin D3 metabolite for both normal bone integrity and healing of tibial fracture in chicks. Endocrinology 1997;138(9):3864–72; Seo EG, Kato A, Norman AW. Evidence for a 24R,25(OH)2-vitamin D3 receptor/binding protein in a membrane fraction isolated from a chick tibial fracture-healing callus. Biochem Biophys Res Commun 1996;225(1):203–8.

population fed with 25(OH)D [137]. These results support a role for 24,25(OH)2D as being an essential vitamin D metabolite important for fracture repair. Circumstantial evidence suggested the presence of a binding protein for 24,25(OH)2D in the chick tibial fracture-healing callus [138,139]. Cell fractionation to isolate a membrane fraction followed by ligand binding studies using hydroxylapatite to separate bound and free ligands described a receptor/binding protein for 24,25(OH)2D in the fracture-healing callus membrane fraction from vitamin D-depleted chicks [139]. These observations were never

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followed through, and to date, there are no published reports of the cloning or characterization of a molecular entity corresponding to this binding activity. Human data concerning the role of 24,25(OH)2D in bone healing are scarce. The available published studies report measured circulating vitamin D metabolites levels at different intervals following fracture. The results are conflicting with increases in serum 24,25(OH)2D reported in young patients [140], whereas no changes were measured in older cohorts [141,142]. It will be interesting to review data on bone healing following fracture in patients with IIH, which have mutations in the CYP24A1 gene. The Cyp24a1-deficient mouse strain [39] represents an invaluable tool to examine the putative role of 24,25(OH)2D in mammalian fracture repair. Cyp24a1 mutant animals that survive past weaning appear to use an alternative pathway of 1,25(OH)2D catabolism to regulate circulating levels of the hormone [41] and are normocalcemic and normophosphatemic when fed regular rodent chow. This allows us to study bone healing in these animals. As a first step, the induction of the expression of the Cyp24a1 gene during fracture repair was confirmed in mice. Wild-type mice were subjected to a stabilized, transverse middiaphysial fracture of the tibia. To stabilize the fracture without disrupting the bone marrow microenvironment, we used a small-scale version of the Ilizarov distraction osteogenesis device used in orthopedic patients [143–145]. RNA was extracted from the fracture callus at 14 days postosteotomy, reverse-transcribed, and analyzed by quantitative reverse-transcription polymerase chain reaction. Cyp24a1 mRNA levels were significantly elevated in the fracture callus as compared to the undamaged contralateral bone (Fig. 6.4, bottom panel). Importantly, these results confirm the data reported previously for chicken [136] and support the hypothesis that the activity of the CYP24A1 enzyme is important for bone fracture repair. Fracture repair was then compared between Cyp24a1−/− mice and wild-type controls. We have observed a defect in callus formation in Cyp24a1−/− mutant animals (data not shown). This phenotype could be rescued by treatment with 24,25(OH)2D but not with administration of exogenous 1,25(OH)2D. Of relevance to these observations are the recently reported results demonstrating that 24,25(OH)2D promotes the osteoblastic differentiation of mesenchymal stem cells [146]. Treatment of human bone marrow–derived mesenchymal stem cells with 24,25(OH)2D inhibited CYP27B1 and VDR expression and promoted osteoblastic differentiation through increased alkaline phosphatase activity and mineralization [146]. These effects of the metabolite to promote mesenchymal stem cell osteoblastic maturation could be involved in the beneficial impact of treatment with 24,25(OH)2D during fracture repair. Cyp24a1-deficient mice were used as a source of tissue to identify differentially expressed genes between wild-type and mutant mouse repair callus. This has led to the identification of a restricted set of genes, which we are currently characterizing for their ability to act as effector of the 24,25(OH)2D signal.

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PERSPECTIVES Idiopathic Infantile Hypercalcemia Diagnosis and  Treatment The development of a rapid serum screening method for IIH due to CYP24A1 mutations [82] allows for the differential diagnosis of various forms of IIH that can then be confirmed by next generation genetic testing. This has enabled us to diagnose patients around the world and observe the high frequency and migration of some of these CYP24A1 mutations for the first time. Knowledge from these types of studies is making endocrinologists and nephrologists increasingly aware of CYP24A1 mutations as an important cause of kidney stones and nephrocalcinosis. It will be interesting to see if individuals with one affected CYP24A1 allele, including relatives of IIH patients, are at increased risk of renal stones, as has been postulated in the literature [77,58]. The treatment of IIH will benefit from studies of the factors that trigger hypercalcemia in the various CYP24A1 knock-in mice described here. There is little doubt that these factors include vitamin D intake [74,56] and the pressures of skeleton formation in the fetus in the pregnant female [61,67,75,80]. But are there additional factors that result in sporadic hypercalcemia? Although treatment with general cytochrome P450 inhibitors such as ketoconazole will block CYP27B1 and the acute rises in 1,25-(OH)2D3 because of lack of CYP24A1, these drugs are not optimal long-term therapies due to their inhibition of other beneficial CYPs. Thus, the discovery of IIH may provide an additional impetus to the development of specific CYP27B1 inhibitors, an initiative begun a couple of decades ago [44–47].

24,25(OH)2D Supplementation for Fracture Repair Published studies examining fracture repair in chicks [136,137] as well as our unpublished studies in Cyp24a1deficient mice support a role for 24,25(OH)2D in optimal bone fracture repair. Although it may be difficult to acquire data with sufficient statistical power, it would be interesting to study how fractured bones heal in patients with CYP24A1 mutations. Because it is a rather lengthy process, it can be argued that repair of long bones has not been optimized by evolution [147]. To survive a fracture and avoid being prey to predators, animals would have needed to restore long bone function within days. Mutations that could shorten healing time, even by half, would not have allowed survival, and thus evolutionary pressure toward optimized fracture repair must have remained slight. But because the process is not optimized, it should be quite possible to enhance it through pharmacological intervention. Efforts to develop suitable preclinical models and beneficial treatments have been described [147]. The observation that mice deficient for Cyp24a1 exhibit impaired callus formation that can be corrected by exogenous administration of 24,25(OH)2D suggests that treatment with vitamin D metabolites hydroxylated at position 24, such as 24,25(OH)2D,

could be useful in the treatment of bone fractures subsequent to trauma or metabolic bone diseases. It can be argued that 24,25(OH)2D is an abundant circulating vitamin D metabolite and that it is present in sufficient amounts to efficiently promote bone healing without the need for additional supplementation. However, it is now recognized that a sizeable proportion of the population suffers from vitamin D insufficiency [148–150], which may have deleterious effects for optimized fracture repair. Thus fracture healing could benefit from supplementation with 24,25(OH)2D or a suitable analog.

CYP24A1 Inhibitors It has long been surmised that inhibition of CYP24A1 activity could be beneficial to enhance 1,25(OH)2D action. The rationale for such an inhibitory treatment was apparent when thinking of improving the efficacy of the vitamin D hormone and D analogs in cancer therapy and in the treatment of hyperproliferative diseases such as psoriasis. Novel findings suggest that CYP24A1 inhibitors could also help in the clinical management of CKD. The first identified inhibitors were antifungal imidazole derivatives, such as ketoconazole and liarozole. They lack specificity because they inhibit steroidogenesis by interfering broadly with cytochrome P450 enzyme systems [151], although this feature could prove beneficial in the treatment of prostate cancer [152]. The prominent phytoestrogen in soy, genistein, was shown to inhibit CYP24A1 in cultured cells [153–157] as well as in mouse colon in vivo [158]. It appears to act through several mechanisms as it was shown to inhibit both CYP24A1 expression (at the transcriptional level) [155] and the activity of the CYP24A1 protein [154]. Phytoestrogens have a beneficial effect on cancers [159,160], and the discovery of their action on vitamin D metabolic pathways suggests that part of their antitumorigenic effects could be mediated by vitamin D [158]. It also raises the possibility that inhibition of CYP24A1 could be achieved in part via nutritional means [161,162]. As part of the effort to chemically synthesize vitamin D analogs with low calcemic activity, potent inhibitors of CYP24A1 based on the 1,25(OH)2D secosteroid structure were identified [109]. These include sulfone [163] and sulfoximine [44] derivatives of 1,25(OH)2D. The 16,23-diene-25sulfone analog (compound CTA018/MT2832) [109] is particularly interesting from a therapeutic standpoint as it exhibits a dual mechanism of action. It acts as a potent and low-calcemic inhibitor of CYP24A1, and in addition it was shown to be a potent activator of VDR-mediated transcription [109]. The compound was shown to exhibit adequate pharmacokinetic and pharmacodynamic profiles and to effectively suppress elevated PTH without affecting calcemia or phosphatemia in a preclinical rodent model of CKD [109]. This new class of analogs with a dual mechanism of action may be able to achieve the desired therapeutic response of preventing SHPT in latestage CKD without leading to acquired resistance to vitamin D analog therapy caused by induction of CYP24A1. It is possible that yet further refinement of pharmacological CYP24A1

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References

inhibitory strategies could be achieved through biorational drug development based on the solved crystal structure of the enzyme [43]. Indeed, the recently described CYP24A1 inhibitor CTA102 shows high potency (IC50 8.5 nM) and selectivity between CYP24A1 and CYP27B1 [107]. Preclinical models of renal phosphate wasting hypophosphatemic states demonstrated the potential of pharmacologic CYP24A1 inhibition as a therapeutic adjunct for their treatment [107].

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I.  HISTORY, CHEMISTRY METABOLISM, CIRCULATION & REGULATION