Potent, Selective and Low-Calcemic Inhibitors of CYP24 Hydroxylase:  24-Sulfoximine Analogues of the Hormone 1α,25-Dihydroxyvitamin D<sub>3</sub>

Kahraman, Mehmet; Sinishtaj, Sandra; Dolan, Patrick; Kensler, Thomas W.; Peleg, Sara; Saha, Uttam Kumar; Chuang, Samuel S.; Bernstein, Galina; Korczak, Bożena; Posner, Gary H.

doi:10.1021/jm040129+

Cited by 109 publications

(66 citation statements)

References 23 publications

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“…25 Therefore, in cells that express both enzymes, MK-24(S)-S(O)(NH)-Ph-1 (but not KETO) can suppress 1,25D 3 degradation while allowing continued 1,25D 3 synthesis, which results in a net increase in 1,25D 3 concentration. Expression of 1a hydroxylase mRNA and 1a hydroxylase enzyme Proteins were resolved by electrophoresis through 10% polyacrylamide gels and transferred to PVDF membranes.…”

Section: Discussionmentioning

confidence: 97%

“…25 1,25D 3 concentration remained constant for 24 hr in tissue culture medium without cells, and no We also observed a statistically significant increase in the 1,25D 3 concentration measured at 6 hr in homogenates prepared from cells treated with 1,25D 3 plus MK-24(S)-S(O)(NH)-Ph-1 (100 6 11 ng/ mL) as compared with the 1,25D 3 concentration measured at 0 hr (59 6 6 ng/mL). This increase was not observed in the presence of KETO.…”

Section: Determination Of the Role Of Cyp24 In Regulating 125d 3 Catmentioning

confidence: 99%

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CYP24, the enzyme that catabolizes the antiproliferative agent vitamin D, is increased in lung cancer

Parise

Egorin

Kanterewicz

et al. 2006

Intl Journal of Cancer

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1a,25-Dihydroxyvitamin D 3 (1,25D 3 ) displays potent antiproliferative activity in a variety of tumor model systems and is currently under investigation in clinical trials in cancer. Studies were initiated to explore its potential in nonsmall cell lung cancer (NSCLC), as effective approaches to the treatment of that disease are needed. In evaluating factors that may affect activity in NSCLC, the authors found that CYP24 (25-hydroxyvitamin D 3 -24-hydroxylase), the enzyme that catabolizes 1,25D 3 , is frequently expressed in NSCLC cell lines but not in the nontumorigenic bronchial epithelial cell line, Beas2B. CYP24 expression by RT-PCR was also detected in 10/18 primary lung tumors but in only 1/11 normal lung tissue specimens. Tumor-specific CYP24 upregulation was confirmed at the protein level via immunoblot analysis of patientmatched normal lung tissue and lung tumor extracts. Enzymatically active CYP24 is expected to desensitize NSCLC cells to 1,25D 3 . The authors therefore implemented a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/ MS) assay for 1,25D 3 and its CYP24-generated metabolites to determine whether NSCLC cells express active enzyme. Analysis of NSCLC cell cultures revealed time-dependent loss of 1,25D 3 coincident with the appearance of CYP24-generated metabolites. MK-24(S)-S(O)(NH)-Ph-1, a specific inhibitor of CYP24, slowed the loss of 1,25D 3 and increased 1,25D 3 half-life. Furthermore, combination of 1,25D 3 with MK-24(S)-S(O)(NH)-Ph-1 resulted in a significant decrease in the concentration of 1,25D 3 required to achieve maximum growth inhibition in NSCLC cells. These data suggest that increased CYP24 expression in lung tumors restricts 1,25D 3 activity and support the preclinical evaluation of CYP24 inhibitors for lung cancer treatment. ' 2006 Wiley-Liss, Inc.Key words: 1a,25-dihydroxyvitamin D 3 ; 25-hydroxyvitamin D 3 -24 hydroxylase; CYP24; nonsmall cell lung cancer Lung cancer is a significant health problem in the United States; it now kills more women than does breast cancer, and more men than do prostate and colon cancers combined.1 Major advances in treatment have been limited, and the prognosis for lung cancer patients remains poor, with overall 5-year survival rates less than 15%.2 Therefore, there is a tremendous need to develop effective therapeutic approaches for this disease. 1a,25-Dihydroxyvitamin D 3 (1,25D 3 ), the biologically active form of vitamin D, has antiproliferative activity in a variety of tumor model systems in vitro and in vivo and is currently under evaluation in clinical trials in cancer (reviewed by Beer and Myrthue 3 ). We initiated studies to explore whether the antiproliferative effects of 1,25D 3 in NSCLC could be increased by inhibiting its metabolism.

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Section: Discussionmentioning

confidence: 97%

Section: Determination Of the Role Of Cyp24 In Regulating 125d 3 Catmentioning

confidence: 99%

CYP24, the enzyme that catabolizes the antiproliferative agent vitamin D, is increased in lung cancer

Parise

Egorin

Kanterewicz

et al. 2006

Intl Journal of Cancer

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“…Thus, CYP24A1 is physiologically essential and potentially an important clinical target for inhibition to extend the action of 1␣,25-(OH) 2 D 3 . Inhibitors designed on azole chemistry (e.g., ketoconazole) (30) and a series of competitive inhibitors based on natural substrates (31) have previously had successes as topical agents in human clinical trials for hyperproliferative conditions such as psoriasis. It remains to be seen whether characterization of the lactone pathway and better understanding of the CYP24A1 active site will facilitate new design strategies for CYP24A1 inhibitors.…”

Section: Discussionmentioning

confidence: 99%

Single A326G mutation converts human CYP24A1 from 25-OH-D ₃ -24-hydroxylase into -23-hydroxylase, generating 1α,25-(OH) ₂ D ₃ -26,23-lactone

Prosser

Kaufmann

O’Leary

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Studies of 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) have demonstrated that it is a bifunctional enzyme capable of the 24-hydroxylation of 1␣,25-(OH) 2D3, leading to the excretory form, calcitroic acid, and 23-hydroxylation, culminating in 1␣,25-(OH) 2D3-26,23-lactone. The degree to which CYP24A1 performs either 23-or 24-hydroxylation is species-dependent. In this paper, we show that the human enzyme that predominantly 24-hydroxylates its substrate differs from the opossum enzyme that 23-hydroxylates it at only a limited number of amino acid residues. Mutagenesis of the human form at a single substrate-binding residue (A326G) dramatically changes the regioselectivity of the enzyme from a 24-hydroxylase to a 23-hydroxylase, whereas other modifications have no effect. Ala-326 is located in the I-helix, close to the terminus of the docked 25-hydroxylated side chain in a CYP24A1 homology model, a result that we interpret indicates that substitution of a glycine at 326 provides extra space for the side chain of the substrate to move deeper into the pocket and place it in a optimal stereochemical position for 23-hydroxylation. We discuss the physiological ramifications of these results for species possessing the A326G substitution, as well as implications for optimal vitamin D analog design. (Fig. 1). Studies using CYP24A1-knockout (8) and VDR-knockout mice (8, 9) demonstrate the total absence of calcitroic acid and 1␣,25-(OH) 2 D 3 -26,23-lactone formation when the vitamin Dinducible, VDR-mediated CYP24A1 is not expressed. The in vivo result of CYP24A1 ablation is decreased viability, with 50% of mice not surviving beyond weaning because of hypercalcemia and nephrocalcinosis (8, 10). Although it has been well established that calcitroic acid is the major biliary product of 1␣,25-(OH) 2 D 3 in the rat (11, 12), there have been claims that intermediates of the C24 oxidation pathway retain some biological activity (13) and that the terminal product of 23-hydroxylation, namely 1␣,25-(OH) 2 D 3 -26,23-lactone, belongs to a family of potent VDR antagonists with potential for use in the treatment of Paget's disease (14, 15).The degree to which CYP24A1 performs either of these pathways varies with the species. It had been shown in the mid-1980s by using isolated kidney mitochondria from a variety of species, that the 24-hydroxylase and 23-hydroxylase activities copurify (16). Although both pathways are used in humans, certain species, such as the guinea pig (17, 18), were shown to 23-hydroxylate, whereas other species, such as the rat, primarily 24-hydroxylate the substrate, 25-OH-D 3 (16). These studies suggested that the two enzyme activities might have different kinetic parameters, but it was not clear whether the activities resided on a single or distinct polypeptide chains. With the cloning and expression of the recombinant protein from different species (19,20), it became clear that a single polypeptide chain was capable of both C23-and C24-hydroxylation activities (5-7).Recent studies of rat and human CYP24A1 hav...

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“…Thus, information on substrate recognition, the reaction mechanism, and the species-based difference of CYP24A1 seems quite useful to develop vitamin D analogs for clinical use. Another area of CYP24A1 research with pharmacological interest is development of specific inhibitor of CYP24A1 (Schuster et al, 2003;Kahraman et al, …”

Section: Introductionmentioning

confidence: 99%

Structure-Function Analysis of Vitamin D 24-Hydroxylase (CYP24A1) by Site-Directed Mutagenesis: Amino Acid Residues Responsible for Species-Based Difference of CYP24A1 between Humans and Rats

Hamamoto¹,

Kusudo²,

Urushino³

et al. 2006

Mol Pharmacol

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Our previous studies revealed the species-based difference of CYP24A1-dependent vitamin D metabolism. Although human CYP24A1 catalyzes both C-23 and C-24 oxidation pathways, rat CYP24A1 shows almost no C-23 oxidation pathway. We tried to identify amino acid residues that cause the speciesbased difference by site-directed mutagenesis. In the putative substrate-binding regions, amino acid residue of rat CYP24A1 was converted to the corresponding residue of human CYP24A1. Among eight mutants examined, T416M and I500T showed C-23 oxidation pathway. In addition, the mutant I500Fshowed quite a different metabolism of 1␣,25-dihydroxyvitamin D 3 [1␣,25(OH) 2 D 3 ] from both human and rat CYP24A1. These results strongly suggest that the amino acid residues at positions 416 and 500 play a crucial role in substrate binding and greatly affect substrate orientation. A three-dimensional model of CYP24A1 indicated that the A-ring and triene part of 1␣,25(OH) 2 D 3 could be located close to amino acid residues at positions 416 and 500, respectively. Our findings provide useful information for the development of new vitamin D analogs for clinical use.The hormonally active form of vitamin D 3 , 1␣,25(OH) 2 D 3 , plays essential roles in calcium homeostasis, immune response, and cell differentiation (Boyle et al., 1971;Holick et al., 1971;Lawson et al., 1971;Norman et al., 1971;Bouillon et al., 1995). A large number of vitamin D analogs have been synthesized for clinical use in type I rickets, osteoporosis, renal osteodystrophy, psoriasis, leukemia, and breast cancer, and their biological activity has been evaluated. Much research has investigated the biological mechanism of vitamin D analogs, revealing that the biological activities of analogs are due not only to the analog itself but also to its metabolites (Binderup et al., 1991;Binderup, 1992;Dilorth et al., 1997). CYP24A1 plays a central role in the metabolism of 1␣,25(OH) 2 D 3 and its analogs in target tissues such as kidneys, intestines, and bones. A species-based difference has been revealed in the CYP24A1-dependent metabolism of 1␣,25(OH) 2 D 3 between rats and humans (Akiyoshi-Shibata et al., 1994;Beckman et al., 1996;Sakaki et al., 1999Sakaki et al., , 2000. Human CYP24A1 demonstrated a remarkable metabolism consisting of both C-23 and C-24 oxidation pathways, whereas rat CYP24A1 showed extreme predominance of C-24 over C-23 oxidation pathways. In addition, a remarkable species-based difference was also observed in the CYP24A1-dependent metabolism of vitamin D analogs Kusudo et al., 2003Kusudo et al., , 2004Abe et al., 2005). These facts suggest that preclinical tests using such animals as rats and mice cannot correctly predict the metabolism of vitamin D analogs in the human body. Thus, information on substrate recognition, the reaction mechanism, and the species-based difference of CYP24A1 seems quite useful to develop vitamin D analogs for clinical use. Another area of CYP24A1 research with pharmacological interest is development of specific inhibitor of CYP...

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Potent, Selective and Low-Calcemic Inhibitors of CYP24 Hydroxylase: 24-Sulfoximine Analogues of the Hormone 1α,25-Dihydroxyvitamin D₃

Cited by 109 publications

References 23 publications

CYP24, the enzyme that catabolizes the antiproliferative agent vitamin D, is increased in lung cancer

CYP24, the enzyme that catabolizes the antiproliferative agent vitamin D, is increased in lung cancer

Single A326G mutation converts human CYP24A1 from 25-OH-D ₃ -24-hydroxylase into -23-hydroxylase, generating 1α,25-(OH) ₂ D ₃ -26,23-lactone

Structure-Function Analysis of Vitamin D 24-Hydroxylase (CYP24A1) by Site-Directed Mutagenesis: Amino Acid Residues Responsible for Species-Based Difference of CYP24A1 between Humans and Rats

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Potent, Selective and Low-Calcemic Inhibitors of CYP24 Hydroxylase: 24-Sulfoximine Analogues of the Hormone 1α,25-Dihydroxyvitamin D3

Cited by 109 publications

References 23 publications

CYP24, the enzyme that catabolizes the antiproliferative agent vitamin D, is increased in lung cancer

CYP24, the enzyme that catabolizes the antiproliferative agent vitamin D, is increased in lung cancer

Single A326G mutation converts human CYP24A1 from 25-OH-D 3 -24-hydroxylase into -23-hydroxylase, generating 1α,25-(OH) 2 D 3 -26,23-lactone

Structure-Function Analysis of Vitamin D 24-Hydroxylase (CYP24A1) by Site-Directed Mutagenesis: Amino Acid Residues Responsible for Species-Based Difference of CYP24A1 between Humans and Rats

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Potent, Selective and Low-Calcemic Inhibitors of CYP24 Hydroxylase: 24-Sulfoximine Analogues of the Hormone 1α,25-Dihydroxyvitamin D₃

Single A326G mutation converts human CYP24A1 from 25-OH-D ₃ -24-hydroxylase into -23-hydroxylase, generating 1α,25-(OH) ₂ D ₃ -26,23-lactone