Targeting of the Mitochondrial TET1 Protein by Pyrrolo[3,2-b]pyrrole Chelators.

Targeting of epigenetic mechanisms, such as the hydroxymethylation of DNA, has been intensively studied, with respect to the treatment of many serious pathologies, including oncological disorders. Recent studies demonstrated that promising therapeutic strategies could potentially be based on the inhibition of the TET1 protein (ten-eleven translocation methylcytosine dioxygenase 1) by specific iron chelators. Therefore, in the present work, we prepared a series of pyrrolopyrrole derivatives with hydrazide (1) or hydrazone (2-6) iron-binding groups. As a result, we determined that the basic pyrrolo[3,2-b]pyrrole derivative 1 was a strong inhibitor of the TET1 protein (IC50 = 1.33 µM), supported by microscale thermophoresis and molecular docking. Pyrrolo[3,2-b]pyrroles 2-6, bearing substituted 2-hydroxybenzylidene moieties, displayed no significant inhibitory activity. In addition, in vitro studies demonstrated that derivative 1 exhibits potent anticancer activity and an exclusive mitochondrial localization, confirmed by Pearson's correlation coefficient of 0.92.

Instability of the Human Cytochrome P450 Reductase A287P Variant Is the Major Contributor to Its Antley-Bixler Syndrome-like Phenotype.

Human NADPH-cytochrome P450 oxidoreductase (POR) gene mutations are associated with severe skeletal deformities and disordered steroidogenesis. The human POR mutation A287P presents with disordered sexual development and skeletal malformations. Difficult recombinant expression and purification of this POR mutant suggested that the protein was less stable than WT. The activities of cytochrome P450 17A1, 19A1, and 21A2, critical in steroidogenesis, were similar using our purified, full-length, unmodified A287P or WT POR, as were those of several xenobiotic-metabolizing cytochromes P450, indicating that the A287P protein is functionally competent in vitro, despite its functionally deficient phenotypic behavior in vivo Differential scanning calorimetry and limited trypsinolysis studies revealed a relatively unstable A287P compared with WT protein, leading to the hypothesis that the syndrome observed in vivo results from altered POR protein stability. The crystal structures of the soluble domains of WT and A287P reveal only subtle differences between them, but these differences are consistent with the differential scanning calorimetry results as well as the differential susceptibility of A287P and WT observed with trypsinolysis. The relative in vivo stabilities of WT and A287P proteins were also examined in an osteoblast cell line by treatment with cycloheximide, a protein synthesis inhibitor, showing that the level of A287P protein post-inhibition is lower than WT and suggesting that A287P may be degraded at a higher rate. Current studies demonstrate that, unlike previously described mutations, A287P causes POR deficiency disorder due to conformational instability leading to proteolytic susceptibility in vivo, rather than through an inherent flavin-binding defect.

Holoenzyme structures of endothelial nitric oxide synthase - an allosteric role for calmodulin in pivoting the FMN domain for electron transfer.

While the three-dimensional structures of heme- and flavin-binding domains of the NOS isoforms have been determined, the structures of the holoenzymes remained elusive. Application of electron cryo-microscopy and structural modeling of the bovine endothelial nitric oxide synthase (eNOS) holoenzyme produced detailed models of the intact holoenzyme in the presence and absence of Ca(2+)/calmodulin (CaM). These models accommodate the cross-electron transfer from the reductase in one monomer to the heme in the opposite monomer. The heme domain acts as the anchoring dimeric structure for the entire enzyme molecule, while the FMN domain is activated by CaM to move flexibly to bridge the distance between the reductase and oxygenase domains. Our results indicate that the key regulatory role of CaM involves the stabilization of structural intermediates and precise positioning of the pivot for the FMN domain tethered shuttling motion to accommodate efficient and rapid electron transfer in the homodimer of eNOS.

Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency.

NADPH-cytochrome P450 oxidoreductase (CYPOR) is essential for electron donation to microsomal cytochrome P450-mediated monooxygenation in such diverse physiological processes as drug metabolism (approximately 85-90% of therapeutic drugs), steroid biosynthesis, and bioactive metabolite production (vitamin D and retinoic acid metabolites). Expressed by a single gene, CYPOR's role with these multiple redox partners renders it a model for understanding protein-protein interactions at the structural level. Polymorphisms in human CYPOR have been shown to lead to defects in bone development and steroidogenesis, resulting in sexual dimorphisms, the severity of which differs significantly depending on the degree of CYPOR impairment. The atomic structure of human CYPOR is presented, with structures of two naturally occurring missense mutations, V492E and R457H. The overall structures of these CYPOR variants are similar to wild type. However, in both variants, local disruption of H bonding and salt bridging, involving the FAD pyrophosphate moiety, leads to weaker FAD binding, unstable protein, and loss of catalytic activity, which can be rescued by cofactor addition. The modes of polypeptide unfolding in these two variants differ significantly, as revealed by limited trypsin digestion: V492E is less stable but unfolds locally and gradually, whereas R457H is more stable but unfolds globally. FAD addition to either variant prevents trypsin digestion, supporting the role of the cofactor in conferring stability to CYPOR structure. Thus, CYPOR dysfunction in patients harboring these particular mutations may possibly be prevented by riboflavin therapy in utero, if predicted prenatally, or rescued postnatally in less severe cases.

Neuronal nitric oxide synthase is phosphorylated in response to insulin stimulation in skeletal muscle.

Type 2 Diabetes (T2DM) is the seventh leading cause of death in the United States, and is quickly becoming a global pandemic. T2DM results from reduced insulin sensitivity coupled with a relative failure of insulin secretion. Reduced insulin sensitivity has been associated with reduced nitric oxide synthase (NOS) activity and impaired glucose uptake in T2DM skeletal muscle. Upon insulin stimulation, NO synthesis increases in normal adult skeletal muscle, whereas no such increase is observed in T2DM adults. Endothelial NOS is activated by phosphorylation in the C-terminal tail in response to insulin. Neuronal NOS (nNOS), the primary NOS isoform in skeletal muscle, contains a homologous phosphorylation site, raising the possibility that nNOS, too, may undergo an activating phosphorylation event upon insulin treatment. Yet it remains unknown if or how nNOS is regulated by insulin in skeletal muscle. Data shown herein indicate that nNOS is phosphorylated in response to insulin in skeletal muscle and that this phosphorylation event occurs rapidly in C2C12 myotubes, resulting in increased NO production. In vivo phosphorylation of nNOS was also observed in response to insulin in mouse skeletal muscle. These results indicate, for the first time, that nNOS is phosphorylated in skeletal muscle in response to insulin and in association with increased NO production.

Intra- and inter-molecular effects of a conserved arginine residue of neuronal and inducible nitric oxide synthases on FMN and calmodulin binding.

Nitric oxide synthases (NOSs) synthesize nitric oxide (NO), a signaling molecule, from l-arginine, utilizing electrons from NADPH. NOSs are flavo-hemo proteins, with two flavin molecules (FAD and FMN) and one heme per monomer, which require the binding of calcium/calmodulin (Ca(2+)/CaM) to produce NO. It is therefore important to understand the molecular factors influencing CaM binding from a structure/function perspective. A crystal structure of the CaM-bound iNOS FMN-binding domain predicted a salt bridge between R536 of human iNOS and E47 of CaM. To characterize the interaction between the homologous Arg of rat nNOS (R753) and murine iNOS (R530) with CaM, the Arg was mutated to Ala and, in iNOS, to Glu. The mutation weakens the interaction between nNOS and CaM, decreasing affinity by ~3-fold. The rate of electron transfer from FMN is greatly attenuated; however, little effect on electron transfer from FAD is observed. The mutated proteins showed reduced FMN binding, from 20% to 60%, suggesting an influence of this residue on FMN incorporation. The weakened FMN binding may be due to conformational changes caused by the arginine mutation. Our data show that this Arg residue plays an important role in CaM binding and influences FMN binding.

Altered human CYP3A4 activity caused by Antley-Bixler syndrome-related variants of NADPH-cytochrome P450 oxidoreductase measured in a robust in vitro system.

NADPH-cytochrome P450 oxidoreductase (CYPOR) variants have been described in patients with perturbed steroidogenesis and sexual differentiation, related to Antley-Bixler syndrome (ABS). It is important to determine the effect of these variants on CYP3A4, the major drug-metabolizing cytochrome P450 (P450) in humans. In this study, 12 CYPOR_ABS variants were separately coexpressed with CYP3A4 in a robust in vitro system to evaluate the effects of these variants on CYP3A4 activity in a milieu that recapitulates the stoichiometry of the mammalian systems. Full-length CYPOR variants were coexpressed with CYP3A4, resulting in relative expression levels comparable to those found in hepatic tissue. Dibenzylfluorescein (DBF), a CYP3A-specific reporter substrate (Biopharm Drug Dispos 24:375-384, 2003), was used to compare the variants and wild-type (WT) CYPOR activities with that of human liver microsomes. CYP3A4, combined with WT CYPOR, demonstrated kinetic parameters (k(cat) and K(m)) equal to those for pooled human liver microsomes. CYPOR variants Y181D, Y459H, V492E, L565P, and R616X all demonstrated maximal loss of CYP3A4 catalytic efficiency, whereas R457H and G539R retained ∼10 and 30% activities, respectively. Conversely, variants P228L, M263V, A287P, and G413S each showed WT-like capacity (k(cat)/K(m)), with the A287P variant being formerly reported to exhibit substantially lower catalytic efficiency. In addition, Q153R exhibited 60% of WT CYPOR capacity to support the DBF O-debenzylation reaction, contradicting increased catalytic efficiency (k(cat)/K(m)) relative to that for the WT, reported previously. Our data indicate the importance of use of simulated, validated in vitro systems, employing full-length proteins with appropriate stoichiometric incorporation of protein partners, when pharmacogenetic predictions are to be made for P450-mediated biotransformation.

Regulation of gap junction function and Connexin 43 expression by cytochrome P450 oxidoreductase (CYPOR).

Cytochrome P450 oxidoreductase (CYPOR) is a microsomal electron-transferring enzyme containing both FAD and FMN as co-factors, which provides the reducing equivalents to various redox partners, such as cytochromes P450 (CYPs), heme oxygenase (HO), cytochrome b(5) and squalene monooxygenase. Human patients with severe forms of CYPOR mutation show bone defects such as cranio- and humeroradial synostoses and long bone fractures, known as Antley-Bixler-like Syndrome (ABS). To elucidate the role of CYPOR in bone, we knocked-down CYPOR in multiple osteoblast cell lines using RNAi technology. In this study, knock-down of CYPOR decreased the expression of Connexin 43 (Cx43), known to play a critical role in bone formation, modeling, and remodeling. Knock-down of CYPOR also decreased Gap Junction Intercellular Communication (GJIC) and hemichannel activity. Promoter luciferase assays revealed that the decrease in expression of Cx43 in CYPOR knock-down cells was due to transcriptional repression. Primary osteoblasts isolated from bone specific Por knock-down mice calvariae confirmed the findings in the cell lines. Taken together, our study provides novel insights into the regulation of gap junction function by CYPOR and suggests that Cx43 may play an important role(s) in CYPOR-mediated bone defects seen in patients.

Mutations of human cytochrome P450 reductase differentially modulate heme oxygenase-1 activity and oligomerization.

Genetic variations in POR, encoding NADPH-cytochrome P450 oxidoreductase (CYPOR), can diminish the function of numerous cytochromes P450, and also have the potential to block degradation of heme by heme oxygenase-1 (HO-1). Purified full-length human CYPOR, HO-1, and biliverdin reductase were reconstituted in lipid vesicles and assayed for NADPH-dependent conversion of heme to bilirubin. Naturally-occurring human CYPOR variants queried were: WT, A115V, Y181D, P228L, M263V, A287P, R457H, Y459H, and V492E. All CYPOR variants exhibited decreased bilirubin production relative to WT, with a lower apparent affinity of the CYPOR-HO-1 complex than WT. Addition of FMN or FAD partially restored the activities of Y181D, Y459H, and V492E. When mixed with WT CYPOR, only the Y181D CYPOR variant inhibited heme degradation by sequestering HO-1, whereas Y459H and V492E were unable to inhibit HO-1 activity suggesting that CYPOR variants might have differential binding affinities with redox partners. Titrating the CYPOR-HO-1 complex revealed that the optimal CYPOR:HO-1 ratio for activity was 1:2, lending evidence in support of productive HO-1 oligomerization, with higher ratios of CYPOR:HO-1 showing decreased activity. In conclusion, human POR mutations, shown to impact P450 activities, also result in varying degrees of diminished HO-1 activity, which may further complicate CYPOR deficiency.

Human cytochrome P450 oxidoreductase deficiency caused by the Y181D mutation: molecular consequences and rescue of defect.

Patients with congenital adrenal hyperplasia, exhibiting combined CYP17 and CYP21 deficiency, were shown by Arlt et al. (2004) to harbor a 541T-->G mutation in exon 5 of POR (encoding NADPH-cytochrome P450 reductase, CYPOR), which resulted in a Y181D substitution that obliterated electron transfer capacity. Using bacterial expression models, we examined catalytic and physical properties of the human CYPOR Y181D variant. As purified, Y181D lacked flavin mononucleotide (FMN) and NADPH-cytochrome c reductase (NCR) activity but retained normal flavin adenine dinucleotide binding and NADPH utilization. Titration of the purified protein with FMN restored 64 of wild-type (WT) NCR activity in Y181D with an activation constant of approximately 2 microM. As determined by FMN fluorescence quenching, Y181D had K(d)(FMN) = 7.3 microM. Biplasmid coexpression of CYPOR and CYP1A2, at the physiological ratio of approximately 1:10 in the engineered MK_1A2_POR Escherichia coli strain, showed the compromised capacity of Y181D to support CYP1A2-catalyzed metabolism of the procarcinogens 2-aminoanthracene, 2-amino-3-methylimidazo(4,5-f)quinoline, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Isolated MK1A2_POR membranes confirmed FMN stimulation of Y181D NCR activity with a 1.6 microM activation constant. CYP1A2 ethoxyresorufin-O-dealkylase activity of the MK1A2_POR(Y181D) membranes, undetectable in the absence of added FMN, increased to 37% of MK1A2_POR(WT) membranes with a 1.2 microM FMN activation constant. Therefore, we conclude that compromised FMN binding is the specific molecular defect causing POR deficiency in patients with Y181D mutation and that this defect, in large part, can be overcome in vitro by FMN addition.

Measurement of membrane-bound human heme oxygenase-1 activity using a chemically defined assay system.

Heme oxygenase (HO) catalyzes heme degradation in a reaction requiring NADPH-cytochrome P450 reductase (CPR). Although most studies with HO used a soluble 30-kDa form, lacking the C-terminal membrane-binding region, recent reports show that the catalytic behavior of this enzyme is very different if this domain is retained; the overall activity was elevated 5-fold, and the K(m) for CPR decreased approximately 50-fold. The goal of these studies was to accurately measure HO activity using a coupled assay containing purified biliverdin reductase (BVR). This allows measurement of bilirubin formation after incorporation of full-length CPR and heme oxygenase-1 (HO-1) into a membrane environment. When rat liver cytosol was used as the source of partially purified BVR, the reaction remained linear for 2 to 3 min; however, the reaction was only linear for 10 to 30 s when an equivalent amount of purified, human BVR (hBVR) was used. This lack of linearity was not observed with soluble HO-1. Optimal formation of bilirubin was achieved with concentrations of bovine serum albumin (0.25 mg/ml) and hBVR (0.025-0.05 microM), but neither supplement increased the time that the reaction remained linear. Various concentrations of superoxide dismutase had no effect on the reaction; however, when catalase was included, the reactions were linear for at least 4 to 5 min, even at high CPR levels. These results not only show that HO-1-generated hydrogen peroxide leads to a decrease in HO-1 activity but also provide for a chemically defined system to be used to examine the function of full-length HO-1 in a membrane environment.

Deletion of the autoregulatory insert modulates intraprotein electron transfer in rat neuronal nitric oxide synthase.

Comparative CO photolysis kinetics studies on wild-type and autoregulatory (AR) insert-deletion mutant of rat nNOS holoenzyme were conducted to directly investigate the role of the unique AR insert in the catalytically significant FMN-heme intraprotein electron transfer (IET). Although the amplitude of the IET kinetic traces was decreased two- to three-fold, the AR deletion did not change the rate constant for the calmodulin-controlled IET. This suggests that the rate-limiting conversion of the electron-accepting state to a new electron-donating (output) state does not involve interactions with the AR insert, but that AR may stabilize the output state once it is formed.

Impairment of human CYP1A2-mediated xenobiotic metabolism by Antley-Bixler syndrome variants of cytochrome P450 oxidoreductase.

Y459H and V492E mutations of cytochrome P450 reductase (CYPOR) cause Antley-Bixler syndrome due to diminished binding of the FAD cofactor. To address whether these mutations impaired the interaction with drug-metabolizing CYPs, a bacterial model of human liver expression of CYP1A2 and CYPOR was implemented. Four models were generated: POR(null), POR(wt), POR(YH), and POR(VE), for which equivalent CYP1A2 and CYPOR levels were confirmed, except for POR(null), not containing any CYPOR. The mutant CYPORs were unable to catalyze cytochrome c and MTT reduction, and were unable to support EROD and MROD activities. Activity was restored by the addition of FAD, with V492E having a higher apparent FAD affinity than Y459H. The CYP1A2-activated procarcinogens, 2-aminoanthracene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and 2-amino-3-methylimidazo(4,5-f)quinoline, were significantly less mutagenic in POR(YH) and POR(VE) models than in POR(wt), indicating that CYP1A2, and likely other drug-metabolizing CYPs, are impaired by ABS-related POR mutations as observed in the steroidogenic CYPs.

Insulin resistance is associated with impaired nitric oxide synthase activity in skeletal muscle of type 2 diabetic subjects.

Type 2 diabetes is an insulin-resistant state characterized by hyperinsulinemia and accelerated atherosclerosis. In vitro and in vivo studies in rodents have suggested that nitric oxide generation plays an important role in glucose transport and insulin action. We determined nitric oxide synthase (NOS) activity in skeletal muscle of 10 type 2 diabetic (hemoglobin A(1C) = 6.8 +/- 0.1%) and 11 control subjects under basal conditions and during an 80 mU/m(2).min euglycemic insulin clamp performed with vastus lateralis muscle biopsies before and after 4 h of insulin. In diabetics, insulin-stimulated glucose disposal (Rd) was reduced by 50%, compared with controls (5.4 +/- 0.3 vs. 10.4 +/- 0.5 mg/kg.min, P < 0.01). Basal NOS activity was markedly reduced in the diabetic group (101 +/- 33 vs. 457 +/- 164 pmol/min.mg protein, P < 0.05). In response to insulin, NOS activity increased 2.5-fold in controls after 4 h (934 +/- 282 pmol/min.mg protein, P < 0.05 vs. basal), whereas insulin failed to stimulate NOS activity in diabetics (86 +/- 28 pmol/min.mg protein, P = NS from basal). Basal NOS protein content in muscle was similar in controls and diabetics and did not change following insulin. In controls, insulin-stimulated NOS activity correlated inversely with fasting plasma insulin concentration (r = -0.58, P = 0.05) and positively with Rd (r = 0.71, P = 0.03). In control and diabetic groups collectively, Rd correlated with insulin-stimulated NOS activity (r = 0.52, P = 0.02). We conclude that basal and insulin-stimulated muscle NOS activity is impaired in well-controlled type 2 diabetic subjects, and the defect in insulin-stimulated NOS activity correlates closely with the severity of insulin resistance. These results suggest that impaired NOS activity may play an important role in the insulin resistance in type 2 diabetic individuals.

Genetic variations in NADPH-CYP450 oxidoreductase in a Czech Slavic cohort.

AIM: Estimating polymorphic allele frequencies of the NADPH-CYP450 oxidoreductase (POR) gene in a Czech Slavic population. METHODS: The POR gene was analyzed in 322 individuals from a control cohort by sequencing and high resolution melting analysis. RESULTS: We identified seven unreported SNP genetic variations, including two SNPs in the 5' flanking region (g.4965C>T and g.4994G>T), one intronic variant (c.1899-20C>T), one synonymous SNP (p.20Ala=) and three nonsynonymous SNPs (p.Thr29Ser, p.Pro384Leu and p.Thr529Met). The p.Pro384Leu variant exhibited reduced enzymatic activities compared with wild-type. CONCLUSION: New POR variant identification indicates the number of uncommon variants might be specific for each subpopulation being investigated, particularly germane to the singular role that POR plays in providing reducing equivalents to all CYP450s in the endoplasmic reticulum. Original submitted 15 September 2014; Revision submitted 17 November 2014.

Conditional deletion of cytochrome p450 reductase in osteoprogenitor cells affects long bone and skull development in mice recapitulating antley-bixler syndrome: role of a redox enzyme in development.

NADPH-cytochrome P450 oxidoreductase (POR) is the primary electron donor for cytochromes P450, dehydrocholesterol reductase, heme oxygenase, and squalene monooxygenase. Human patients with specific mutations in POR exhibit severe developmental malformations including disordered steroidogenesis, sexual ambiguities and various bone defects, similar to those seen in patients with Antley-Bixler syndrome (ABS). To probe the role of POR during bone development, we generated a conditional knockout mouse (CKO) by cross breeding Por (lox/lox) and Dermo1 Cre mice. CKO mice were smaller than their littermate controls and exhibited significant craniofacial and long bone abnormalities. Differential staining of the CKO mice skull bases shows premature fusion of the sphenooccipital and basioccipital-exoccipital synchondroses. Class III malocclusion was noted in adult knockout mice with an unusual overgrowth of the lower incisors. Shorter long bones were observed along with a reduction in the bone volume fraction, measured by microCT, in the Por-deleted mice compared to age- and sex-matched littermate controls. Concerted up- or down-regulation of proteins in the FGF signaling pathway observed by immunohistochemistry in the tibia samples of CKO mice compared to wild type controls shows a decrease in the FGF signaling pathway. To our knowledge, this is the first report of a mouse model that recapitulates both skull and long bone defects upon Por deletion, offering an approach to study the sequelae of POR mutations. This unique model demonstrates that P450 metabolism in bone itself is potentially important for proper bone development, and that an apparent link exists between the POR and FGF signaling pathways, begging the question of how an oxidation-reduction flavoprotein affects developmental and cellular signaling processes.

Differential calmodulin-modulatory and electron transfer properties of neuronal nitric oxide synthase mu compared to the alpha variant.

Neuronal nitric oxide synthase µ (nNOSµ) contains 34 additional residues in an autoregulatory element compared to nNOSα. Cytochrome c and flavin reductions in the absence of calmodulin (CaM) were faster in nNOSµ than nNOSα, while rates in the presence of CaM were smaller. The magnitude of stimulation by CaM is thus notably lower in nNOSµ. No difference in NO production was observed, while electron transfer between the FMN and heme moieties and formation of an inhibitory ferrous-nitrosyl complex were slower in nNOSµ. Thus, the insert affects electron transfer rates, modulation of electron flow by CaM, and heme-nitrosyl complex formation.

Nitric oxide synthases activation and inhibition by metallacarborane-cluster-based isoform-specific affectors.

A small library of boron-cluster- and metallacarborane-cluster-based ligands was designed, prepared, and tested for isoform-selective activation or inhibition of the three nitric oxide synthase isoforms. On the basis of the concept of creating a hydrophobic analogue of a natural substrate, a stable and nontoxic basic boron cluster system, previously used for boron neutron capture therapy, was modified by the addition of positively charged moieties to its periphery, providing hydrophobic and nonclassical hydrogen bonding interactions with the protein. Several of these compounds show efficacy for inhibition of NO synthesis with differential effects on the various nitric oxide synthase isoforms.

Identification of six novel P450 oxidoreductase missense variants in Ashkenazi and Moroccan Jewish populations.

BACKGROUND: The enzyme NADPH-P450 oxidoreductase (POR) is the main electron donor to all microsomal CYPs. The possible contribution of common POR variants to inter- and intra-individual variability in drug metabolism is of great pharmacogenetic interest. AIM: To search for POR polymorphic alleles and estimate their frequencies in a Jewish population. MATERIALS & METHODS: We analyzed the POR gene in 301 Ashkenazi and Moroccan Jews. RESULTS: A total of 30 POR SNPs were identified, nine in the noncoding regions and 21 in the protein-coding regions (ten synonymous, 11 missense). Six of these missense variants are previously undescribed (S102P, V164M, V191M, D344N, E398A and D648N). CONCLUSION: The data collected in this study on missense POR SNPs, interpreted in light of the crystallographic structure of human POR, indicate that some POR missense variants may be potential biomarkers for future POR pharmacogenetic screening.