UGT1A1 promoter polymorphism increases risk of nilotinib-induced hyperbilirubinemia.

Nilotinib is a novel BCR-ABL inhibitor with significantly improved potency and selectivity over imatinib. In Phase I and Phase II clinical studies of nilotinib in patients with a variety of leukemias, infrequent instances of reversible, benign elevation of bilirubin were observed. Uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) glucuronidates bilirubin in humans, and a polymorphism in the promoter of the gene that encodes it has been associated with hyperbilirubinemia during treatment with a number of drugs. Pharmacogenetic analysis of that TA-repeat polymorphism found an association between the (TA)7/(TA)7 genotype and risk of hyperbilirubinemia in Phase I patients with imatinib-resistant/intolerant chronic myeloid leukemia (CML) or relapsed/refractory Ph+ acute lymphoblastic leukemia (ALL); this result was replicated in two separate analyses of the chronic phase (CP) and accelerated phase (AP) CML arms of a Phase II study. As nilotinib is not known to be glucuronidated by UGT1A1, the combined impact of inhibition of UGT1A1 activity by nilotinib and genetic polymorphism is the most likely cause of the increased rate of hyperbilirubinemia.

Characterization of human A(2B) adenosine receptors: radioligand binding, western blotting, and coupling to G(q) in human embryonic kidney 293 cells and HMC-1 mast cells.

Recombinant human A(2B) adenosine receptors (A(2B)ARs) and receptors extended on the amino terminus with hexahistidine and the FLAG epitope, DYKDDDDK (H/F-A(2B)) were stably overexpressed (to >20,000 fmol/mg protein) in human embryonic kidney 293 cells (HEK-A(2B)). By Western blotting, the H/F-A(2B) receptor runs as a 34.8-kDa glycoprotein. Pharmacological properties of A(2B)ARs were characterized with (125)I-3-aminobenzyl-8-phenyl-(4-oxyacetic acid)-1-propylxanthine (K(D), 36 nM). In competition binding assays, the affinity of agonists is reduced by substitution on either the N(6)- or the C-2 position of the adenine ring, whereas 5'-substitutions increase affinity, resulting in the potency order: 5'-N-ethylcarboxamidoadenosine (NECA) >> N(6)-aminobenzyl-NECA approximately 2-chloroadenosine > 2-[4-(2-carboxyethyl)phenethylamino]-NECA (CGS21680) > N(6)-aminobenzyladenosine. The A(2B)AR is potently blocked by the A(2A)-selective antagonist 4-(2-[7-amino-2-[2-furyl][1,2, 4]triazolo-[2,3-a][1,3,5] triazin-5-yl-amino]ethyl)phenol (ZM241385; K(I), 32 nM for A(2B), 1.4 nM for A(2A)) and the A(1) selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (K(I), 50.5 nM for A(2B); 2.5 nM for A(1)). The K(I) values for the antiasthmatic xanthines, theophylline (7.8 microM) and enprofylline (6.4 microM), are below their therapeutic plasma concentrations (20 to 50 microM), and agree with K(I) determinations for inhibition of NECA-stimulated cAMP accumulation in HEK-A(2B) cells. NECA or N(6)-(2-iodo)benzyl-5'-N-methylcarboxamidodoadenosine (IB-MECA) stimulate inositol trisphosphates and calcium accumulation in HEK-A(2B) or HEK-A(3) cells, respectively, but only the A(3) response is prevented by pertussis toxin. In human HMC-1 mast cells, A(2B)AR activation stimulates calcium mobilization and cAMP accumulation. We conclude that HEK-A(2B) cells and HMC-1 mast cells possess A(2B)AR glycoproteins that are coupled to both G(q/11) and G(s).

Purification of A1 adenosine receptor-G-protein complexes: effects of receptor down-regulation and phosphorylation on coupling.

We examined the effects of exposing A1 adenosine receptors (A1ARs) to an agonist on the stability and phosphorylation state of receptor-guanine nucleotide-binding regulatory protein (R-G-protein) complexes. Non-denatured recombinant human A1ARs extended on the N-terminus with hexahistidine (His6) and the FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) epitope (H/F) were purified to near homogeneity from stably transfected Chinese-hamster ovary (CHO)-K1 cells. Purified receptors have pharmacological properties similar to receptors in membranes. G-proteins were co-purified with 15+/-2% of H/F-A1AR unless receptor-G-protein (R-G) complexes were uncoupled by pre-treating cell membranes with GTP. By silver staining, purified A1AR-G-protein complexes contain receptors, G-protein alpha and beta subunits and an unidentified 97 kDa protein. Pretreating intact cells with N6-cyclopentyladenosine (CPA) for 24 h decreased both the total number of receptors measured in membranes and the number of purified A1ARs by about 50%. In contrast, pretreating cells with CPA decreased the number of R-G complexes measured in membranes (54+/-6%) significantly less than it decreased the number of purified R-G complexes (78+/-3%) as detected by 125I-N6-(4-aminobenzyl)adenosine binding or by Western blotting Gialpha2. The effect of CPA to decrease the fraction of receptors purified as R-G complexes was not associated with any change in low-level A1AR phosphorylation (found on serine), or low-level phosphorylation of G-protein alpha or beta subunits or the 97 kDa protein. These experiments reveal a novel aspect of agonist-induced down-regulation, namely a diminished stability of receptor-G-protein complexes that is manifested as uncoupling during receptor purification.

Double tagging recombinant A1- and A2A-adenosine receptors with hexahistidine and the FLAG epitope. Development of an efficient generic protein purification procedure.

An expression plasmid for mammalian cells (CLDN10B) has been modified to add nucleotides encoding hexahistidine and the FLAG peptide (H/F) to cDNAs. The new mammalian expression plasmid has been named pDoubleTrouble (pDT). The plasmid and a recombinant baculovirus were used to produce native-and H/F-human A1 and A2A adenosine receptors, optimally expressed in CHO-K1 and Sf9 cells, respectively. Binding to recombinant H/F-A1 receptors (Bmax = 30 pmol/mg protein) was characterized using [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]CPX) and 125I-N6-aminobenzyladenosine (125I-ABA). Binding to H/F-A2A receptors (Bmax = 48 pmol/mg protein) was characterized using [3H]5'-N-ethylcarboxamidoadenosine ([3H]NECA) and [3H]2-[4-(2-carboxyethyl)phenethylamino]-NECA ([3H]CGS21680). By comparison to native receptors, the addition of H/F to the amino termini of these receptors had no effect on the binding affinities cyclic AMP accumulation in intact cells was not affected by the H/F extension. Anti-FLAG and Ni-nitrilotriacetic acid affinity chromatography resulted in high yield ( >50% overall recovery) of nearly homogeneous deglycosylation with N-glycosidase F. We anticipate that pDT will be generally useful for facilitating the purification in high yield of recombinant receptors and other proteins by single or sequential affinity chromatography steps.

A1 adenosine receptors. Two amino acids are responsible for species differences in ligand recognition.

Species differences in ligand binding to A1 adenosine receptors were localized to the seventh transmembrane (TM7) region based on the binding of [8-3H]cyclopentyl-1, 3-dipropylxanthine and three other ligands to wild type and six bovine/canine interspecies receptor chimeras expressed in COS-1 cells. Subsequent site-directed mutagenesis experiments identified amino acid 270 (isoleucine/methionine, bovine/canine) as being primarily responsible for species differences in the binding of N6-adenine-substituted compounds, R-N6-phenylisopropyladenosine (R-PIA) and (S)-N6-endonorbornan-2-yl-9-methyladenine, and the C-8-substituted xanthine, [3H]cyclopentyl-1,3-dipropylxanthine. These data are consistent with the hypothesis that the N6 region of adenines and the C-8-region of xanthines bind to the same region of the receptor. A second TM7 amino acid, 277 (serine/threonine, bovine/canine), selectively influences the binding of the ribose-substituted adenosine analog, 5'-N-ethylcarboxamidoadenosine to a variable extent, depending on the nature of amino acid 270. We hypothesize that amino acid 270 of the A1 receptor interacts with the N6 region of adenosine, while amino acid 277 is important, especially in the absence of an N6 substitution, for interactions with a distinct nucleoside region, possibly on the ribose.

Molecular cloning and functional expression of a sheep A3 adenosine receptor with widespread tissue distribution.

Using the polymerase chain reaction, an A3 adenosine receptor has been cloned from the hypophysial par tuberalis of sheep. The clone encodes a 317-amino acid protein that is 72% identical to the rat A3 adenosine receptor. In contrast to rat, where abundant A3 mRNA transcript is found primarily in testis, the sheep transcript is most abundant in lung, spleen, and pineal gland and is present in moderate levels in brain, kidney, and testis. The agonist N6-amino[125I]iodobenzyladenosine binds with high affinity (Kd congruent to 6 nm) and specificity to recombinant A3 adenosine receptors expressed transiently in COS-1 cells or stably in CHO K1 cells. The potency order of agonists is N6-aminoiodobenzyladenosine > N-ethylcarboxamidoadenosine > or = (R)-phenylisopropyladenosine >> cyclopentyladenosine. Little or no binding of purine nucleotides was detected. The potency order of antagonists is 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1- propylxanthine (I-ABOPX) (Ki = 3 nM) > 1,3-dipropyl-8-(4-acrylate)phenylxanthine (BW-A1433) > 1,3-dipropyl-8-sulfophenylxanthine = xanthine amine cogener >> 8-cyclopentyl-1,3-dipropylxanthine. Enprofylline does not bind. These data indicate that, in contrast to A1 adenosine receptors, A3 adenosine receptors preferentially bind ligands with aryl rings in the N6-position of adenine and in the C8-position of xanthine. Among antagonists, the A3 adenosine receptor preferentially binds 8-phenylxanthines with acidic versus basic para-substituents (I-ABOPX > BW-A1433 > 1,3-dipropyl-8-sulfophenylxanthine = xanthine amine cogener). Agonists reduce forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells stably transfected with recombinant sheep A3 adenosine receptors; the reduction is blocked by BW-A1433 but not by 8-cyclopentyl-1,3-dipropylxanthine. These data suggest that (i) A3 adenosine receptors display unusual structural diversity for species homologs, (ii) in contrast to rat, sheep A3 adenosine receptors have a broad tissue distribution, and (iii) some xanthines with acidic side chains bind with high affinity to A3 adenosine receptors.

Cloning and expression of a bovine adenosine A1 receptor cDNA.

A bovine brain adenosine A1 receptor cDNA encoding a 326 amino acid protein has been identified. This cDNA, which encodes a protein greater than 90% identical to analogous rat and dog receptors, was transiently expressed in COS-1 cells. Recombinant receptors exhibited the features of bovine A1 receptors that distinguish it from rat and canine receptors, including subnanomolar Ki for 1,3-dipropyl-8-cyclopentylxanthine, R-phenylisopropyl- adenosine (R-PIA) and xanthine amino conjugate, and the distinct potency order: R-PIA greater than S-PIA much greater than 5'-N-ethylcarboxamidoadenosine greater than 2'-chloroadenosine. The results indicate that the pharmacological differences between A1 adenosine receptors among species result from only minor differences in receptor structures.