2-Oxoglutarate analogue inhibitors of prolyl hydroxylase domain 2

Analogues of the 2-oxoglutarate cosubstrate of the human oxygen sensing enzyme prolyl hydroxylase domain 2 (PHD2) with variations in the potential iron-chelating group were screened as inhibitors and for binding (using non-denaturing electrospray ionization mass spectrometry) to PHD2.     

An intramembranous reductant which participates in the proline hydroxylation reaction with intracisternal prolyl hydroxylase and unhydroxylated procollagen in isolated microsomes from L-929 cells

We previously have described a substance present in crude sonicates of L-929 cells which replaced ascorbate in vitro as a reductant for prolyl hydroxylase (B. Peterkofsky, D. Kalwinksy and R. Assad, 1980, Arch. Biochem. Biophys.199, 362–373). In the present study we found that almost 90% of the substance was particulate after differential centrifugation of stationary phase L-929 cell homogenates. The substance was not localized in nuclei or mitochondria and was found in the same fractions as microsomes, but these fractions also contained lysosomes and cell membranes. The reductant could not be solubilized from particles by Brij-35, indicating that it is an intrinsic component of a membrane rather than intracisternally located. The intramembranous cofactor, in the absence of ascorbate, participated in the in vitro hydroxylation of [4-³H]proline in radio-actively labeled, intracisternal unhydroxylated procollagen in isolated microsomes which also contained prolyl hydroxylase. Hydroxylation was determined by measuring tritiated water formed from release of the 4-trans tritium atom. Since it is unlikely that such participation could occur if the cofactor were located within the membrane of another subcellular organelle, we have concluded that it is in the same particle as prolyl hydroxylase and unhydroxylated procollagen, that is, the microsome. With the endogenous reductant the reaction was slower than with saturating ascorbate and was increased by NADH. Maximum hydroxylation with the endogenous reductant was close to that which could be achieved with ascorbate. These results provide strong evidence that the endogenous reductant alone can account for the phenomenon of ascorbate-independent proline hydroxylation in L-929 cells. As in the case of ascorbate, the microsomal reductant functioned only in the presence of α-ketoglutarate and Fe²⁺ and served as reductant for lysyl hydroxylase. It also was detected in the particulate fraction of virally transformed BALB 3T3 cells and in purified microsomes from bones of intact chick embryos. Since ascorbate could be taken up and concentrated in bone microsomes, it is unlikely that the endogenous reductant serves as an intermediary between ascorbate and intracisternal prolyl hydroxylase.     

Submicrosomal localization of prolyl hydroxylase from chick embryo limb bone.

A fraction greatly enriched in microsomes was prepared from chick embryo limb bone tissue homogenates by differential centrifugation in a high density solution of Metrizamide. This fraction was used to determine the submicrosomal localization of prolyl hydroxylase. At a low concentration (0.05%) of the non-ionic detergents Triton X-100 and Brij-35, 90 to 93% of prolyl hydroxylase activity was released from microsomes. Concentrations of Triton X-100 greater than 0.1% were required to solubilize the intrinsic membrane enzyme NADH-ferricyanide reductase and to release membrane-bound ribosomes, while Brij-35 did not extensively solubilize membrane components even at concentrations up to 0.4%. In addition, prolyl hydroxylase activity which could subsequently be released from microsomes by Brij-35 was relatively resistant to trypsin proteolysis at concentrations which removed more than 50% of the ribosomes and approximately 40% of the protein from microsomes. These results suggest that 90 to 93% of prolyl hydroxylase activity in connective tissue is located within the cisternae of the endoplasmic reticulum. Gel filtration of prolyl hydroxylase released from microsomes or found in the soluble fraction of limb bone homogenates revealed two peaks of activity corresponding to molecular weights of 230,000 and 450,000 to 500,000. The latter is twice the value reported for purified chick embryo prolyl hydroxylase. A fraction of the total prolyl hydroxylase activity (generally 20 to 35%) in microsome preparations could be measured in the absence of detergent, although the microsomal membrane should be impermeable to the large unhydroxylated collagen chains used as substrate. On the basis of experimental data, it was concluded that detergent-independent activity was most likely due to damaged microsomal membranes and that this damage was sufficient to allow substrate and trypsin to enter the cisternae but not to allow prolyl hydroxylase to be released.     

The role of ascorbate in the prolyl hydroxylase reaction.

Binding isotherms of some anthracyclic derivatives with DNA have been determined by modern electrochemical techniques(Voltammetry - ac polarography). These techniques making use of the difference between the diffusion coefficients of DNA-drug complex and of free molecules of drug in solution allow the direct determination of the later ones. No striking differences can be noticed between the tested anthracyclines. No correlation can be established between the affinity of daunorubicin and some of its analogs for DNA and their more or less antitumoral activity.     

Stimulatory effect of soluble supernatant on hydroxylase activity of rat testis microsomes.

Addition of soluble supernatant to testis microsomes results in 42% increase in steroid 17,20-lyase activity and a 65% increase in 17alpha-hydroxylase activity. This stimulatory activity could be partially purified by salt fractionation. The activating factor(s) was not removed by dialysis nor did it appear to be lipid. It was destroyed by trypsin. Differential effects of heat were observed with the hydroxylase and lyase activators. The activation did not affect Km but only increased Vmax. The supernatant could be added to each enzyme to the point of maturation. No binding of steroids by the supernatant could be detected. Corpus luteum and placental supernatant did not stimulate enzymic activity, but supernatant from an adrenal adenoma was active.     

Antioxidative galloyl esters as enzyme inhibitors of p-hydroxybenzoate hydroxylase

Gallic acid and its esters were evaluated as enzyme inhibitors of recombinant p-hydroxybenzoate hydroxylase (PHBH), a NADPH-dependent flavin monooxygenase from Pseudomonas aeruginosa. n-Dodecyl gallate (DG) (IC(50)=16 microM) and (-)-epigallocatechin-3-O-gallate (EGCG) (IC(50)=16 microM), a major component of green tea polyphenols, showed the most potent inhibition, while product-like gallic acid did not inhibit the enzyme significantly (IC(50)>250 microM). Inhibition kinetics revealed that both DG and EGCG inhibited PHBH in a non-competitive manner (K(I)=18.1 and 14.0 microM, respectively). The enzyme inhibition was caused by specific binding of the antioxidative gallate to the enzyme, and by scavenging reactive oxygen species required for the monooxygenase reaction. Molecular modeling predicted that EGCG binds to the enzyme in the proximity of the FAD binding site via formation of three hydrogen bonds.     

Design and synthesis of substituted pyridine derivatives as HIF-1alpha prolyl hydroxylase inhibitors

Structure-guided de novo drug design led to the identification of a novel series of substituted pyridine derivatives as HIF-1alpha prolyl hydroxylase inhibitors. Pyridine carboxyamide derivatives bearing a substituted aryl group at the 5-position of the pyridine ring show appreciable activity, while constraining the side chain by placing a pyrazole carboxylic acid generated a potent lead series with consistent activity against EGLN-1.     

Human prolyl hydroxylase. Purification, partial characterization and preparation of antiserum to the enzyme.

Prolyl hydroxylase was purified from human foetal skin and from a mixture of human foetal tissues by the affinity chromatography procedure using poly(L-proline). The enzyme from both sources was pure, when examined by polyacrylamide gel electrophoresis, as a native protein or in the presence of sodium dodecylsulphate, and enzyme activity recovery varied from 38% to 70% with seven enzyme preparations. The enzyme synthesized from 61.0 mumol to 82.7 mumol hydroxyproline mg protein-1 h-1 degrees C with a saturating concentration of (Pro-Pro-Gly)5 as substrate. The molecular weight of the enzyme was identical with that of the chick prolyl hydroxylase when studied by gel filtration, and the molecular weights of the subunits of the enzyme were about 61000 and 64000 as determined by sodium dodecylsulphate-polyacrylamide gel electrophoresis. The amino acid composition of the human enzyme was very similar to that of the chick prolyl hydroxylase. Antisera to human and chick prolyl hydroxylases were prepared in rabbits. A single precipitin line was seen between the antiserum to human prolyl hydroxylase and the human enzyme in double immunodiffusion, and no cross-reactivity was detected between the human chick enzymes by this technique. However, a distinct cross-reactivity was observed between the human and chick enzymes in inhibition experiments.     

Turnover of prolyl hydroxylase tetramers and the monomer-size protein in chick-embryo cartilaginous bone and lung in vivo

The turnover of prolyl hydroxylase and an immunoreactive protein that corresponds in size to the smaller subunit of the enzyme was studied in vivo after injection of [(3)H]leucine into 11-day chick embryos. The specific radioactivity and total radioactivity of the monomer-size protein were much higher than those of the enzyme tetramers in the cartilaginous bone at 3h and 12h after the radioisotope injection, indicating that the monomer-size protein represents precursors rather than degradation products of the enzyme tetramers. Between 24 and 144h after the injection the specific radioactivity and total radioactivity of the two forms of the enzyme protein showed essentially identical decay rates, the observed specific radioactivity of the monomer-size protein being about 120-130% and total radioactivity about 80% of that of the enzyme tetramers. The true half-life, when corrected for dilution caused by tissue growth and re-utilization of the [(3)H]leucine, was 37.9h for the monomer-size protein and 39.0h for the tetramers. The results obtained in the lung were less reliable owing to high blank radioactivity values in the immunoprecipitation, but even so some definite differences were found between this tissue and the cartilaginous bone. The specific radioactivity of both forms of the enzyme protein at 24h was only about 20-25% of that in the cartilaginous bone. The total radioactivity of the monomer-size protein in the lung remained about 5 times that of the enzyme tetramers, whereas it was only about 0.8 times that of the tetramers in the cartilaginous bone. As in the cartilaginous bone, the decay rates of both forms of the enzyme protein were essentially identical in the lung, with a true half-life of about 46h. The results suggest that the rate of prolyl hydroxylase synthesis is slower in the lung than in the cartilaginous bone, whereas the degradation rates are fairly similar in these two tissues. The data further suggest that, in the lung at least, a large part of the monomer-size protein became degraded without being converted into enzyme tetramers.     

Assay of prolyl hydroxylase in cultured fibroblast monolayers

Prolyl hydroxylase may be assayed in cultured cell monolayers after a cold acetone treatment or air-drying. Enzyme activity of dried cells is stable when cell monolayers are stored frozen and desiccated, thus permitting simultaneous assay of cells harvested at different times, e.g., on different days of a culture cycle. Uniformity of cell monolayers permits use of separate dishes for assay of protein or DNA to determine specific enzyme activity. This procedure greatly facilitates harvesting and assay of cells for a variety of biochemical measurements.     

Involvement of superoxide in the prolyl and lysyl hydroxylase reactions

Four superoxide dismutase active copper chelates, Cu(acetylsalicylate)₂, Cu(salicylate)₂, Cu(lysine)₂ and Cu(tyrosine)₂, proved to be inhibitors of prolyl and lysyl hydroxylase. The kinetics of the inhibition are consistent with the proposal that these compounds dismutated ${}^{\mathrm{?}}\text{O}{}_2{}^{\mathrm{staggered?}}$ at the active site of the enzymes. The data strongly suggest that ${}^{\mathrm{?}}\text{O}{}_2{}^{\mathrm{staggered?}}$ is the active form of O₂ in the prolyl and lysyl hydroxylase reactions.     

Use of soluble, collagenous peptides from medium of chick calvaria cultures as substrate for prolyl hydroxylase

Underhydroxylated collagenous proteins accumulate in the media of embryonic chick calvaria cultured in the presence of α,α′-dipyridyl for 24 h. These soluble collagenous proteins, when labeled with radioactive proline, were shown to be a specific, stable, and highly efficient substrate for in vitro measurement of prolyl hydroxylase. The ability of the media proteins to serve as a substrate for prolyl hydroxylase was abolished by digestion with purified bacterial collagenase. This method of substrate preparation provides a soluble, efficient, economical substrate for routine prolyl hydroxylase assays, and permits the accumulation of sufficient quantities of substrate of one specific activity to serve for extended periods of time.     

Inhibition of prolyl hydroxylase by poly(ADP-ribose) and phosphoribosyl-AMP. Possible role of ADP-ribosylation in intracellular prolyl hydroxylase regulation

Poly(ADP-ribose) prepared by incubating NAD+ with rat liver nuclei inhibited the hydroxylation reaction catalyzed by purified prolyl hydroxylase (proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) in vitro. Near complete inhibition of the enzyme was seen in the presence of 6 nM (ADP-Rib)18 with a Ki(app) of 1.5 nM. The monomer unit of poly(ADP-ribose), adenosine diphosphoribose (ADP-Rib), was found to be a weak inhibitor. On the other hand, poly(ADP-ribose)-derived phosphoribosyl-AMP (PRib-AMP) and its dephosphorylated product, ribosyl-ribosyl-adenine (Rib-RibA), inhibited the enzyme in nanomolar concentrations (Ki(app) 16.25 nM). The order of inhibition was (ADP-Rib)18 greater than PRib-AMP, Rib-RibA much greater than ADP-Rib. These results suggested that the 1"----2' ribosyl-ribosyl moiety in these compounds was involved in the inhibition of the enzyme. The possibility that intracellular prolyl hydroxylase is regulated by the involvement of ADP-ribosylation reactions was examined in confluent cultures of skin fibroblast treated with 20 mM lactate. The activity of prolyl hydroxylase was stimulated by 145% over that of untreated cultures. In the lactate-treated cells, the level of NAD+ was lowered and the total ADP-ribosylation of cellular proteins reduced by 40%. These observations imply that the lactate-induced activation of cellular prolyl hydroxylase is mediated by a reduction in ADP-ribosylation and that the synthesis and degradation of ADP-ribose moiety(ies) may possibly regulate prolyl hydroxylase activity in vivo.     

Design and synthesis of a series of novel pyrazolopyridines as HIF-1alpha prolyl hydroxylase inhibitors

Recently resolved X-ray crystal structure of HIF-1alpha prolyl hydroxylase was used to design and develop a novel series of pyrazolopyridines as potent HIF-1alpha prolyl hydroxylase inhibitors. The activity of these compounds was determined in a human EGLN-1 assay. Structure-based design aided in optimizing the potency of the initial lead (2, IC(50) of 11 microM) to a potent (11l, 190 nM) EGLN-1 inhibitor. Several of these analogs were potent VEGF inducers in a cell-based assay. These pyrazolopyridines were also effective in stabilizing HIF-1alpha.