Novel inhibitors of prolyl 4-hydroxylase. 3. Inhibition by the substrate analogue N-oxaloglycine and its derivatives.

N-Oxaloglycine (3) is an alpha-ketoglutarate (1) analogue that is a competitive inhibitor of prolyl 4-hydroxylase (EC 1.14.11.2). A study of the structure-activity relationships of some other oxalo derivatives shows that substitution on the glycine moiety modulates activity stereoselectively and that if the omega-carboxylate is homologated or replaced by either acylsulfonamides or anilide, then activity is sharply reduced. This sensitivity to these changes is contrasted with the relative insensitivity of another putative alpha-ketoglutarate analogue, pyridine-2,5-dicarboxylic acid (2), and the implication is discussed that compounds of both series are unlikely to bind to prolyl hydroxylase in the same way even though both inhibit the enzyme competitively.

Assay of prolyl 4-hydroxylase by the chromatographic determination of [14C]succinic acid on ion-exchange minicolumns.

An assay for prolyl 4-hydroxylase (EC 1.14.11.2) is described which measures succinic acid produced during the decarboxylation of 2-oxoglutaric acid in the presence of poly(L-Pro-Gly-L-Pro). [1-14C]Succinic acid was separated from its precursor 2-oxo[5-14C]glutaric acid by using ion-exchange minicolumns. The contamination of succinic acid by 2-oxoglutaric acid was approx. 1%, and the recovery of succinic acid was 100%. Kinetic parameters of prolyl 4-hydroxylase measured by the assay showed good agreement with published values. Our experience indicates that the measurement of prolyl 4-hydroxylase by the production of succinic acid is especially suited to investigations involving large numbers of assays.

Inhibition of prolyl 4-hydroxylase by hydroxyanthraquinones.

Prolyl 4-hydroxylase (EC 1.14.11.2) is an essential enzyme in the post-translational modification of collagen. Inhibitors of this enzyme are of potential interest for the treatment of diseases involving excessive deposition of collagen. We have found that anthraquinones with at least two hydroxy groups ortho to each other are potent inhibitors of this enzyme. Kinetic studies revealed that 2,7,8-trihydroxyanthraquinone (THA) competitively inhibited the co-substrate, 2-oxoglutarate, but was non-competitive with regard to ascorbate and was tentatively considered to be uncompetitive with regard to protocollagen. The inhibition by THA was greatly enhanced in the absence of added Fe2+ and was partially reversed by the addition of concentrations of Fe2+ in excess of the optimum for the enzymic reaction. Binding studies indicated that THA is an effective chelating agent for Fe2+. Several non-quinoidal compounds bearing the catechol moiety also inhibited the enzyme. The results suggest that THA inhibited prolyl 4-hydroxylase by binding to the enzyme at the site for 2-oxoglutarate possibly involving the Fe2+ atom, rather than by complexing with Fe2+ in free solution. The inhibition of prolyl 4-hydroxylase by THA exhibited strong positive co-operativity and may involve three distinct but non-independent binding sites.

Anti-proliferative properties of Clozic, a disease-modifying anti-arthritic agent.

Cytostatic drugs have been used in the treatment of rheumatoid arthritis but are of limited clinical application due to their severe toxic side-effects. We have discovered that 'Clozic' (ICI 55897), an agent with disease-modifying properties in rheumatoid arthritis patients, inhibits the growth of a variety of mammalian cell types including a matrix-secreting cell culture derived from neonatal rat hearts. The inhibition of growth was reversible and no loss of cell viability occurred when measured by lactate dehydrogenase released into the medium or by vital staining, suggesting a cytostatic rather than a cytotoxic mechanism. Cytostatic activity was observed at ICI 55897 concentrations within the reported therapeutic plasma concentration range and was related to the concentration of unbound compound, since the effect could be reduced by increasing the albumin concentration in the medium. Other oxyalkanoic acids inhibited cell growth. Their inhibitory potency correlated with lipophilicity. The anti-proliferative potencies of R and S enantiomers of two oxyalkanoic acids containing asymmetric centres were similar. These observations suggest that the anti-proliferative effect of the oxyalkanoic acids is due to their interaction with lipophilic cell target sites.