The potency of oxalyl amino acid derivatives as inhibitors of prolyl 4-hydroxylase was studied in vitro, in isolated microsomes and in chicken embryonic-tissue culture. These compounds represent structural analogues of 2-oxoglutarate in which the -CH2- moiety at C-3 is replaced by -NH-, with or without further structural modifications. The most efficient inhibitor of purified prolyl 4-hydroxylase was oxalylglycine. Its mode of inhibition was competitive with respect to 2-oxoglutarate. The Ki value varied between 1.9 and 7.8 microM, depending on the variable substrate used. Oxalylalanine inhibited purified enzyme with a Ki of 40 microM. Other oxalyl amino acid derivatives showed little inhibitory activity. In microsomes isolated from embryonic chicken bone, oxalylglycine and oxalylalanine inhibited prolyl hydroxylation with IC50 values of 23 and 120 microM respectively. Dimethyloxalylglycine was not an inhibitor of purified prolyl 4-hydroxylase and only weakly active in the microsomal system, but efficiently suppressed hydroxyproline synthesis in embryonic chicken calvaria and lung. The data suggest that dimethyloxalyl amino acids are converted into active inhibitors in intact cells, most likely in the cytoplasmic compartment.

The biochemical and morphological consequences of procollagen prolyl 4-hydroxylase inhibition by pyridine-2,4-dicarboxylic acid (2,4-PDCA) and its diethyl ester (diethyl-2,4-PDC) were studied in chick-embryo calvaria, which predominantly synthesize type I collagen. Half-maximal inhibition of tissue hydroxyproline formation required 650 microM-2,4-PDCA, whereas the Ki with respect to chicken prolyl 4-hydroxylase in vitro was 2 microM. In contrast, half-maximal inhibition was caused by 10 microM-diethyl-2,4-PDC in the intact calvaria, although chicken prolyl 4-hydroxylase in vitro was not inhibited even at 1 mM. The collagenous material produced in the presence of diethyl-2,4-PDC showed an altered 'melting' profile and a lowering of the transition temperature by 10 degrees C, indicating misalignment and thermal instability of its triple-helical structure. Amount and electrophoretic mobility of procollagen type I chains were increased in a dose-dependent manner. The amounts of partially processed species and alpha-chains were decreased, without change in mobility. This marked effect on procollagen-collagen conversion in the intact calvaria suggests that the underhydroxylated collagenous material generated in the presence of diethyl-2,4-PDC is resistant to or acts as endogenous secondary inhibitor of type I procollagen N-proteinase. Electron microscopy of treated calvaria cells showed dilated rough endoplasmic reticulum and numerous phagolysosomes, indicating intracellular retention and lysosomal degradation of the newly synthesized underhydroxylated collagenous material. In summary, these results identify 2,4-PDCA and diethyl-2,4-PDC as the first prolyl 4-hydroxylase-directed inhibitor/proinhibitor pair that affects intra- and extra-cellular events during collagen formation.

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A series of 7-(1H-pyrrol-3-yl)-substituted-3,5-dihydroxyhept-6(E)- enoates (-heptanoates) 1 and 2 have been prepared and tested for inhibiti 3-hydroxy-3-methylglutaryl-coenzyme A reductase. The most potent compounds exceeded mevinolin's activity in vitro and in vivo.

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