ABSTRACT
Peptidyl alpha-hydroxylating monooxygenase (PHM) functions in vivo towards the biosynthesis of alpha-amidated peptide hormones in mammals and insects. PHM is a potential target for the development of inhibitors as drugs for the treatment of human disease and as insecticides for the management of insect pests. We show here that relatively simple ground state analogs of the PHM substrate hippuric acid (C(6)H(5)-CO-NH-CH(2)-COOH) inhibit the enzyme with K(i) values as low as 0.5microM. Substitution of sulfur atom(s) into the hippuric acid analog increases the affinity of PHM for the inhibitor. Replacement of the acetylglycine moiety, -CO-NH-CH(2)-COOH with an S-(thioacetyl)thioglycolic acid moiety, -CS-S-CH(2)-COOH, yields compounds with the highest PHM affinity. Both S-(2-phenylthioacetyl)thioglycolate and S-(4-ethylthiobenzoyl)thioglycolic acid inhibit the proliferation of cultured human prostate cancer cells at concentrations >100-fold excess of their respective K(i) values. Comparison of K(i) values between mammalian PHM and insect PHM shows differences in potency suggesting that a PHM-based insecticide with limited human toxicity can be developed.
Subject(s)
Enzyme Inhibitors/chemistry , Hippurates/chemistry , Hippurates/pharmacology , Insecticides/chemistry , Mixed Function Oxygenases/antagonists & inhibitors , Multienzyme Complexes/antagonists & inhibitors , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Hippurates/chemical synthesis , Humans , Inhibitory Concentration 50 , Insecticides/metabolism , Insecticides/pharmacology , Mixed Function Oxygenases/metabolism , Models, Molecular , Multienzyme Complexes/metabolism , Rats , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
Thiols, simple and complex, including polypeptide and protein thiols, react rapidly and selectively with esters of propiolic acid under very mild conditions (aqueous buffer, room temperature, pH 7) to give thioacrylates. These stable derivatives exhibit strong, characteristic ultraviolet spectra, maximal at 280-290 nm, molar absorbance ca 12,500. In a single, simple experiment the thiol is detected, its amount is estimated, it is stabilized against oxidation and disulfide scrambling, it is converted into a derivative amenable to isolation and structure elucidation procedures, and it is tagged with recognizable ultraviolet and NMR characteristics generated de novo. The reagents are stable and inexpensive and the procedures are quick, easy, sensitive and selective.
Subject(s)
Alkynes/chemistry , Propionates/chemistry , Sulfhydryl Compounds/analysis , Indicators and Reagents , Molecular Probe Techniques , Spectrophotometry, Ultraviolet , Sulfhydryl Compounds/chemistryABSTRACT
The influence of three important cytokines (IL-8, TNF-alpha, and HuIFN-alpha) on ongoing rhinovirus infections has been examined in vitro, individually or as combinations. TNF-alpha was able to transform traces of HRV infections into full-blown infections. Furthermore, TNF-alpha was able to down-regulate the antiviral action of HuIFN-alpha completely, even at levels of just a few pg/ml. This suggests that the induction of TNF-alpha by HRV may be part of the virus's strategy to minimize the interferon response which is part of the host's immune defence system. However, troxerutin (a flavonoid) was able to neutralize the downregulatory action of TNF-alpha on the HuIFN-alpha system at low levels and re-establish the antiviral activity ascribed to IFN-alpha. IL-8 exerted a minor influence on the interferon system, and had no influence on rhinovirus infections. The in vitro findings are supported, in part, by recent in vivo findings in a common cold pilot study.
Subject(s)
Interferon-alpha/pharmacology , Interleukin-8/pharmacology , Rhinovirus/drug effects , Tumor Necrosis Factor-alpha/pharmacology , Cell Line , Common Cold/drug therapy , Common Cold/immunology , Humans , Hydroxyethylrutoside/analogs & derivatives , Hydroxyethylrutoside/pharmacology , Rhinovirus/physiology , Virus Replication/drug effectsABSTRACT
The title enzyme, peptidylglycine alpha-hydroxylating monooxygenase (PHM), is essential to the in vivo generation of a wide variety of physiologically significant alpha-amidated peptide hormones from the corresponding C-terminal glycine-extended prohormones. Over a 20-year period of time a massive amount of experimental information about the enzyme has accumulated, but its mechanism of action has remained obscure. A major stumbling block to proposed mechanisms is the fact that the two copper atoms found in the active site are fixed 11 A apart. A novel mechanism is now proposed which accommodates and, indeed, requires this separation and proceeds through energetically accessible steps. It is proposed that hydroxylation at the terminal glycine residue of the C-terminal glycine-extended prohormone proceeds first by a concerted sequence of single-electron electromeric shifts, whereby both copper atoms are oxidized to Cu(II), oxygen is reduced to peroxide coordinated to Cu(M), and the glycyl group is tautomerized to its enolate coordinately bound to Cu(H). Upon subsequent reversion to the carbonyl tautomer, by a sequence of two-electron shifts, the enolate as nucleophile reacts with peroxide as electrophile, generating product alpha-hydroxyglycine, decoordinated from Cu(H), reopening the mouth of the active-site pocket to egress of product and ingress of substrates.
