Hydroxamic Acid Moiety Research Articles

Assembly of Coordination Nanostructures via Ligand Derivatization of Oxide Surfaces

A scheme is presented for the construction of coordination nanostructures on oxide surfaces (glass, Si/SiO2, quartz), based on application of epoxy-terminated monolayers as anchors for covalent grafting of ligands. Two ligands bearing amine groups were reacted with epoxysilane monolayers on oxide surfaces, providing ligand-terminated substrates. The ligands employed were (i) a pyridine moiety, used for subsequent binding of cobalt tetraphenylporphine (CoTPP), and (ii) deferoxamine (DFX), which contains hydroxamic acid moieties, used for subsequent construction of various Zr4+-based coordination layers. The results suggest that a dense ligand layer was obtained in both cases, allowing the formation of coordination overlayers on the oxide surfaces. The growth of coordinated layers was similar to analogous overlayers assembled on Au substrates, indicating that high ligand coverage is achieved by the epoxy-amine surface reaction. Epoxy-based functionalization of oxide substrates is a mild and efficient method for preparing high-quality coordination overlayers. Moreover, the method makes use of commercially available silane and amine reactants, providing the basis for wide application.

New Hydroxamic Acid Derivative Prevents Sq-OOH Induced Wrinkle

Lipid peroxidation has been suggested to play a key role in damaging biological processes resulting from excessive exposure to UV light. Aging of skin has been associated with increased peroxidation of skin lipid. In particular, squalene, which is a main component of skin surface polyunsaturated lipids, is easily peroxidized. Squalene-monohydroperoxide (Sq-OOH), the initial product of peroxidized squalene, is produced at the human skin by natural exposure to sunlight during day time activities. Recently, a few researchers reported on the damages of hairless mice skin by the topical application of Sq-OOH. Repeated application of Sq-OOH to the hairless mice induced its the skin wrinkles with clear and fine appearance. Histologically were seen, more keratinocyte hypertrophy with Sq-OOH treated skin than with chronic UVB irradiated group in epidermis. In dermis, characteristics, found in UVB irradiated skin, such as the loss of polarity, fractured collagen bundle and elastosis, were not observed severely. The alteration of epidermal layer might be the main factor for the formation of wrinkle caused by Sq-OOH. From the previous works, Sq-OOH is considered as an important source of damage in the early stage of aging process to result in epidermal wrinkle. Thus, it is very important to minimize such damage that would be induced mainly by Sq-OOH against healthy skin condition. Therefore, we tried to develop new anti-aging materials for protecting skin damage by Sq-OOH. Hydroxamic acids, the naturally occurring products exhibit low toxicities in general, and are of interest for many therapeutic applications. Recently, hydroxamic acids and derivatives were reported largely as effective antibacterial and antifungal agents. Their activities originate from their ability to coordinate on metal ions. That is, hydroxamic acid group is regarded as an one electron donor. Hydroxamic acid moiety has also drawn much attention as a reactive oxygen species (ROS) scavenger. In this study, a new skin protecting compound, (4-(Nhydroxycarbamoyl)phenyl)-N-adamantanylcarboxamide (1) is synthesized to show inhibitory activities of lipid peroxidation both in vitro and in vivo. The targeted compound 1 is a hydroxamic acid derivative which possesses an amide linker between hydroxamic acid and adamantane moiety. Adamantane group was adopted as a hydrophobic moiety to optimize antioxidant activity of hydroxamic acid in animal test. Compound 1 is believed to be adequate in skin permeation because of its balance in hydrophilic and hydrophobic character.

Solid-Phase Synthesis of Biologically Interesting Compounds Containing Hydroxamic Acid Moiety

Chemical strategies developed for the solid-phase synthesis of hydroxamates are divided into four groups: (i) the traditional synthesis of hydroxamates via cleavage of resin-bound esters by hydroxylamine and its derivatives, (ii) introduction of hydroxamic acid moiety on the resin-bound precursor, (iii) transformation of polymer-supported hydroxylamine, attached to a solid supported linker either by oxygen (O-linking strategy) or by nitrogen (N-linking strategy), and (iv) synthesis of N-alkyl hydroxamates. The scope and limitation of individual approaches are discussed.

Heterocyclic zinc-binding groups for use in next-generation matrix metalloproteinase inhibitors: potency, toxicity, and reactivity

In an effort to improve the zinc-chelating portion of matrix metalloproteinase (MMP) inhibitors, we have developed a family of heterocyclic zinc-binding groups (ZBGs) as alternatives to the widely used hydroxamic acid moiety. Elaborating on findings from an earlier report, we performed in vitro inhibition assays with recombinant MMP-1, MMP-2, and in a cell culture assay using neonatal rat cardiac fibroblast cells. In both recombinant and cell culture assays, the new ZBGs were found to be effective inhibitors, typically 10-100-fold more potent than acetohydroxamic acid. The toxicity of these chelators was examined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt cytotoxicity assays, which demonstrate that most of these compounds are nontoxic at concentrations of almost 100 microM. To address the possible interaction of sulfur-containing ZBGs with biological reductants, the reactivity of these chelators with 5,5'-dithiobis(2-nitrobenzoic acid) was examined. Finally, thione ZBGs were shown to be effective inhibitors of cell invasion through an extracellular matrix membrane. The data presented herein suggest these heterocyclic ZBGs are potent, nontoxic, and biocompatible compounds that show promise for incorporation into a new family of MMP inhibitors.

A quantitative structure–activity relationship study on some series of anthranilic acid-based matrix metalloproteinase inhibitors

A quantitative structure–activity relationship (QSAR) study has been made on four different series of anthranilic acid-based matrix metalloproteinase (MMP) inhibitors, in which two substituted aryl rings, one bearing the hydroxamic acid moiety that binds with the zinc atom of MMPs, are joined through a bridge group of sulfonamide. The QSAR results indicate that the sulfonamide group plays a very important role in the inhibition activity of the inhibitors and that the effectiveness of this sulfonamide group can be increased by the presence at the aryl rings or at the sulfonamide nitrogen itself of nitrogen-containing or some such substituents that can increase the electronic character of the sulfonamide group. The hydrophobic character of the molecules is not found to be of any advantage; rather in most of the cases it is shown to have detrimental effect, suggesting that MMPs provide little opportunity to the inhibitors to have a any hydrophobic interactions with them. On the other hand, polarizability of the molecules has been found to be conducive to activity in some cases. Thus the inhibition mechanism seems to predominantly involve the electronic interactions between the inhibitors and the enzymes.

Iron chelation properties of an extracellular siderophore exochelin MS.

The coordination chemistry of an extracellular siderophore produced by Mycobacterium smegmatis, exochelin MS (ExoMS), is reported along with its pK(a) values, Fe(III) and Fe(II) chelation constants, and aqueous solution speciation as determined by spectrophotometric and potentiometric titrations. Exochelin MS has three hydroxamic acid groups for Fe(III) chelation and has four additional acidic protons from a carboxylic acid group and three primary amine groups, on the backbone of the molecule. The pK(a) values for the three hydroxamic acid moieties, the carboxylic acid group and the alkylammonium groups on ExoMS, correspond well with the literature values for these moieties. Equilibrium constants for proton-dependent Fe(III)-ExoMS equilibria were determined using a model involving the sequential protonation of the Fe(III)-ExoMS complexes at the first and second coordination shells. The equilibrium constants (beta) for the overall formation of Fe(III)ExoMS(H(3))(2+) and Fe(II)ExoMS(H(3))(+) from Fe((aq))(3+) or Fe((aq))(2+) and the deprotonated hydroxamate coordinating group form of the siderophore, ExoMS(H(3))(-), are calculated as log beta(III) = 28.9 and log beta(II) = 10.1. A calculated pFe value of 25.0 is very similar to that of other linear trihydroxamic acid siderophores, and indicates that ExoMS is thermodynamically capable of removing Fe(III) from transferrin. The E(1/2) for the Fe(III)-ExoMS/Fe(II)-ExoMS couple was determined from quasi reversible cyclic voltammograms at pH = 6.5 and found to be -380 mV.

Vibratilicin: a Novel Compound from the Basidiomycete Cortinarius vibratilis

AbstractA novel N‐containing compound, vibratilicin (=3‐[3‐(dimethylamino)‐4‐(hydroxyamino)‐4‐oxobutoxy]‐2‐(palmitoyloxy)propyl (9E,12E)‐octadeca‐9,12‐dienoate; 1), was isolated from the fruiting bodies of the basidiomycete Cortinarius vibratilis Fr. Compound 1 is a representative of the rare natural products containing hydroxamic acid moieties, and can be viewed as a derivative of neoengleromycin (2). Also isolated from the same fungus were five known compounds: ergosta‐5,7,22‐trien‐3β‐ol, 5α,8α‐epidioxyergosta‐6,22‐dien‐3β‐ol, p‐anisic acid, N‐(2‐hydroxyhexadecanoyl)‐4‐sphingenine, and (4E,8E)‐2‐N‐(2′‐hydroxypalmitoyl)‐1‐O‐(β‐D‐glucopyranosyl)‐9‐methyl‐4,8‐sphingadienine. Their structures were determined mainly spectroscopically, including 2D‐NMR techniques (HMBC, HMQC, 1H,1H‐COSY).

Histone deacetylase inhibitors: the Abbott experience.

Histone deacetylase inhibitors have generated significant interest as anti-cancer agents due to their ability to cause growth arrest, terminal differentiation and/ or apoptosis in carcinoma cells. Abbott entered this area after the serendipitous discovery of the biaryl hydroxamate A-161906 in a TGF beta mimetic screen and the subsequent identification of this compound as an inhibitor of selected HDACs. The complex biology of these enzymes became evident when cloning and expression of the HDACs demonstrated that they were present as multiprotein and, in some cases, multi-HDAC containing complexes in their active forms. This discovery suggested that any selectivity determinations would have to be considered in the context of these multi-protein/HDAC complexes. However, siRNA gene knockdown studies did demonstrate that reduction of the Class I HDACs resulted in a phenotype similar to that observed with small molecule HDAC inhibitors. Evaluation of the Abbott small molecule HDAC inhibitors utilized a Class I HDAC (HDAC 1/2) preparation and antiproliferation assays using HT1080 fibrosarcoma and MDA435 breast carcinoma cells. Characterization of several series of hydroxamic acids indicated that while many of these analogs possessed potent enzymatic and cellular activity, in general these compounds had unacceptable pharmacokinetic profiles and marginal antitumor effects. Replacement of the potentially labile hydroxamic acid moiety with a trifluoromethyl ketone or a ketooxazole gave measurable HDAC potency but only modest cellular and in vivo activity. However, hydroxamate replacement with an alpha-ketoamide moiety provided potent HDAC inhibitors (IC(50) values as low as 3 nM) with excellent cellular activity (IC50 values < 0.2 microM) and measurable anti-tumor activity in a flank tumor growth model.

Molecular diversity of hydroxamic acids: part II. Potential therapeutic applications.

A hydroxamic acid moiety has been demonstrated as the key structural element in many highly potent and selective inhibitors against a variety of metalloprotease enzymes, such as MMPs, TACE, HDAC, PDF, etc. Over the last several years, there has been a rapid growth of literature and patent applications dealing with the development of the hydroxamic acid-based inhibitors. This review highlights the most recent examples to show their potential therapeutic applications.

Iron chelation properties of an extracellular siderophore exochelin MN.

The coordination chemistry of an extracellular siderophore produced by Mycobacterium neoaurum, exochelin MN (ExoMN), is reported along with its pK(a) values, Fe(III) and Fe(II) chelation constants, and aqueous solution speciation as determined by spectrophotometric and potentiometric titration techniques. Exochelin MN is of particular interest as it can efficiently transport iron into pathogenic M. leprae, which is responsible for leprosy, in addition to its own parent cells. The Fe(III) coordination properties of ExoMN are important with respect to understanding the Fe(III) acquisition and uptake mechanism in pathogenic M. leprae, as the siderophores from this organism are very difficult to isolate. Exochelin MN has two hydroxamic acid groups and an unusual threo-beta-hydroxy-l-histidine available for Fe(III) chelation. The presence of threo-beta-hydroxy-l-histidine gives rise to a unique mode of Fe(III) coordination. The pK(a) values for the two hydroxamic acid moieties, the histidine imidazole ring and the alkylammonium groups on ExoMN, correspond well with the literature values for these moieties. Proton-dependent Fe(III)- and Fe(II)-ExoMN equilibrium constants were determined using a model involving sequential protonation of the Fe(III)- and Fe(II)-ExoMN complexes. These data were used to develop a model whereby deprotonation reactions on the surface of the complex in the second coordination shell result in first coordination shell isomerization. The overall formation constants were calculated: log beta(110) = 39.12 for Fe(III)-ExoMN and 16.7 for Fe(II)-ExoMN. The calculated pFe value of 31.1 is one of the highest among all siderophores and their synthetic analogues and indicates that ExoMN is thermodynamically capable of removing Fe(III) from transferrin. The E(1/2) for the Fe(III)ExoMN/Fe(II)ExoMN(-) couple was determined to be -595 mV from quasi-reversible cyclic voltammograms at pH = 10.8, and the pH-dependent E(1/2) profile was used to determine the Fe(II)-ExoMN protonation constants.

Oxal hydroxamic acid derivatives with inhibitory activity against matrix metalloproteinases

Several amines, amino acid derivatives and low molecular weight peptides containing an amide-bound oxal hydroxamic acid moiety have been synthesized and tested for their inhibitory effects towards native human gelatinase B (MMP-9) and the catalytic domains of the membrane type MT1-MMP (MMP-14) and of neutrophil collagenase (MMP-8). A number of these compounds exhibited considerable inhibitory activity against the tested metalloproteinases.

Histone deacetylase inhibitors as new cancer drugs.

Histone deacetylase inhibitors are potent inducers of growth arrest, differentiation, or apoptotic cell death in a variety of transformed cells in culture and in tumor bearing animals. Histone deacetylases and the family of histone acetyl transferases are involved in determining the acetylation of histones, which play a role in regulation of gene expression. Radiograph crystallographic studies reveal that the histone deacetylase inhibitors, suberoylanilide hydroxamic acid and trichostatin A, fit into the catalytic site of histone deacetylase, which has a tubular structure with a zinc atom at its base. The hydroxamic acid moiety of the inhibitor binds to the zinc. Histone deacetylase inhibitors cause acetylated histones to accumulate in both tumor and peripheral circulating mononuclear cells. Accumulation of acetylated histones has been used as a marker of the biologic activity of the agents. Hydroxamic acid-based histone deacetylase inhibitors limit tumor cell growth in animals with little or no toxicity. These compounds act selectively on genes, altering the transcription of only approximately 2% of expressed genes in cultured tumor cells. A number of proteins other than histones are substrates for histone deacetylases. The role that these other targets play in histone deacetylase inducement of cell growth arrest, differentiation, or apoptotic cell death is not known. This review summarizes the characteristics of a variety of inhibitors of histone deacetylases and their effects on transformed cells in culture and tumor growth in animal models. Several structurally different histone deacetylase inhibitors are in phase I or II clinical trials in patients with cancers.

Regio- and stereochemically controlled formation of hydroxamic acid containing anti- or syn-1,4-cycloalkenols from acylnitroso-derived Diels-Alder adducts.

Treatment of acylnitroso hetero Diels-Alder cycloadducts 2 with iron(III) or copper(II) in an alcohol solvent induces ring opening to afford predominantly monocyclic anti-1,4-hydroxamic acids 3. However, treatment of cycloadducts 2 with copper(II) in toluene reverses the stereoselectivity of the ring opening to afford syn-1,4-hydroxamic acids 4. These regio- and stereoselective processes separately provide anti-1,4- and syn-1,4-disubstituted cyclopentenes while regenerating a hydroxamic acid moiety, thus enhancing the chemical versatility of the Diels-Alder cycloadducts.

ChemInform Abstract: A Strategy for the Solution-Phase Parallel Synthesis of N-(Pyrrolidinylmethyl)hydroxamic Acids.

Both five- and six-membered iminocyclitols have proven to be useful transition-state analogue inhibitors of glycosidases. They also mimic the transition-state sugar moiety of the nucleoside phosphate sugar in glycosyltransferase-catalyzed reactions. Described here is the development of a general strategy toward the parallel synthesis of a five-membered iminocyclitol linked to a hydroxamic acid group designed to mimic the transition state of GDP-fucose complexed with Mn(II) in fucosyltransferase reactions. The iminocyclitol 8 containing a protected hydroxylamine unit was prepared from d-mannitol. The hydroxamic acid moiety was introduced via the reaction of 8 with various acid chlorides. The strategy is generally applicable to the construction of libraries for identification of glycosyltransferase inhibitors.

A strategy for the solution-phase parallel synthesis of N-(pyrrolidinylmethyl)hydroxamic acids.

Both five- and six-membered iminocyclitols have proven to be useful transition-state analogue inhibitors of glycosidases. They also mimic the transition-state sugar moiety of the nucleoside phosphate sugar in glycosyltransferase-catalyzed reactions. Described here is the development of a general strategy toward the parallel synthesis of a five-membered iminocyclitol linked to a hydroxamic acid group designed to mimic the transition state of GDP-fucose complexed with Mn(II) in fucosyltransferase reactions. The iminocyclitol 8 containing a protected hydroxylamine unit was prepared from D-mannitol. The hydroxamic acid moiety was introduced via the reaction of 8 with various acid chlorides. The strategy is generally applicable to the construction of libraries for identification of glycosyltransferase inhibitors.

Binding of salicylhydroxamic acid and several aromatic donor molecules to Arthromyces ramosus peroxidase, investigated by X-ray crystallography, optical difference spectroscopy, NMR relaxation, molecular dynamics, and kinetics.

The X-ray crystal structure of the complex of salicylhydroxamic acid (SHA) with Arthromyces ramosus peroxidase (ARP) has been determined at 1.9 A resolution. The position of SHA in the active site of ARP is similar to that of the complex of benzhydroxamic acid (BHA) with ARP [Itakura, H., et al. (1997) FEBS Lett. 412, 107-110]. The aromatic ring of SHA binds to a hydrophobic region at the opening of the distal pocket, and the hydroxamic acid moiety forms hydrogen bonds with the His56, Arg52, and Pro154 residues but is not asscoiated with the heme iron. X-ray analyses of ARP-resorcinol and ARP-p-cresol complexes failed to identify the aromatic donor molecules, most likely due to the very low affinities of these aromatic donors for ARP. Therefore, we examined the locations of these and other aromatic donors on ARP by the molecular dynamics method and found that the benzene rings are trapped similarly by hydrophobic interactions with the Ala92, Pro156, Leu192, and Phe230 residues at the entrance of the heme pocket, but the dihedral angles between the benzene rings and the heme plane vary from donor to donor. The distances between the heme iron and protons of SHA and resorcinol are similar to those obtained by NMR relaxation. Although SHA and BHA are usually considered potent inhibitors for peroxidase, they were found to reduce compound I and compound II of ARP and horseradish peroxidase C in the same manner as p-cresol and resorcinol. The aforementioned spatial relationships of these aromatic donors to the heme iron in ARP are discussed with respect to the quantum chemical mechanism of electron transfer in peroxidase reactions.

Thalidomide and metabolites: indications of the absence of `genotoxic' carcinogenic potentials

Because of the reintroduction into human therapeutics of thalidomide, a recognized developmental toxicant in humans, there has been concern about its potential for inducing other health effects as well. The present study is concerned with the possible mutagenicity and carcinogenicity of this chemical. Using the expert system, META, a series of putative metabolites of thalidomide was generated. In addition to the known or hypothesized metabolites of thalidomide ( N=12), a number of additional putative metabolites ( N=131) were identified by META. The structures of these chemicals were subjected to structure–activity analyses using predictive CASE/MULTICASE models of developmental toxicity, rodent carcinogenicity and mutagenicity in Salmonella. While thalidomide and some of its putative metabolites were predicted to be developmental toxicants, none of them were predicted to be rodent carcinogens. Putative metabolites containing the hydroxamic acid or hydroxylamine moieties were predicted to be mutagens. None of the `known' metabolites of thalidomide contained these reactive moieties. Whether such intermediates are indeed generated or whether they are generated and are either unstable in the presence of oxygen or react rapidly with nucleophiles is unknown.

Isolation and Partial Characterization of an Extracellular Low-Molecular Mass Component with High Phenoloxidase Activity fromThermoascus aurantiacus

An extracellular low-molecular mass component (LMMC) with catalytic properties was isolated from liquid cultures containing wheat bran of ascomycete thermophilicThermoascus aurantiacus.The partially purified LMMC showed very high activity with typical phenoloxidase substrates in the absence of hydrogen peroxide at acidic pH (2.8). However, in this pH range, the phenoloxidase (PO) activity was quickly lost. The LMMC showed a high optimum temperature (80°C) and an elevated thermostability. The molecular mass of the component estimated by gel filtration chromatography was 530 Da. IR and1H- and13C-NMR spectra indicated the presence of hydroxamic acid moiety. Qualitative determination of metal ions by several techniques revealed the presence of mainly iron associated with this structure. Iron may be the responsible for the ability for catalyze oxidation reactions, such as o-dianisidine oxidation, by the LMMC. These results suggested the existence of a hydroxamate-type metal-binding component, most likely hydroxamate siderophore. In addition, the chrome azurol S (CAS) universal assay for noncomplexed siderophores detection revealed the production of these compounds byT.aurantiacusin solid and liquid media.

NMR Studies of the Role of Hydrogen Bonding in the Mechanism of Triosephosphate Isomerase

Triosephosphate isomerase (TIM) catalyzes the reversible interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP), with Glu-165 removing the pro-R proton from C1 of DHAP and neutral His-95 polarizing the carbonyl group of the substrate. TIM and its complexes with the reactive intermediate analogs, phosphoglycolic acid (PGA) and phosphoglycolohydroxamic acid (PGH), were studied by 1H NMR at 600 MHz and at low temperature (-4.8 degrees C). His-95 shows an N epsilon H resonance at 13.1 ppm which shifts to 13.3 ppm in the TIM-PGA complex and to 13.5 ppm in the TIM-PGH complex. In the TIM-PGH complex, His-95 N epsilon H shows a slow, pH-independent exchange rate with water (kex = 80 s-1 at 30 degrees C, Eact = 19 kcal/mol), which is 44-fold slower than that of an exposed histidine suggesting partial shielding from bulk solvent, and a fractionation factor phi = 0.71 +/- 0.02 consistent with its donation of a normal hydrogen bond. The formation of the TIM-PGH complex results in the appearance of several deshielded proton resonances, including one at 14.9 ppm and one at 10.9 ppm which overlaps with another resonance. The resonance at 14.9 ppm is absent and the resonance at 10.9 ppm is much weaker in the TIM complex of PGA, which lacks the hydroxamic acid (-NHOH) moiety. 15N-labeled PGH was synthesized and the NH proton of free [15N]PGH shows a single 1H-15N HMQC cross peak with delta (1H) = 10.3 ppm and delta (15N) = 168 ppm which shifts to delta (1H) = 10.9 ppm and delta (15N) = 174 ppm in the TIM complex of [15N]PGH. The 15N-1H coupling in the complex indicates covalent N-H bonding, and the deshielded delta (15N) indicates a significant contribution of the imidate resonance form of PGH. The 14.9 ppm resonance is assigned to the NOH proton of bound PGH. This resonance shows a pH-independent exchange rate with water (kex = 3900 s-1 at 30 degrees C, Eact = 8.9 kcal/mol) which may reflect the dissociation of the TIM-PGH complex, and meets the criteria for a low-barrier hydrogen bond on the basis of the significant downfield shift of 6.2 ppm from the NOH proton of the model compound acetohydroxamic acid, and a very low fractionation factor phi = 0.38 +/- 0.06. In the X-ray structure of the TIM-PGH complex [Davenport, R. C., Bash, P. A., Seaton, B. A., Karplus, M., Petsko, G. A., and Ringe, D. (1991) Biochemistry 30, 5821], the NOH proton of bound PGH is hydrogen bonded to Glu-165. A low-barrier hydrogen bond from PGH NOH to Glu-165 suggests a dual role for Glu-165 in catalysis of proton transfer not only between the C1 and C2 carbons but also between the O1 and O2 oxygens in the interconversion of DHAP and GAP in wild type TIM. Such a mechanism, together with the measured exchange rate of the His-95 N epsilon H proton with solvent protons can accommodate the classical measurements of tritium incorporation from DHAP into GAP.

Angiotensin-converting enzyme secretase is inhibited by zinc metalloprotease inhibitors and requires its substrate to be inserted in a lipid bilayer.

Mammalian angiotensin-converting enzyme (ACE; EC 3.4.15.1) is one of several proteins that exist in both membrane-bound and soluble forms as a result of a post-translational proteolytic processing event. For ACE we have previously identified a metalloprotease (secretase) responsible for this proteolytic cleavage. The effect of a range of structurally related zinc metalloprotease inhibitors on the activity of the secretase has been examined. Batimastat (BB94) was the most potent inhibitor of the secretase in pig kidney microvillar membranes, displaying an IC50 of 0.47 microM, whereas TAPI-2 was slightly less potent (IC50 18 microM). Removal of the thienothiomethyl substituent adjacent to the hydroxamic acid moiety or the substitution of the P2' substituent decreased the inhibitory potency of batimastat towards the secretase. Several other non-hydroxamate-based collagenase inhibitors were without inhibitory effect on the secretase, indicating that ACE secretase is a novel zinc metalloprotease that is realted to, but distinct from, the matrix metalloproteases. The full-length amphipathic form of ACE was labelled selectively with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine in the membrane-spanning hydrophobic region. Although trypsin was able to cleave the hydrophobic anchoring domain from the bulk of the protein, there was no cleavage of full-length ACE by a Triton X-100-solubilized pig kidney secretase preparation when the substrate was in detergent solution. In contrast, the Triton X-100-solubilized secretase preparation released ACE from pig intestinal microvillar membranes, which lack endogenous secretase activity, and cleaved the purified amphipathic form of ACE when it was incorporated into artificial lipid vesicles. Thus the secretase has an absolute requirement for its substrate to be inserted in a lipid bilayer, a factor that might have implications for the development of cell-free assays for other membrane protein secretases. ACE secretase could be solubilized from the membrane with Triton-X-100 and CHAPS, but not with n-octyl beta-D-glucopyranoside. Furthermore trypsin could release the secretase from the membrane, implying that like its substrate, ACE, it too is a stalked integral membrane protein.