Introduction

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Histamine is an endogenous substance that has profound effects on a variety of cells and tissues (Falus, 1994; Hill, 1990). With specific reference to the intestinal lining, it has been shown to have marked stimulant effects in vitro that could result in diarrhoea in vivo (Rangachari et al., 1992). This has been confirmed in cases of histamine poisoning where diarrhoea is a prominent feature (Taylor, 1991). Under normal conditions, several mechanisms exist to rapidly inactivate the autacoid. Interference with these inactivating mechanisms leads to potentiation of the effects of histamine. Among these, the rapid inactivation by the ubiquitous diamine oxidase is particularly significant (Kusche and Lorenz, 1983; Beaven, 1982; Jarisch and Wantke, 1996; Sessa and Perin, 1994). Inhibition of this enzyme as in instances of scombroid fish poisoning can have serious consequences (Taylor, 1991; Murray et al., 1982). Diamine oxidase can be inhibited by a variety of compounds including guanidine derivatives, aromatic diamidines, several antihistaminics, hydroxylamine and a variety of drugs used in clinical practice (Sattler et al., 1985; Beaven, 1982; Jarisch and Wantke, 1996).

Diamine oxidase, originally termed histaminase (Kusche and Lorenz, 1983), is a member of a family of amine oxidases (EC 1.4.4.6.) that are homodimers of 60 to 105 kDa subunits and contain tightly bound copper and a carbonyl cofactor (Janes et al., 1990). The enzyme, which is widely distributed, catalyses the oxidative deamination (RCH₂NH₂ + O₂ + H₂O  RCHO + H₂O₂ + NH₃) of diamines with three to six carbon atoms as well as histamine, which could be considered a cyclic diamine (Sessa and Perin, 1994). In a variety of mammals it is found in tissues such as the placenta, intestines, kidney and thymus. High activities of the enzyme are present in the mucosal layer of the intestine, its location suggesting a protective function, because endogenously released histamine can have profound pathological effects if degradation does not occur promptly (Beaven, 1982; Rangachari et al., 1992).

Earlier (Rangachari et al., 1992), we showed that a series of O-alkylhydroxylamines markedly potentiated the responses of the canine colonic epithelium to histamine. The underlying mechanism was the inhibition of diamine oxidase, because: 1) the potentiation was seen only with those agonists that possessed an imidazole nucleus (i.e., histamine, 2-methyl and 4-methylhistamine); 2) the compounds delayed the disappearance of histamine from the bathing solutions; 3) the compounds inhibited a preparation of colonic diamine oxidase; 4) selective inhibitors of diamine oxidase (aminoguanidine, semicarbazide) mimicked the effects; 5) putrescine, a substrate for the enzyme, also produced similar effects when added at a high concentration and 6) the effects were not seen with sodium nitroprusside, NaNO₂ or derivatives of cGMP excluding NO or cGMP from a possible role.

Preliminary structure-activity relations suggested that whereas O-substituted aliphatic compounds were effective, N-substitution led to loss of activity. Thus O-methyl was fully active whereas N-methyl was totally inactive. These activities were defined using the responses of the intact tissue (Rangachari et al., 1992). However, it was possible that compounds labeled inactive may have been termed so merely because they were unable to penetrate the tissue to get access to the enzyme located in the epithelial cells.

In our study, we have conducted a more systematic study and explored these relations further. We compared and contrasted the effects of the synthesized compounds on the intact tissue as well as on an enzyme preparation from the same tissue. With the second generation of O-alkylhydroxylamines, we assessed the effects of increasing the length of the aliphatic chains, introducing double bonds and substituting bulky/charged aromatic groups.

The strategy we adopted was as follows: 1) all compounds were tested for their abilities to inhibit diamine oxidase activity in a preparation obtained from the canine colon. 2) The same compounds were screened rapidly using the "secondary rise" screening procedure we developed in the previous study (Rangachari et al., 1992). Serosal addition of histamine elicited a rapid increase in short-circuit current across the tissue. This response attained a peak and faded within a matter of minutes. The addition of an "active" hydroxylamine produced a sharp rapid secondary increase in I_sc. Compounds that produced this response also potentiated responses to histamine when added prior to the addition of the agonist. 3) Following this rapid screen, selected compounds were used to obtain a quantitative estimate of potentiation by estimating changes in pD₂ to exogenous histamine.

Based on the data obtained, we attempted to develop a structure-activity relationship for the derivatives synthesized. Using this information we have also used molecular modeling techniques in an effort to define the nature of the active site of diamine oxidase.

	Methods and Materials

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Tissue Preparation

Many of the procedures used in our study were described in detail in our earlier publications (Rangachari et al., 1992), so only the salient points will be emphasized. Briefly, adult dogs of either sex were killed rapidly by i.v. pentobarbitone sodium (100 mg/kg) and the proximal colon quickly excised and immersed in warm, oxygenated modified Krebs solution. An initial dissection was carried out to remove the circular and longitudinal muscles, followed by a finer dissection to remove the muscularis mucosa and attendant submucosal plexuses as well. The resulting preparation was set up in conventional Ussing chambers for recording short-circuit currents.

The tissues were bathed on both sides with warm, oxygenated modified Kreb's solution having the following composition (in mM): 116 NaCl, 4.6 KCl, 1.2 MgCl₂, 1.5 CaCl₂, 22 NaHCO₃, 1.2 NaH₂PO₄ and 10 glucose. Short-circuit currents (I_sc) were measured in µA using a World Precision Instruments (Sarasota, FL) dual voltage clamp. The data were collected and analyzed using the Acknowledge MP100 data acquisition system (Biopac, Santa Barbara, CA).

Diamine Oxidase Preparation

Colonic tissues were collected as described above. To prepare the enzyme, all the muscle layers were removed to provide sheets of the epithelial tissue. The epithelial tissues were weighed, homogenized in 9 volumes of phosphate buffer (v:w:w) and centrifuged for 30 min (48,000 × g, 4°C). The supernatant was collected and used as the source of the enzyme. The supernatants from five dogs were pooled and frozen in small aliquots for use as the test solution in these experiments. The enzyme was found to be stable for at least 6 wk.

Ussing Chamber Experiments

Two sets of experiments were carried out in Ussing chambers. In both cases, changes in I_sc were measured as indices of tissue responsiveness. All tissues were allowed to stabilize for 45 min to 1 h before experiments were begun.

"Secondary Rise" Protocol. The secondary-rise screening protocol was adopted from an earlier study (Rangachari et al., 1992) to identify active hydroxylamine compounds. Briefly, the tissues were exposed to a single concentration of histamine (10⁴ M) and when the initial response to the agonist had begun to fade, the hydroxylamine compound was added at a concentration of 10⁴ M. The presence or absence of a secondary increase in I_sc in response to the addition of hydroxylamine was noted.

Concentration-Response Studies. Multiple pieces of tissue from the same dog were set up for each experiment. Cumulative concentration response curves were constructed using histamine. Control tissues were treated with histamine alone. Test tissues were pre-treated with selected hydroxylamines (at a fixed concentration of 10⁴ M) for 15 min before addition of histamine. On each day all the five test compounds and the two reference compounds N-methyl and O-methyl) were studied.

Determination of Diamine Oxidase Activity

The procedure used was the method of Okuyama and Kobayashi (1961) as modified by Kusche et al. (1975). The assay mixture for the test consisted of 1) 0.6 ml of 0.1 M sodium phosphate buffer (pH 7.2), 2) 0.1 ml of test sample solution (diamine oxidase ± inhibitors), 3) 0.05 ml of substrate solution (containing 4.5 mM putrescine dihydrochloride and 1.0 µCi/ml of [1,4-¹⁴C]putrescine dihydrochloride). Based on our previous experience, we used an incubation period of 10 min at 37°C at which time the reaction was stopped by adding 1.0 ml of alkaline buffer (800 mM NaOH, 600 mM NaHCO₃ at pH 12.2). The labeled reaction product ([C¹⁴]-¹-pyrroline) was directly extracted into 6 ml of scintillation fluid (toluene containing 3.5 g/liter of 2,5-diphenyloxazole). The radioactivity present in 5 ml of the scintillation fluid was measured using a Beckman LS-5801 liquid scintillation counter. Three blanks were routinely used. These were 1) a sample blankperchloric acid added before substrate solution (zero time incubation), 2) an enzyme blank0.1 mM aminoguanidine added to inactivate the enzyme and a 3) reagent blankwhere buffer was used instead of the enzyme. The data from these experiments were expressed as percentages of the control diamine oxidase activity (dpm of ¹⁴C · min¹ · g¹ · protein¹) (see Rangachari et al., 1992).

General Experimental Methods for Preparation of O-Alkylhydroxylamines

Melting points were measured on a Gallenkamp capillary tube melting point apparatus and are uncorrected. Proton and ¹³C nuclear magnetic resonance spectra were recorded on a Bruker AC-200 (200 MHz) or Bruker AC-300 (300 MHz) spectrometer. Solvents used were chloroform-d, DMSO-d₆ or deuterium oxide with TMS as an internal standard. The abbreviations (s) = singlet, (d) = doublet, (t) = triplet, (qr) = quartet, (pn) = pentet, (sx) = sextet, (sp) = septet and (m) = multiplet are used to describe spin-spin coupling patterns. Proton spectra were collected in sixteen scans in 16K data points. The FID patterns were processed using exponential multiplication (line broadening = 0.3) and were zero-filled to 32K before Fourier transformation. The ¹³C spectra were collected in adequate scans to determine all carbon signals in 16K data points. The FID patterns were processed as with the proton spectra (line broadening = 3.0) and zero-filled to 32K before Fourier transformation.

DMF was dried by distilling a known volume of benzene to remove the water as an azeotrope. The DMF was then distilled under reduced pressure and stored over molecular sieves under a nitrogen atmosphere. ACN was distilled from CaH₂ under dry nitrogen immediately before use. All other solvents used were of reagent grade. NMR solvents (chloroform-d, DMSO-d₆) were stored over molecular sieves (4 Å) before use.

The general method used for synthesis of O-alkyl and O-benzylhydroxylamines (compounds 1-16, Fig. 1) was a modification of the Gabriel procedure for synthesis of primary amines (Gabriel, 1887) using N-hydroxyphthalimide followed by removal of the phthaloyl group by hydrazinolysis (Ing and Manske, 1926; Drain, 1965; Rangachari et al., 1992). The major changes in the present work were the use of dimethylformamide as solvent and sodium hydride to generate the N-hydroxyphthalimide anion. Product hydroxylamines were normally converted to their hydrochloride salts, lyophilized and stored as the dry solid before use. The dry hydrochlorides were found to be quite stable over long time periods.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Shown are the structures and numbering system for all of the O-alkylhydroxylamines synthesized for this study. Details regarding the preparation and chemical properties for each of these compounds are given in the Appendix.

The most difficult hydroxylamines to prepare were the O-p-hydroxybenzyls (compounds 11 and 12) because of the tendency for the intermediates, and the final product, to undergo elimination and form quinonemethides. The procedure of Drain et al. (1965) proved acceptable for these two compounds, although the yield of purified 11 from the hydrazinolysis and hydrolysis steps was only 15%.

The intermediate O-alkyl-N-hydroxyphthalimides were decomposed to give the free O-alkylhydroxylamines with hydrazine in slight excess (1.1-1.34 mole equivalents). Because hydrazine and substituted hydrazides (e.g., semicarbazide) are inhibitors of diamine oxidase it was important to remove as much hydrazine as possible from the prepared compounds. This was accomplished by taking advantage of the different pK_as of hydrazine (8.23) and hydroxylamine (6.03) and extraction of a dichloromethane solution of the reaction products with equal volumes of a buffer of pH 7.4. Routinely two or three such extractions were carried out, depending on the excess of hydrazine used, leaving the residual hydrazine at a maximum in the parts per thousand range. For hydrazine at this level to have an effect on our experiments, the concentrations of O-alkylhydroxylamines would need to be in the 10³ M range, a concentration that is two orders of magnitude above the 10⁵ M cut-off that we accept for a compound to be active.

The synthesis of O-2-butyl (3) is described in detail in the Appendix as a typical example. Spectroscopic data, as well as any variations in the general method, for the remaining compounds listed in figure 1 are also included in the Appendix.

Data Analysis and Statistics

All data obtained were analyzed using distribution-free methods (analysis of variance-Kruskal Wallis). Mean effective dose values (pD₂) were determined by non-linear regression analysis (Fig.P, ver. 6.0; Biosoft, MA). A four-parameter logistic equation of the following form was used: Y represents the magnitude of the response to the agonist at a particular concentration, x. A-D are computer-generated parameters reflecting maximal responses (A), slope factor (B), pD₂ (C) and minimal response (D), respectively. Based on this expression, pD₂ values were estimated from the values of C obtained for each concentration-response curve. pD₂ values from treated tissues were compared to those from the control tissues to evaluate statistical significance. Molecular modeling was performed using PC-Model (Serena Software, Bloomington, IN).

Drugs and Reagents

All chemical reagents used for making physiological salt solutions were of analar grade and purchased from either Sigma Chemical Co. (St. Louis, MO) or Aldrich (Milwaukee, WI). Histamine dihydrochloride, aminoguanidine hemisulfate and semicarbazide dihydrochloride were obtained from Sigma. [¹⁴C]Putrescine dihydrochloride was obtained from New England Nuclear (Wilmington, DE). Commercially available hydroxylamine derivatives were bought from Sigma or Aldrich. All other hydroxylamines were synthesized as described above (see Appendix for details). All of the chemicals used to synthesize these compounds were also purchased from either Sigma or Aldrich.

	Results

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Effect on Diamine Oxidase Activity. All the compounds synthesized were tested for their abilities to inhibit diamine oxidase. We also tested a known inhibitor of diamine oxidase (aminoguanidine) as well as the reference compounds, O-methyl and N-methyl. The data for the aliphatic and the benzylic hydroxylamines are shown separately (Fig. 2, A and B). In figure 2A we have highlighted (in black) the inhibitions produced by all compounds at a concentration of 10⁶ M. At this concentration, the standard inhibitor, aminoguanidine, produces a complete inhibition of diamine oxidase activity.

View larger version (41K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent inhibition of diamine oxidase activity (expressed as a percentage of DAO activity in untreated controls) by a series of A, aliphatic hydroxylamines and B, benzylic hydroxylamines. For the sake of comparison, we have included one positive control, the standard inhibitor, aminoguanidine, as well as a negative control, N-methyl in each figure. Concentrations of interest have been highlighted for further discussion. Refer to Table 1 for individual IC50 values.

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"Secondary-Rise" Experiments. These experiments were carried out using the protocol described earlier. Serosal addition of histamine (10⁴ M) produced a sharp transient increase in I_sc. The addition of an active compound to the serosal solutions after the fade in the response produced a sharp increase in I_sc that exceeded the original peak. Those compounds that produced no change in the slope were termed inactive. There was an interim category that had less definitive effects. To this class belonged compounds that either produced a flattening of the curve or a slow secondary increase in I_sc. We termed these compounds marginally active. Examples of these patterns are shown in figure 3. In Table 1, we summarize the results of this exploratory screen. Shown alongside that are the data culled from the enzyme studies showing the inhibitory potencies of the same compounds in comparison to the standard inhibitor aminoguanidine.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Typical response patterns observed in the "secondary rise" experiments. A, In the first panel, we show an example of a compound that produced an active response. The addition of O-1-allyl hydroxylamine at 104 M produces a sharp increase that is rapid and is significantly larger than the original response to histamine. B, The addition of these compounds produces either a mere flattening of the fade [O-(1-E-Cinnamyl)] or a slow weak secondary increase (O-1-octyl) in Isc. Compounds eliciting this type of response were grouped together and termed marginally active. C, the addition of the O-1-decyl produced no secondary increase in Isc after the initial histamine response. Such compounds were termed negative.

Potency Experiments. Changes in pD₂ to histamine produced by selected compounds were estimated from cumulative concentration-response curves. The compounds selected for detailed study were two aliphatic compounds, O-1-pentyl and O-1-octyl, and three of the aromatic derivatives, O-1-benzyl, O-p-methoxybenzyl and O-p-hydroxybenzyl. In addition, we tested two reference compounds. These were the contrasting methylhydroxylamines, where the O-substituted compound is fully active whereas the N-substituted compound is inactive. All compounds were tested at 10⁴ M. In these experiments, the protocol described in "Materials and Methods" was followed. For illustrative purposes, the results obtained with O-p-hydroxybenzyl hydroxylamine are shown (see Fig. 4). As can be seen, there is a clear shift to the left with no significant change in the maximal responses obtained. The data obtained with the hydroxylamines tested are tabulated (see Table 2). A one-way analysis of variance was performed on the changes in maximal responses and the pD₂ values. The maxima did not show any significant changes; however, significant differences were found among the pD₂ values. A post hoc analysis of the mean pD₂ values was carried out.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Pretreatment of epithelial tissues with 104 M O-p-hydroxybenzylhydroxylamine produces a significant leftward shift in the cumulative dose-response to histamine (IC50 = 2.6 × 106 vs. 1.4 × 105 for the control), with no significant change in maximal responses (262.8 vs. 247.9 µA/cm2). Values shown are the means of five animals. Tissues were set up in pairs, with one member of the pair used as a control. Differences were determined using the Wilcoxon paired-sample test with a significance level of 0.05.

	Discussion

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The responses of the canine colonic epithelium to histamine are markedly potentiated by O-alkylhydroxylamines. Our earlier studies suggested strongly that the underlying mechanism was the inhibition of diamine oxidase (Rangachari et al., 1992).

In this study, we sought to explore further the structure-activity relations involved. We synthesized a second generation of hydroxylamines and assessed their biological activity using an enzyme preparation of diamine oxidase from the canine colonic epithelium as well as an in vitro preparation from the same tissue.

All compounds were also tested using a preparation of soluble DAO. Here it was assumed that access to the enzyme would not be a significant factor. Using DAO activity as an index, a difference appeared between the "active" aliphatic compounds and the benzylic hydroxylamines (Fig. 2, A and B). The former produced inhibitory effects at concentrations tenfold lower (10⁶ M) than the "active" benzylic ones. This may reflect the greater steric bulk associated with the added benzyl group and its substituents. At 10⁵ M several of the benzylhydroxylamines became active, and the trends seen with the aliphatic compounds at 10⁶ M became more pronounced. At the highest concentration tested (10⁴ M) selectivity was essentially lost and even the clearly negative control (N-methyl) began to exert some inhibitory effects.

To assess biological activity on the intact tissue, we set up an initial screening procedure (secondary rise experiments) that enabled us to compare these compounds with our previous studies and to categorize the compounds into three groupsactive, marginally active and inactive (see Table 1; Fig. 3).

In these experiments, we were concerned that access to the enzyme may have played a role in the resulting classification of compounds. In contrast to the active hydroxylamines that produce large, rapid secondary increases in I_sc (Fig. 3A), compounds that were labeled marginally active produced a mere "flattening" of the I_sc profile or a small slowly increasing secondary rise in I_sc that often appeared somewhat delayed in onset (Fig. 3B). It was possible, in part this could arise from poorer penetration of these compounds to the location of the enzyme rather than an inability to inhibit the enzyme itself.

Further experiments were carried out to determine the potency (shift in pD₂) of compounds selected (at least two from each category) to added histamine in tissues that had been pretreated with selected hydroxylamines. These data indicated that compounds that were initially classified as either active or inactive retained their identity; however, under these conditions where the tissue received a prolonged exposure (pretreatment) to the hydroxylamine, marginally active compounds exhibited potentiating effects. This suggests that access to the enzyme may be a limiting factor for these compounds.

The generally accepted mechanism for the deamination carried out by diamine oxidase (Hartman and Klinman, 1991; Hartman et al., 1993) is summarized in figure 5 with putrescine (17) acting as substrate. Amine oxidases contain a modified amino acid residue 6-hydroxydopa, or TOPA quinone, which is an active site prosthetic group that plays a crucial role in deamination. A series of quinone imine and quinol-amine intermediates are produced before putrescine is converted to 4-aminobutanal which cyclises to form ¹-pyrroline.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   The generally accepted mechanism for the catalyzed deamination of putrescine (17) into 1-pyrroline by the TOPA-Cu(II) active site of diamine oxidase is shown. A schematic representation of this process is given with all relevant details being illustrated in the box shown immediately below. The free carbonyl group of enzyme bound TOPA quinone reacts with one nitrogen of putrescine (17) giving the intermediate aminol (18), which eliminates water to form the intermediate quinone-imine (19) [arbitrarily drawn here in the anticonformation (to the TOPA-OH group) for convenience]. Intermediate 19 aromatizes by loss of a proton to an enzyme bound base to give an aliphatic iminium species (20) which then hydrolyses via attack by water to the amino-quinol (21) and 4-aminobutanal (22). Aldehyde 22 cyclises separately to form 1-pyrroline (not shown) and the amino-quinol 21 proceeds through the Cu(II)-O2 redox cycle to regenerate TOPA quinone and ammonium ion. In the presence of excess substrate, the intermediate quinone-imine (23) can react alternatively with putrescine to form the diamino analogue of 18 which eliminates NH3 instead of H2O giving imine 19 (Mure and Klinman, 1993). All compounds and intermediates have been numbered to facilitate their discussion in the text.

The intermediate aminol (18) has been included to point out firstly the curious partial reversibility of many carbonyl reagents that affect diamine oxidases (Pec et al., 1992). We have modified slightly the accepted scheme, shown in detail in figure 5, by representing the enzyme bound base [B-Enz, now known to be a histidine (Shah and Ali, 1994)] as initially protonated. We have done so, because it can act as a general acid catalyst for the quinone oxygen as the amine begins its nucleophilic attack on the carbonyl group. The quinone-aminol equilibrium is fast in both directions but the aminol-iminium ion equilibrium can be very slow in the reverse direction and in addition pH dependent (Tamura et al., 1989). And second, the change in geometry from tetrahedral in (18) to planar in (19) is basis of the spatial model of the TOPA portion of the active site of diamine oxidase we present below.

The reaction mechanism rationale for the inhibition of diamine oxidases by hydroxylamines, and other carbonyl reagents like semicarbazides and aminoguanidine, is illustrated in figure 6. In general, when a hydroxylamine like O-1-pentyl reacts with a carbonyl group an oxime is formed. As an example, the oxygen of O-1-pentyloxime (24) occupies the site of the CH₂ alpha to the nitrogen in (19) and the enzyme histidine base has no protons to remove, halting the whole oxidation process at this point (step C, Fig. 6). A number of other compounds such as polyamines, aryl diamidines and amiloride are inhibitors of diamine oxidase but they are noncompetitive and generally require higher concentrations (>10⁵ M) to be effective.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Comparison of the normal formation of the putrescine iminium ion (19) with that of O-1-pentyloxime-H+ ion 24 which forms when an O-1-pentylhydroxylamine interacts with the active site of diamine oxidase. The replacement of the -carbon of 19 by an oxygen in 24 (see the circled region on each compound) leaves the enzyme bound base (Enz-B:) with no protons of the correct acidity to remove thus effectively stopping the reaction at step C and inhibiting the enzyme. This illustrates the mechanism of O-alkylhydroxylamine inhibition of diamine oxidase.

For our proposed model for the TOPA portion of diamine oxidase's active site, we have taken the simple view that the TOPA quinone fragment is somewhat restricted in its rotation about the C-C(ring) bond by the coordinating copper (or via any ligand between it and the copper). An alternative, induced fit view, where bulky groups cause enzyme conformational changes which either prevent access of the ONH₂ or slow the rate of the elimination step (step B in Fig. 6), is however, equally plausible. We consider our model to be consistent with recent x-ray crystallographic studies on pea seedling DAO (Kumar et al., 1996) where the copper atom is found to be in a solvent-inaccessible site; i.e., the enzyme must undergo conformational changes before an amine can interact with the active TOPA carbonyl. The results from our study of the hydroxylamines, together with use of molecular modeling and energy minimization of the conformations of the O-alkylhydroxylamine inhibitors, form the basis of the model. Because putrescine is believed to be the endogenous substrate for this enzyme, and it was used to determine diamine oxidase activity, we have chosen it rather than histamine for the model shown in figure 7.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   A diagrammatic representation of the proposed substrate trough of the TOPA quinone portion of diamine oxidase is shown. The trough extends away from the TOPA and is in the same plane as the TOPA ring. The syn (to the OH of TOPA) form of the iminium ion is represented here. Our data offer no information on whether the putrescine iminium ion is syn or anti (molecular modeling showed the two forms to have similar energies). What our data does suggest is that if the proposed trough lies on the same surface side as the enzyme bound TOPA, then our oximes are probably syn because of the data from O-(1-E-Cinnamyl). Panel a shows a side view of the trough from within the enzyme and perpendicular to the TOPA ring, b shows a view from above the enzyme surface and in the plane of the TOPA ring and c shows the same top view as for (b) but with the atoms represented as solid spheres.

At the point when the aminol is eliminating toward the imine [i.e., (18)  (19)], the active site may be visualized as a trough-like region extending away from the TOPA carbonyl. Figure 7a represents a side view from within the enzyme and perpendicular to the TOPA ring. Figure 7b is a view from above the enzyme and in the plane of the TOPA ring. Figure 7c shows the same top view as figure 7b but with the atoms represented as solid spheres. The illustrated trough is in the same plane as the TOPA ring and wide enough to accommodate an extended, straight hydrocarbon chain; that is of the order of 4 to 4.5 Å in width and a depth of 4.8 to 5 Å if the chain were to be fully enfolded. From about 4.2 to 6.5 to 7 Å out from the TOPA carbonyl carbon the trough becomes increasingly resistant to accommodating any branches in the hydrocarbon chain (e.g., methyl groups), but from about 8 Å and further, larger groups or atoms have minimal effects. The trough walls out to about 6.5 to 7 Å would most likely be composed of hydrophobic residues. In the region of 7 to 8 Å the enzyme contains a charged basic site, e.g., an aspartate or a glutamate residue, which acts to bind ionically the cationic terminus of a putrescine, a cadaverine or histamine. The presence of such a charged base, suggested in general terms by Bardsley et al. (1971), helps rationalize the strong selective binding of putrescine to diamine oxidase and inhibition of the enzyme by positively charged species such as guanidines, amidines and isothioureas. The immediate region around the TOPA carbonyl is probably relatively flexible, because the initial nucleophilic attack of the amine must take place perpendicular to the TOPA ring.

The assumption of hydroxylamine side chain extension and coplanarity may seem arbitrary but the concept of a "binding trough" extending from the TOPA carbonyl fits well with the natural substrates, for on modeling the putrescine iminium cation (19) (see Fig. 8). The NH₃⁺ nitrogen was found to be 7.4 Å from the carbon of the quinone carbonyl as illustrated and, as noted above, an enzyme base at ca. 8 Å from the carbonyl carbon would ionically anchor the NH₃⁺ group and increase enzyme specificity. Such a suggestion is supported by the observation that shorter (e.g., 1,2-diaminoethane) and longer diamines (e.g., 1,6-diaminohexane) are less active substrates than putrescine (Pec and Frebort, 1992). Most likely the base has some flexibility in accommodating intermediate chain lengths and compounds with no charges along the chain should offer few problems, but compounds with negative charges such as 2-(O-oxyamino)ethanoic acid should be, and in fact are, very poor substrates (Rangachari et al., 1992). It is noteworthy that histamine, which is a good substrate for diamine oxidase, has in a planar extended conformation the most distant N of which is 6.7 Å from the TOPA carbonyl carbon and if the N is protonated, it should also be anchored by the enzyme-bound base.

View larger version (52K): [in this window] [in a new window]   Fig. 8.   Top views of selected TOPA O-alkyl and O-benzylhydroxylamines illustrating how atoms protruding sideways into the proposed trough wall result in decreased inhibitory effects (see Fig. 6c). Atoms that tend to lie above the trough, as in O-(1-E-Cinnamyl), enable retained activity. a, TOPA O-1-pentyloxime; b, TOPA O-2-butyloxime; c, TOPA O-1-(2methyl)propyl; d, TOPA O-benzyloxime; e, TOPA O-1-Cinnamyloxime.

The idea of the trough having increased resistance to large groups comes from the reduction in inhibition of DAO activity produced by branched chain O-alkylhydroxylamines. In initial screening tests O-1-propyl and O-1-butylhydroxylamines were effective inhibitors, with O-2-butyl being less effective, and tert-butyl having no effect on DAO activity (Rangachari et al., 1992). In the studies reported here on DAO inhibition, the straight chain O-1-pentylhydroxylamine (2) produced greater inhibition at 10⁶ M than the branched compounds [O-2-butyl (3), O-1(2-methyl)propyl (6) and O-1(3-methyl)butyl (9)].

To illustrate how methyl substitution increases steric bulk, models of TOPA-O-1-pentyloxime (24) and TOPA-O-2-butyloxime, together with geometric data, are shown in figure 8, a and b. When both -Hs of O-1-butyl are replaced by CH₃s, as in tert-butylhydroxylamine, the steric effects become so large the oxime will not form at all. A model of O-1(2-methyl)propyl (6), illustrated in figure 7c, shows the size of a methyl group on the  carbon and how the enzyme resists steric bulk in this region. Moving large groups further away beyond the  carbon of a chain (7.33 Å away from the TOPA carbonyl carbon) results in a decrease in enzyme resistance to steric bulk and a consequence increase in inhibitory activity [see the discussion on O-(1-E-Cinnamyl) below].

The O-benzyl group of compounds contributes further to the "binding trough" concept. As a group they are appreciably less effective than the aliphatic hydroxylamines and they are poor inhibitors of the enzyme at 10⁶ M, but at 10⁵ M they show a clear distinction of activities as noted above. The parent compound, O-1-benzyl (16) shows only marginal inhibition at 10⁵ M. However, a para hydroxyl substituent on the ring causes a remarkable increase in activity. The flat, aromatic ring leads to it having large steric effects in the ring plane but small effects perpendicular to the ring. As a consequence the conformations of the ring about adjacent bonds are important and molecular modeling of O-1-benzyl gave a twisted conformation as the lowest energy conformer, where the phenyl ring is turned by 45° from the TOPA-N-O plane. This conformer is illustrated as a top view of a solid sphere structure in figure 8d (and should be compared with O-1-pentyl in Fig. 8a). The twist of the phenyl places ortho and meta hydrogens in regions similar to where methyl groups on the ,  and  carbons of a straight chain contribute steric bulk and unfavorable enzyme interactions. Consequently inhibitory activity is decreased to levels similar to that of O-1-(3-methyl)butyl (9). For a hydroxyl on the phenyl ring to cause increased inhibitory activity, there must be an additional factor, or factors, which favor binding of the ring near the active site. We suggest that hydrogen bonding, either directly or via an intermediate water, between the para oxygen and either a nearby enzyme backbone NH-C = O or a side chain OH, as in a serine or a threonine, is responsible for the extra binding.

O-(1-E-Cinnamyl) (10) was at first sight surprising because it showed inhibitory activity at 10⁶ M. However, molecular modeling illustrated by the solid sphere structure shown in figure 8e, showed the phenyl ring adopts conformations where it is approximately perpendicular to the TOPA-N-O plane and beyond the position where an  carbon of a straight chain would be (7.33 Å away). That is, steric bulk this far away from the TOPA carbonyl carbon seems to have fewer enzyme interactions.

	Summary

Top Abstract Introduction Methods and materials Results Discussion Summary Appendix References

O-Alkylhydroxylamines inhibit diamine oxidase by reacting with the carbonyl of the TOPA quinone at the active site to form an oxime of diamine oxidase. The ability of each member of the series to inhibit the enzyme is determined by their ability to access the TOPA quinone a "trough-like" region of the enzyme. However, in the tissue another determinant appears to be the access to the location of the enzyme. Thus the pharmacological responses observed in canine colon (histamine potentiation) reflect both the ability of the O-alkylhydroxylamine to penetrate the tissue and the ability to interact with the trough-like region of the enzyme and the TOPA-carbonyl of the active site.