Spermine prevent iron accumulation and depress lipofuscin accumulation in cultured myocardial cells.

The present work demonstrates that spermine prevents aging in cultured myocytes exposed to oxidative stress. It is found that physiological levels of spermine reduce lipofuscin accumulation with 20%, and that the antioxidative effect compares with vitamin E. By autometallography we also demonstrate that spermine prevent accumulation of free iron in the myocytes, probably by acting as a chelating agent. The effect compares to that of deferoxamine. These data provide additional insight into the antioxidative mechanism of spermine, and suggest that spermine may prevent diseases related to the Fenton reaction, as well as retard aging reactions.

Effect of ambient oxygen concentration on lipofuscin accumulation in cultured rat heart myocytes--a novel in vitro model of lipofuscinogenesis.

The objective of this study was to elucidate the factors involved in the accumulation of lipofuscin in post-mitotic cells. The hypothesis that oxidative stress accelerates the rate of lipofuscin accumulation was tested by examining the effects of 5%, 20%, and 40% ambient oxygen concentration on lipofuscin content in cultured rat cardiac myocytes. Lipofuscin was quantified by microspectrofluorometry at 7 and 12 days of in vitro age. Lipofuscin-emitted yellow autofluorescence increased in direct relationship to ambient oxygen concentration with age. Transmission electron microscopic examination of the cells after 3, 8, and 12 days in culture indicated a progressive time and oxygen dependent increase in the frequency and size of lipofuscin organelles. The results are interpreted to suggest that oxidative stress is one of the causal factors in the accumulation of lipofuscin.

Effect of ethanol on lipofuscin accumulation in cultured rat cardiac myocytes.

The objective of this study was to determine the effect of ethanol on in vitro life span, rate of contraction and lipofuscin content of neonatal rat cardiac myocytes. Lipofuscin was quantified by microspectrofluorometry. The effects of 0, 3.1, 6.5, and 12.5 mM ethanol on myocytes, kept under an ambient oxygen concentration of 20% and 40%, were studied. Exposure to low concentrations of ethanol resulted in a decrease in the amount of lipofuscin whereas exposure to high concentration of ethanol caused an increase in the level of lipofuscin. The length of cell survival in controls and 3.1 mM ethanol exposed myocytes was similar under 20% oxygen, but was longer in the latter group under 40% oxygen, as compared to controls. The total number of contractions in 3.1 mM ethanol-exposed myocytes were, respectively, 4% and 8% higher under 20% and 40% oxygen atmosphere than in control cells.

Heavy metals and lipofuscinogenesis. A study on myocardial cells cultured under varying oxidative stress.

The aim of this study was to examine the influence on lipofuscinogenesis of a number of transition and non-transition heavy metals in cultured post-mitotic cells (neonatal rat myocytes) at varying oxidative stress. The effects of Al, Cd, Cr, Cu, Hg, Pb and Zn, added to the medium as chlorides, were examined after 14 days in culture under 5, 20 and 40% ambient oxygen. Lipofuscin was quantified by microspectrofluorometry of individual cells. The addition of Al (40 microM), Cd (40 nM), Hg (30 microM) and Pb (40 microM) to the culture growth medium markedly increased the amount of intracellular lipofuscin, whereas Cr (40 microM), Cu (40 microM) and Zn (40 microM) had the opposite effect. Transmission electron microscopic examination of the myocytes showed greatly increased numbers of autophagic vacuoles in cells exposed to those heavy metals that increased lipofuscin formation. This effect was most pronounced when cells were grown at high (40%) oxygen tension. Possible explanations for the metal augmented pigment formation may be (i) inhibition of lysosomal enzymes, (ii) catalytic interference with peroxidative reactions, or (iii) general toxicity with unspecifically increased autophagocytosis. The decreased pigment accumulation after the addition of Zn, Cr and Cu may, at least partly, be related to the replacement of iron, which has catalytic activity in Fenton reactions.

Indenoindole depresses lipofuscin formation in cultured neonatal rat myocardial cells.

Lipofuscin accumulation in cultured rat myocardial cells is considered an index of intra-lysosomal oxidative reactions and was registered by autofluorescence measurements. Lipofuscinogenesis in secondary lysosomes is thought to be a consequence of Fenton reactions, and is much enhanced by oxidative stress obtained by culturing the cells in an atmosphere containing 40% oxygen. The influence of the synthetic antioxidant indenoindole (DHII), as compared to control cells, was a dose-responsive depression of lipofuscinogenesis to a degree of 19% and 17% with 20 microM DHII and to 25% and 23% with 40 microM DHII after 7 and 14 days in culture, respectively. This demonstrates a significant quenching of oxidative stress and suggests the therapeutic value of DHII and related antioxidants in protecting against oxygen radical-related diseases. It is also suggested that neonatal cardiac myocytes in culture are a suitable model system for the evaluation of oxygen radical-induced myocardial damage.

Water and ion distributions in myocytes cultured under oxidative stress mimic changes found in the process of aging.

Element concentrations and local water content were measured in cytoplasm, nuclei, mitochondria and lipofuscin granula (LG) of isolated cardiac myocytes of the rat. Cells were cultured for 14 days under either 5%, 20% or 40% ambient oxygen concentration. LG were found to contain less cations and more phosphorous than mitochondria, which might be related to their lower protein and higher lipid content. Under oxidative stress, dehydration of mitochondria occurred, while their cation content remained constant. This is the same pattern of changes as found in cells in situ of aging rats. Therefore, it is concluded that peroxidative damage via oxygen derived free radicals is the reason for the mitochondrial water loss in the process of aging.

Effect of ferric iron and desferrioxamine on lipofuscin accumulation in cultured rat heart myocytes.

The general objective of this study was to examine the role of oxidative stress and transition metal catalysis in lipofuscinogenesis in postmitotic cells. Effects of 30 microM ferric chloride and different concentrations of desferrioxamine, ranging from 12.5 to 50 microM, on lipofuscin content were examined in rat cardiac myocytes at 6 and 12 days of in vitro age, under 20% and 40% ambient oxygen concentration. Lipofuscin was quantified by microspectrofluorometry of individual cells. Using X-ray microanalysis, iron was found to be sequestered in the secondary lysosomes that also contain the fluorescent lipofuscin material. Augmentation of iron in the culture medium markedly increased the level of lipofuscin accumulation while desferrioxamine had the opposite effect. Both of these effects were more pronounced at higher oxygen tension. Iron and oxygen had a cumulative effect on lipofuscin accumulation. Results are interpreted to indicate that oxidative stress and iron, in loosely bound form, are causal factors in lipofuscinogenesis. The possibility of employing microspectrofluorometry of lipofuscin as an indicator of oxidative stress is emphasized.

Design and synthesis of novel alpha(1)(a) adrenoceptor-selective antagonists. 3. Approaches to eliminate opioid agonist metabolites by using substituted phenylpiperazine side chains.

Dihydropyrimidinones, such as 1, represent a novel class of alpha(1a) adrenoceptor antagonists with potential for the treatment of benign prostatic hyperplasia (BPH) (see part 1 of this series). Analysis of the metabolites of 1 revealed that 4-methoxycarbonyl-4-phenylpiperidine is formed as the major metabolite and is an agonist at the mu-opioid receptor. To circumvent any potential liability resulting from the metabolite, we decided to identify alternate templates devoid of agonist activity at the mu-opioid receptor to replace the 4-methoxycarbonyl-4-phenylpiperidine moiety. The present study describes the synthesis and SAR of dihydropyrimidinones linked to substituted 4-phenylpiperazine containing side chains. Compound (+)-38 was identified as a lead compound with a binding and functional profile comparable to that of 1. The putative metabolite 2-carboxamidophenylpiperazine has negligible affinity for the mu-opioid receptor.

Design and synthesis of novel alpha1a adrenoceptor-selective dihydropyridine antagonists for the treatment of benign prostatic hyperplasia.

We report the synthesis and evaluation of novel alpha1a adrenoceptor subtype-selective antagonists. Systematic modification of the lipophilic 4,4-diphenylpiperidinyl moiety of the dihydropyridine derivatives 1 and 2 provided several highly selective and potent alpha1a antagonists. From this series, we identified the 4-(methoxycarbonyl)-4-phenylpiperidine analogue SNAP 5540 (-) [(-)-63] for further characterization. When examined in an isolated human prostate tissue assay, this compound was found to have a Ki of 2.8 nM, in agreement with the cloned human receptor binding data (Ki = 2.42 nM). Further evaluation of the compound in isolated dog prostate tissue showed a Ki of 3.6 nM and confirmed it to be a potent antagonist (Kb = 1.6 nM). In vivo, this compound effectively blocked the phenylephrine-stimulated increase in intraurethral pressure (IUP) in mongrel dogs, at doses which did not significantly affect the arterial pressure (diastolic blood pressure, DBP), with a DBP Kb/IUP Kb ratio of 16. In addition, (-)-63 also showed greater than 40 000-fold selectivity over the rat L-type calcium channel and 200-fold selectivity over several G protein-coupled receptors, including histamine and serotonin subtypes. These findings prove that alpha1a adrenoceptor-subtype selective antagonists such as (-)-63 may be developed as uroselective agents for an improved treatment of BPH over nonselective alpha1 antagonists such as prazosin and terazosin, with fewer side effects.

Identification of a dihydropyridine as a potent alpha1a adrenoceptor-selective antagonist that inhibits phenylephrine-induced contraction of the human prostate.

A number of novel dihydropyridine derivatives based upon 1, 4-dihydro-3-(methoxycarbonyl)-2, 6-dimethyl-4-(4-nitrophenyl)-5-((3-(4, 4-diphenylpiperidin-1-yl)propyl)aminocarbonyl)pyridine (4) have been synthesized and tested at cloned human alpha adrenoceptors as well as the rat L-type calcium channel. Within this compound series, 5-(aminocarbonyl)-1,4-dihydro-2, 6-dimethyl-4-(4-nitrophenyl)-3-((3-(4, 4-diphenylpiperidin-1-yl)propyl)aminocarbonyl)pyridine (19) displayed good binding affinity and selectivity for the alpha1a adrenoceptor (pKi = 8.73) and potently inhibited (pA2 = 9.23) phenylephrine-induced contraction of the human prostate.

Design and synthesis of novel alpha(1)(a) adrenoceptor-selective antagonists. 1. Structure-activity relationship in dihydropyrimidinones.

Dihydropyrimidinones such as compound 12 exhibited high binding affinity and subtype selectivity for the cloned human alpha(1a) receptor. Systematic modifications of 12 led to identification of highly potent and subtype-selective compounds such as (+)-30 and (+)-103, with high binding affinity (K(i) = 0.2 nM) for alpha(1a) receptor and greater than 1500-fold selectivity over alpha(1b) and alpha(1d) adrenoceptors. The compounds were found to be functional antagonists in human, rat, and dog prostate tissues. Compound (+)-103 exhibited excellent selectively to inhibit intraurethral pressure (IUP) as compared to lowering diastolic blood pressure (DBP) in mongrel dogs (K(b)(DBP)/K(b)(IUP) = 40) suggesting uroselectivity for alpha(1a)-selective compounds.

Design and synthesis of novel alpha(1)(a) adrenoceptor-selective antagonists. 2. Approaches to eliminate opioid agonist metabolites via modification of linker and 4-methoxycarbonyl-4-phenylpiperidine moiety.

We have previously described compound 1a as a high-affinity subtype selective alpha(1a) antagonist. In vitro and in vivo evaluation of compound 1a showed its major metabolite to be a mu-opioid agonist, 4-methoxycarbonyl-4-phenylpiperidine (3). Several dihydropyrimidinone analogues were synthesized with the goal of either minimizing the formation of 3 by modification of the linker or finding alternative piperidine moieties which when cleaved as a consequence of metabolism would not give rise to mu-opioid activity. Modification of the linker gave several compounds with good alpha(1a) binding affinity (K(i) = < 1 nM) and selectivity (>300-fold over alpha(1b) and alpha(1d)). In vitro analysis in the microsomal assay revealed these modifications did not significantly affect N-dealkylation and the formation of the piperidine 3. The second approach, however, yielded several piperidine replacements for 3, which did not show significant mu-opioid activity. Several of these compounds maintained good affinity at the alpha(1a) adrenoceptor and selectivity over alpha(1b) and alpha(1d). For example, the piperidine fragments of (+)-73 and (+)-83, viz. 4-cyano-4-phenylpiperidine and 4-methyl-4-phenylpiperidine, were essentially inactive at the mu-opioid receptor (IC(50) > 30 microM vs 3 microM for 3). Compounds (+)-73 and (+)-83 were subjected to detailed in vitro and in vivo characterization. Both these compounds, in addition to their excellent selectivity (>880-fold) over alpha(1b) and alpha(1d), also showed good selectivity over several other recombinant human G-protein coupled receptors. Compounds (+)-73 and (+)-83 showed good functional potency in isolated human prostate tissues, with K(b)s comparable to their in vitro alpha(1a) binding data. In addition, compound (+)-73 also exhibited good uroselectivity (DBP K(b)/IUP K(b) > 20-fold) in the in vivo experiments in dogs, similar to 1a.

Design and synthesis of novel alpha(1)(a) adrenoceptor-selective antagonists. 4. Structure-activity relationship in the dihydropyrimidine series.

We have previously disclosed dihydropyridines such as 1a,b as selective alpha(1a) antagonists as a potential treatment for benign prostatic hyperplasia (BPH). The propensity of dihydropyridines toward an oxidation led us to find suitable replacements of the core unit. The accompanying papers describe the structure-activity relationship (SAR) of dihydropyrimidinones 2a,b as selective alpha(1a) antagonists. We report herein the SAR of dihydropyrimidines such as 4 and highlight the similarities and differences between the dihydropyrimidine and dihydropyrimidinone series of compounds.

Lipofuscinogenesis in a model system of cultured cardiac myocytes.

Lipofuscin, or age pigment, is a yellowish-brown, autofluorescent, protein, and lipid containing pigment that accumulates in the lysosomal vacuome of a variety of post-mitotic cells in man and animals during aging. Lipofuscin seems to be an end product of peroxidation, fragmentation and polymerization of proteins and lipids. Protein and lipid containing materials are regularly sequestered to the lysosomal system by means of endocytosis and autophagocytosis. Lysosomes and acidified endosomes constitute cellular compartments where iron for short periods of time may exist in reactive form allowing for peroxidative processes. The importance was demonstrated in model systems of cultured cells subjected to oxidative stress and augmented amounts of FeCl3 in the culture medium. This combination was considered to result in increased intracellular, mainly mitochondrial, production of hydrogen peroxide as well as increased intralysosomal concentration of iron. One of the results of this situation was dramatically increased lipofuscinogenesis.

Mechanisms of lipofuscinogenesis: effect of the inhibition of lysosomal proteinases and lipases under varying concentrations of ambient oxygen in cultured rat neonatal myocardial cells.

The objective of this study was to analyse the relative roles of oxidative stress and lysosomal lytic enzymes in lipofuscin formation in cultured neonatal rat cardiac myocytes. Myocytes were exposed to E-64 (an inhibitor of lysosomal cathepsins L. D and H), netilmicin (an inhibitor of lysosomal phospholipases A1 and C) and leupeptin (an inhibitor of cytosolic and lysosomal thiolproteinases) under varied conditions of oxidative stress (20% and 40% ambient oxygen) for up 14 days. Lipofuscin was quantified by microspectrofluorometry. The amount of lipofuscin accumulation was enhanced by the lytic enzyme inhibitors as well as by the increase in the ambient oxygen concentration. However, the effects of enzyme inhibitors was less obvious under 40% ambient oxygen than under 20% oxygen. Data are interpreted as suggesting that, under high levels of oxidative stress, intralysosomal peroxidative changes related to lipofuscin formation occur so rapidly that lytic activity assumes a minor role in lipofuscinogenesis whereas, under low oxidative stress, inhibition of lytic activity makes a greater contribution to lipofuscinogenesis by allowing a longer period of time for peroxidative changes. The results further substantiate the hypotheses that (a) lipofuscinogenesis is strongly related to oxidative stress, and (b) lipofuscin forms intralysosomally as a result of processes involving incomplete degradation of heterophagocytosed and or autophagocytosed material.

In vitro studies on L-771,688 (SNAP 6383), a new potent and selective alpha1A-adrenoceptor antagonist.

L-771,688 (SNAP 6383, methyl(4S)-4-(3, 4-difluorophenyl)-6-[(methyloxy)methyl]-2-oxo-3-[(¿3-[4-(2-pyridin yl)-1-piperidinyl]propyl¿amino)carbonyl]-1,2,3, 4-tetrahydro-5-pyrimidine carboxylate) had high affinity (Ki less than or = 1 nM) for [3H]prazosin binding to cloned human, rat and dog alpha1A-adrenoceptors and high selectivity (>500-fold) over alpha1B and alpha1D-adrenoceptors. [3H]Prazosin / (+/-)-beta-[125I]-4-hydroxy-phenyl)-ethyl-aminomethylteralone ([125I]HEAT) binding studies in human and animal tissues known to contain alpha1A and non-alpha1A-adrenoceptors further demonstrated the potency and alpha1A-subtype selectivity of L-771,688. [3H]L-771,688 binding studies at the cloned human alpha1A-adrenoceptors and in rat tissues indicated that specific [3H]L-771,688 binding was saturable and of high affinity (Kd=43-90 pM) and represented binding to the pharmacologically relevant alpha1A-adrenoceptors. L-771,688 antagonized norepinephrine-induced inositol-phosphate responses in cloned human alpha1A-adrenoceptors, as well as phenylephrine or A-61603 (N-[5-4,5-dihydro-1H-imidazol-2yl)-2-hydroxy-5,6,7, 8-terahydro-naphthlen-1-yl] methanesulfonamide hydrobromide) induced contraction in isolated rat, dog and human prostate, human and monkey bladder neck and rat caudal artery with apparent Kb values of 0.02-0.28 nM. In contrast, the contraction of rat aorta induced by norepinephrine was resistant to L-771,688. These data indicate that L-771,688 is a highly selective alpha1A-adrenoceptor antagonist.

Efficacy of the MCHR1 antagonist N-[3-(1-{[4-(3,4-difluorophenoxy)phenyl]methyl}(4-piperidyl))-4-methylphenyl]-2-methylpropanamide (SNAP 94847) in mouse models of anxiety and depression following acute and chronic administration is independent of hippocampal neurogenesis.

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide that plays a role in the modulation of food intake and mood. In rodents, the actions of MCH are mediated via the MCHR1 receptor. The goal of this study was to investigate the effects of acute (1 h) and chronic (28 days) p.o. dosing of a novel MCHR1 antagonist, N-[3-(1-{[4-(3,4-difluorophenoxy)-phenyl]methyl}(4-piperidyl))-4-methylphenyl]-2-methylpropanamide (SNAP 94847), in three mouse models predictive of antidepressant/anxiolytic-like activity: novelty suppressed feeding (NSF) in 129S6/SvEvTac mice and light/dark paradigm (L/D) and forced swim test (FST) in BALB/cJ mice. A significant increase in the time spent in the light compartment of the L/D box was observed in response to acute and chronic treatment with SNAP 94847. An anxiolytic/antidepressant-like effect was found in the NSF test after acute and chronic treatment, whereas no effect was observed in the FST. Because neurogenesis in the dentate gyrus has been shown to be a requirement for the effects of antidepressants in the NSF test, we investigated whether neurogenesis was required for the effect of SNAP 94847. We showed that chronic treatment with SNAP 94847 stimulated proliferation of progenitors in the dentate gyrus. The efficacy of SNAP 94847 in the NSF test, however, was unaltered in mice in which neurogenesis was suppressed by X-irradiation. These results indicate that SNAP 94847 has a unique anxiolytic-like profile after both acute and chronic administration and that its mechanism of action is distinct from that of selective serotonin reuptake inhibitors and tricyclic antidepressants.

Effect of alpha-tocopherol and some metal chelators on lipofuscin accumulation in cultured neonatal rat cardiac myocytes.

The objective of this study was to test further the hypothesis that oxidative stress is a major causal factor in lipofuscin formation. We have previously shown that cultured cardiac myocytes constitute a suitable model system for the study of factors influencing lipofuscinogenesis. The specific aim of the present study was to elucidate the effects of the chain-breaking free radical scavenger alpha-tocopherol, and the chelators desferrioxamine, EDTA and DTPA on the accumulation of lipofuscin. The effects were examined at different degrees of oxidative stress, obtained by varying the ambient oxygen concentration from 5 to 40%. Lipofuscin was quantified by microspectrofluorometry. Lipofuscin-specific, yellow autofluorescence increased with time in culture, and with enhanced oxidative stress. Increasing concentration of alpha-tocopherol, up to 40 microM, had an inhibitory effect on lipofuscin accumulation that was most pronounced at high oxidative stress. Desferrioxamine and DTPA, both caused a pronounced reduction in lipofuscin formation, while EDTA had no significant effect. The findings are interpreted to support the concept that oxidative stress is a causal factor in lipofuscinogenesis, and that lipofuscin is a product of autophagocytosed, membrane-rich material subjected to free radical-induced, metal-catalyzed peroxidation, fragmentation, and polymerization within the lysosomal vacuome.

Steady-state kinetic evaluation of the reverse reaction for Escherichia coli 5-enolpyruvoylshikimate-3-phosphate synthase.

Recently it has been found that the kinetic mechanism for Escherichia coli 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS) in the forward direction is random with synergistic binding of substrates and inhibitors (K. J. Gruys, M. C. Walker, and J. A. Sikorski, 1992, Biochemistry 31, 5534). This work, however, did not address the reverse reaction with 5-enolpyruvoylshikimate-3-phosphate (EPSP) and phosphate (Pi) as substrates where a similar question of random versus ordered addition of substrates remained. Previous transient-state kinetic results led to a proposal for an equilibrium-ordered mechanism, where binding of EPSP occurs first followed by Pi (K. S. Anderson, and K. A. Johnson, 1990, Chem. Rev. 90, 1131). Steady-state kinetic results of the reverse reaction presented here suggest that, like the forward reaction, addition of substrates occurs randomly. Initial velocity studies with EPSP and Pi show a normal intersecting pattern in the reciprocal plots, consistent with a random or steady-state-ordered mechanism, but not with equilibrium-ordered addition of substrates. Inhibition of the EPSPS reverse reaction by 5-amino-S3P or the S3P-glyphosate hybrid molecule gave the expected competitive patterns versus EPSP, but mixed noncompetitive patterns versus Pi. These results also disfavor an equilibrium-ordered model, but again are consistent with a random or steady-state-ordered mechanism. A more quantitative mechanistic analysis of the inhibition data to determine the true rather than apparent Ki values provides evidence for a random over a steady-state-ordered addition of substrates. These results in combination with previous findings lead to the conclusion that the mechanism is random addition of EPSP and Pi since it is the only possible model for substrate addition that is consistent with the cumulative data from all kinetic (transient- as well as steady-state) and direct binding studies.

An EPSP synthase inhibitor joining shikimate 3-phosphate with glyphosate: synthesis and ligand binding studies.

A novel EPSP synthase inhibitor 4 has been designed and synthesized to probe the configurational details of glyphosate recognition in its herbicidal ternary complex with enzyme and shikimate 3-phosphate (S3P). A kinetic evaluation of the new 3-dephospho analog 12, as well as calorimetric and (31)P NMR spectroscopic studies of enzyme-bound 4, now provides a more precise quantitative definition for the molecular interactions of 4 with this enzyme. The very poor binding, relative to 4, displayed by the 3-dephospho analog 12 is indicative that 4 has a specific interaction with the S3P site. A comparison of Ki(calc) for 12 versus the Ki(app) for 4 indicates that the 3-phosphate group in 4 contributes about 4.8 kcal/mol to binding. This compares well with the 5.2 kcal/mol which the 3-phosphate group in S3P contributes to binding. Isothermal titration calorimetry demonstrates that 4 binds to free enzyme with an observed Kd of 0.53 +/- 0.04 microM. As such, 4 binds only 3-fold weaker than glyphosate and about 150-fold better than N-methylglyphosate. Consequently, 4 represents the most potent N-alkylglyphosate derivative identified to date. However, the resulting thermodynamic binding parameters clearly demonstrate that the formation of EPSPS x 4 is entropy driven like S3P. The binding characteristics of 4 are fully consistent with a primary interaction localized at the S3P subsite. Furthermore, (31)P NMR studies of enzyme-bound 4 confirm the expected interaction at the shikimate 3-phosphate site. However, the chemical shift observed for the phosphonate signal of EPSPS x 4 is in the opposite direction than that observed previously when glyphosate binds with enzyme and S3P. Therefore, when 4 occupies the S3P binding site, there is incomplete overlap at the glyphosate phosphonate subsite. As a glyphosate analog inhibitor, the potency of 4 most likely arises from predominant interactions which occur outside the normal glyphosate binding site. Consequently, 4 is best described as an S3P-based substrate-analog inhibitor. These combined results corroborate the previous kinetic model [Gruys, K. J., Marzabadi, M. R., Pansegrau, P. D., & Sikorski, J. A. (1993) Arch. Biochem. Biophys. 304, 345-351], which suggested that 4 interacts well with the S3P subsite but has little, if any, interaction at the expected glyphosate phosphonate or phosphoenolpyruvate-Pi subsites.