The Relationship of Enzyme Inhibitory Activity to the Structure of n-Alkylphosphonate and Phenylalkylphosphonate Esters<sup>*</sup>

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Studies in the time course of the response of rabbit polymorphonuclear leukocytes (PMN's) to the complement-associated chemotactic factor have revealed that the response is virtually complete by 60 min with less than 15% additionally responding cells thereafter. Phosphonate esters with a well defined capacity to inhibit serine esterases have been used to study the cell-associated enzymes of the rabbit PMN required for the chemotactic response. Two types of inhibition of the cell response to the chemotactic factor have been found: (a) cell-dependent inhibition occurring as a result of pretreatment of PMN's with phosphonate esters; and (b) chemotactic factor-dependent inhibition demonstrated only when the phosphonate ester is present during the chemotactic response. Differences were found in these two modes of inhibition caused by various phosphonates, in terms of their time course of inhibition, in the dose response curves, and in the structure-activity relationships. It has been conclusively demonstrated that the phosphonate esters have no direct inhibitory effect on the chemotactic factor. This has been shown by retention of activity of the chemotactic factor following incubation with phosphonate esters and subsequent removal by dialysis. In addition, the activity of the chemotactic factor and its physical-chemical characteristics in density gradient ultracentrifugation were unaltered in the continued presence of a potent phosphonate inhibitor of chemotaxis. The uptake of the dye trypan blue was studied in cells treated with phosphonate in such a manner to induce cell-dependent inhibition of chemotaxis. Even when 84% cell-dependent inhibition of chemotaxis occurred, no uptake of the dye by leukocytes was found. Thus, phosphonate-induced inhibition of cell responsiveness in chemotaxis was not associated with generalized cell damage as defined by exclusion of the dye. It is concluded that cell-dependent inhibition is due to the presence of a cell-bound esterase which is already activated and thus susceptible to inhibition by phosphonate esters before contact of the cell with the chemotactic factor. The second type of inhibition, chemotactic factor-dependent inhibition, is considered due to a cell-bound esterase which becomes susceptible to inhibition by phosphonate esters only after contact of the PMN with the chemotactic factor. It is postulated that the chemotactic factor activates this phosphonate-resistant precursor making it susceptible to the inhibitory action of the phosphonate ester.

Section: Discussionmentioning

confidence: 98%

“…Phosphonate Inhibitors.--The p-nitrophenylethyl phosphonates of the general structure O where R represents the alkyl, phenylalkyl, ¢o-aminoalkyl, or ¢o-chloroalkyl chain were used) They have been described previously (5). The phosphonate esters were dissolved in acetone diluted 1:10 or 1:100 (in medium 199 or appropriate phosphate buffer).…”

Section: Methodsmentioning

confidence: 99%

Mechanisms of the Inhibition of Chemotaxis by Phosphonate Esters

Ward

Becker

1967

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“…It was dissolved directly in Tyrode's gelatin immediately before use. The p-nitrophenylethylphosphonate esters were described earlier (2,6,13,14,22). The cyclohexyl phosphonofluoridate esters were described in references 7 and 8.…”

Section: Methodsmentioning

confidence: 99%

Activation of stimulus-specific serine esterases (proteases) in the initiation of platelet secretion. I. Demonstration with organophosphorus inhibitors.

Henson¹,

Gould²,

Becker³

1976

The effect of organophosphorus inhibitors of serine esterases (proteases) on secretion from washed rabbit platelets was examined. Five noncytotoxic stimuli were employed: collagen, thrombin, heterologous anti-platelet antibody (in the absence of complement), rabbit C3 bound to zymosan, and platelet activating factor derived from antigen-stimulated, IgE-sensitized rabbit basophils. Diisoprophyl phosphofluoridate, three series of p-nitrophenyl ethyl phosphonates, and a series of cyclohexyl phenylalkylphosphonofluridates were all found to be inhibitory to the platelet secretion. These are irreversible inhibitors of serine proteases but in this system were only inhibitory if added to the platelets concurrently with the stimuli. Pretreatment of either the platelets or the stimuli with the inhibitors followed by washing, was without effect on the subsequent reaction. This suggested the involvement of stimulus-activatable serine proteases in the secretory process. The concept was supported by finding that nonphosphorylating phosphonates or hydrolyzed phosphonates or phosphonofluoridates were without inhibitory action. The effect of a series of phosphonates or phosphonoflouridates in inhibiting each stimulus exhibited a unique activity-structure profile. The demonstration of such unique profiles with four series of inhibitors for each of the five stimuli was interpreted as demonstrating that a specific activatable serine protease was involved in the platelet secretory response to each stimulus.

“…Phosphonate Inhibitors.--The p-nitrophenylethyl alkyl, phenyl alkyl, and the ¢o-chloroalkyl phosphonates have been described previously (7,14,15). The synthesis and properties of p-nitrophenylethy160-aminoalkyl phosphonates will be described subsequently by Mr. Robert Stat~, Mr. Dennis Werber, and Mr. Gim Tijoe of American University.…”

Section: Methodsmentioning

confidence: 99%

“…The low ionic strength prevented dissociation of C'la from the cell during the reaction (17), and pH 8.0 was used in order for the results to be directly comparable with previous work on other enzymes (7,15).…”

Section: Methodsmentioning

confidence: 99%

A Comparison of the Specificity of Inhibition by Phosphonate Esters of the First Component of Complement and the Antigen-Induced Release of Histamine From Guinea Pig Lung

Becker

Austen

1964

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The ability of a number p-nitrophenylethyl alkyl, phenyl alkyl, chloroalkyl, and aminoalkyl phosphonates to inhibit the activated first component (C'1a) of guinea pig complement, and the antigen-induced release of histamine from sliced, perfused guinea pig lung has been compared. C'1a in its reactivity with these phosphonates is distinctly more similar to trypsin than to any of the other enzymes studied previously. It is suggested that both trypsin and C'1a possess an anionic group in the active center of the respective enzyme, but the distance between the anionic and esteratic site in C'1a might be less than in trypsin. The pattern of inhibition of histamine relase by the alkyl, phenyl alkyl, and chloroalkyl phosphonates is similar to the inhibition of C'1a by these compounds, although distinct differences are apparent. The aminoalkyl phosphonates are distinctly less active inhibitors of histamine release than the corresponding alkyl phosphonates, whereas the reverse is true of the inhibition of C'1a. On the basis of these differences, it is tentatively concluded that the organophosphorus-inhibitable enzymes in the guinea pig systems studied here are similar but not identical.