Aminoalkylphosphinate inhibitors of D-Ala-D-Ala adding enzyme

Miller, David J.; Hammond, Stephen M.; Anderluzzi, Daniela; Bugg, Timothy D. H.

doi:10.1039/a704097k

Cited by 86 publications

(43 citation statements)

References 22 publications

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“…EF6265 was synthesized in our laboratory with consideration of the previously reported methods (Baylis et al, 1984;Miller et al, 1998;Vassiliou et al, 1999). Detailed procedures and spectroscopic data will be reported on another occasion.…”

Section: Methodsmentioning

confidence: 99%

Enhancement of Fibrinolysis by EF6265 [(S)-7-Amino-2-[[[(R)-2-methyl-1-(3-phenylpropanoylamino)propyl]hydroxyphosphinoyl] methyl]heptanoic Acid], a Specific Inhibitor of Plasma Carboxypeptidase B

Suzuki¹,

Muto²,

Fushihara³

et al. 2004

J Pharmacol Exp Ther

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Plasma procarboxypeptidase B, also known as thrombin-activatable fibrinolysis inhibitor (TAFI), is converted by thrombin into the active enzyme, carboxypeptidase B (CPB)/activated TAFI. Plasma CPB down-regulates fibrinolysis by removing carboxy-terminal lysines, the ligands for plasminogen and tissue-type plasminogen activator (tPA), from partially degraded fibrin. To target thrombosis in a new way, we have identified and optimized a phosphinic acid-containing inhibitor of CPB,heptanoic acid] and determined both the pharmacological profile and pathophysiological role of CPB in rat thrombolysis. EF6265 specifically inhibited plasma CPB activity with an IC 50 (50% inhibitory concentration) of 8.3 nM and enhanced tPA-mediated clot lysis in a concentrationdependent manner. EF6265 decreased detectable thrombi (percentage of glomerular fibrin deposition; control, 98 Ϯ 1.1; EF6265, 0.1 mg/kg, 27 Ϯ 9.1) that had been generated by tissue factor in a rat microthrombosis model with concomitant increases in plasma D-dimer concentration (control, Ͻ0.5 g/ml; EF6265, 0.1 mg/kg, 15 Ϯ 3.5 g/ml). EF6265 reduced plasma ␣2-antiplasmin activity to a lesser extent than tPA. In an arteriovenous shunt model, EF6265 (1 mg/kg) enhanced exogenous tPA-mediated thrombolysis under the same conditions that neither EF6265 nor tPA (600 kIU/kg) alone reduced thrombi. EF6265 (1 and 30 mg/kg) did not affect the bleeding time in rats. Moreover, it did not prolong the bleeding time evoked by tPA (600 kIU/kg). These results confirm that circulating procarboxypeptidase B functions as a fibrinolysis inhibitor's zymogen and validates the use of CPB inhibitors as both an enhancer of physiological fibrinolysis in microcirculation and as a novel adjunctive agent to tPA for thromboembolic diseases while maintaining a small effect on primary hemostasis.Thrombosis-related diseases, including myocardial infarction, cerebral infarction, and disseminated intravascular coagulation are life-threatening and the search for treatments remains challenging. Although thrombolytics including tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator and anticoagulants including heparin have been developed over the last few decades, the risk of hemorrhaging with these antithrombotic agents restricts their clinical use. The dose of tPA must be high enough to overcome the inhibitory effects of plasminogen activator inhibitor-1 in the plasma, and results in the generation of plasmin in circulating blood (Rijken and Sakharov, 2001). Consequently, these large quantities of generated plasmin can induce thrombolysis and result in hemorrhaging as a side effect (Bloom et al., 1988). Many efforts have therefore been made to identify and develop pharmacologically distinct antithrombotic agents while maintaining a low risk of hemorrhaging.Plasma procarboxypeptidase B (proCPB; EC 3.4.17.20), also known as thrombin-activatable fibrinolysis inhibitor (TAFI) or procarboxypeptidase U, is produced in the liver and Article, publication date, and citation informa...

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Section: Methodsmentioning

confidence: 99%

Enhancement of Fibrinolysis by EF6265 [(S)-7-Amino-2-[[[(R)-2-methyl-1-(3-phenylpropanoylamino)propyl]hydroxyphosphinoyl] methyl]heptanoic Acid], a Specific Inhibitor of Plasma Carboxypeptidase B

Suzuki¹,

Muto²,

Fushihara³

et al. 2004

J Pharmacol Exp Ther

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“…Phosphinate transition-state analogs of the MurC to -F enzymes were synthesized by various groups in the 1990s, and some of these showed potent enzyme inhibition (at low nanomolar levels for some, illustrating druggability of the targets) but no antibacterial activity, presumably due to a lack of cell entry (127,254,310,362,403). A number of inhibitors of MurB, -C, -D, and -F, found via screens for enzyme inhibition or binding, had micromolar (in some cases, low micromolar) 50% inhibitory concentrations (IC 50 s) and no (or weak in one case [370]) antibacterial activity (11,30,103,141,364,370).…”

Section: Murb To Murf Enzymes As Antibacterial Targetsmentioning

confidence: 99%

Challenges of Antibacterial Discovery

Silver¹

2011

Clin Microbiol Rev

1,135

961

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SUMMARY The discovery of novel small-molecule antibacterial drugs has been stalled for many years. The purpose of this review is to underscore and illustrate those scientific problems unique to the discovery and optimization of novel antibacterial agents that have adversely affected the output of the effort. The major challenges fall into two areas: (i) proper target selection, particularly the necessity of pursuing molecular targets that are not prone to rapid resistance development, and (ii) improvement of chemical libraries to overcome limitations of diversity, especially that which is necessary to overcome barriers to bacterial entry and proclivity to be effluxed, especially in Gram-negative organisms. Failure to address these problems has led to a great deal of misdirected effort.

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“…Previously described inhibitors of MurF include phosphinate transition state analogs (K i ϭ 200 to 700 M) (14) and the nonhydrolyzable ATP analog AMP-PCP [adenylyl 5Ј-(␤,␥-methylenediphosphonate)] (K ii ϭ 4 M) (1). Aside from per- Gu et al (10) and the thiazolylaminopyrimidine series described herein lacked measurable antibacterial activity.…”

Section: Discussionmentioning

confidence: 99%

Utility of Muropeptide Ligase for Identification of Inhibitors of the Cell Wall Biosynthesis Enzyme MurF

Baum

Crespo-Carbone

Abbanat

et al. 2006

Antimicrob Agents Chemother

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MurF is a key enzyme in the biosynthesis of the bacterial cell wall in both gram-positive and gram-negative bacteria. This enzyme has not been extensively exploited as a drug target, possibly due to the difficulty in obtaining one of the substrates, UDP-MurNAc-L-Ala-␥-D-Glu-meso-diaminopimelate, which is usually purified from bacteria. We have identified putative inhibitors of Escherichia coli MurF by a binding assay, thus bypassing the need for substrate. Inhibition of enzymatic activity was demonstrated in a high-performance liquid chromatography-based secondary assay with UDP-MurNAc-L-Ala-␥-D-Glu-diaminopimelate substrate prepared in a novel way by using muropeptide ligase enzyme to add UDP-MurNAc to synthetic L-Ala-␥-D-Glu-diaminopimelate; the substrate specificity of muropeptide ligase for peptides containing L-Lys in place of diaminopimelate was also investigated. Using the muropeptide ligase-generated MurF substrate, a thiazolylaminopyrimidine series of MurF enzyme inhibitors with 50% inhibitory concentration values as low as 2.5 M was identified.The steps involved in the synthesis of the bacterial cell wall have long been considered to be good targets for antibacterial agents, as evidenced by drugs such as ␤-lactams and vancomycin (18). The MurF enzyme catalyzes the last cytoplasmic step of bacterial cell wall synthesis, the ligation of D-Ala-D-Ala to UDP-MurNAc-tripeptide with the concomitant hydrolysis of ATP. In gram-negative bacteria, the tripeptide is L-Ala-␥-DGlu-meso-A 2 pm (where A 2 pm represents diaminopimelate); in gram-positive bacteria, L-Lys replaces meso-A 2 pm (23).MurF is an attractive antibacterial drug target for several reasons: (i) it carries out an essential step of cell wall biosynthesis as demonstrated by the study of a temperature-sensitive lethal mutation in this gene in Escherichia coli (12); (ii) it is a single-copy gene in both gram-positive and gram-negative bacteria with extensive amino acid sequence conservation, raising the possibility of broad-spectrum inhibitors; and (iii) an earlier step in this pathway, MurA, is the target of the antibacterial drug fosfomycin (9), suggesting that interference with MurF function would likewise disrupt bacterial replication. In addition, normal MurF activity has been shown to be necessary for ␤-lactam resistance in methicillin-resistant Staphylococcus aureus (20).Despite these attractive features, MurF has not been used extensively as a target in high-throughput screening, possibly due to the difficulty in obtaining sufficient quantities of its substrate, UDP-MurNAc-tripeptide. Previous efforts to assay MurF that bypassed the need for substrate included the use of a coupled reaction containing the enzymes MurA, B, C, D, E, and F (8, 24) or permeabilized cells (2). A more direct approach would be an assay to detect compounds that bind to MurF. We have recently reported the use of capillary electrophoresis to identify compounds that bind to E. coli MurF.Similarly, Gu et al. (10) utilized an unspecified affinity selection screening technol...

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Aminoalkylphosphinate inhibitors of D-Ala-D-Ala adding enzyme

Cited by 86 publications

References 22 publications

Enhancement of Fibrinolysis by EF6265 [(S)-7-Amino-2-[[[(R)-2-methyl-1-(3-phenylpropanoylamino)propyl]hydroxyphosphinoyl] methyl]heptanoic Acid], a Specific Inhibitor of Plasma Carboxypeptidase B

Enhancement of Fibrinolysis by EF6265 [(S)-7-Amino-2-[[[(R)-2-methyl-1-(3-phenylpropanoylamino)propyl]hydroxyphosphinoyl] methyl]heptanoic Acid], a Specific Inhibitor of Plasma Carboxypeptidase B

Challenges of Antibacterial Discovery

Utility of Muropeptide Ligase for Identification of Inhibitors of the Cell Wall Biosynthesis Enzyme MurF

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