Inhibition of Thrombin by Peptides Containing Lysyl-α-Keto Carbonyl Derivatives

Abstract

SummarySeveral H-N-Me-D-Phe-Pro-Lysyl-α-keto carbonyl derivatives were shown to be potent thrombin inhibitors (Ki 0.2 to 27 nM). The inhibitory potencies of these compounds toward tissue plasminogen activator, plasmin and factor Xa were minimal; however, substantial cross-reactivity versus trypsin was observed (Ki values from 0.5 to 1500 nM). Inhibition of thrombin by α-keto carbonyl compounds appeared to occur via a one-step reversible reaction. The α-keto carbonyl inhibitors bound thrombin with a second order rate constant (k, 1–4 μM-1s-1) that was 10–100-fold slower than that expected for a diffusion-controlled reaction. Certain α-kelo earbonyl inhibitors were as potent (on a weight basis) as hirudin when evaluated in a rat arterial thrombosis model. The modest oral bioavailability (10–19%) in rats demonstrated for three of the α-keto carbonyl thrombin inhibitors suggests the possibility that α-keto amide containing thrombin inhibitors may have utility as orally-active antithrombotic agents.

Characterization of the Two-step Pathway for Inhibition of Thrombin by α-Ketoamide Transition State Analogs

“…Determination of Inhibition Constants (K i )-The inhibition constants were determined as described previously (5,8,9). When total inhibitor ([I t ]) and enzyme ([E t ]) concentrations were comparable, the quadratic equation (18 -20) …”

“…Determination of Inhibition Constants (K i )-The inhibition constants were determined as described previously (5,8,9 …”

“…The sources of other materials were described previously (5, 8). Proteinase assays were performed at room temperature in 50 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% polyethylene glycol 8000 unless otherwise indicated.Determination of Inhibition Constants (K i )-The inhibition constants were determined as described previously (5,8,9 …”

“…Cyclotheonamide A is a potent (K i ϭ 1 nM) reversible inhibitor of thrombin (5,6) and several other trypsin-like proteinases (5,7). Various ␣-ketoamide derivatives of tripeptide substrates of thrombin have been prepared in an attempt to identify selective, potent, reversible inhibitors of thrombin (8,9). Placement of a t-4-AChxGly 2 residue at the P1 position in ␣-ketoamide thrombin inhibitors provided excellent selectivity for thrombin, relative to tryspin (10 -12), probably because the S1 specificity pocket of thrombin is larger than that of trypsin (13,14).…”

“…Concentrations of DAPA were determined from measurements of absorbance at 330 nm using an extinction coefficient of 4.01 cm Ϫ1 mM Ϫ1 (17). The sources of other materials were described previously (5,8). Proteinase assays were performed at room temperature in 50 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% polyethylene glycol 8000 unless otherwise indicated.…”