Direct thrombin inhibitors built on the azaphenylalanine scaffold provoke degranulation of mast cells

Abstract

The main structural feature of direct thrombin inhibitor LK-732 responsible for the appropriate interaction at the thrombin active site is a strong basic group. A possibility that a strong basic group of LK-732 might contribute to the mast cell degranulation effect and consequent reduction of tracheal air flow (TAF) and fall of mean arterial blood pressure (MAP) in rats was investigated in the present study. At doses up to 5 mg/kg (i.v.), LK-732 did not cause significant changes of TAF and MAP. At 7 mg/kg (i.v.), a sudden reduction of TAF and a fall of MAP was observed within 5 min after LK-732 administration (75% mortality, p = 0.007). A less basic direct thrombin inhibitor LK-658 (21 mg/kg, i.v.) did not significantly disturb TAF and MAP. A reduction of TAF and a fall of MAP caused by LK-732 (7 mg/kg, i.v.) was almost completely abolished in rats with degranulated mast cells (0% mortality, p = 0.008). LK-732 concentration-dependently degranulated rat peritoneal mast cells in vitro (pEC(50) = 1.92 +/- 0.05 muM). A structure-activity relationship (SAR) study revealed that the terminal basic groups attached to the aromatic ring are responsible for the mast cell degranulation effect. A good correlation was observed between mast cell degranulation and pK(b) of analogues of LK-732 (R(2) = 0.49), but not between mast cell degranulation and thrombin K(i) (R(2) = 0.23). LK-732-induced reduction of TAF, the fall of MAP and high mortality originate from LK-732-induced mast cell degranulation. As judged by the SAR study, this effect could be overcome by reducing the basicity of LK-732.

Fragment‐Based Design, Synthesis, and Characterization of Aminoisoindole‐Derived Furin Inhibitors

“…Hence, we suspected that an as‐yet‐unknown off‐target is responsible for the toxicity of these inhibitors, rather than the furin inhibition. As other groups have reported toxic effects of benzamidine‐derived protease inhibitors through a G i ‐dependent histamine release associated with a drop in blood pressure, [30,31] we speculated, if the MRGPRX2 could be an off‐target for these furin inhibitors. Our results reveal that the toxic effects of the strongly multibasic inhibitor MI‐1148 in mice may be explained at least in part by activation of MRGPRX2.…”

“…This suggested a different, but so far unknown mechanism, underlying the toxicity of these compounds. Other groups have also reported toxic effects via a sharp drop of arterial blood pressure for various benzamidine‐derivatives in rats, which were developed as thrombin inhibitors [29,30] . A benzamidine‐induced mast cell degranulation leading to a strong histamine release was described as reason for the drop of blood pressure and reduced tracheal airflow causing the mortality [30] .…”

“…Other groups have also reported toxic effects via a sharp drop of arterial blood pressure for various benzamidine‐derivatives in rats, which were developed as thrombin inhibitors [29,30] . A benzamidine‐induced mast cell degranulation leading to a strong histamine release was described as reason for the drop of blood pressure and reduced tracheal airflow causing the mortality [30] . In subsequent work, one of the groups reported that benzamidines provoke a release of histamine from mast cells via an activation of G i proteins, because the release could be blocked by treatment with pertussis toxin (PTX), an inhibitor of the G i protein [31] .…”

“…[29,30] A benzamidine-induced mast cell degranulation leading to a strong histamine release was described as reason for the drop of blood pressure and reduced tracheal airflow causing the mortality. [30] In subsequent work, one of the groups reported that benzamidines provoke a release of histamine from mast cells via an activation of G i proteins, because the release could be blocked by treatment with pertussis toxin (PTX), an inhibitor of the G i protein. [31] Therefore, we were interested in assessing whether the PTXsensitive G i -coupled Mas-related GPCR-X2 (MRGPRX2) could be a potential off-target of our benzamidine-derived furin inhibitors.…”

Recent Research Developments in the Direct Inhibition of Coagulation Proteinases – Inhibitors of the Initiation Phase

“…Although ximelagatran was approved for clinical use in Europe, the US FDA rejected its application in 2004 due to concerns of hepatotoxicity [67]. Recent studies suggest that direct inhibitors that use a basic group as a P1-arginine mimic Fondaparinux for recognizing the enzyme appear to have another side effect -the degranulation of mast cells leading to histamine release [68,69]. Finally, trials with ximelagatran, and other direct inhibitors, e.g., dabigatran, DX9065a and razaxaban, show the persistence of bleeding risk [56,57,[60][61][62][63][64].…”