Structural binding evidence of the trypanocidal drugs Berenil® and Pentacarinate® active principles to a serine protease model

Abstract

Bovine trypsin is a model system for the serine protease class of enzymes, which is an important target for contemporary medicinal chemistry. Some structural and thermodynamic reports are available on its interaction with benzamidine-based compounds but no structural information is available so far on its binding modes to the active principles of the trypanocidal drugs Pentacarinate (pentamidine) and Berenil (diminazene). The crystallographic structures of bovine beta-trypsin in complex with the ligands were determined to a resolution of 1.57 A (diminazene) and 1.70 A (diminazene and pentamidine). The second benzamidine moieties in these inhibitors are bound to the enzyme in different hot spots and only few hydrogen bonds mediate these interactions. Thermodynamic parameters for the association of pentamidine with beta-trypsin reveal that this inhibitor has about 1.3-fold lower affinity than diminazene. Moreover its binding mode resembles other benzamidine-based compounds that assess the aryl binding pocket of the enzyme; however, with almost 2.5-fold higher affinity. This is the first structural evidence of the binding of Berenil and Pentacarinate active principles trypanocidal drugs to serine proteases.

Small molecule inhibitors of mesotrypsin from a structure-based docking screen

“…Although our virtual screen had not employed any prior knowledge about trypsin inhibitory compounds, a literature search of these compounds revealed that diminazene and pentamidine, another bis-benzamidine analog, are both trypanocidal drugs with known inhibitory activity towards trypanosome serine oligopeptidase [30], while also displaying activity against bovine trypsin [30, 31]. Upon obtaining pentamidine, we assayed the three bis-benzamidine compounds against mesotrypsin over multiple substrate and compound concentrations to determine the inhibition mode: competitive, non-competitive, or mixed inhibition.…”

“…We anticipate the conformational disorder observed in diminazene could arise in part from cis-to-trans isomerization of the triazene linker [32, 33]. Diminazene has been previously observed as two distinct conformational isomers bound to bovine trypsin in PDB IDs: 3GY2, 3GY6 (Fig 6B) [31]. …”

Ultrafast Dynamics of a Triazene: Excited-State Pathways and the Impact of Binding to the Minor Groove of DNA and Further Biomolecular Systems

“…8 Nevertheless, the two fitted lifetimes are only slightly increased to 0.28 and 1.41 ps. The amplitude of the latter again increases with wavelength (83:17 at 480 nm, 76:24 at 540 nm, inset of Figure 2).…”

“…1−6 However, little is known about the underlying mechanism and how the properties of berenil change upon interaction with biomolecules. Berenil shows a high binding affinity to enzymes like β-trypsin 7,8 or diamine oxidase, 9 it is an efficient minor-groove binder of AT-rich DNA, 1,10−20 and it also strongly binds to Gquadruplex DNA. 21,22 A characteristic blue fluorescence emitted by berenil after UV excitation is observable when it is added to cell cultures that are in the stationary or lag phase of a growth cycle, yet it is absent when berenil is added to cells in the log phase.…”

“…7,8,13,21 The binding between the particular system and berenil is reflected in characteristic shifts 10 in the absorption spectra ( Figure S2 in the Supporting Information). Time-resolved fluorescence spectroscopy (see Supporting Information for experimental details) provides insight into the decay behavior, as shown in Figure 2 for berenil in buffer solution and bound to the different biomolecules as well as in neat water and ethylene glycol, respectively.…”