A new class of competitive inhibitors for the cysteine
protease papain is described. These inhibitors are
based upon a 4-heterocyclohexanone ring and are designed to react with
the enzyme active site nucleophile to give
a reversibly formed hemithioketal. The electrophilicity of the
ketone in these inhibitors is enhanced by ring strain
and by through-space electrostatic repulsion with the heteroatom at the
1-position of the ring. Equilibrium constants
for addition of water and 3-mercaptopropionic acid to several
4-heterocyclohexanones were measured by 1H NMR
spectroscopy. These reactions model addition of the active site
nucleophile to the corresponding inhibitors. The
equilibrium constants give a linear correlation with the field
substituent constant F for the functional group at
the
1-position of the heterocyclohexanone. These equilibrium constants
also correlate well with the inhibition constants
for the 4-heterocyclohexanone-based inhibitors, which range from 11 to
120 μM. Thus, the model system can be
used to predict the potency of structurally related enzyme
inhibitors.
Three inhibitors that are based upon a 4-heterocyclohexanone nucleus were synthesized and evaluated for activity against the serine protease plasmin. Inhibitors of plasmin have potential as cancer chemotherapeutic agents that act by blocking both angiogenesis and metastasis. Inhibitor 1 has moderate activity against plasmin but shows good selectivity for this enzyme compared to other serine proteases including trypsin, thrombin, and kallikrein. Inhibitor 2 shows both good activity and selectivity for plasmin. Inhibitor 3, which does not incorporate an aminohexyl group that can interact with the S1 subsite, has poor activity. These results, along with previous work, demonstrate that the 4-heterocyclohexanone nucleus can effectively serve as the basis for designing inhibitors of both serine and cysteine proteases.
␣-Bungarotoxin, the classic nicotinic antagonist, has high specificity for muscle type ␣1 subunits in nicotinic acetylcholine receptors. In this study, we show that an 11-amino-acid pharmatope sequence, containing residues important for ␣-bungarotoxin binding to ␣1, confers functional ␣-bungarotoxin sensitivity when strategically placed into a neuronal non-␣ subunit, normally insensitive to this toxin. Remarkably, the mechanism of toxin inhibition is allosteric, not competitive as with neuromuscular nicotinic receptors. Our findings argue that ␣-bungarotoxin binding to the pharmatope, inserted at a subunit-subunit interface diametrically distinct from the agonist binding site, interferes with subunit interface movements critical for receptor activation. Our results, taken together with the structural similarities between nicotinic and GABA A receptors, suggest that this allosteric mechanism is conserved in the Cys-loop ion channel family. Furthermore, as a general strategy, the engineering of allosteric inhibitory sites through pharmatope tagging offers a powerful new tool for the study of membrane proteins.
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