TMC-95A

Noncovalent proteasome inhibitors introduce an alternative mechanism of inhibition to that of covalent inhibitors used in cancer therapy. Starting from a noncovalent linear mimic of TMC-95A, a series of dimerized inhibitors using polyaminohexanoic acid spacers has been designed and optimized to target simultaneously two of the six active sites of the eukaryotic 20S proteasome. The homodimerized compounds actively inhibited chymotrypsin-like (Ki = 6-11 nM) and trypsin-like activities, whereas postacid activity was poorly modified. The noncovalent binding mode was ascertained by X-ray crystallography of the inhibitors complexed with the yeast 20S proteasome. The inhibition of proteasomal activities in human cells was evaluated. The use of the multivalency inhibitor concept has produced highly efficient and selective noncovalent compounds (no inhibition of calpain and cathepsin) that have potential therapeutic advantages compared to covalent binders such as bortezomib and carfilzomib.

We have designed and evaluated 45 linear analogues of the natural constrained cyclopeptide TMC-95A. These synthetically less demanding molecules are based on the tripeptide sequence Y-N-W of TMC-95A. Structural variations in the amino acid side chains and termini greatly influenced both the efficiency and selectivity of action on a given type of active site. Inhibition constants were submicromolar (Ki approximately 300 nM) despite the absence of the entropically favorable constrained conformation that is characteristic of TMC-95A and its cyclic analogues. These linear compounds were readily prepared and reasonably stable in culture medium and could be optimized to inhibit one, two, or all three proteasome catalytic sites. Cytotoxicity assays performed on a series of human tumor cell lines identified the most potent inhibitors in cells.

The complex thermodynamics that govern noncovalent protein-ligand interactions are still not fully understood, despite the exponential increase in experimental structural data available from X-ray crystallography and NMR spectroscopy. The eukaryotic 20S proteasome offers an ideal system for such studies as it contains in duplicate three proteolytically active sites with different substrate specificities. The natural product TMC-95A inhibits these proteolytic centers noncovalently with distinct affinities. X-ray crystallographic analysis of the complexes of the yeast proteasome core particle with this natural inhibitor and two synthetic analogues clearly revealed highly homologous hydrogen-bonding networks involving mainly the peptide backbone despite the strongly differentiated binding affinities to the three active sites of the 20S proteasome. The natural product and the two analogues are constrained in a rigid beta-type extended conformation by the endocyclic biaryl clamp, which preorganizes the peptide backbone for optimal adaptation of the ligands to the active site clefts and thus favors the binding processes entropically. However, the biaryl clamp also dictates the orientation of the P1 and P3 residues and their mode of interaction with the protein binding subsites. This limitation is optimally solved in TMC-95A with the conformationally restricted (Z)-prop-1-enyl group acting as P1 residue, at least for the chymotrypsin-like active site; however, it critically affects the inhibitory potencies of the analogues, thus suggesting the use of less-rigid endocyclic clamps in the design of proteasome inhibitors that allow for a better presentation of residues interacting with the active site clefts of the enzyme.