Structure-based design and synthesis of bimodal proteasome inhibitors as therapeutic agents

The proteasome is a multicatalytic enzyme responsible for degrading damaged, misfolded, and uneeded intracellular proteins. Since many vital cellular pathways rely on the degradative function of the proteasome, its dysregulation is implicated in the pathology of several diseases, thus making it an attractive target for inhibition. Here we report two novel tetrapeptide aldehyde inhibitors based on the structure of natural product TMC‐95A, a potent noncovalent inhibitor of the proteasome. Conversion of the carbonyl of the C-terminal residue to an aldehyde enabled these inhibitors to covalently bind to the catalytic Thr residue of the proteasome’s active site. The P1 and P3 residues, which engage with the primary active site pockets, were varied to mimic TMC‐95A and MG‐132, a well known peptide aldehyde proteasome inhibitor. The combination of covalent and noncovalent mechanisms of binding to the active site allow these compounds to bimodally inhibit the proteasome. Additionally, the P2 and P4 residues of two of the inhibitors were cyclized via a biaryl ether moiety in order to mimic the synthetically inaccessible macrocycle found in TMC‐95A. The compounds were then tested in vitro with 20S proteasome using fluorometric reversible kinetics. The two macrocyclic lead compounds were also compared to linear peptide aldehyde analogs that were prepared and tested in the same fashion to the macrocycles. The resulting inhibition constants were used to evaluate the effect of the macrocycle, the addition of the aldehyde functionality, and to compare the different amino acid sequences. The aldehyde was found to greatly increase potency compared to noncovalent inhibitors with a similar structure. The compounds with amino acid sequences similar to MG‐132 were the most potent (Ki̳ = 33 nM, 76 nm) while the cyclic compounds were found to be slightly less potent than their linear analogues. These compounds were also tested with chymotrypsin, calpain, and cruzain to investigate their inhibitory specificity. The kinetic data demonstrated that the general structure of the inhibitors showed much greater affinity for the proteasome than for the other proteases tested.