Proteasome plays a fundamental role in the protein turnover by degrading misfolded, abnormal or damaged proteins, previously labeled through addition of a polyubiquitin chain. An aberration of this proteolytic system could result in different types of hematological malignancies. An overexpression of protesome has been detected in multiple myeloma (MM) cells, and, in this context, selectively targeting the chymotrypsin-like (ChT-L) activity of 20S proteasome has emerged as a promising approach for anticancer therapy, in particular for the treatment of MM. To date, three proteasome inhibitors have been approved by the FDA for the treatment of MM: the peptide boronates bortezomib and ixazomib, and the peptide epoxyketone carfilzomib. Despite the wide use of these proteasome inhibitors in the clinic, their toxicity and off-targets interactions remain a great limitation for their use. On the contrary, noncovalent inhibitors, devoid of a reactive warhead, may lack of several drawbacks that are related to covalent inhibition.

Fig. 1. Flow chart of the multistep hierarchical SBVS strategy implemented in this work.

In an effort to identify novel noncovalent inhibitors of the mammalian 20S proteasome, we screened the NCI (National Cancer Institute) lead-like library (~65,375 compounds) using a multistep hierarchical structure-based virtual screening (SBVS) approach (Fig. 1). Using this strategy, we discovered two compounds (1and2, Fig. 2) that noncovalently inhibit the ChT-L activity of the mammalian 20S proteasome withK_ivalues of 2.18 µM and 2.12 µM respectively. Since a co-inhibition of another proteasome proteolytic activity is strongly required to enhance an antitumor response, we identified compound1as promising lead compound, because of its ability to co-inhibit the caspase-like (C-L) activity withK_i=8.59 µM, differently to compound2which inhibited the sole ChT-L activity.

Fig. 2. Molecular structures of the new identified 20S proteasome inhibitors1-6.

A subsequent hierarchical similarity search over the full NCI database with the most active tripeptide-based inhibitor1led to the identification of an additional set of 12 ligands, four of which (3-6,Fig. 2) exhibitedK_ivalues in the low micromolar range for the ChT-L activity. Compound6was the most active inhibitor, with a binding affinity towards the ChT-L activity in the submicromolar range (K_i=0.42 µM). We solved the structure in solution of the most active inhibitor6by NMR spectroscopy and elucidated its binding mode into the ChT-L, C-L and T-L active sites of the yeast 20S proteasome by applying docking experiments. Substrate selectivity of compound6for the ChT-L subunit seems to be dictated by the interaction of the inhibitor’s P1 side chain with the active site’s S1 specificity pocket. On the contrary, the S1 pocket in C-L site is positively charged by the guanidinium group of R45 and therefore does not favor the hydrophobic methyl ester group of6. In T-L subunit, G45 results in a more spacious unstructured S1 pocket in comparison with C-L and ChT-L subunits, allowing for motion and flexibility of the P1 side chains of the bound ligand. The cytotoxic effects of the active compounds were also evaluated on dexamethasone resistant (MM.1R) human multiple myeloma cells, which are less responsive to chemotherapy, and are representative of patients in the later stages of the disease. The obtained IC₅₀values ranged from 16.2 µM to 29.8 µM, with the most active compound being compound6(IC₅₀=16.2 µM). A western blot analysis of ubiquitin detected changes in total ubiquitin levels due to an increase of ubiquitinated protein levels that accounts for proteasome inhibition. Also in this case, compound6was proven to be the most effective at 20 µM, thus being worthy to be considered a promising lead compound for the development of new effective drugs for the treatment of MM.

Carmen Di Giovanni¹,Roberta Ettari²,Serena Sarno¹,Archimede Rotondo³,Alessandra Bitto⁴,Francesco Squadrito⁴,Domenica Altavilla³,Tanja Schirmeister⁵,Ettore Novellino¹,Silvana Grasso²,Maria Zappalà²,Antonio Lavecchia^1
1Department of Pharmacy, “Drug Discovery” Laboratory, University of Naples Federico II, Naples, Italy
²Department of Chemical, Biological, Pharmaceutical and Environmental Sciences,
University of Messina, Viale Annunziata, Messina, Italy
³Department of Biomedical Sciences, Dentistry, and Morpho-functional Sciences,
University of Messina, Via C. Valeria, Messina, Italy
⁴Department of Clinical and Experimental Medicine, University of Messina, Via C. Valeria, Messina, Italy
⁵Institute of Pharmacy and Biochemistry, University of Mainz, Mainz, Germany

Publication

Identification of noncovalent proteasome inhibitors with high selectivity for chymotrypsin-like activity by a multistep structure-based virtual screening.
Di Giovanni C, Ettari R, Sarno S, Rotondo A, Bitto A, Squadrito F, Altavilla D, Schirmeister T, Novellino E, Grasso S, Zappalà M, Lavecchia A
Eur J Med Chem. 2016 Oct 4

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