Exploring EZH2-Proteasome Dual-Targeting Drug Discovery through a Computational Strategy to Fight Multiple Myeloma

Multiple myeloma is an incurable plasma cell neoplastic disease representing about 10–15% of all haematological malignancies diagnosed in developed countries. Proteasome is a key player in multiple myeloma and proteasome inhibitors are the current first-line of treatment. However, these are associated with limited clinical efficacy due to acquired resistance. One of the solutions to overcome this problem is a polypharmacology approach, namely combination therapy and multitargeting drugs. Several polypharmacology avenues are currently being explored. The simultaneous inhibition of EZH2 and Proteasome 20S remains to be investigated, despite the encouraging evidence of therapeutic synergy between the two. Therefore, we sought to bridge this gap by proposing a holistic in silico strategy to find new dual-target inhibitors. First, we assessed the characteristics of both pockets and compared the chemical space of EZH2 and Proteasome 20S inhibitors, to establish the feasibility of dual targeting. This was followed by molecular docking calculations performed on EZH2 and Proteasome 20S inhibitors from ChEMBL 25, from which we derived a predictive model to propose new EZH2 inhibitors among Proteasome 20S compounds, and vice versa, which yielded two dual-inhibitor hits. Complementarily, we built a machine learning QSAR model for each target but realised their application to our data is very limited as each dataset occupies a different region of chemical space. We finally proceeded with molecular dynamics simulations of the two docking hits against the two targets. Overall, we concluded that one of the hit compounds is particularly promising as a dual-inhibitor candidate exhibiting extensive hydrogen bonding with both targets. Furthermore, this work serves as a framework for how to rationally approach a dual-targeting drug discovery project, from the selection of the targets to the prediction of new hit compounds.

The YΦ Motif Defines the Structure-Activity Relationships of Human 20S Proteasome Activators

The 20S proteasome (20S) facilitates turnover of most eukaryotic proteins. Substrate entry into the 20S first requires opening of gating loops through binding of HbYX motifs that are present at the C-termini of certain proteasome activators (PAs). The HbYX motif has been predominantly characterized in the archaeal 20S, whereas little is known about the sequence preferences of the human 20S (h20S). Here, we synthesized and screened ∼120 HbYX-like peptides, revealing unexpected differences from the archaeal system and defining the h20S recognition sequence as the Y-F/Y (YΦ) motif. To gain further insight, we created a functional chimera of the optimized sequence, NLSYYT, fused to the model activator, PA26E102A.A cryo-EM structure of PA26E102A-h20S identified key interactions, including non-canonical contacts and gate-opening mechanisms. Finally, we demonstrated that the YΦ sequence preferences are tuned by valency, allowing multivalent PAs to sample greater sequence space. These results expand the model for termini-mediated gating and provide a template for the design of h20S activators.

A Peptidomimetic Fluorescent Probe to Detect the Trypsin β2 Subunit of the Human 20S Proteasome

This work describes the chemical synthesis, combinatorial selection, and enzymatic evaluation of peptidomimetic fluorescent substrates specific for the trypsin-like (β2) subunit of the 20S human proteasome. After deconvolution of a library comprising nearly 6000 compounds composed of peg substituted diaminopropionic acid DAPEG building blocks, the sequence ABZ–Dap(O2(Cbz))–Dap(GO1)–Dap(O2(Cbz))–Arg–ANB–NH2, where ABZ is 2-aminobenzoic acid, and ANB- 5 amino 2- nitro benzoic acid was selected. Its cleavage followed sigmoidal kinetics, characteristic for allosteric enzymes, with Km = 3.22 ± 0.02 μM, kcat = 245 s−1, and kcat/Km = 7.61 × 107 M−1 s−1. This process was practically halted when a selective inhibitor of the β2 subunit of the 20S human proteasome was supplemented to the reaction system. Titration of the substrate resulting in decreased amounts of proteasome 20S produced a linear signal up to 10−11 M. Using this substrate, we detected human proteasome 20S in human urine samples taken from the bladders of cancer patients. This observation could be useful for the noninvasive diagnosis of this severe disease.