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Understanding the correlation between structural features of small-molecule drugs and their mode of action is a fascinating topic and crucial for the drug-discovery process. However, in many cases, knowledge of the exact parameters that dictate the mode of action is still lacking. Following a large screening for ubiquitin specific protease 2 (USP2) inhibition, an effective para-quinone-based inhibitor with an unclear mode of action was identified. To gain a deeper understanding of the mechanism of inhibition, a set of para-quinones were prepared and studied for USP2 inhibition, electrocatalysis, and reactive oxygen species (ROS) quantification. The excellent correlation obtained from the above-mentioned studies disclosed a distinct pattern of "N-C=O-N" in the bicyclic para-quinones to be a crucial factor for ROS generation, and demonstrated that minor changes in such a skeleton drastically altered the ROS-generating ability. The knowledge acquired herein would serve as an important guideline for future medicinal chemistry optimization of related structures to select the preferred mode of action.

Deubiquitinases (DUBs) counteract ubiquitination by removing or trimming ubiquitin chains to alter the signal. Their diverse role in biological processes and involvement in diseases have recently attracted great interest with regard to their mechanism and inhibition. It has been shown that some DUBs are regulated by reactive oxygen species (ROS) in which the catalytic Cys residue undergoes reversible oxidation, hence modulating DUBs activity under oxidative stress. Reported herein for the first time, the observation that small molecules, which are capable of generating ROS efficiently, inhibit DUBs by selective and nonreversible oxidation of the catalytic Cys residue. Interestingly, the small molecule beta-lapachone, which is currently in clinical trials for cancer, is among the potent inhibitors, thus suggesting possible new cellular targets for its therapeutic effects. Our study describes a novel mechanism of DUBs inhibition and opens new opportunities in exploiting them for cancer therapy.

Ubiquitination is a key posttranslational modification, which affects numerous biological processes and is reversed by a class of enzymes known as deubiquitinases (DUBs). This family of enzymes cleaves mono-ubiquitin or poly-ubiquitin chains from a target protein through different mechanisms and mode of interactions with their substrates. Studying the role of DUBs in health and diseases has been a major goal for many laboratories both in academia and in industry. However, the field has been challenged by the difficulties in obtaining native substrates and novel reagents using traditional enzymatic and molecular biology approaches. Recent advancements in the synthesis and semisynthesis of proteins made it possible to prepare several unique ubiquitin conjugates to study various aspects of DUBs such as their specificities and structures. Moreover, these approaches enable the preparation of novel activity based probes and assays to monitor DUB activities in vitro and in cellular contexts. Efforts made to bring new chemical entities for the selective inhibition of DUBs based on these tools are also highlighted with selected examples.

A multidisciplinary approach, composed of organic synthesis, electrochemistry, electrocatalysis and cellular studies, for correlating the molecular features of a 1,2-naphthoquinone scaffold with its ROS generating ability., Recent studies have suggested that selective targeting of overexpressed enzymes in cancer cells by small molecules that induce the formation of reactive oxygen species (ROS) could be a viable approach in cancer therapy. One such example is the inactivation of ubiquitin specific protease-2 (USP2)—an emerging drug target to combat prostate cancer—by β-lapachone, which has been identified to involve oxidation of the catalytic cysteine's thiol residue to sulfinic acid. A rational design of β-lapachone analogs with improved activity requires a much better understanding of the variables that determine ROS production by this class of molecules. This crucial aspect was addressed via modulation of its 1,2-naphthoquinone scaffold and establishment of a structure/activity relationship, regarding its ability to reduce molecular oxygen to a ROS. The same series of compounds was also examined in terms of their inhibitory effect on the enzymatic activity of USP2. One deduction from these investigations was that the ortho-quinone motif in β-lapachone is much better suited for the catalytic reduction of oxygen than the para-quinone motif and some approved quinone based drugs. A broader conclusion, obtained from the series of compounds with ortho-quinone motifs, is that only the agents whose redox potential is in the narrow range of –0.3 ± 0.1 V (vs. Ag/AgCl in pH 7.5 aqueous buffer) induce the formation of ROS. The excellent correlation between the ROS production ability and the USP2 inhibition potency emphasizes that the relatively easy, fast, and reliable testing of electrocatalytic oxygen reduction by small molecules might be applied to screening and evaluating new drug candidates for similar targets.

Ubiquitination/ubiquitylation is involved in a wide range of cellular processes in eukaryotes, such as protein degradation and DNA repair. Ubiquitination is a reversible post-translational modification, with the removal of the ubiquitin (Ub) protein being catalyzed by a family of enzymes known as deubiquitinases (DUBs). Approximately 100 DUBs are encoded in the human genome and are involved in a variety of regulatory processes, such as cell-cycle progression, tissue development, and differentiation. DUBs were, moreover, found to be associated with several diseases and as such are emerging as potential therapeutic targets. Several directions have been pursued in the search for lead anti-DUB compounds. However, none of these strategies have delivered inhibitors reaching advanced clinical stages due to several challenges in the discovery process, such as the absence of a highly sensitive and practically available high-throughput screening assay. In this study, we report on the design and preparation of a FRET-based assay for DUBs based on the application of our recent chemical method for the synthesis of Ub bioconjugates. In the assay, the ubiquitinated peptide was specifically labeled with a pair of FRET labels and used to screen a library comprising 1000 compounds against UCH-L3. Such analysis identified a novel and potent inhibitor able to inhibit this DUB in time-dependent manner with k(inact) = 0.065 min(-1) and K(i) = 0.8 μM. Our assay, which was also found suitable for the UCH-L1 enzyme, should assist in the ongoing efforts targeting the various components of the ubiquitin system and studying the role of DUBs in health and disease.

Studying and targeting deubiquitinases is of high importance due to their various roles in cellular processes and involvement in diseases such as cancer. The recent development of unique probes and reagents using chemical synthesis of proteins became very instrumental in supporting these efforts. Here, we present a protein synthetic approach that enables the rapid synthesis of differently modified labeled-ubiquitinated peptides to facilitate rapid optimization of deubiquitinase substrates.

Chemical synthesis of a peptide-ubiquitin conjugate linked by an N-hydroxy isopeptide bond to determine what effect the N-hydroxy group has on the enzymatic hydrolysis of the isopeptide linkage by deubiquitinases is reported. This conjugate was subjected to proteolysis by UCH-L3 in the presence and absence of various metal ions, and no substantive difference in hydrolysis was seen compared to a control lacking the N-hydroxy group. The accessibility of N-hydroxy ubiquitinated substrates may find uses to study other deubiquitinases in particular those which use a zinc ion as a part of their catalytic mechanism.