Current Pharmaceutical Design - Volume 19, Issue 18, 2013

The ubiquitin-proteosome system (UPS) regulates a wide range of cellular processes including protein degradation, DNA repair, transcription regulation, and cell signaling. Alterations and mutations in UPS components give rise to various human diseases, most prominently cancer and neurodegenerative disorders. Here, we review recent advances in UPS-based drug discovery highlighting the emerging relationships between the UPS and disease and discuss potential future therapeutic interventions. In particular, we focus on recent structural approaches in UPS and explain how the knowledge of protein structural details can guide the design of specifically targeted inhibitors.

The proteasomal pathway of protein degradation involves two discrete steps: ubiquitination and degradation. Blocking protein degradation by inhibiting the proteasome has well described biologic effects and proteasome inhibitors are approved for the treatment of multiple myeloma and mantle cell lymphoma. In contrast, the biological effects and potential therapeutic utility of inhibiting the ubiquitination cascade and the initiating enzyme UBA1 are less well understood. UBA1 is the initial enzyme in the ubiquitination cascade and initiates the transfer of ubiquitin molecules to target proteins where they are degraded by the proteasome. Here, we review the biological effects of UBA1 inhibition and discuss UBA1 inhibitors as potential anti-cancer agents. Similar to proteasome inhibition, blocking UBA1 elicits an unfolded protein response and induces cell death in malignant cells over normal cells. Chemical UBA1 inhibitors have been developed that target different regions of the enzyme and inhibit its function through different mechanisms. These molecules are useful tools to understand the biology of UBA1 and highlight the potential of inhibiting this target for the treatment of malignancy.

The Anaphase Promoting Complex (APC) has been characterized to play pivotal roles in regulating the timely cell cycle progression by forming two functionally distinct E3 ubiquitin ligase sub-complexes, APCCdc20 and APCCdh1. Interestingly, recent studies have shown that Cdh1 is functioning as a tumor suppressor whereas Cdc20 may function as an oncoprotein to promote the development and progression of human cancers. In this review, we will discuss the physiological role of Cdc20 and its downstream substrates in vitro and in the transgenic mouse model reminiscent of the pathogenesis of human cancers. Furthermore, we summarize recent findings to indicate that Cdc20 may represent a promising therapeutic target, thus development of Cdc20 inhibitors could be useful for achieving better treatment outcome of cancer patients.

The ubiquitin-proteasome system (UPS) promotes the timely degradation of short-lived proteins with key regulatory roles in a vast array of biological processes, such as cell cycle progression, oncogenesis and genome integrity. Thus, abnormal regulation of UPS disrupts the protein homeostasis and causes many human diseases, particularly cancer. Indeed, the FDA approval of bortezomib, the first class of general proteasome inhibitor, for the treatment of multiple myeloma, demonstrated that the UPS can be an attractive anti-cancer target. However, normal cell toxicity associated with bortezomib, resulting from global inhibition of protein degradation, promotes the focus of drug discovery efforts on targeting enzymes upstream of the proteasome for better specificity. E3 ubiquitin ligases, particularly those known to be activated in human cancer, become an attractive choice. Cullin-RING Ligases (CRLs) with multiple components are the largest family of E3 ubiquitin ligases and are responsible for ubiquitination of ∼20% of cellular proteins degraded through UPS. Activity of CRLs is dynamically regulated and requires the RING component and cullin neddylation. In this review, we will introduce the UPS and CRL E3s and discuss the biological processes regulated by each of eight CRLs through substrate degradation. We will further discuss how cullin neddylation controls CRL activity, and how CRLs are being validated as the attractive cancer targets by abrogating the RING component through genetic means and by inhibiting cullin neddylation via MLN4924, a small molecule indirect inhibitor of CRLs, currently in several Phase I clinical trials. Finally, we will discuss current efforts and future perspectives on the development of additional inhibitors of CRLs by targeting E2 and/or E3 of cullin neddylation and CRL-mediated ubiquitination as potential anti-cancer agents.

Smad ubiquitylation regulatory factor-1 (Smurf1) is a HECT-type ubiquitin ligase. The role of Smurf1 in cell development and migration, viral autophagy and immune responses has been the subject of intensive study in recent years. Smurf1 regulates multiple biological networks, including TGF-β and BMP signaling pathways, the non-canonical pathway and the Toll-like receptor pathway, and is linked to certain diseases and disorders, such as bone formation and embryonic development disorders. Increasing evidence suggests that Smurf1 could be a good candidate for further translational studies and a potential target for novel drug design. In this review, we summarize the physiological functions of Smurf1 and its associated disorders; and discuss the current state of drug discovery in the context of the ubiquitin-proteasomal system, and feasible pharmaceutical strategies toward Smurf1 and its regulators, as well as RNA interference and structure-based chemical drug selection.

Deubiquitinating enzymes (DUBs) remove ubiquitin and ubiquitin-like modifications from proteins and they have been known to contribute to processes relevant in microbial infection, such as immune responses pathways. Numerous viral and bacterial DUBs have been identified, and activities of several host DUBs are known to be modulated during the infection process, either by a pathogen or by a host. Recently there have been attempts to take advantage of this feature and design therapeutic inhibitors of DUBs that can be used to limit the spread of infection. This review is focused on exploring the potential of DUBs in the treatment of infectious diseases.

Within the past decade, there has been a revolution in the types of drugs developed to treat cancer. Therapies that selectively target cancer-specific aberrations, such as kinase inhibitors, have made a dramatic impact on a subset of patients. In spite of these successes, there is still a dearth of treatment options for the vast majority of patients. Therefore, there is a need to design therapies with broader efficacy. The p53 tumor suppressor pathway is one of the most frequently altered in human cancers. However, about half of all cancers retain wild-type p53, yet through various mechanisms, the p53 pathway is otherwise inactivated. Targeting this pathway for reactivation truly represents the " holy grail" in cancer treatment. Most commonly, destabilization of p53 by various components of ubiquitin- proteasome system, notably the ubiquitin ligase MDM2 and its partner MDMX as well as various deubiquitinating enzymes (DUBs), render p53 inert and unresponsive to stress signals. Reinstating its function in cancer has been a long sought-after goal. Towards this end, a great deal of work has been devoted to the development of compounds that either interfere with the p53-MDM2 and p53- MDMX interactions, inhibit MDM2 E3 activity, or target individual DUBs. Here we review the current progress that has been made in the field, with a special emphasis on both MDM2 and DUB inhibitors. Developing inhibitors targeting the upstream of the p53 ubiquitination pathway will likely also be a valuable option.

During the last two decades, the studies on ubiquitination in regulating transcription factor NF-κB activation have elucidated the expanding role of ubiquitination in modulating cellular events by non-proteolytic mechanisms, as well as by proteasomal degradation. The significance of ubiquitination has also been recognized in regulating gene transcription, epigenetic modifications, kinase activation, DNA repair and subcellular translocation. This progress has been translated into novel strategies for developing anti-cancer therapeutics, exemplified by the success of the first FDA-approved proteasome inhibitor drug Bortezomib. Here we discuss the current understanding of the ubiquitin-proteasome system and how it is involved in regulating NF-κB signaling pathways in response to a variety of stimuli. We also focus on the recent progress of anti-cancer drug development targeting various steps of ubiquitination process, and the potential of these drugs in cancer treatment as related to their impact on NF-κB activation.

Muscle wasting is a prevalent and disabling condition in chronic disease and cancer and has been associated with increased mortality and impaired efficacy of surgical and medical interventions. Pharmacological therapies to combat muscle wasting are currently limited but considered as an important unmet medical need. Muscle wasting has been attributed to increased muscle proteolysis, and in particular ubiquitin 26S-proteasome system (UPS)-dependent protein breakdown. However, rates of muscle protein synthesis are also subject to extensive (patho) physiological regulation, and the balance between synthesis and degradation ultimately determines net muscle protein turnover. As multinucleated muscle fibers accommodate threshold changes in muscle protein content by the accretion and loss of muscle nuclei, myonuclear turnover may additionally determine muscle mass. Current insights in the mechanisms dictating muscle mass plasticity not only reveal intricate interactions and crosstalk between these processes, but imply the existence of signaling molecules that act as molecular switchboards, which coordinate and integrate cellular responses upon conditions that evoke changes in muscle mass. These “master regulators” of skeletal muscle mass plasticity are preferred targets for pharmacological modulation of skeletal muscle wasting. In this review Glycogen synthase kinase-3β (GSK-3β) is highlighted as a master regulator of muscle mass plasticity since, in addition to its role in UPS-mediated muscle protein degradation, it also controls protein synthesis, and influences myonuclear accretion and cell death. Moreover, the regulation of GSK-3β activity as well as currently available pharmacological inhibitors are described and discussed in the context of multimodal treatment strategies aimed at the inhibition of GSK-3β, and optimal exploitation of its potential role as a central regulator of skeletal muscle mass plasticity for the treatment of muscle wasting.

The proteasome inhibition has been proved to be effective in the treatment of multiple myeloma and other malignancies. In addition to direct antitumor effects, proteasome inhibition also exerts strong effects on immune cells, such as T cells, B cells and DCs. Therefore, proteasome inhibition, through the utilization of small molecule drugs like bortezomib, could be used therapeutically to modulate immune responses in transplantation. In the current review, we discuss the emerging data, both preclinical and clinical, of using proteasome inhibition in treating complications of transplantation, such as antibody-mediated organ rejection (AMR) and graft-versus-host disease (GVHD). The therapy based on proteasome inhibition may present substantial opportunities as new therapeutic paradigms in transplantation.

Neurodegenerative diseases are severe disorders characterized by progressive neurodegeneration in specific brain regions. The ubiquitin-proteasome system (UPS) is closely linked to neurodegenerative disease. In most cases, UPS impairment and dysregulation of the UPS components are frequently observed. Moreover, toxin-induced neurodegeneration produces neuronal cell death accompanied by decreased UPS function. These studies suggest an involvement of the UPS in these diseases. In this review, we summarize the changes to UPS components in neurodegenerative diseases and the association between the UPS and disease pathology. Dysfunction of the UPS results in the abnormal accumulation of proteins; thus, the UPS plays a critical role in disease pathogenesis. Drugs targeting specific components of the UPS may provide promising strategies for disease treatment.

Alterations of the ubiquitin proteasome system (UPS) contribute to the progression of many diseases, such as cancer, neurodegenerative diseases, immunological disorders, and inflammation. Pharmacologic inhibition of specific ubiquitin regulatory enzymes and ubiquitination events is an important challenge in drug discovery. Identifying the substrates of the various enzymes that participate in the UPS is needed to determine which enzymes are potential drug candidates. Additionally, identifying the ubiquitination events regulated by pharmacological drugs can potentially discover new applications. In this review we describe mass spectrometry-based proteomic approaches for the identification of ubiquitinated proteins and their modification sites on a proteome-wide scale, focusing on the ubiquitin remnant profiling, a newly developed ubiquitination profiling technique. We then discuss the application of this approach for the profiling of ubiquitination events regulated by cell signaling pathways and explore its future applications for drug discovery in the UPS.

Prostate cancer is a critical public health problem in USA and Europe. New non-invasive imaging methods are urgently needed, due to the low accuracy and specificity of current screen methods and the desire of localizing primary prostate cancer and bone metastasis. Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) are the non-invasive and sensitive imaging methods which have been widely used for diagnosing diseases in the clinic. Lack of suitable radiotracers is the major issue for nuclear imaging of prostate cancer, although radiolabeled bombesin (BN) peptides targeting the Gastrin-Releasing Peptide Receptor (GRPR) on tumor cells are widely investigated. In this review we discuss the recent trends in the development of GRPRtargeted radiopharmaceuticals based on BN analogs with regard to their potential for imaging and therapy of GRPR-expressing malignancies. Following a brief introduction of GRPR and bombesin peptides, we summarize the properties of prostate cancer specific radiolabeled bombesins. New bombesin tracers published in the last five years are reviewed and compared according to their novelties in biomolecules, radionuclides, labeling methods, bifunctional chelators and linkers. Hot topics such as multimerization, application of agonists and antagonists are highlighted in the review. Lastly, a few clinical trials of cancer nuclear imaging with radiolabeled bombesin have been discussed.

Ultrasound-based multimodal molecular imaging and functional ultrasound contrast agents (UCAs) have garnered great interest in biomedical imaging over the past few years. With the rapid development of molecular imaging technique, UCAs with singlefunction and single imaging modality can no longer satisfy increasingly growing medical diversification and hominization demand. The device of functional UCAs including multimodality and multifunction is of great value for disease diagnosis, treatment and research, and greatly accelerate the development of ultrasound-based multimodal molecular imaging. Functional UCAs can not only be applied for multimodality imaging to compensate for the disadvantages of each imaging modality whilst taking advantage of their individual strengths to acquire more biological information, but also can act as a vector carrying drugs or therapeutic genes to accomplish the combination of diagnosis and treatment, which is an existing study hotspot and has achieved initiatory success. In this paper, we reviewed the advances of ultrasound-based multimodal imaging, multimodal and multifunctional UCAs.