A selection of Proteasomal and De-ubiquitinase inhibitors

In 2014, the U.S. Food and Drug Administration (FDA) approved the proteasome inhibiting drug Velcade (Bortezomib) for the retreatment of adult patients with Multiple Myeloma. My colleague Philippe Fixe discussed the use of Bortezomib in his post Proteasome inhibitor approved by FDA for Myeloma retreatment. In this post, I’d like to give you an overview of the compounds which can be used to inhibit proteasome activity and furthermore the activity of De-ubiquitinases (DUB, Isopeptidases), enzymes which catalyze the de-ubiquitination of ubiquitinated proteins and which are seen as very promising drug targets especially in cancer drug development.

Therapeutical potential of proteasome and DUB inhibitors

Ubiquitination (as well as modifications with ubiquitin-like proteins such as SUMO) are well known as important means of regulating protein trafficking, cell division, apoptosis, and metabolism in cells. Turn-over of most proteins in cells is regulated by ubiquitin tagging, setting a mark for their transport to and subsequent degradation to the 26S proteasome.

Ubiquitination: The process of adding ubiquitin proteins (see Fig) to target proteins is carried out by a complex enzymatic path which is mastered by three types of players – E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme), and E3 (ubiquitin ligase).

Proteasom and DUB inhibitors - cartoon

Ubiquitin (de)conjugation pathway

De-Ubiquitination: The removal of Ubiquitin on the other hand is carried out by DUBs which can be grouped into five families, depending on sequence homology: 1) the ubiquitin-C terminal hydrolases (UCH); 2) the ubiquitin specific processing proteases (USPs); 3) Machado Joseph Disease Domain proteases (MJD); 4) Otubain Proteases (OTU); and 5) JAMM Domain Proteases (JAMM). Removal of ubiquitin or ubiquitin-like moieties can affect cellular physiology in a number of ways, and isopeptidases and E3 ligases, have been linked to numerous pathologies.

Proteasomal pathway: The 26S proteasome, a multi-protein complex, is the central protease in ubiquitin-mediated protein degradation. The complex is responsible for cell quality control by eliminating misfolded proteins from the cytosol and endoplasmic reticulum.

Both the enzymes of the ubiquitin pathway as well as the proteasome are emerging as pioneer drug targets for many therapeutic areas, because (mis)controlling degradation of key proteins that are involved in apoptosis, cell cycle and signal transduction is involved in a number of diseases including cancer, neurodegenerative disease, metabolic disease, and cardiovascular disease.

Compounds inhibiting the proteasomal pathway and (de)-ubiquitination enzymes

Proteasome inhibitors

  • MG-132 is a specific, potent, reversible, and cell-permeable proteasome inhibitor ((Ki= 4 nM). It reduces the degradation of ubiquitin-conjugated proteins in mammalian cells and permeable strains of yeast by the 26S complex without affecting its ATPase or isopeptidase activities. Most commonly used proteasome inhibitor.
  • Bortezomib: Potent, selective and reversible proteasome inhibitor (Ki=0.6 nM). Inhibits proliferation of a number of tumor cell lines (IC50=7 nM).
  • PS-341 (Pyz-Phe-boroLeu) the active ingredient in Bortezomib (Velcade). PS-341 is a reversible, cell permeable, potent, and selective proteasome inhibitor (Ki=0.6 nM)
  • Epoxomicin is a naturally-occuring selective inhibitor with anti-inflammatory activity. It was originally discovered in 1992.  This molecule is a potent, selective and cell permeable irreversible inhibitor of the 20S proteasome.
  • Lactacystin binds and inhibits specific three proteolytic activities of the proteasome. It was the first non-peptidic proteasome inhibitor discovered and is widely used as a research tool in biochemistry and cell biology.
  • Celastrol is a chemical compound isolated from the root extracts of Tripterygium wilfordii and Celastrus regelii. This inhibitor is a pentacyclic triterpenoid and belongs in the family of quinone methides. It exhibits antioxidant, anti-inflammatory, anticancer and insecticidal activities. Its effects in humans have not been studied clinically.
  • Aclacinomycin A – originally identified as an anti-tumor drug produced by Streptomyces galilaeus. This molecule is the first described non-peptidic inhibitor showing discrete specificity for the chymotrypsin-like activity of the 20S proteasome.
  • Carfilzomib is analog of epoxomicin, Carfilzomib is a selective proteasome inhibitor currently approved as a therapeutic for multiple myeloma.
  • MLN2238 inhibits the chymotrypsin-like proteolytic (β5) site of the 20S proteasome with IC50 and Ki of 3.4 nM and 0.93 nM, respectively.
  • MLN9708 is a selective inhibitor of chymotrypsin-like proteolytic (β5) site of the 20S proteasome with IC50 and Ki of 3.4 nM and 0.93 nM.
  • Oprozomib is a clinical-stage proteasome inhibitor that selectively inhibits the chymotrypsin-like activity of the 20S proteasome β5/LMP7 with IC50 of 36 nM/82 nM.

DUB inhibitors

  • WP-1130 is a novel DUB inhibitor directly inhibiting USP9x, USP5, USP14 and UCH37. This molecule induces rapid accumulation of polyubiquitinated proteins and triggers tumor cell apoptosis.
  • NSC-632839 is an inhibitor of ubiquitin isopeptidase activity that displays no effect on the proteolytic activity of the proteasome. This molecule induces apoptosis via a Bcl-2-dependent and apoptosome-independent pathway of caspase activation.
  • TCID is a potent and selective inhibitor of ubiquitin C-terminal hydrolase-L3 (UCH-L3). TCID can diminish glycine transporter GlyT2 ubiquitination in brain stem and spinal cord primary neurons
  • LDN-57444 inhibits ubiquitin C-terminal hydrolase (UCH-L1). It decreases proteasome activity and increases levels of ubiquitinated proteins. This inhibitor induces apoptosis. Treatment with LDN-57444 causes dramatic alterations in synaptic protein distribution and spine morphology in vivo.
  • PR-619 is a nonselective, reversible inhibitor of DUBs (5-20 μM) that is highly useful for preserving ubiquitinated proteins during cell lysis.
  • P5091 is a Selective inhibitor of USP7 (IC50=4.2 μM). Induces apoptosis in multiple myeloma cells.
  • IU1 is a cell-permeable, reversible and selective inhibitor of the deubiquitinating activity of human USP14 . It promotes cell survival during proteotoxic stress and selectively stimulates ubiquitin-dependent protein degradation in vitro.
  • 1,10-phenanthroline is a chelator with high affinity for divalent metal ions.  While not specific for DUBs, it has been shown to be a potent inhibitor of JAMM-type isopeptidases through chelation of the active site Zn2+ ion.
  • b-AP15 abrogates the deubiquitinating activity of the 19S regulatory particle by inhibiting UCHL5 and USP14. b-AP15 induces tumor cell apoptosis in four different solid tumor models.
  • LDN-57444 is a cell permeable ubiquitin C-terminal hydrolase (UCH-L1) inhibitor (Ki=0.4 µM) with approximately 28-fold greater selectivity over UCH-L3.
  • P22077 is a cell permeable inhibitor of the ubiquitin-specific protease USP7 (IC50=8.6μM), and the closely related deubiquitinase (DUB) USP47.
  • ML323 binds to the complex of USP1-UAF1 resulting in the inhibition of USP1 deubiquitylase activity. The IC50 is reported to be 76nM against Ub-Rho110 and 174nM against K63-linked diubiquitin.

Compounds inhibiting other Ubiquitination related enyzmes

E3 Ubiquitin ligase inhibitors

  • SMER3 is a selective inhibitor of Skp1-Cullin-F-box (SCF)MET30 ubiquitin ligase, an E3 ligase that regulates transcription, cell-cycle control, and immune response.
  • Thalidomide is an immunomodulatory drug and the prototype of the thalidomide class of drugs. This inhibitor is being seen mainly as a treatment of certain cancers (multiple myeloma) and of complication of leprosy.
  • TAME is a competitive inhibitor of the ubiquitin ligase anaphase-promoting complex/cyclosome and promotes Cdc20 autoubiquitination. This molecule inhibits cyclin proteolysis in mitotic Xenopus oocyte extracts and blocks cyclin degradation in interphase extract.
  • NCS-66811 is a cell-permeable, non-peptide inhibitor of  MDM2-p53 interactions (Ki=120nM).
  • Nutlin-3 is a cis-imidazoline analog that inhibits the interaction between p53 and its negative regulator MDM2 (IC50 = 0.09 μM).
  • SL-01 inhibits the p53-MDM2 interaction (20 µM).
  • SKPin C1 inhibits the cullin-RING ubiquitin E3 ligase SCF-Skp2.
  • SMER3 is a small molecule enhancer of Rapamycin 3 (SMER3) is a specific inhibitor of the ubiquitin E3 ligase SCFMET30.
  • PTC-209 treatment of cells leads to the selective reduction in the levels of BMI-1 protein (Kreso, van Galen et al. 2014). BMI-1 is a component of the polycomb PRC complex and interacts directly with the E3 ligase RING1A and/or RING2.
  • Heclin is an inhibitor of HECT-domain containing Ubiquitin E3 Ligases.

Ubiquitin E1 inhibitor

  • PYR-41 is a cell-permeable pyrazone compound that irreversibly inhibits ubiquitin-activating enzyme E1 activity. PYR-41 is also known to block ubiquitination-dependent protein degradation and other ubiquitination-mediated cellular activities. E1 inactivation by PYR-41 or other means has been shown to result in an overall elevation of sumoylation.

p97 inhibitor

p97 is a chaperone-like protein guiding protein substrates to the 26S proteasome for degradation.

  • DBeQ is a cell permeable, potent, selective and reversible inhibitor of the AAA-ATPase p97. This inhibitor blocks both ubiquitin dependent and endoplasmic reticulum-associated degradation pathways, protein clearance pathways, and activates caspases -3 and -7 inducing apoptosis.
  • MDBN is an irreversible inhibitor of p97/valosin-containing protein (VCP)/cdc48 (IC50=1.7 µM) identified in a cell-based protein degradation assay. Cell permeable.
  • NMS-873 is an allosteric inhbitor of ATPase activity of p97, binding to the D1 and D2 domains of adjacent subunits. NMS-873 is reported to have an IC50 for p97 of 20-30nM.

Interested in any of these inhibitors? Let me know through the form below!

Written by Ali El Baya, PhD
Ali el Bayâ is the Sales Manager at tebu-bio for the North of Europe.