Will Targeting the Ubiquitin Proteosome System Compare to Protein Phosphorylation Drug Discovery

Post-translational modifications (PTMs) modulate protein function in most eukaryotes and contribute to the functional diversity of the proteome. In 2011, the number of post-translational modifications listed in the Swiss-Prot database far exceeded the number of mutations identified and it is estimated that soon they will even exceed the number of protein sequences. Despite the growing volume of research pertaining to post-translational modifications, we are still unraveling their complexity and tremendous impact on normal development and disease.

Post-Translational Modifications are Involved in Almost All Cellular Events Including

Number 1

Gene expression

Number 2

Signal transduction

Number 3

Protein-protein interactions

Number 4

Cellular metabolism

Number 5

Protein turnover and localization

Number 6

DNA repair

Number 7

Cell-cell interactions

Number 8

Communication between internal and external cellular environment

Number 9

Translocation of proteins across biological membranes

Role of Post-Translational Modifications in Disease and Drug Development

An understanding of post-translational modifications is vital, not only due to their role in a wide array of normal cellular functions, but also due to their drug-targeting potential for life-threatening diseases. Some of the more common post-translational modifications and their role in disease can be found in Table 1. Currently, post-translational modifications are tracked as disease markers or used as molecular targets for developing target-specific therapies. They have been found to have a profound effect on the stability, activity, and pharmacokinetics of many therapeutic proteins, thus characterization of post-translational modifications and the establishment of their biological influence has become a critical step in early stage biopharmaceutical drug development.

Table 1

Linked Diseases

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  • Cancer
  • Neurodegenerative diseases (Parkinson's and dementia)
  • Inflammatory and autoimmune diseases
  • Dementia and Alzheimer's disease
  • Melanoma
  • Leukemia
  • Cancer
  • Metabolic disorders
  • Lipofuscinosis
  • Cancers
  • Metabolic disorders
  • Cancer
  • Viral infection
  • Neurodegenerative disorders
  • Muscle wasting
  • Diabetes
  • Inflammation
  • Pathogenic infection
  • Cancer
  • Neurodegenerative disorders

Protein phosphorylation, first discovered in the 1950's, is one of the most common post-translational modification mechanisms in eukaryotes. In fact, it has been suggested that 20-30% of all eukaryotic proteins can be phosphorylated by over 500 kinases, bringing the estimated number of phosphorylation sites to greater than 170,000. Aberrant phosphorylation directly causes or is a consequence of many human diseases including cancer, neurodegenerative disorders, inflammatory and autoimmune diseases. Current estimates indicate the field of protein phosphorylation accounts for almost 30% of pharmaceutical research and development.

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Protein phosphorylation drug discovery revealed that it can take years, if not decades, before a field of research can reach a stage where it can be exploited for therapeutic purposes. To demonstrate; protein phosphorylation was first identified in the 1950's as a mechanism for controlling glycogenolysis. The 70's brought forth the idea that it could function as a control switch for metabolism, but it was only during the late 1980's to early 90's when researchers finally realized protein phosphorylation regulates most aspects of cell life. Even still, as late as 1998, many believed there was little future in kinase drug discovery. That is, until the remarkable discovery of Gleevec, a tyrosine kinase inhibitor for treating chronic myelogenous leukemia, which catapulted protein kinases into becoming the most popular class of drug targets for the pharmaceutical industry. While the field of developing kinase inhibitors has encountered a number of challenges over the years, none have proven insurmountable.

One of the benefits of targeting protein kinases for drug development is the fact that the same technologies and small-molecule libraries can be effectively used to develop inhibitors spanning a variety of protein kinases that play roles in numerous therapeutic areas. The question which lingers in the minds of many researchers, however, is whether the most important drug targets in the protein phosphorylation arena have finally become exhausted? Enter the promising field of the ubiquitin-proteosome system, or UPS.


Unlike phosphorylation, the role of ubiquitination in human disease has only recently garnered attention. Ubiquitination is a highly dynamic, enzymatically-catalyzed post-translational modification that targets proteins for degradation and recycling. It is thought to be critical to almost every cellular function and has been implicated in numerous diseases and disorders. The process of ubiquitination involves the covalent attachment of a small, regulatory protein, ubiquitin (Ub), to specific residues of a target protein, most often lysine. Three classes of enzymes, acting in a precise sequence, are required for this to occur. They include: Ub-activating enzymes, Ub-conjugating enzymes, and Ub-ligases, and are commonly referred to as E1, E2, and E3, respectively. In most cases, Ub-tagged proteins are recognized by the 26S proteasome and targeted for degradation. Alternatively, Ub can be released by deubiquitinating enzymes (DUBs), demonstrating Ub conjugation is a reversible process.

Cellular Functions Involving Ubiquitin

Cell proliferation



Immune response

DNA repair

Neural degeneration


Stress response

The Ubiquitin System and Disease

The ubiquitin-proteosome system plays an important role in both cellular proliferation and survival. It is involved in regulating the turnover of proteins integral to cell-cycle progression, such as cyclins p27 and p53. In addition, the ubiquitin-proteosome system plays a vital role in regulating the NF-κB pathway, perhaps the most important cell survival pathway. These functions have made the ubiquitin-proteosome system a popular route for cancer cells to exploit in order to achieve aberrant growth and resistance to apoptosis. They have also made the ubiquitin-proteosome system a popular target for the discovery of novel drugs in the fight against cancer.

Specific Roles of the Ubiquitin System and Cancer

  • Breast and ovarian cancers: BRCA1 mutation; loss of tumor suppressor function
  • Lung cancer, clear cell carcinoma, VHL-disease cancers: VHL mutation; loss of tumor suppressor function
  • Various malignancies: MDM2 overexpression; loss of p53 tumor suppressor function
  • Prostate cancer: USP2a overexpression; increased cMYC oncogene expression; SPOP mutation
  • Leukemia, cholangiocarcinomas, GI and endometrial cancers: FBW7 mutation; loss of tumor suppressor function; potential oncogene
  • Colorectal, breast, and prostate: SKP2 overexpression; loss of tumor suppressor function of p27
  • Cervical cancer: HPV-mediated degradation of p53
Therapeutics targeting the ubiquitin-proteosome system for cancer therapy
Proteosome Inhibitors
  • Peptide boronates (bortezomib, delanzomib and MLN9708)
  • Peptide epoxyketones (carfilzomib, oprozomib, ONX-0914, and PR-924)
  • Peptide aldehydes (MG132 and IPSI-001)
  • Non-peptide βlactones (Marizomib)
  • Natural products
Ub Activating Enzyme Inhibitor
  • PYR-41 (1st cell-permeable inhibitor to specifically inhibit Uba1)
  • PYZD-4409

E1 Inhibitors
(NEDD8 Activating Enzyme (NAE) Inhibitors)

  • MLN4924 (inhibits cullin neddylation)
E2 Inhibitors
  • Cdc34 inhibitor (CCO651)
  • Ubc13-Uev1A inhibitors (NSC697923)
  • Rad6B inhibitor (SMI#8 and SMI#9)
E3 Inhibitors

HDM2-p53 Inhibitors

  • Cisimidazoline analogs (Nutlin-3, RG7112, and RO5503781)
  • Spiro-oxindoles (MI-63, AT-219, MI-319, SAR405838)
  • Isoquinolinones (PXN727 and PXN822)
  • Benzodiazepinediones (TDP521252 and TDP665759)
  • JNJ-26854165
  • HLI98 series
  • SJ-172550 - 1st small molecule inhibitor of HDMX
  • Indolyl hydantoins (RO-2443 and RO-5963) – Dual HDM2/HDMX antagonists
  • MK-8242 and CGM097 (structures undisclosed)

SCF (Skp2-p27 axis) Inhibitors

  • CpdA
  • Small molecule inhibitors specific to SCFSkp2 activity

APC/C Inhibitors

  • TAME
Analogous Mechanisms Ubiquitination and Phosphorylation

Interestingly, a number of similarities exist between phosphorylation and ubiquitination. This is not surprising given that interplay between the two systems is critical for the regulation of many cellular processes. For example, phosphorylation regulates a number of E3 Ub ligases and DUBs. In addition, a number of kinases can be activated or inhibited by interactions with polyubiquitin chains or polyubiquitination. Unlike phosphorylation, however, ubiquitination may have greater flexibility due to the presence of "ubiquitin-like" proteins and the fact that ubiquitin can form up to eight different polyubiquitin chains. Furthermore, while both systems are reversible, the number of deubiquitinases, along with E1-activating enzymes, E2-conjugating enzymes and E3 ligases exceeds the number of protein kinases. Will this affect ubiquitin-proteosome system-targeted drug development? Fully understanding these characteristics and other interactions is likely to become increasingly more important for drug development targeting the ubiquitin system in the next decade.

Future Considerations for Ubiquitin-Proteosome System-Targeted Drug Discovery

It is clear that protein phosphorylation and the ubiquitin-proteosome pathway play significant roles in regulating diverse biological processes. Manipulating the ubiquitin-proteosome system in cancer cells, however, may prove to be the biggest challenge for scientists in the coming years. Specifically, researchers must decipher the effects of targeting proteins within the system which serve dual roles as tumor suppressors and oncogenes. The complexity of such studies is staggering given the fact that such proteins can be influenced by a myriad of factors, many of which are still largely unknown. Thus, until the full effects of inhibition within the ubiquitin-proteosome system can be predicted, we will not realize the full-scope potential for UPS-targeted drug discovery.

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