A selective inhibitor of the UFM1-activating enzyme, UBA5.

Protein conjugation with ubiquitin and ubiquitin-like small molecules, such as UFM1, is important for promoting cancer cell survival and proliferation. Herein, the development of the first selective micromolar inhibitor of the UBA5 E1 enzyme that initiates UFM1 protein conjugation is described. This organometallic inhibitor incorporates adenosine and zinc(II)cyclen within its core scaffold and inhibits UBA5 noncompetitively and selectively over other E1 enzymes and a panel of human kinases. Furthermore, this compound selectively impedes the cellular proliferation (above 50µM) of cancer cells containing higher levels of UBA5. This inhibitor may be used to further probe the intracellular role of the UFM1 pathway in disease progression.

Retraction of "Identification of Purine-Scaffold Small-Molecule Inhibitors of Stat3 Activation by QSAR Studies".

[This retracts the article DOI: 10.1021/ml100224d.].

Mutations in UBA3 confer resistance to the NEDD8-activating enzyme inhibitor MLN4924 in human leukemic cells.

The NEDD8-activating enzyme (NAE) initiates neddylation, the cascade of post-translational NEDD8 conjugation onto target proteins. MLN4924, a selective NAE inhibitor, has displayed preclinical anti-tumor activity in vitro and in vivo, and promising clinical activity has been reported in patients with refractory hematologic malignancies. Here, we sought to understand the mechanisms of resistance to MLN4924. K562 and U937 leukemia cells were exposed over a 6 month period to MLN4924 and populations of resistant cells (R-K562(MLN), R-U937(MLN)) were selected. R-K562(MLN) and R-U937(MLN) cells contain I310N and Y352H mutations in the NAE catalytic subunit UBA3, respectively. Biochemical analyses indicate that these mutations increase the enzyme's affinity for ATP while decreasing its affinity for NEDD8. These mutations effectively contribute to decreased MLN4924 potency in vitro while providing for sufficient NAE function for leukemia cell survival. Finally, R-K562(MLN) cells showed cross-resistance to other NAE-selective inhibitors, but remained sensitive to a pan-E1 (activating enzyme) inhibitor. Thus, our work provides insight into mechanisms of MLN4924 resistance to facilitate the development of more effective second-generation NAE inhibitors.

Exploring a new frontier in cancer treatment: targeting the ubiquitin and ubiquitin-like activating enzymes.

The labeling of proteins with small ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers regulates a plethora of activities within the cell, such as protein recycling, cell cycle modifications, and protein translocation. These processes are often overactive in diseased cells, leading to unregulated cell growth and disease progression. Therefore, in systems where Ub/Ubl protein labeling is dysregulated, the development of drugs to selectively and potently disrupt Ub/Ubl protein labeling offers a targeted molecular approach for sensitizing these diseased cells. This Perspective outlines the progress that has been made in the context of inhibitor development for targeting Ub/Ubl pathways.

Targeting the ubiquitin E1 as a novel anti-cancer strategy.

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.

Identification of Purine-Scaffold Small-Molecule Inhibitors of Stat3 Activation by QSAR Studies.

To facilitate the discovery of clinically useful Stat3 inhibitors, computational analysis of the binding to Stat3 of the existing Stat3 dimerization disruptors and quantitative structure-activity relationships (QSAR) were pursued, by which a pharmacophore model was derived for predicting optimized Stat3 dimerization inhibitors. The 2,6,9-trisubstituted-purine scaffold was functionalized in order to access the three subpockets of the Stat3 SH2 domain surface and to derive potent Stat3-binding inhibitors. Select purine scaffolds showed good affinities (K(D), 0.8-12 µM) for purified, nonphosphorylated Stat3 and inhibited Stat3 DNA-binding activity in vitro and intracellular phosphorylation at 20-60 µM. Furthermore, agents selectively suppressed viability of human prostate, breast and pancreatic cancer cells, and v-Src-transformed mouse fibroblasts that harbor aberrant Stat3 activity. Studies herein identified novel small-molecule trisubstituted purines as effective inhibitors of constitutively active Stat3 and of the viability of Stat3-dependent tumor cells, and are the first to validate the use of purine bases as templates for building novel Stat3 inhibitors.

Identification of NAE Inhibitors Exhibiting Potent Activity in Leukemia Cells: Exploring the Structural Determinants of NAE Specificity.

MLN4924 is a selective inhibitor of the NEDD8-activating enzyme (NAE) and has advanced into clinical trials for the treatment of both solid and hematological malignancies. In contrast, the structurally similar compound 1 (developed by Millennium: The Takeda Oncology Company) is a pan inhibitor of the E1 enzymes NAE, ubiquitin activating enzyme (UAE), and SUMO-activating enzyme (SAE) and is currently viewed as unsuitable for clinical use given its broad spectrum of E1 inhibition. Here, we sought to understand the determinants of NAE selectivity. A series of compound 1 analogues were synthesized through iterative functionalization of the purine C6 position and evaluated for NAE specificity. Optimal NAE specificity was achieved through substitution with primary N-alkyl groups, while bulky or secondary N-alkyl substituents were poorly tolerated. When assessed in vitro, inhibitors reduced the growth and viability of malignant K562 leukemia cells. Through this study, we have successfully identified a series of sub-10 nM NAE-specific inhibitors and thereby highlighted the functionalities that promote NAE selectivity.