Insights Into the Allosteric Inhibition of the SUMO E2 Enzyme Ubc9

Abstract

Conjugation of the small ubiquitin-like modifier (SUMO) to protein substrates is an important disease-associated posttranslational modification, although few inhibitors of this process are known. Herein, we report the discovery of an allosteric small-molecule binding site on Ubc9, the sole SUMO E2 enzyme.A nX -rayc rystallographic screen was used to identify two distinct small-molecule fragments that bind to Ubc9 at as ite distal to its catalytic cysteine.T hese fragments and related compounds inhibit SUMO conjugation in biochemical assays with potencies of 1.9-5.8 mm.Mechanistic and biophysical analyses,c oupled with molecular dynamics simulations,p oint toward ligand-induced rigidification of Ubc9 as amechanism of inhibition.The posttranslational modification of protein substrates with as mall ubiquitin-like modifier (SUMO) tag occurs through at ightly regulated E1/E2/E3 enzymatic cascade and modulates ab road variety of cellular functions.[1] Ubc9 is the only E2 enzyme involved in the conjugation of all three SUMO isoforms to many diverse substrates within the proteome.As acentral enzyme in the SUMOylation cycle,Ubc9 is required for normal embryonic development, and it is dysregulated in av ariety of disease states such as cancer [2] and ischemia.[3]Ubc9 has been suggested as ap otential anticancer target for small-molecule inhibitors,n otably for MYC-driven [4] and RAS/Raf-driven [5] cancers,a sw ell as multiple myeloma.[6]Despite its relevance to disease,i th as been challenging to identify small molecules that modulate the function of Ubc9.In related ubiquitin-like signaling pathways,inhibitors of E1 [7] and E3 [8] enzymes are now clinically used as anticancer chemotherapeutics.Bycontrast, very few reversible inhibitors of any of the approximately 40 known ubiquitin and ubiquitin-like E2 conjugating enzymes have been discovered, [9] only one of which has been characterized in complex with the protein.[10] Thus,n ew experimentally validated chemical inhibitors of Ubc9 would provide substantial insight into targeting this important enzyme,a nd potentially E2 enzymes in general.One powerful approach to identify ligands for challenging protein targets is fragment-based inhibitor discovery.[11] In general, fragment-based approaches leverage sensitive techniques such as NMR, thermal shift, surface plasmon resonance (SPR), or X-ray crystallography to identify lowmolecular-weight ligands that bind weakly but specifically to target proteins.[12] Weak leads identified through fragmentbased approaches can provide excellent starting points for the development of highly active inhibitors,e ven in cases where the targets are considered to be challenging,such as proteinprotein interactions or so-called "undruggable" targets. [13] As part of al arger program aimed toward identifying chemical inhibitors of Ubc9, we elected to pursue an X-ray crystallographic fragment screening strategy (see the Supporting Information).[14] Ubc9 crystals diffracting to aresolution of 1.12 ( PDB ID:5 F6E) were soaked with as eries ...

Expression of SUMO associated proteins in the mouse endometrium is regulated by ovarian hormones throughout the estrous cycle

“…The E2 ligase UCB9 is responsible for the conjugation of SUMO to target molecules (35). The present study found that UBC9 was highly expressed throughout the endometrium, with no obvious changes throughout the estrous cycle.…”

A Small-Molecule Microarray Approach for the Identification of E2 Enzyme Inhibitors in Ubiquitin-Like Conjugation Pathways

“…In addition to wild-type enzyme, two Ubc9 mutants (K59A and E42A) that disrupt a recently identified allosteric site were evaluated. 6 Compound 2 was able to inhibit SUMO conjugation to the fluorescent peptide substrate for all three enzymes without any apparent change in potency ( Suppl. Fig.…”

“…The in vitro sumoylation assay was performed in 20 µL reaction buffer (50 mM Tris [pH 9], 5 mM MgCl 2 , 1 mM DTT) containing SUMO E1 (0.1 µM), Ubc9 (0.15 µM), SUMO-1 (His-tag, 1.4 µM), and a fluorescent peptide (FL-AR) substrate 5 (1 µM) incubated with various concentrations of small molecule in DMSO or DMSO alone (4% final concentration). Experiments with mutant Ubc9, obtained as previously described, 6 were conducted according to the same procedure using K59A or E42A Ubc9 in place of the wild-type E2. The reaction was then initiated by the addition of ATP (2 mM final concentration) and incubated at room temperature for 90 min.…”

“…The in vitro sumoylation assay was executed as previously described. 5,6 The assay was performed in a 384-well plate format in 20 µL Tris buffer (50 mM Tris [pH 9], 5 mM MgCl 2 , and 1 mM DTT) with SUMO E1 (0.1 µM), SUMO-1 (His-tag, 1.4 µM), Ubc9 (0.15 µM), fluorescent peptide FL-AR (1 µM), and small molecule (4% DMSO final). For experiments with detergent, either Triton X-100 or Tween 20 (0.01%, v/v) was incorporated into the assay buffer as well.…”

“…5 A more recent fragment-based approach revealed an allosteric small-molecule binding site on Ubc9 that inhibits thioester formation, with fragments having IC 50 values in the low millimolar range. 6 Inhibitors that target the SUMO E1 enzyme, including ginkgolic acid and tannic acid, have also been reported. To date, these existing inhibitors of sumoylation suffer from weak potency in biochemical or cell-based assays, polyphenolic structures, redox activity, pleiotropic mechanisms of action, or a combination of these issues.…”