Suramin Inhibits Cullin-RING E3 Ubiquitin Ligases PNAS PLUS

Suramin inhibits cullin-RING E3 ubiquitin ligases PNAS PLUS

Kenneth Wua, Robert A. Chonga, Qing Yub, Jin Baic, Donald E. Sprattd, Kevin Chinga, Chan Leea, Haibin Miaoe,f, Inger Tapping, Jerard Hurwitzg,1, Ning Zhenge,f, Gary S. Shawd, Yi Sunb,h, Dan P. Felsenfeldi,2, Roberto Sanchezj, Jun-nian Zhengc, and Zhen-Qiang Pana,c,1

aDepartment of Oncological Sciences, The Icahn School of Medicine at Mount Sinai, New York, NY 10029; bInstitute of Translational Medicine, School of Medicine, Zhejiang University, Hangzhou 310029, Zhejiang, China; cJiangsu Center for the Collaboration and Innovation of Cancer Biotherapy, Cancer Institute, Xuzhou Medical College, Xuzhou, 221002, China; dDepartment of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London N6A 5C1, ON, Canada; eDepartment of Pharmacology, University of Washington, Seattle, WA 98195; fHoward Hughes Medical Institute, University of Washington, Seattle, WA 98195; gProgram in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021; hDepartment of Radiation Oncology, University of Michigan, Ann Arbor, MI 48109; iDepartment of Regenerative Biology, The Icahn School of Medicine at Mount Sinai, New York, NY 10029; and jDepartment of Structural and Chemical Biology, The Icahn School of Medicine at Mount Sinai, New York, NY 10029

Contributed by Jerard Hurwitz, January 26, 2016 (sent for review December 4, 2015; reviewed by Serge Y. Fuchs and Pengbo Zhou) Cullin-RING E3 ubiquitin ligases (CRL) control a myriad of biological substrate, after which the E2 Cdc34 builds the K48-linked poly- processes by directing numerous protein substrates for proteasomal ubiquitin chain (10, 11). degradation. Key to CRL activity is the recruitment of the E2 In an effort to target CRL-mediated K48 polyubiquitination, we ubiquitin-conjugating enzyme Cdc34 through electrostatic inter- developed a FRET-based in vitro reporter assay system. On the actions between E3′s cullin conserved basic canyon and the acidic basis of this assay format, a high-throughput screen (HTS) was C terminus of the E2 enzyme. This report demonstrates that a small- performed, resulting in the identification of a potent inhibitor of molecule compound, suramin, can inhibit CRL activity by disrupting CRL activity, suramin. Interestingly, suramin is a century-old its ability to recruit Cdc34. Suramin, an antitrypansomal drug that antitrypansomal drug that also possesses antitumor activity (12). also possesses antitumor activity, was identified here through a fluorescence-based high-throughput screen as an inhibitor of Results ’ ubiquitination. Suramin was shown to target cullin 1 s conserved Tracking CRL-Mediated K48 Ubiquitination by FRET. To target CRL- basic canyon and to block its binding to Cdc34. Suramin inhibits mediated K48 ubiquitination, we developed a FRET K48 di-Ub the activity of a variety of CRL complexes containing cullin 2, 3, assay that measures the linkage-specific Ub–Ub transfer reaction and 4A. When introduced into cells, suramin induced accumulation by fluorescence. The assay has five components (Fig. 1A): (i)E1; of CRL substrates. These observations help develop a strategy of – (ii) E2 Cdc34 specific for K48 linkage; (iii) the E3 subcomplex regulating ubiquitination by targeting an E2 E3 interface through – – “ – small-molecule modulators. ROC1 CUL1 (411 776) [called ROC1 CUL1 C-terminal domain (CTD)”] required for E2 activation; (iv) donor Ub carrying a protein degradation | K48-polyubiquitination | E3 ligase | E2 enzyme | K48R substitution and fluorescent dye iFluor 555 at position suramin Q31C (Fig. S1A); and (v) receptor Ub bearing the G75G76 de-

letion and fluorescent dye iFluor 647 at position E64C (Fig. S1A). BIOCHEMISTRY > B n eukaryotes selective protein degradation requires a cellular These five proteins are of 95% purity (Fig. S1 ). The reaction Iprogram known as the “ubiquitin (Ub)-proteasome system.” was modified from previously reported versions (10), allowing only The Ub-proteasome system achieves target selection by tagging one nucleophilic attack to produce a single Ub–Ub isopeptide protein substrates with lysine-48 (K48)-linked polyubiquitin bond (Fig. 1A) exclusively carrying a K48 linkage (13). ROC1 chains that drive the modified substrates for destruction by the binds to the Cdc34 catalytic core domain (14), whereas the CUL1 26S proteasome (1). K48-polyubiquitination is driven by the CTD provides a basic pocket that interacts with Cdc34’sacidicC concerted action of three enzymes: a Ub-activating enzyme (E1), terminus, thereby facilitating E2 recruitment (15, 16). a Ub-conjugating enzyme (E2) such as Cdc34, and a Ub ligase (E3), such as the SCF (Skp1–Cullin1–F-box–ROC1/Rbx1) complex Significance (2, 3). The SCF complex belongs to the cullin-RING Ub E3 ligase ∼ (CRL) family that comprises 300 members, about half of all E3s Interactions between E2 and E3 enzymes are key for ubiquiti- identified in humans (3). nation, but whether such a dynamic association is susceptible Typically, ubiquitination with the SCF E3 begins with the ac- to perturbation by small-molecule modulators remains elusive. tivation of a substrate degradation signal (degron) by post- By demonstrating that suramin can inhibit cullin-RING E3 translational modification such as phosphorylation that often is ubiquitin ligase by disrupting its ability to recruit E2 Cdc34, this triggered in response to a diversified array of cellular signaling work suggests that the E2–E3 interface may be druggable. pathways (4, 5). The resulting active degron drives affinity in- In addition, suramin is an antitrypansomal drug that also teractions with the F-box substrate receptor protein within the possesses antitumor activity. Our findings have linked the E3 complex. A well-studied example is the complex formed be- ubiquitin-proteasome pathway to suramin and suggest addi- tween the β-TrCP F-box protein and the DpSGϕXpS degron tional biochemical mode of action for this century-old drug. motif present on IκBα and β-catenin (6). Through a mechanism yet to be defined, the E3-substrate association triggers covalent Author contributions: K.W., J.H., D.P.F., R.S., and Z.-Q.P. designed experiments; K.W., modification of Nedd8 to the E3′s cullin 1 (CUL1) scaffold R.A.C., Q.Y., J.B., K.C., C.L., Y.S., J.-n.Z., and Z.-Q.P. conducted experiments; K.W., J.B., D.E.S., H.M., I.T., N.Z., G.S.S., and J.-n.Z. contributed new reagents/analytic tools; and subunit at its conserved lysine residue in the winged-helix B K.W. and Z.-Q.P. wrote the paper. “ ” domain (WHB) (also known as ECTD ), converting the E3 Reviewers: S.Y.F., University of Pennsylvania; and P.Z., Weill Cornell Medical College. – from a restrained state into an active conformation (7 9). Last, The authors declare no conflict of interest. SCF, bound substrate, and Ub-charged E2-conjugating enzyme 1 To whom correspondence may be addressed. Email: [email protected] or (s) are engaged in multifaceted interactions that produce K48- [email protected]. linked polyubiquitin chains. In this context, emerging studies 2Present address: CHDI Management/CHDI Foundation, Princeton, NJ 08540. have revealed the actions of two E2 enzymes: the E2 UbcH5 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. acts as an initiator E2 and attaches a single Ub molecule to a 1073/pnas.1601089113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1601089113 PNAS | Published online March 21, 2016 | E2011–E2018 Downloaded by guest on September 24, 2021 Fig. 1. FRET K48 di-Ub assay. (A) Reaction scheme. (B) Detection by gel-based analysis. The FRET K48 di-Ub assay was carried out in a test tube as described in detail in Materials and Methods. Shown is an image of fluorescent reaction products detected by a Typhoon 9500 scanner. Only the complete reaction in which all components of the assay were present supported di-Ub formation. (C) FRET. Aliquots of the above reaction mixture were spotted onto a 384-well plate, which was subjected to analysis with the Synergy-H1 reader. The fluorescence signals ranging from 545 to 700 nm are shown. Only the complete reaction yielded FRET. (D)Z′ factor. Three independent FRET experiments were carried out. The graph represents an average of three independent experiments; error bars indicate SD. the Z′ factor at each time point was calculated and is shown. “Control” refers to reactions lacking ATP.

The FRET K48 di-ubiquitin (di-Ub) assay formed di-Ub of FRET K48 di-Ub synthesis with an IC50 of 460 nM (Fig. 2A). Fig. expected size only in the complete reaction (Fig. 1B, lane 1). The 2B shows a discharge reaction (11) in which the E3 ROC1–CUL1 reaction was abolished by the removal of any of the five com- CTD complex stimulated the discharge of the preformed E2 ponents (Fig. 1B, lanes 2–7), demonstrating that the formation of Cdc34∼S-Ub to form a di-Ub product (compare lanes 1 and 2). di-Ub is absolutely dependent on the presence of the E1/E2/E3 Increasing amounts of suramin prevented the E3-dependent dis- enzymes. For FRET, the reaction aliquot was subjected to charge and diminished the production of di-Ub (lanes 4–8). The spectroscopic analysis that revealed a robust FRET signal only in inhibitory effects by suramin were observed regardless of whether the complete reaction (Fig. 1C). Thus, the results from the gel CUL1 CTD was modified by Nedd8 (Fig. S3). Finally, we de- and spectroscopic assays are in complete agreement, showing termined whether suramin inhibited ubiquitination of IκBα-Ub by β that the observed FRET truly reflects the formation of the K48- E2 Cdc34, which required the holo-E3 complex SCF TrCP and linked di-Ub chain as a result of enzymatic synthesis. Nedd8 (Fig. S4). Suramin inhibited the ubiquitination of IκBα–Ub An HTS assay is judged by Z’ factor, which measures statistical in a dose-dependent fashion (Fig. 2C). Together, these results es- effect size based on the means and SDs of both the positive and tablish the inhibitory activity by suramin on ubiquitination catalyzed negative control samples, with a value in the range of 0.5–1 in- by E2 Cdc34 and E3 ROC1–CUL1. dicating an excellent assay for screening (17). The calculated Z′ factor of the FRET K48 di-Ub assay was 0.86 (Fig. 1D), dem- Suramin Targets CUL1’s Conserved Basic Canyon. We explored the onstrating a sufficient window for HTS. Providing further proof-of- mechanistic action of suramin and found that it did not inhibit principle evidence of the utility of this FRET assay for an inhibitor the formation of Ub thiol ester with either E1 (Fig. S5A)orCdc34 screen, a recently discovered Cdc34a inhibitor (CC0651) (18) was (Fig. S5B). However, titration experiments revealed that the in- showntoinhibitdi-Ubformationasdetectedbyeithergelassay hibitory effect by suramin is strictly dependent on the concentra- – (Fig. S1C)orFRETwithanIC50 of 3.92 μM(Fig. S1D). tion of ROC1 CUL1 CTD, because higher levels of the RING complex, but not of E1 or E2 Cdc34, reversed the inhibition (Fig. HTS Identification of Suramin as an Inhibitor of CRL-Mediated K48 S6). Together these results suggested that ROC1–CUL1 CTD Ubiquitination. On the basis of the optimized FRET K48 di-Ub mediates suramin’s inhibitory effect. assay format (Fig. S2A), an HTS was performed that identified Indeed isothermal titration calorimetry (ITC) experiments suramin as an inhibitor of CRL-mediated K48-linked ubiquitination showed direct binding of suramin to ROC1–CUL1 CTD with a (Fig. S2 B and C). Titration experiments with freshly purchased Kd of 2.80 ± 0.42 μM (Fig. 3A). However, the binding affinity suramin reproducibly demonstrated that this compound inhibits with suramin decreased markedly when mutant forms of CUL1

E2012 | www.pnas.org/cgi/doi/10.1073/pnas.1601089113 Wu et al. Downloaded by guest on September 24, 2021 CTD were used (Fig. 3B). Such CUL1 mutants, containing charge- PNAS PLUS swapping substitutions K431E/K432E/K435E or K678E/K679E/ R681E, disrupted the CUL1 basic canyon (15), which is a conserved region that functions to bind the acidic tail of E2 Cdc34. The inhibitory effects of the K431E/K432E/K435E or K678E/K679E/ R681E mutants were confirmed by di-Ub synthesis experiments (Fig. S7). Note that neither ROC1 nor Cdc34 alone exhibited significant binding for suramin (Fig. 3B). These results strongly suggest that suramin targets the basic canyon of CUL1. Consistent with these findings, immunoprecipitation experiments revealed that suramin inhibited the binding of Cdc34 to ROC1–CUL1 CTD by 90% (Fig. 3C, lanes 3–6). Of note, immunoprecipitation did not detect interactions between ROC1– CUL1 CTD and the Cdc34 C-terminal tail alone. Together, these results suggest that suramin’s mechanism of action involves its binding to the CUL1 conserved basic canyon, thereby com- petitively blocking the recruitment of the E2 Cdc34 to the E3 complex (Fig. 4). A demonstration of how suramin occupies the CUL1 basic pocket awaits future high-resolution structural work. In addition to Cdc34, the E3 ROC1–CUL1 core complex can work with the E2 enzymes UbcH5 and Ubc12, producing mono- ubiquitinated species and neddylated CUL1 (11), respectively. Fig. β S8 examined the ubiquitination of β-catenin by SCF TrCP and E2 UbcH5c in an assay similar to that shown in Fig. 2C. It was evident that at least threefold higher levels of suramin were required for inhibition of UbcH5c activity than in the Cdc34 reaction (compare Fig. 2C and Fig. S8). Moreover, suramin levels as high as 10 μM inhibited the transfer of Nedd8 to ROC1–CUL1 CTD by <50% (Fig. S9). Together, these data suggest that Cdc34-mediated ubiquitination is more susceptible to suramin than is UbcH5 or Ubc12. Our ITC experiments demonstrate that suramin binds ROC1– CUL1 CTD with a Kd of 2.8 μM (Fig. 3A), an affinity that appears to be weaker than expected considering that this compound inhibits the FRET K48 di-Ub synthesis with an IC50 of 0.46 μM(Fig. 2A). However, we should note that the Kd measurement by ITC was carried out with PBS, which has a significantly higher ionic BIOCHEMISTRY strength than used in the FRET K48 di-Ub synthesis assay (Materials and Methods). This difference in ionic strength may account for the higher-than-expected Kd, because the suramin– CUL1 CTD interactions are charge-based and therefore are likely to be sensitive to salt. Moreover, it is noteworthy that a similar discrepancy between IC50 and affinities has been reported for the Cdc34 inhibitor CC0651. In the previous study, CC0651 had an IC50 of 2.5 μM in the ubiquitination assay, but an EC50 of 51 μM was observed in the binding assay to Cdc34-Ub (18).

Suramin Is an Inhibitor of CRL. Cullins 2–5 (CUL2–5) share a conserved, C-terminally located basic canyon originally identified in CUL1 (15). In support, a more recent work revealed direct binding of the CUL2 basic canyon to the Cdc34 C-terminal tail (16). Given the evidence that suramin targets CUL1’s basic pocket (Fig. 3), we reasoned that suramin inhibits other CRL complexes as well. To test this hypothesis, we performed di-Ub synthesis reactions catalyzed by Cdc34 in the presence of ROC1– CUL2, ROC1–CUL3, or ROC1–CUL4A. Suramin inhibited all reactions tested (Fig. 5A). ROC1–CUL2 and ROC1–CUL4A appeared to be less sensitive to suramin than the ROC1–CUL1 CTD and ROC1–CUL3 complexes (Figs. 2A and 5A). The basis for these differences is presently unclear. However, note that in contrast to SCF/CRL1, whose E3 ligase activity can be measured in many reconstituted assays, well-developed in vitro systems for quantifying other CRL complexes are lacking in general. Such Fig. 2. Suramin inhibits K48 ubiquitination by CRL complexes. (A)TheIC50 of limitation precludes in-depth comparison of various CRL com- suramin. The graph represents the average of three independent experiments with calculated SD. (B) Suramin blocks the discharge of preformed Cdc34∼Ub by plexes in their responses to suramin. ROC1–CUL1 CTD to form the di-Ub product. The 32P-di-Ub synthesis reaction We next attempted to examine the impact of suramin on the was carried out as described in SI Materials and Methods.(C) Suramin inhibits stability of CRL substrates. Because of its predominantly anionic the ubiquitination of IκBα-Ub. Suramin, in the amounts indicated, was added to structure, suramin was proposed to be membrane impermeant, the ubiquitination reaction and performed as described in Fig. S4. and limited cellular uptake of suramin may occur by the process

Wu et al. PNAS | Published online March 21, 2016 | E2013 Downloaded by guest on September 24, 2021 Fig. 3. Suramin binds to CUL1’s conserved basic canyon. (A) ITC was carried out as described in Materials and Methods.(B) Mutations in the CUL1 basic canyon impair binding to suramin. ITC was carried out with suramin (or its analog NF449) and ROC1–CUL1 CTD with wild-type or substituted CUL1 to disrupt the CUL1 basic canyon, as specified. ROC1 alone or Cdc34 showed no significant binding to suramin. It appears that two molecules of suramin bind to one CUL1 molecule. In support of this binding mode, NF449, an analog that resembles two suramin molecules, bound to ROC1–CUL1 CTD in a 1:1 fashion. However, conclusive proof for this claim requires future high-resolution structural studies. The ROC1/Rbx1–CUL1 structure and the zoomed in basic cleft of CUL1 were derived from Protein Data Bank (PDB) ID code 1LDJ (43) and were visualized using PyMOL. ROC1/Rbx1 is shown in magenta, and the basic residues in the basic cleft of CUL1 (K431, K432, K435, K678, K679, R681) that were replaced by glutamates are highlighted in yellow. The CDC34 catalytic core (residues 7–182) was derived from PDB ID code 2OB4 and was visualized using PyMOL. The RING domain of ROC1/Rbx1 was derived from PDB ID code 2LGV (14) and was visualized using PyMOL. (C) Suramin blocks the binding of ROC1–CUL1 CTD to Cdc34. Immunoprecipitation experiments were carried out with HA-Cdc34 and ROC1–CUL1 CTD in the presence of the indicated compounds, in amounts specified and as described in SI Materials and Methods. A darker exposure of the gel is shown to visualize ROC1 better, because this smaller protein stained poorly.

of receptor-mediated fluid phase endocytosis (19). Subsequently, We used MLN4924, a well-characterized inhibitor of neddyla- Baghdiguian et al. (20) performed quantitative auto-radiographic tion that inactivates CRL (23), as a positive control for the im- analysis revealing that in the absence of serum albumin labeled munoblots analysis (Fig. 5B). In agreement with previous findings suramin was found to enter the human colon adenocarcinoma cells (23), MLN4924 caused stabilization of SCF(CRL1)Skp2 substrate and distribute over the nucleus, the Golgi apparatus, and the mi- p27, CRL3Keap1 substrate Nrf2, and CRL4Cdt2 substrate CDT1 tochondria. In addition, using an indirect acridine orange reporter (Fig. 5B), albeit with varying degree of effects. As shown, suramin assay, Huang et al. (21) reported that suramin enters and accu- at 62.5 μM accumulated p27 and CDT1 (Fig. 5B). Accumulation of mulates in low-pH intracellular compartments (endosomes, lyso- Nrf2 was observed in cells treated with higher concentrations of somes, and transGolgi complex) of normal and v-sis–transformed suramin (250–500 μM). It thus appeared difficult to correlate these NIH 3T3 cells. To facilitate the cellular analysis of suramin, we used cellular effects of suramin and the in vitro response shown by a serum-starvation protocol developed by Baghdiguian et al. (20) to variousCRLcomplexes(Figs.2and5A). On the other hand, increase the uptake of suramin and treated U2OS cells with this cellular effects are complex and likely are influenced by many compound in concentrations ranging from 62.5 to 500 μM. The se- factors, including subcellular localization. In this context, note lection of this range of compound concentration was in line with that although both CDT1 and p27 are nuclear proteins (24, 25), previous studies showing that suramin at high concentrations (200– CRL3Keap1 appears to function in the cytoplasm (26). Given 400 μM) inhibits the growth of a variety of cancer cell lines (22). the previous observations that suramin is preferentially enriched in

E2014 | www.pnas.org/cgi/doi/10.1073/pnas.1601089113 Wu et al. Downloaded by guest on September 24, 2021 Discussion PNAS PLUS The FRET K48 di-Ub Assay: A Strategy for Targeting CRL-Mediated K48 Ubiquitination. The Food and Drug Administration (FDA)-ap- proved antimyeloma and anti-CRL4cereblon drug lenalidomide/ thalidomide (29, 30) provides proof-of-concept evidence in favor of promoting CRL-based therapies. This study describes the development of an in vitro ubiquitination reporter system, the FRET K48di-Ubassay,thatcanbeusedeffectivelyforanHTScampaign to discover small-molecule chemical probes that target the CRL subcomplex containing ROC1 and its cullin partner as well as E1 and E2 Cdc34. Our FRET strategy is innovative, and easily adaptable for other K48-dependent ubiquitination HTS assays. First, by allowing only one nucleophilic attack, it produces a single Ub–Ub isopeptide bond (Fig. 1A). In contrast, previous assays have used the wild-type fluorescent Ub that yields polyubiquitination (31, 32). The restriction to a single Ub–Ub linkage eliminates the complexity associated with polyubiquitin Fig. 4. A model of suramin competing with Cdc34 for binding to the con- chains and allows straightforward quantification of each assay. served basic canyon of CUL1. The ROC1/Rbx1–CUL1 structure was derived This innovation ensures a high degree of reproducibility that is from PDB ID code 1LDJ (43) and was visualized using PyMOL. critical for HTS success, as validated in our present screen (Fig. 1D and Fig. S2A). Second, FRET is produced by two Ub-linked fluorophores that become juxtaposed upon Ub–Ub conjugation the nucleus (20), we can speculate that targeted degradation of (Fig. S1A). Each fluorophore is uniquely engineered to either nuclear proteins p27 and CDT1 by SCF(CRL1)Skp2 and CRL4Cdt2, donor or receptor Ub at a specific site (Fig. S1A). Note that optimal respectively, is sensitive to suramin. In comparison, nucleus- positioning of the fluorophore in Ub is critical, because N-terminally located suramin wound be ineffective in blocking the activity of Keap1 labeled Ub, commonly used in previous publications (31, 33), was cytoplasmic CRL3 . inactive in our system. Commercial fluorescent Ub proteins do not In summary, these data are consistent with a hypothesis that specify the location of the fluorescent dye in Ub, and it is unclear if suramin is a general inhibitor of CRL-mediated ubiquitination. such reagents can be generally optimal for HTS FRET assays. On Notably, suramin caused stabilization of CDT1 and p27 at con- the other hand, our FRET strategy should be applicable to other centrations well within the range required to inhibit the growth HTS campaigns that depend on K48 ubiquitination but require of a variety of cancer cell lines (22). Thus, these findings raised different E3/E2 enzyme combinations. the possibility that the inhibition of CRL by suramin might contribute to its antiproliferative activity. Cullin Conserved Basic Canyon: An Emerging Target of Regulation ∼ by Chemical Probes. The CRL superfamily, which contains 300 BIOCHEMISTRY Structure and Activity Relationship Analysis of Suramin. Suramin is a members and accounts for nearly half of the E3s identified in hu- polysulfonated naphthylurea. A panel of commercially available mans, affects a myriad of biological processes by directing numerous suramin analogs was analyzed with both CRL subcomplexes, protein substrates for proteasomal degradation (2, 3). Key to CRL – – ROC1 CUL1 CTD and ROC1 CUL3 (Fig. 6). A minimal phar- activity is the recruitment of the E2 Cdc34 through electrostatic macophore appears to be a structure with two assemblies of highly interactions between E3’s cullin conserved basic canyon and the acidic groups within a certain distance. The end of the molecule acidic C terminus of the E2 enzyme (15, 16). Cdc34 is the only E2 must be an aromatic ring (naphthyl/benzyl) with at least one required for cell viability in yeast (34). Using a chimera strategy, anionic substituent, preferably sulfonates. It also must contain yeast genetic studies have shown that fusion of the Cdc34 acidic at least one aromatic spacer on either side of the central urea. C-terminal tail to another E2 (such as Ubc2/Rad6) can transform Comparison of NF340, NF023, and NF110 revealed intriguing thechimericenzymeintoaCdc34-like protein essential for sup- properties. When tested in the FRET K48 di-Ub assay with porting cell growth, underscoring an indispensable role for the E2’s – μ ROC1 CUL1 CTD, NF340 exhibited an IC50 of 7.26 M, 17-fold negatively charged C terminus (35, 36). Subsequent biochemical μ less inhibitory than NF023, which possessed an IC50 of 0.42 M experiments have demonstrated the interactions between Cdc34 (Fig. 6). Previous studies have shown that NF340 inhibits the and E3 SCF (CRL1) (37), mapped the binding activity to the E2 ligand binding to the P2Y11 receptor (27) and angiogenesis (28). acidic C terminus and E3’sROC1–CUL1 CTD subcomplex (38), Although NF023 and NF340 are structurally similar overall, they and finally localized the contacts to CUL1 CTD’s basic canyon and differ in the number of sulfonate groups (six in NF023 and four the E2 negatively charged C-terminal tail (15, 16). Canonical cullins in NF340) and in the position of sulfonates on the naphthyl ring 1–5 share a conserved, C-terminally located basic canyon (15). In- (4, 6, and 8 in NF023; 3 and 7 in NF340). On the other hand, it deed, recent work has demonstrated direct binding of the CUL2 should be noted that NF110, which contains four sulfonate basic canyon to the Cdc34 C-terminal tail (16). In further support, – μ groups, inhibits ROC1 CUL1 CTD with an IC50 of 1.03 M (Fig. the biochemical reconstitution assays have revealed that a variety of 6). Together, these results suggest that the position of sulfonates CRL complexes tested, including ROC1–CUL2, ROC1–CUL3, and on the naphthyl ring impacts selectivity. It is possible that NF340 ROC1–CUL4A, can support Cdc34 for di-Ub synthesis (Fig. 5A). binds to the P2Y11 receptor but binds poorly to CUL1 because In the present work we demonstrate that the cullin conserved of slight differences in the basic pocket in P2Y11 and CUL1. basic canyon is a target of regulation by chemical probes. Suramin, Overall, ROC1–CUL3 appeared more sensitive than ROC1– a small-molecule inhibitor identified through an HTS using the CUL1 CTD in response to suramin and its analogs (Fig. 6). FRET K48 di-Ub assay format (Fig. 2), is shown to bind the Although cullins share a conserved basic canyon, subtle differ- ROC1–CUL1 CTD complex directly (Fig. 3A), most likely at ences may result in the differential abilities of suramin and an- the CUL1’s conserved basic canyon (Fig. 3B). Consequently, alogs to interact with the cullins. These results suggest that suramin is found to block the binding of Cdc34 to the ROC1– compounds that selectively target distinct classes of CRL com- CUL1 CTD complex in vitro (Fig. 3C) and to accumulate CRL plexes might be developed in the future. substrates in vivo (Fig. 5B).

Wu et al. PNAS | Published online March 21, 2016 | E2015 Downloaded by guest on September 24, 2021 A B 0.50 0.45 ROC1-CUL3 (0.5 µM) 0.40

0.35 IC50 0.22 M 0.30

0.25

0.20

0.15 FRET relative activity 0.001 0.01 0.1 1 10 100 1000

ROC1-CUL2 (1 µM)

ROC1-CUL4A (1 µM)

Fig. 5. Suramin is an inhibitor of CRL. (A) Suramin inhibits CRL complexes containing CUL2-4. The FRET K48 di-Ub assay was used to test suramin inhibition with ROC1–CUL3. The 32P-di-Ub synthesis assay was used with ROC1–CUL2 and ROC1–CUL4A. Both assays are described in SI Materials and Methods. In all cases, Cdc34 was used. Detected di-Ub products in the presence or absence of suramin were quantified and are shown graphically. (B) Suramin accumulates CRL substrates. Extracts from U2OS cells treated or not treated with compounds were subject to immunoblot analyses using the indicated antibodies, as described in SI Materials and Methods. The Nedd8 inhibitor MLN4924 is known to accumulate CRL substrates (23) and was used as a positive control. Detected CRL substrate signals were quantified by Odyssey infrared imaging (LI-COR); the relative values are presented under each blot.

One caveat is that suramin has many cellular targets (12), SCF or its subcomplexes. Suramin inhibited the ROC1–CUL1 presumably because of its ability to bind highly positively CTD-dependent FRET K48 di-Ub synthesis or discharge reaction charged regions that are analogous to the cullin conserved basic with similar potency, exhibiting an IC50 of 0.46 μMor1μM, re- canyon. However, analyses of suramin analogs (Fig. 6) may spectively (compare Fig. 2 A and B). However, the ubiquitination β have shed some light on the prospect of improving selectivity. of IκBα-Ub by SCF TrCP appeared less sensitive to suramin (Fig. The differential inhibition observed among suramin target 2C). A possible explanation of this paradox is the existence of protein–protein complexes suggests that subtle differences in suramin-insensitive mechanisms for the recruitment of Cdc34 by β basic pockets of the cullins might result in measurable differ- the holo-SCF TrCP E3 complex. Ubiquitination of IκBα–Ub re- β ences in their affinity to suramin and analogs. Such postulated quires the assembly of the SCF TrCP-IκBα-Ub complex. It is pos- differences, if validated, might be exploited for developing se- sible that, in addition to using electrostatic interactions mediated lective suramin analogs with differential abilities to target dis- by CUL1’s basic canyon and E2’s acidic C terminus, which is tinct classes of CRL complexes. sensitive to suramin, the IκBα-linked Ub may bind and help re- β We have reproducibly observed that suramin exhibits differential cruit Cdc34. Furthermore, in the context of the SCF TrCP-IκBα- inhibitory potency toward ubiquitination reactions mediated by Ub complex, the ROC1 RING domain may exhibit affinity to

E2016 | www.pnas.org/cgi/doi/10.1073/pnas.1601089113 Wu et al. Downloaded by guest on September 24, 2021 has been attributed to its ability to antagonize the effects of var- PNAS PLUS ious growth factors, including fibroblast growth factors, vascular endothelial growth factor, epidermal growth factor, platelet- derived growth factor, transforming growth factor-β,andinsulin- like growth factors (12). However, our findings that suramin blocks the ability of CRL to recruit Cdc34 suggest an alternative mechanism. By inducing the accumulation of CRL substrates such as CDT1 (Fig. 5B), which causes DNA rereplication, suramin potentially could exert antitumor effects, at least in part, through sensitizing cancer cells for apoptosis in a mechanism that is analogous to that of the Nedd8 inhibitor MLN4924 (23). However, whether suramin can target intracellular proteins effectively is uncertain because of the compound’slimitedcell permeability (19). On the other hand, the concentrations of suramin required to mediate growth inhibitory activity are reported to be in the range of 200–400 μM (22), similar to the levels of this compound needed to cause accumulation of CRL substrates (62.5– 250 μM) found in this work (Fig. 5B). Clearly, future structural and activity analyses are necessary to establish whether the inhibition of CRL activity by suramin contributes to antitrypansomal and/or tumor-suppression effects. If validated, the development of suramin analogs that can selectively target CRLs potentially could provide novel therapeutics with desirable pharmacokinetic properties and minimal toxicity. Materials and Methods Detailed procedures for the HTS experiment, protein preparation, assays, and cell-based experiments are provided in SI Materials and Methods.

Preparation of Fluorescently Labeled Ub. Site-directed mutagenesis was used to create Ub-Q31C/K48R (donor Ub) and Ub-E64C/ΔGG (acceptor Ub), each containing a single cysteine residue for labeling with maleimide-based fluorescent dyes. To prepare fluorescent Ub, purified Ub-Q31C/K48R and Ub-E64C/ΔGG, 150 mg each, were treated with 10 mM Tris(2-carboxyethyl) phosphine (TCEP) (Sigma) in degassed PBS for 15 min at room temperature and then were bound to Ni-NTA agarose (Qiagen) for 1 h at 4 °C. After binding, the BIOCHEMISTRY Fig. 6. Analysis of the structure and activity relationship of suramin. Various beads were washed with degassed PBS to remove the TCEP and any unbound suramin analogs, as indicated, were subject to the FRET K48 di-Ub assay, as protein. The resulting beads were incubated with 15 mg of iFluor 555 maleimide Δ described in Materials and Methods, in the presence of either ROC1–CUL1 (UbQ31C/K48R) or iFluor 647 maleimide (UbE64C/ GG) (AAT Bioquest), dissolved in dimethylformamide (Sigma), for 1 h at room temperature and then overnight CTD or ROC1–CUL3. The IC50 is shown. Compounds 7 and 8 (up to 100 μM) showed no inhibition. at 4 °C, in the dark. Note that the above handling was performed inside an anaerobic glove box (Captair Pyramid; Erlab). The subsequent steps were performed in normal air. Following incubation, beads were washed with PBS to the Cdc34 core domain at levels significantly higher than those remove unreacted dye. Then the labeled proteins were eluted with PBS plus displayed by a subcomplex (ROC1–CUL1 CTD). Both E2-Ub 250 mM imidazole. The eluted proteins were dialyzed at 4 °C in the dark, against buffer containing 25 mM Tris·HCl (pH 7.4), 1 mM EDTA, 10% glycerol, 150 mM and RING-E2 interactions are driven by hydrophobic interac- NaCl, 0.01% Nonidet P-40, and 1 mM DTT. The final yields were ∼108 mg labeled tions (39) and thus are unlikely to be sensitive to suramin. Ub-Q31C/K48R and ∼129 mg for labeled Ub-E64C/ΔGG. However, despite subtle differences, suramin effectively inhibits SCF/CRL1 and other CRL complexes (Figs. 2 and 5A). FRET K48 di-Ub Assay. The reaction mixture (15 μL) was assembled onto a Recent studies have revealed that Cdc34 activity can be regu- 384-well microtiter plate (or in a test tube). Each well contained 33 mM · lated by a protein inhibitor or small-molecule compound. Glo- Tris HCl (pH 7.4), 1.7 mM MgCl2, 0.33 mM DTT, 0.07 mg/mL BSA, 14 nM Ub mulin, a HEAT-repeat–containing protein, was shown to bind the E1, 124 nM E2 Cdc34, 1 μM ROC1–CUL1 CTD, 0.93 μM Ub C31-iFluor 555 μ E2-interacting surface of the ROC1/RBX1 RING domain and to (donor), and 1.62 M Ub C64-iFluor 647 (receptor) in the absence or presence inhibit CRL-mediated chain formation by Cdc34 (40, 41). Like- of a compound such as suramin, in amounts as indicated. ATP (0.66 mM) was added to the mixture followed by a brief centrifugation to settle the mix- wise, the small-molecule inhibitor of Cdc34a, CC0651, stabilizes a ture. The resulting plate was incubated at 30 °C in a Synergy-H1 reader noncovalent complex between the E2 and the donor Ub, thereby (BioTek), and the fluorescence signal was monitored. Ubiquitination was blocking Ub discharge (18). To our knowledge, disrupting the quantified based on the ratio of acceptor:donor fluorescence (excitation CRL–Cdc34 interaction by suramin represents the first experi- 515 nm; donor emission 570 nm, acceptor emission 670 nm). mental evidence that an E2–E3 interface might be susceptible to manipulation by chemical probes. This finding reveals an IC50 Determination. Compound stock solutions in DMSO were titrated into opportunity/strategy for developing therapeutic drugs targeting 384-well plates using an HP D300 digital dispenser. Assays were performed in a key ubiquitination enzymes at an unconventional site. 384-well microtiter plate as described above using the Synergy-H1 reader. Results were analyzed and graphed using SigmaPlot software (SyStat). The IC50 curve was determined using four-parameter logistic standard curve analysis. Suramin Therapeutics: A Role for CRL Inhibition? Suramin has been used as the drug of choice for the treatment of African try- ITC. ITC was performed on a MicroCal iTC200 system (GE) at 25 °C. Samples panosomiasis since 1924. It possesses antitumor activity (12) and were prepared in degassed PBS. Typically 0.15–0.3 mM suramin was titrated also has been observed to reverse autism-like behaviors in mice in 2.5-μL injections, administered at 3-min intervals, into the sample cell (42). The mechanism of suramin in the treatment of trypano- containing 10–30 μM protein (or protein complex). The data were analyzed somiasis is presently unknown. The antitumor activity of suramin in Origin software (OriginLab).

Wu et al. PNAS | Published online March 21, 2016 | E2017 Downloaded by guest on September 24, 2021 ACKNOWLEDGMENTS. We thank Thimo Kurz for reagents and Sharmila the Natural Sciences and Engineering Research Council of Canada Fellow- Sivendran, John Tseng, Andres Maldonado, Vincent Anguiano, and Avin ships (to D.E.S.), Canadian Institutes of Health Research Grant MOP-14606 Dar for technical assistance. Z.-Q.P. is the recipient of the 2013 Jiangsu (to G.S.S.), and NIH Grants GM61051/CA095634, National Nature Science Special Medical Expert Award. This work was supported by NIH Fellowship Foundation of China Grant 81572710, and a Mount Sinai Technology and 1F30DK095572-01 (to R.A.C.), Canadian Institutes of Health Research and Development Fund Award (to Z.-Q.P.).

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