P53 upregulation by USP7-engaging molecular glues

Molecular glues are typically small chemical molecules that act on the interface between the target protein and the degradation machinery to trigger ternary complex formation. Identi�cation of molecular glues is challenging, and there has been a lack of target-upregulating molecular glues, which are desired for many targets such as tumor suppressor proteins (TSPs). TSPs are usually degraded by the proteasome through polyubiquitination (poly-ub) by speci�c E3 ligases, whereas deubiquitinases (DUBs) are capable of removing poly-ub conjugates to counteract these E3 ligases. Thus, small molecular glues that enhance the anchoring of TSPs to DUBs may stabilize them through deubiquitination. Here, through small-molecule microarray-based technology and unbiased screening, we identi�ed three potential molecular glues that may tether P53 to the DUB USP7 and elevate the P53 level. Among them, bromocriptine (BC) is an FDA-approved drug showing the most robust effects. We further demonstrated that BC increased P53 stability via the predicted molecular glue mechanism engaging USP7. To con�rm the generality of the screening platform, we identi�ed another USP7-engaging molecular glue that upregulates PTEN, which is another well-known TSP. Taken together, we established a potential screening platform that may facilitate the discovery of novel molecular glues stabilizing TSPs via engaging the DUB USP7. Similar strategies could be applied to the identi�cation of other types of molecular glues that may bene�t drug discovery and chemical biology studies.


Introduction
Targeted protein degradation (TPD) technologies such as proteolysis-targeting chimeras (PROTACs) and lysosome-engaging degrader technologies have been booming in the last decade, leading to promising drug discovery strategies and candidate drugs that selectively degrade pathogenic proteins 1,2 .
Meanwhile, many diseases are caused by the loss-of-function mechanism due to the depletion or reduced activities of speci c protein targets, and TPD technologies are incapable of tackling them.Instead, technologies that can selectively upregulate or restore the level or function of these targets are desired for these diseases.Gene therapy approaches over-expressing the target protein may achieve this goal, but they typically require delivery of adeno-associated viruses (AAVs), which could raise safety concerns and be prohibitively expensive 3,4 .In addition, there is a limitation of the gene size that could be packaged into AAVs, precluding the expression of relatively large proteins 5 .Thus, strategies to identify small-molecule compounds that selectively upregulate the target protein are highly desired yet largely unexplored.
A recent study reports the deubiquitinase-targeting chimeras (DUBTACs) technology stabilizes the target protein by heterobifunctional molecules consisting of a covalent DUB recruiter linked to a proteintargeting ligand 6 .DUBs are catalytic enzymes that can speci cally disassemble the ubiquitin-protein conjugates by cleaving the isopeptide bonds formed between the C-terminus of ubiquitin and the target proteins, protecting them from degradation and inactivation 7 .DUBTACs engaging the DUB OTUB1 successfully up-regulated ΔF508-CFTR and WEE1 proteins 6 .Meanwhile, DUBTACs are chimeric compounds that are typically large and less druglike according to Lipinski's Rule of Five 8 .The reported DUBTACs covalently interact with the DUB and may have certain advantages, such as high e ciency of DUB engagement.Meanwhile, this may lead to possible limitations such as a "permanent" occupancy of the DUB that may in uence the DUB's function and prevent recycling of the DUB.In comparison, molecular glues are typically small chemical molecules that act on the interface between the target protein and the degradation machinery to trigger ternary complex formation 9 .They do not have separate chemical moieties interacting with each of the two proteins and the linker connecting the two moieties.Thus, they are usually smaller and more druglike than chimeric compounds 9 .Therefore, molecular glues may exhibit advantages over DUBTACs in certain aspects.The targeted-protein degradation molecular glue lenalidomide has already achieved great clinical success and provided therapeutic bene ts for cancer patients 10 .Thus, the identi cation of small molecular glues upregulating the target protein via engaging speci c DUBs (DUBEGs, for DUB-engaging glues) is highly desired to expand the landscape and possibly enhance the drug-likeness of targeted-protein upregulation compounds.
Before the publication of the DUBTAC study, we made an independent attempt to identify DUBEGs that regulate speci c targets, especially tumor suppressor proteins (TSPs).TSPs regulate cell proliferation and play an essential role in preventing cancer development 11 .Upregulating TSPs may inhibit cancer cell proliferation and the growth of tumors.TSPs are categorized into ve major classes based on their mechanisms of action that are not necessarily mutually exclusive 12 .These classes include proteins controlling the progression of cell cycle stages (e.g., pRB and p16) 13 , receptor protein or signal transducers orchestrating signals that inhibit cell proliferation (e.g., transforming growth factor (TGF)-β) 14 , checkpoint-control proteins triggering cell cycle arrest in case of DNA damage or chromosomal defects (e.g., p16, p14, and breast cancer type 1 susceptibility protein (BRCA1)) 15 , proteins inducing apoptosis (e.g., P53) 16 , and proteins involved in the repair of DNA (e.g., DNA mismatch repair protein 2 (MSH2) and P53) 17 .
Among TSPs, P53 is a promising yet challenging target for cancer treatment.P53 is a potent transcription factor that controls a major pathway protecting cells from malignant transformation, inhibiting cell proliferation, and inducing cell apoptosis mutations 18 .Its malfunctioning mutations may contribute to ~ 50% of tumorigenesis, and its paramount role in tumor suppression has attracted great interest in drug development 19,20 .Meanwhile, targeting P53 is extremely di cult due to at least two reasons.First, agonists of the wild-type P53 function are desired, whereas classical drug discoveries typically prefer inhibitors; second, P53 is a transcription factor, which is "undruggable" due to a lack of enzymatic activities and activity-in uencing "pockets" for binding of small molecules.The P53 reactivator PC14586 exhibited encouraging effects in clinical trials, but it targets only the P53 Y220C mutant 21 .Upregulation of the wild-type P53 may offer a more universal therapeutic strategy that inhibits cancer growth and tumorigenesis 22 .The previously reported P53 upregulating compounds function via inhibition or lowering of its E3 ligase MDM2.Under normal conditions, the P53 level is usually kept low due to the poly-ub by the E3 ligase MDM2 or MDMX.Thus, MDM2 inhibitors such as Nutlin-3a may increase the P53 level and decrease cancer cell viability in vitro and in vivo in wild-type P53 models [23][24][25] .Thus, the Nutlin-3a derivative RG7388 entered clinical trials 26 .Upregulating P53 could also be achieved by inhibition of the ubiquitin-speci c protease 7 (USP7), which is a MDM2-targeting DUB that may deubiquitinate MDM2 27,28 .USP7 inhibitors have been developed to upregulate P53 and degrade MDM2, without causing a feedback upregulation of MDM2 29 .Meanwhile, E3 ligase inhibition or lowering may in uence all its different substrates and increase the risk of side effects.In fact, MDM2 sustains STAT5 stability and contributes to T cell-mediated antitumor immunity, and thus MDM2 de ciency in T cells promotes cancer growth 30 .A very recent study also attempted to upregulate P53 by DUBTACs, but it requires an additional P53-binding DNA oligo in addition to DUBTACs and the mechanism requires quaternary complex formation 31 .Thus, small molecular compounds that upregulate P53 directly are highly desired.Beside MDM2, USP7 has also been reported as a potential P53-targeting DUB that may deubiquitinate endogenous P53 32,33 .Thus, we hypothesized that DUBEGs enhancing the P53-USP7 interaction without in uencing the USP7 enzymatic activity may provide a new way to upregulate P53 and provide proof-ofconcept evidence for DUBEGs.
Molecular glues were identi ed primarily from serendipitous discoveries, and identifying them by screening approaches is emerging but still highly challenging 34 .Notably, no previous studies have been reported to identify upregulating molecular glues.To resolve this problem, we established an unbiased screening platform that may detect potential ternary complex formation in vitro and used it to screen for potential P53 DUBEGs.We further investigated their functions and mechanisms of action.Finally, to validate the system's generality, we used it to screen for potential DUBEGs targeting another TSP, PTEN 35 .

Identi cation of potential P53-USP7 molecular glues
To identify compounds that may enhance P53-USP7 interaction via binding to these proteins, we stamped a small molecule microarray (SMM) consisting of 3,375 compounds in duplicates onto isocyanate-functionalized glass slides, on which small molecules were immobilized through covalent bonds.We then ew through the recombinant puri ed USP7 catalytic domain with the N-terminal maltose binding protein (MBP) tag (MBP-USP7CD) and MBP-P53 (Extended Data Fig. 1a-b) proteins sequentially (Fig. 1a).The binding of the ow-through protein to speci c compound spots could then be detected by a label-free oblique-incidence re ectivity difference (OI-RD) microscope 36,37 .By owing through the two proteins sequentially, the potential USP7-compound-P53 ternary complex formation could be detected on the SMM (Fig. 1a).Meanwhile, compounds that interact with P53 or USP7 alone were excluded.The owing through of MBP (Extended Data Fig. 1c) was performed as a negative control for non-speci c or the MBP tag-binding compounds.
Based on the screening with two SMMs (4 repeats of each compound), we identi ed three compounds, bromocriptine (BC) (4L10), liothyronine (8C10), and conivaptan (7E4), that may interact with MBP-USP7 and enhance subsequent MBP-P53 tethering while exhibit no binding to MBP (Fig. 1b).Phenyltoloxamine (5K18) also exhibited binding signals for MBP-USP7 and MBP-P53, but it is likely a false positive because it may also interact with the control protein MBP (Fig. 1b).

Potential P53-usp7 Molecular Glues Increased P53 Levels In A Proteasome-And Usp7-Dependent Manner
We then investigated whether these potential P53-USP7 molecular glues may elevate endogenous P53 levels.All three hit compounds (4L10, 8C10, and 7E4) signi cantly upregulated P53 in a dose-dependent manner in HeLa cells at ~ µM concentrations (Fig. 2a-c).We then discovered that these compounds interact with both P53 and USP7 as detected by Bio-Layer Interferometry (BLI) (Extended Data Fig. 2).5K18 failed to elevate the P53 level (Fig. 2d), consistent with the speculation that it is a false positive as suggested by its possible binding to MBP (Fig. 1b).The hit compounds' 2D structures and information are presented in Fig. 2e.
We further investigated the mechanism of actions of these potential P53-USP7 molecular glues.The P53expressing mRNA level was not in uenced by the treatment of these compounds (Fig. 3a), suggesting that the effects were post-transcriptional.Meanwhile, the half-life of P53 was signi cantly prolonged (Fig. 3b & Extended Data Fig. 4f, measured by the decay of P53 after the cycloheximide treatment to block protein synthesis), con rming that the upregulation effects were mediated by protein stabilization.Finally, treatment of the proteasome inhibitor epoxomicin signi cantly blocked P53 upregulation induced by these compounds (Fig. 3c), suggesting that the effects were mediated by the proteasomal degradation of P53.Taken together, the mechanism of action of these potential P53-USP7 molecular glues is consistent with the predicted inhibition of P53's proteasomal degradation.
We then investigated the predicted USP7-dependence of the compound-induced P53 upregulation.USP7 knockout in HeLa cells largely blocked the compounds' effects on P53 (Fig. 3d), con rming the involvement of USP7.Expressing USP7 cDNA in the USP7 knockout HeLa cells stored compounds' capability of upregulating P53 (Fig. 3e-g), further con rming the USP7-dependent mechanism.The baseline level of P53 was increased in the USP7 KO cells (Fig. 3d), consistent with previous reports 27,28 , although we observed variation of the P53 baseline level in different clones (not shown).

The Potential P53-usp7 Molecular Glues Decreased Poly-ub Of P53
The P53-USP7 molecular glues are supposed to stabilize P53 by decreasing its poly-ub by tethering it to the DUB USP7.Thus, we tested the poly-ub of P53 by immunoprecipitating it in HeLa cells and detected its poly-ub by Western blots.Since the poly-ub form of P53 is rapidly degraded by the proteasome, we applied the proteasome inhibitor epoxomicin to the cells to enable its detection.As a low-abundance protein, the poly-ub signal of P53 is extremely weak.Thus, we over-expressed His-tagged ubiquitin (Hisub) in the cells to further facilitate its detection.The His-ub transfection was performed in a large plate to ensure the same His-ub transfection e ciency, and the cells were then suspended and plated into individual wells for the compounds' treatment.All three potential P53-USP7 molecular glues drastically reduced the poly-ub level (Fig. 3h), consistent with the predicted deubiquitylation mechanism.

Bromocriptine (Bc) May Tether P53 To Usp7 Via Interacting With Both Proteins
Among the three hits, BC exhibited the most potent P53 upregulation effects and is an FDA-approved drug for treating type 2 diabetes, pituitary prolactinomas, acromegaly and Parkinson's disease 38 , and thus we focused on it for further characterization and mechanistic studies.The dose dependence of BC exhibited a "hook" effect with an optimal dose at 10 µM (Fig. 4a), suggesting that excessive BC may interact with the two proteins separately, consistent with a bifunctional mechanism.After the BC treatment at 10 µM, P53 elevation appeared at ~ 8 hours and reached a plateau at ~ 24 hours, which is much longer than the endogenous half-life of P53 (Fig. 4b) 39 , consistent with the stabilization mechanism.Besides HeLa cells, BC also increased the P53 level in SJSA-1 cells (Fig. 4c), another cancer cell line expressing wild-type P53 25 .
BC interacts with both P53 and USP7 at ~ µM a nity as validated by BLI (Extended Data Fig. 2) and Isothermal Titration Calorimetry (ITC) (Extended Data Fig. 3a) using recombinant puri ed proteins (Extended Data Fig. 1b & Extended Data Fig. 1d).Thus, we further investigated their potential binding sites for BC.We performed hydrogen-deuterium exchange (HDX) analyses 40 and revealed BC's potential binding site at the FLQKTDPKDPAN peptide region in the USP7's catalytic domain (Extended Data Fig. 3bc).Such studies were technically challenging for P53, which contains exible regions.Thus, we expressed different P53 protein fragments (Extended Data Fig. 1e) and tested their potential binding with BC, guided by computational docking analyses.We revealed that BC likely interacts with the TAD I-TAD II domain (LP47) in the N-terminus of P53 (Extended Data Fig. 3d-e).The binding sites revealed by these experiments are also consistent with the ones predicted by docking (Extended Data Fig. 3f).To further validate the potential molecular glue mechanism, we performed the pull-down experiments using recombinant puri ed proteins (Extended Data Fig. 1b-d) and con rmed that BC signi cantly enhanced the P53-USP7 interaction (Fig. 4d-e).

Bc Did Not In uence Usp7 Enzymatic Activities
The binding of BC to USP7 may enable BC's molecular glue's function by engaging USP7.Meanwhile, this may also lead to possible changes in the USP7's enzymatic activity.Thus, we tested the other substrates of UPS7, DAXX and PTEN.We observed no changes in their levels (Extended Data Fig. 4a), suggesting that the USP7 activity was unchanged.We further con rmed this by the in vitro enzymatic assay measuring the enzymatic activity of USP7 and observed no changes (Extended Data Fig. 4b).The MDM2 level and activity may also in uence the level of P53 and its poly-ub.We thus investigated its level and observed a signi cant increase rather than a decrease in the HeLa cells (Extended Data Fig. 4c).The increase of MDM2 cannot be the contributor to the increased P53 level because the direction of its change is predicted to lower P53.The observed MDM2 elevation is likely due to the compensatory transcriptional feedback response to the P53 elevation, which has been reported previously 41,42 .
The D2-like Dopamine Receptors Are Not Involved BC is known as a selective D2-like dopamine receptor agonist.Meanwhile, BC is unlikely to regulate P53 through the D2-like dopamine receptors, because the observed hook effects and effective concentration range (several µM) are inconsistent with the D2-like receptor activation mechanism (EC50 ~ nM) 43 .To further exclude this possibility, we tested another potent D2-like dopamine receptor agonist quinpirole hydrochloride and observed no signi cant changes in the P53 level (Extended Data Fig. 4d), validating the D2-like dopamine receptor-independent mechanism.We also blocked D2-like dopamine receptors with tri uoperazine, and then treated the cells with BC versus the DMSO control.The P53 upregulation by BC was unaffected (Extended Data Fig. 4e), further con rming that D2-like dopamine receptors were not involved.

The P53 Upregulation Effects Could Be Competitively Inhibited
In contrast to quinpirole, the D2-like dopamine receptor agonist that shares similar activities with BC on the known target but not P53, a BC's structural analog BC-AN1 was capable of elevating P53 with similar e cacy to the one of BC (Fig. 5a).In comparison, we also identi ed another structural analog BC-AN2, which was incapable of elevating the P53 level (Fig. 5b).These structural analogs (Fig. 5c) provide chemical biology tools to further elucidate the mechanism of action of BC as a potential P53-USP7 molecular glue.
To elucidate why BC and BC-AN1 could elevate P53 whereas BC-AN2 could not, we tested their binding with P53 or USP7 by the Cell Thermal Shift Assay (CETSA) for intracellular compound-protein interactions.All three compounds obviously shifted the thermal stability of P53 but not TUBB (Extended Data Fig. 5a-c), suggesting that all compounds may interact with P53 in the cells.Meanwhile, only BC and BC-AN1 but not BC-AN2 shifted the thermal stability of USP7 (Extended Data Fig. 5a-c), suggesting that BC-AN2 may not interact with USP7 in the cells.We further con rmed these observations and measured the compound-protein a nities by real time OI-RD (Extended Data Fig. 6a-c) 44 .Taken together, that BC and BC-AN1 can interact with both P53 and USP7, whereas BC-AN2 only interacts with P53 but not USP7CD.These results possibly explain why only BC and BC-AN1 but not BC-AN2 could elevate the P53 level, suggesting that interacting with both proteins might be required.
Taking advantage of BC-AN2's binding to only P53 but not USP7, we performed a competition experiment to test if the inactive structural analog BC-AN2 may compete with the active ones for the binding of P53 to inhibit their P53 upregulation effects.We pre-incubated the cells with BC-AN2 at 50 µM for two hours, and then treated the cells with BC or BC-AN1 at 10 µM.In this case, BC and BC-AN1 could no longer upregulate p53 (Fig. 5d-e).The observed competition effects con rmed that BC and BC-AN1 upregulated P53 via the molecular glue mechanism and the intracellular binding to both P53 and USP7 was required for their regulation of P53.
Bc And Bc-an1 Activated P21 Expression And Inhibited Cancer Cell Proliferation p21 (WAF1/CIP1) is a major downstream factor of P53 and a potent tumor suppressor that prevents uncontrolled cell proliferation by inducing cell cycle arrest 45 .It is an inhibitor of Cyclin-dependent kinases (CDK) and is transcriptionally activated by P53 46 .Consistent with the observed P53 upregulation, treatment of BC signi cantly increased the level of p21-expressing mRNA (Fig. 6a), illustrating the potential functional impacts of these compounds via P53 upregulation.
We further investigated the compounds' effects on the proliferation of cancer cells.BC and BC-AN1 signi cantly suppressed the proliferation of HeLa cells (Fig. 6b).Such effects were obviously reduced in USP7-KO HeLa cells or HeLa cells with P53 knocked down (Fig. 6c-d).The data demonstrate that the effects at the cellular level depend on the USP7-mediated P53 upregulation, con rming the functional impact of the molecular glue mechanism.The suppression of cell proliferation by BC or BC-AN1 was also observed in another cancer cell line SJSA-1, with a signi cant elevation of apoptosis signals as well (Fig. 6e-f).

Discovery Of A Potential Pten-usp7 Molecular Glue Using The Same Screening Platform
To validate the generality of our screening platform for upregulating molecular glues, we performed a similar screen aiming at identifying DUBEGs for another TSP, PTEN.Through highly similar screening procedures except replacing MBP-P53 with MBP-PTEN, we identi ed one possible PTEN-USP7 molecular glue, WZ8040.WZ8040 signi cantly upregulated PTEN in the cancer cell line DU145 at ~ µM concentrations (Fig. 7a) and it interacted with both PTEN and USP7 (Extended Data Fig. 7a-b).We further investigated the mechanism of action of this potential PTEN-USP7 molecular glue.Treatment of the proteasome inhibitor epoxomicin or MG132 largely blocked the PTEN upregulation induced by WZ8040.In contrast, the treatment of the autophagy inhibitor chloroquine (CQ) did not (Fig. 7b & Extended Data Fig. 7c), con rming the proteasome-dependence of the effect.Note that the caspase inhibitor Z-VAD-FMK (FMK) was added to prevent drastic cell death of DU145 cells caused by epoxomicin (Fig. 7b).USP7 knockdown in DU145 cells largely blocked the WZ8040's effects on PTEN (Fig. 7c), con rming the USP7dependence.
Since the WZ8040 is an inhibitor of a mutated form of EGFR, T790M, we investigated whether EGFR is involved.Knock-down of EGFR did not block the PTEN upregulation effect of WZ8040 (Extended Data Fig. 7e), suggesting that WZ8040 regulates PTEN in an EGFR-independent manner.
Treatment of WZ8040 signi cantly suppressed the proliferation of several cancer cell lines, including DU145, C33A, and Hec-1-a, in a dose-dependent manner (Fig. 7d-f).It also signi cantly elevated the apoptosis signals of DU145 cells (Extended Data Fig. 7d).The 2D structure and information of WZ8040 is presented in Fig. 7g.
Taken together, WZ8040 may function as a PTEN-USP7 molecular glue that upregulates PTEN and suppresses relevant cancer cell growth.

Discussion
Our study identi ed potential P53 DUBEGs through an in vitro screening platform.As the famous "guardian of the genome", P53 is a promising drug target for cancer and other related diseases.While certain P53 upregulating compounds have been discovered and entered clinical trials, our study provides the proof-of-concept of upregulating P53 via a novel molecular glue mechanism, which may open a direction for P53-targeted drug discovery.Besides cancer, P53 also contributes to the etiology of other non-neoplastic diseases and rare hereditary diseases such as the Li-Fraumeni Syndrome 47 , which may all bene t from new P53-targeting strategies.
While there are many P53 mutations related to tumorigenesis, we utilized the wild-type P53 protein for the screening based on several considerations.First, there are many P53 mutations associated with cancer risks and many cancer cell types carry more than one P53 mutation.Screenings using all the different P53 mutants may be ine cient.Second, while somatic TP53 mutations occur in almost every kind of cancer at a rate from 5-50%, most of the cancer cell types have at least one copy of wild-type P53 48 .Thus, elevating the wild-type P53 level is feasible in most cases.Third, while several P53 mutations may function through a gain-of-function (GOF) mechanism, most P53 mutants still function via the loss-offunction or the dominant negative mechanisms 48 , justifying the rationale of increasing wild-type P53.Nonetheless, it is desirable to test the compounds' binding to different P53 mutants or to screen for mutant-speci c DUBEGs in future studies.Based on our mapping of the compound's binding site in P53 (Extended Data Fig. 3d-f), we may predict that mutations outside the LP47 region may not abolish the compound's effects, although direct experiments are needed to con rm the results.
We chose USP7 as the potential DUB for DUBEGs mainly because it is relatively easy to purify with its catalytic domain's crystal structure reported and it is a potential DUB for P53 deubiquitylation 32,33,49,50 .
USP7 inhibitors can also upregulate P53, likely via lowering MDM2 29 .Meanwhile, the activity of USP7 may also in uence many other substrates and USP7-engaging molecular glues may provide an additional strategy to upregulate P53.
The compounds that we identi ed likely upregulate P53 through the molecular glue mechanism.They upregulated P53 in a proteasome and USP7-dependent manner (Fig. 3c-e) and decreased the polyubiquitination of P53 (Fig. 3h).The top hit compound BC also enhanced the USP7-P53 interaction (Fig. 4d-e).Its P53-regulating effect is competitively blocked by an inactive structural analog that only interacts with P53 but not USP7 (Fig. 5d), further con rming the molecular glue mechanism in cells.Consistent with this, BC seems to interact with both P53 at its TAD I-TAD II domain and USP7 at its FLQKTDPKDPAN peptide region in the catalytic domain (Extended Data Fig. 3), exhibiting a hook effect of P53 upregulation (Fig. 4a).This differs from immunomodulatory imide drugs, which are molecular glues that mainly interact with the E3 ligases but not the target proteins 51,52 .The critical next step in developing this concept would be resolving the compound-P53 and compound-USP7 interaction interfaces, which may further con rm the mechanism and enable the development of potential platform technologies for targeted protein upregulation.The activity of BC and its active/inactive analogs provide some preliminary clues, but extensive medicinal chemistry and structural biology work are necessary to elucidate the answers in the future.
BC is indicated in the symptomatic treatment of idiopathic or postencephalitic Parkinson's disease, likely via its dopamine receptor agonist activity 53 .BC may also bene t prolactin-secreting adenomas and reduce the tumor size as observed decades ago while the mechanisms were unclear 54 .The P53 upregulating activity of BC may also contribute to tumor size reduction, although this has not been studied.The potency and e cacy of BC or BC-AN1 are still low compared to the MDM2 inhibitor that entered clinical trials (Nutlin-3a and its analogs) 25 .Extensive medicinal chemistry efforts are needed to further improve the potency and e cacy of the compounds.
While both our DUBEGs and the reported DUBTACs function through DUBs, their mechanisms are distinct.Besides having distinct chemical structures and functioning through different DUBs, the compoundprotein interaction mechanisms are also different.DUBTACs interact with the DUB OTUB1 covalently, triggering persistent occupation and allosteric changes of OTUB1.While this leads to the high-a nity binding that may contribute to the substantial effect size observed 6 , it may also lead to long-term alterations of the substrates and prevent the possible recycling of degraders inside the cells.Meanwhile, the DUBEGs we identi ed have weaker but more reversible interactions with the DUB, and thus likely have less in uence on the DUB's substrate and allow the compounds to function in a catalytic manner.Finally, the DUBTACs are designed by assembly of different chemical moieties and are typically large, and DUBEGs could be smaller than DUBTACs.
We validated the generality of the screening platform by identifying DUBEGs of another target PTEN.Taking advantage of SMM and OI-RD, the screening system can detect potential ternary complex formation in vitro (Fig. 1a), which is critical to identify molecular glues that induce or enhance the ternary complex formation.We realize the potential caveat that the system may capture false positives due to the in vitro setting, but this could be addressed by cellular experiments including the CETSA assay that we performed to validate the binding (Extended Data Fig. 5).In addition, a strong in vitro baseline binding of the target and DUB without compounds' treatment may also lead to false positives.In this case, almost all the positive compounds showing signals when owing through the rst protein will also exhibit signals when owing through second protein, which may raise alerts of the screening results and more detailed measurement of compound-induced a nity changes could be performed to address this.
In summary, we have identi ed DUBEGs capable of stabilizing P53 or PTEN through engaging USP7 and established a screening platform that could be utilized to screen for DUBEGs or potentially other types of molecular glues for disease-relevant proteins.

Declarations Figures
Figure 1 Identi cation of potential P53-USP7 molecular glues by in vitro screenings.a, The schematic illustration of the screening design.The SMM was stamped in duplicates onto isocyanate-functionalized glass slides.OI-RD screenings were performed by owing through the indicated recombinant puri ed proteins, including MBP, MBP-USP7 catalytic domain (MBP-USP7CD), and MBP-P53.Flowing through of the BSAcontaining buffer was applied before and after owing through of each tested protein to wash out nonspeci c binding.The catalytic domain of USP7 was used to facilitate the protein puri cation and enable direct engagement of the domain by potential molecular glues.The compounds binding to each protein could be identi ed in the difference images obtained by subtracting the image before owing through from one after owing through, as illustrated by the bright circles.Compounds that exhibit positive signals in both difference images of MBP-USP7CD and MBP-P53 but not MBP were selected as hits.b, The representative screening results (from two replicated SMM chips) showing the three positive hits (in solid red rectangles) and one false positive hit that also binds with MBP (in the dashed red rectangle).
The compounds were annotated by their positions in the microplates used to print SMM.

Supplementary Files
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