Biology subjects

Dovala, D.

Publications and source records attributed to Dovala, D..

18 recordsLinked to original sources

bioRxiv2025

Targeted Transcriptional Repression by Induced Proximity

Most cancer-driving proteins remain "undruggable" due to the absence of ligandable pockets and their reliance on intrinsically disordered or protein-DNA/protein-protein interactions. Transcription factors, which orchestrate oncogenic gene expression programs, are particularly challenging: they turn over rapidly, evade durable pharmacological inhibition, and resist even emerging targeted protein degradation strategies. Here, we describe a new induced-proximity therapeutic modality, Transcriptional Regulation via Active Control of Epigenetic Reprogramming (TRACER), that enforces locus-specific transcriptional silencing by recruiting endogenous corepressor complexes to transcription factor binding sites. We developed small-molecule TRACERs that tether methyl-CpG binding domain protein 2 (MBD2) and the Nucleosome Remodeling and Deacetylase (NuRD) complex to transcription factor-directed ligands. An estrogen receptor (ER) TRACER potently suppressed ER transcriptional activity in breast cancer cells, downregulated canonical ER target genes, and required MBD2 and histone deacetylase (HDAC1/2) for activity, confirming on-target epigenetic repression. Extending this approach to prostate cancer, an androgen receptor (AR) TRACER transcriptionally repressed both full-length AR and the drug-resistant truncation variant AR-V7, achieving >90% inhibition of AR transcriptional activity in androgen-independent prostate cancer cells with locus-specific gene repression. We further show that TRACERs can be modularly reprogrammed to recruit alternative repressors, including PRC2. Collectively, these findings establish TRACERs as a generalizable modality to pharmacologically silence undruggable transcription factors through targeted epigenetic reprogramming, offering a powerful new strategy for treating cancers refractory to existing therapies.

biochemistry↗

bioRxiv2025

Cooperativity and Communication between the Active Sites of the Dimeric SARS-CoV-2 Main Protease

The coronaviral main protease (Mpro) is essential for the replication of the virus, and has been the subject of various biochemical, structural and enzymatic studies, as well as a drug target against SARS-CoV-2 infections. SARS-CoV-2 Mpro is known to be active as a dimer, with the N terminus of one protomer completing a key active site pocket of the other protomer. Despite apparent cooperativity in catalytic activity, how the two distal active sites in the dimer communicate and might be modulating binding and/or catalysis at the other remain to be clarified. Here, we have investigated the interplay between cooperativity, dimerization, and substrate cleavage in SARS-CoV-2 Mpro through a combination of enzymatic assays, crystal structures, and protein characterization. To disentangle the contribution of each active site to the observed enzymatic activity, we developed a cleavage assay involving heterodimers of active and inactive (C145A or inhibitor-bound) monomers. Strikingly, we found that heterodimerization increased cleavage efficiency per active monomer. Additionally, we mapped a network of critical residues bridging the two active sites and probed this network through engineered mutations. By dissecting the cooperativity and communication between the active sites, we provide new insights into the Mpro reaction cycle and functional significance of its dimeric architecture.

biochemistry↗

bioRxiv2025

Sulfinyl Aziridines as Stereoselective Covalent Destabilizing Degraders of the Oncogenic Transcription Factor MYC

While MYC is a significant oncogenic transcription factor driver of cancer, directly targeting MYC has remained challenging due to its intrinsic disorder and poorly defined structure, deeming it "undruggable." Whether transient pockets formed within intrinsically disordered and unstructured regions of proteins can be selectively targeted with small molecules remains an outstanding challenge. Here, we developed a bespoke stereochemically-paired spirocyclic oxindole aziridine covalent library and screened this library for degradation of MYC. Through this screen, we identified a hit covalent ligand KL2-236, bearing a unique sulfinyl aziridine warhead, that engaged MYC in vitro as pure MYC/MAX protein complex and in situ in cancer cells to destabilize MYC, inhibit MYC transcriptional activity and degrade MYC in a proteasome-dependent manner through targeting intrinsically disordered C203 and D205 residues. Notably, this reactivity was most pronounced for specific stereoisomers of KL2-236 with a diastereomer KL4-019 that was largely inactive. Mutagenesis of both C203 and D205 completely attenuated KL2-236-mediated MYC degradation. We have also optimized our initial KL2-236 hit compound to generate a more durable MYC degrader KL4-219A in cancer cells. Our results reveal a novel ligandable site within MYC and indicate that certain intrinsically disordered regions within high-value protein targets, such as MYC, can be interrogated by isomerically unique chiral small molecules, leading to destabilization and degradation.

cancer biology↗

bioRxiv2025

Molecular Mechanisms of Drug Resistance and Compensation in SARS-CoV-2 Main Protease: The Interplay Between E166 and L50

The SARS-CoV-2 main protease (Mpro) is essential for viral replication, and a primary target for COVID-19 antivirals. Direct-acting antivirals such as nirmatrelvir, the active component of Paxlovid, target the Mpro active site to block viral polyprotein cleavage and thus replication. However, drug resistance mutations at the active site residue Glu166 (E166) have emerged in in vitro selection studies, raising concerns about the durability of current antiviral strategies. Here, we investigate the molecular basis of drug resistance conferred by E166A and E166V mutations against nirmatrelvir and the related PF-00835231, individually and in combination with the distal mutation L50F. We found that E166 mutations reduce nirmatrelvir potency by up to 3000-fold while preserving substrate cleavage, with catalytic efficiency reduced by only up to 2- fold. This loss of catalytic efficiency was compensated for by the addition of L50F in the double- mutant variants. We have determined three cocrystal structures of the E166 variants (E166A, E166V, and E166V/L50F) bound to PF-00835231. Comparison of these structures with wild- type demonstrated that E166 is crucial for dimerization and for shaping the substrate-binding S1 pocket. Our findings highlight the mutability of E166, a prime site for resistance for inhibitors that leverage direct interactions with this position, and the potential emergence of highly resistant and active variants in combination with the compensatory mutation L50F. These insights support the design of inhibitors that target conserved protease features and avoid E166 side- chain interactions to minimize susceptibility to resistance.

biochemistry↗

bioRxiv2024

A small molecule VHL molecular glue degrader for cysteine dioxygenase 1

The Von Hippel-Lindau Tumor Suppressor gene product (pVHL) is an E3 ligase substrate receptor that binds proline-hydroxylated HIF1-, leading to its ubiquitin-dependent degradation. By using protein arrays, we identified a small molecule that binds the HIF1- binding pocket on pVHL and functions as a molecular glue degrader of the neosubstrate cysteine dioxygenase (CDO1) by recruiting it into the VHL-cullin-ring E3 ligase complex and leading to its selective degradation. The CDO1 binding region involved in VHL recruitment was characterized through a combination of mutagenesis and protein-protein docking coupled with molecular dynamics-based solvation analysis. The X-ray structure of the ternary complexes of VHL, CDO1, and degrader molecules confirms the binding region prediction and provides atomic insights into key molecular glue interactions.

biochemistry↗

bioRxiv2023

Contributions of hyperactive mutations in Mpro from SARS-CoV-2 to drug resistance

The appearance and spread of mutations that cause drug resistance in rapidly evolving diseases, including infections by SARS-CoV-2 virus, are major concerns for human health. Many drugs target enzymes, and resistance-conferring mutations impact inhibitor binding and/or enzyme activity. Nirmatrelvir, the most widely used inhibitor currently used to treat SARS-CoV-2 infections, targets the main protease (Mpro) preventing it from processing the viral polyprotein into active subunits. Our previous work systematically analyzed resistance mutations in Mpro that reduce binding to inhibitors; here we investigate mutations that affect enzyme function. Hyperactive mutations that increase Mpro activity can contribute to drug resistance but had not been thoroughly studied. To explore how hyperactive mutations contribute to resistance, we comprehensively assessed how all possible individual mutations in Mpro affect enzyme function using a mutational scanning approach with a FRET-based yeast readout. We identified hundreds of mutations that significantly increased Mpro activity. Hyperactive mutations occurred both proximal and distal to the active site, consistent with protein stability and/or dynamics impacting activity. Hyperactive mutations were observed three times more than mutations which reduced apparent binding to nirmatrelvir in recent studies of laboratory grown viruses selected for drug resistance. Hyperactive mutations were also about three times more prevalent than nirmatrelvir-binding mutations in sequenced isolates from circulating SARS-CoV-2. Our findings indicate that hyperactive mutations are likely to contribute to the natural evolution of drug resistance in Mpro and provide a comprehensive list for future surveillance efforts.

biochemistry↗

bioRxiv2023

Ophiobolin A Covalently Targets Complex IV Leading to Mitochondrial Metabolic Collapse in Cancer Cells

SummaryOphiobolin A (OPA) is a sesterterpenoid fungal natural product with broad anti-cancer activity. While OPA possesses multiple electrophilic moieties that can covalently react with nucleophilic amino acids on proteins, the proteome-wide targets and mechanism of OPA remain poorly understood in many contexts. In this study, we used covalent chemoproteomic platforms to map the proteome-wide reactivity of OPA in a highly sensitive lung cancer cell line. Among several proteins that OPA engaged, we focused on two targets--cysteine C53 of HIG2DA and lysine K72 of COX5A--that are part of complex IV of the electron transport chain and contributed significantly to the anti-proliferative activity. OPA activated mitochondrial respiration in a HIG2DA and COX5A-dependent manner, led to an initial spike in mitochondrial ATP, but then compromised mitochondrial membrane potential leading to ATP depletion. We have used chemoproteomic strategies to discover a unique anti-cancer mechanism of OPA through activation of complex IV leading to compromised mitochondrial energetics and rapid cell death.

biochemistry↗

bioRxiv2023

Systematic analyses of the resistance potential of drugs targeting SARS-CoV-2 main protease

Drugs that target the main protease (Mpro) of SARS-CoV-2 are effective therapeutics that have entered clinical use. Wide-scale use of these drugs will apply selection pressure for the evolution of resistance mutations. To understand resistance potential in Mpro, we performed comprehensive surveys of amino acid changes that can cause resistance in a yeast screen to nirmatrelvir (contained in the drug Paxlovid), and ensitrelvir (Xocova) that is currently in phase III trials. The most impactful resistance mutation (E166V) recently reported in multiple viral passaging studies with nirmatrelvir showed the strongest drug resistance score for nirmatrelvir, while P168R had the strongest resistance score for ensitrelvir. Using a systematic approach to assess potential drug resistance, we identified 142 resistance mutations for nirmatrelvir and 177 for ensitrelvir. Among these mutations, 99 caused apparent resistance to both inhibitors, suggesting a strong likelihood for the evolution of cross-resistance. Many mutations that exhibited inhibitor-specific resistance were consistent with distinct ways that each inhibitor protrudes beyond the substrate envelope. In addition, mutations with strong drug resistance scores tended to have reduced function. Our results indicate that strong pressure from nirmatrelvir or ensitrelvir will select for multiple distinct resistant lineages that will include both primary resistance mutations that weaken interactions with drug while decreasing enzyme function and secondary mutations that increase enzyme activity. The comprehensive identification of resistance mutations enables the design of inhibitors with reduced potential of developing resistance and aids in the surveillance of drug resistance in circulating viral populations.

molecular biology↗

bioRxiv2023

Crystal Structures of Inhibitor-Bound Main Protease from Delta- and Gamma-Coronaviruses

With the spread of SARS-CoV-2 throughout the globe to cause the COVID-19 pandemic, the threat of zoonotic transmissions of coronaviruses (CoV) has become even more evident. As human infections have been caused by alpha- and beta-CoVs, structural characterization and inhibitor design mostly focused on these two genera. However, viruses from the delta and gamma genera also infect mammals and pose potential zoonotic transmission threat. Here, we determined the inhibitor-bound crystal structures of the main protease (Mpro) from the delta-CoV porcine HKU15 and gamma-CoV SW1 from beluga whale. Comparison with the apo structure of SW1 Mpro, which we also present here, enabled identifying structural arrangements upon inhibitor binding at the active site. The binding modes and interactions of two covalent inhibitors, PF-00835231 (lufotrelvir) bound to HKU15 and GC376 bound to SW1 Mpro, reveal features that may be leveraged to target diverse coronaviruses and toward structure-based design of pan-CoV inhibitors.

biochemistry↗

bioRxiv2022

Targeted Protein Degradation through E2 Recruitment

Targeted protein degradation (TPD) with Proteolysis Targeting Chimeras (PROTACs), heterobifunctional compounds consisting of protein targeting ligands linked to recruiters of E3 ubiquitin ligases, has arisen as a powerful therapeutic modality to induce the proximity of target proteins with E3 ligases to ubiquitinate and degrade specific proteins in cells. Thus far, PROTACs have primarily exploited the recruitment of E3 ubiquitin ligases or their substrate adapter proteins but have not exploited the recruitment of more core components of the ubiquitin-proteasome system (UPS). In this study, we used covalent chemoproteomic approaches to discover a covalent recruiter against the E2 ubiquitin conjugating enzyme UBE2D--EN67--that targets an allosteric cysteine, C111, without affecting the enzymatic activity of the protein. We demonstrated that this UBE2D recruiter could be used in heterobifunctional degraders to degrade neo-substrate targets in a UBE2D-dependent manner, including BRD4 and the androgen receptor. Overall, our data highlight the potential for the recruitment of core components of the UPS machinery, such as E2 ubiquitin conjugating enzymes, for TPD, and underscore the utility of covalent chemoproteomic strategies for identifying novel recruiters for additional components of the UPS.

biochemistry↗

bioRxiv2022

Rational Chemical Design of Molecular Glue Degraders

Targeted protein degradation with molecular glue degraders has arisen as a powerful therapeutic modality for eliminating classically undruggable disease-causing proteins through proteasome-mediated degradation. However, we currently lack rational chemical design principles for converting protein-targeting ligands into molecular glue degraders. To overcome this challenge, we sought to identify a transposable chemical handle that would convert protein-targeting ligands into molecular degraders of their corresponding targets. Using the CDK4/6 inhibitor Ribociclib as a prototype, we identified a covalent handle that, when appended to the exit vector of Ribociclib, induced the proteasome-mediated degradation of CDK4 in cancer cells. Covalent chemoproteomic profiling of this CDK4 degrader revealed covalent interactions with cysteine 32 of the RING family E3 ubiquitin ligase RNF126. Structural modification of our initial covalent scaffold led to an improved CDK4 degrader with the development of a but-2-ene, 1,4-dione ("fumarate") handle that showed improved interactions with RNF126. Thereafter, we worked to identify the minimum covalent motif required for interaction with RNF126, which we then transplanted onto chemically related and un-related protein-targeting ligands. This strategy successfully produced molecules which induced the degradation of several proteins across diverse protein classes, including BRD4, BCR-ABL and c-ABL, PDE5, AR and AR-V7, BTK, LRRK2, and SMARCA2. Our study undercovers a design strategy for converting protein-targeting ligands into covalent molecular glue degraders.

biochemistry↗

bioRxiv2022

Chemoproteomics-Enabled Discovery of a Covalent Molecular Glue Degrader Targeting NF-κB

Targeted protein degradation using heterobifunctional Proteolysis-Targeting Chimeras (PROTACs) or molecular glues has arisen as a powerful therapeutic modality for degrading disease targets. While PROTAC design is becoming more modular and straightforward, the discovery of novel molecular glue degraders has been more challenging. While several recent studies have showcased phenotypic screening and counter-screening approaches to discover new molecular glue degraders, mechanistically elucidating the ternary complex induced by the small-molecule that led to the initial phenotype--i.e. identifying the degraded target and relevant components of the ubiquitin-proteasome system--has remained cumbersome and laborious. To overcome these obstacles, we have coupled the screening of a covalent ligand library for anti-proliferative effects in leukemia cells with quantitative proteomic and chemoproteomic approaches to rapidly discover both novel covalent molecular glue degraders and their associated ternary complex components and anti-proliferative mechanisms. We have identified a cysteine-reactive covalent ligand EN450 that impairs leukemia cell viability in a NEDDylation and proteasome-dependent manner. Chemoproteomic profiling revealed covalent interaction of EN450 with an allosteric C111 in the E2 ubiquitin ligase UBE2D. Follow-up quantitative proteomic profiling revealed the proteasome-mediated degradation of the oncogenic transcription factor NFKB1 as a putative degradation target. Subsequent validation studies demonstrated that EN450 induced the ternary complex formation between UBE2D and NFKB1 and that both UBE2D and NFKB1 were important for the anti-proliferative mechanisms of EN450. Our study thus puts forth the discovery of a novel molecular glue degrader that uniquely induced the proximity of an E2 ligase with a transcription factor to induce its degradation and anti-proliferative effects in cancer cells. Taken more broadly, our study showcases a rapid and modular approach for discovering novel covalent molecular glue degraders and their respective ternary complex components in an unbiased fashion.

biochemistry↗

bioRxiv2022

Discovery of Potent Pyrazoline-Based Covalent SARS-CoV-2 Main Protease Inhibitors

While vaccines and antivirals are now being deployed for the current SARS-CoV-2 pandemic, we require additional antiviral therapeutics to not only effectively combat SARS-CoV-2 and its variants, but also future coronaviruses. All coronaviruses have relatively similar genomes that provide a potential exploitable opening to develop antiviral therapies that will be effective against all coronaviruses. Among the various genes and proteins encoded by all coronaviruses, one particularly "druggable" or relatively easy-to-drug target is the coronavirus Main Protease (3CLpro or Mpro), an enzyme that is involved in cleaving a long peptide translated by the viral genome into its individual protein components that are then assembled into the virus to enable viral replication in the cell. Inhibiting Mpro with a small-molecule antiviral would effectively stop the ability of the virus to replicate, providing therapeutic benefit. In this study, we have utilized activity-based protein profiling (ABPP)-based chemoproteomic approaches to discover and further optimize cysteine-reactive pyrazoline-based covalent inhibitors for the SARS-CoV-2 Mpro. Structure-guided medicinal chemistry and modular synthesis of di- and tri-substituted pyrazolines bearing either chloroacetamide or vinyl sulfonamide cysteine-reactive warheads enabled the expedient exploration of structure-activity relationships (SAR), yielding nanomolar potency inhibitors against Mpro from not only SARS-CoV-2, but across many other coronaviruses. Our studies highlight promising chemical scaffolds that may contribute to future pan-coronavirus inhibitors.

biochemistry↗

bioRxiv2022

Defining the Substrate Envelope of SARS-CoV-2 Main Protease to Predict and Avoid Drug Resistance

Coronaviruses, as exemplified by SARS-CoV-2, can evolve and spread rapidly to cause severe disease morbidity and mortality. Direct acting antivirals (DAAs) are highly effective in decreasing disease burden especially when they target essential viral enzymes, such as proteases and polymerases, as demonstrated in HIV-1 and HCV and most recently SARS-CoV-2. Optimization of these DAAs through iterative structure-based drug design has been shown to be critical. Particularly, the evolutionarily conserved molecular mechanisms underlying viral replication can be leveraged to develop robust antivirals against rapidly evolving viral targets. The main protease (Mpro) of SARS-CoV-2, which is evolutionarily constrained to recognize and cleave 11 specific sites to promote viral maturation, exemplifies one such target. In this study we define the substrate envelope of Mpro by determining the molecular basis of substrate recognition, through nine high-resolution cocrystal structures of SARS-CoV-2 Mpro with the viral cleavage sites. These structures enable identification of evolutionarily vulnerable sites beyond the substrate envelope that may be susceptible to drug resistance and compromise binding of the newly developed Mpro inhibitors.

biochemistry↗

bioRxiv2022

Comprehensive fitness landscape of SARS-CoV-2 Mpro reveals insights into viral resistance mechanisms

With the continual evolution of new strains of SARS-CoV-2 that are more virulent, transmissible, and able to evade current vaccines, there is an urgent need for effective anti-viral drugs. SARS-CoV-2 main protease (Mpro) is a leading target for drug design due to its conserved and indispensable role in the viral life cycle. Drugs targeting Mpro appear promising but will elicit selection pressure for resistance. To understand resistance potential in Mpro, we performed a comprehensive mutational scan of the protease that analyzed the function of all possible single amino acid changes. We developed three separate high-throughput assays of Mpro function in yeast, based on either the ability of Mpro variants to cleave at a defined cut-site or on the toxicity of their expression to yeast. We used deep sequencing to quantify the functional effects of each variant in each screen. The protein fitness landscapes from all three screens were strongly correlated, indicating that they captured the biophysical properties critical to Mpro function. The fitness landscapes revealed a non-active site location on the surface that is extremely sensitive to mutation making it a favorable location to target with inhibitors. In addition, we found a network of critical amino acids that physically bridge the two active sites of the Mpro dimer. The clinical variants of Mpro were predominantly functional in our screens, indicating that Mpro is under strong selection pressure in the human population. Our results provide predictions of mutations that will be readily accessible to Mpro evolution and that are likely to contribute to drug resistance. This complete mutational guide of Mpro can be used in the design of inhibitors with reduced potential of evolving viral resistance.

molecular biology↗

bioRxiv2021

Deubiquitinase-Targeting Chimeras for Targeted Protein Stabilization

Targeted protein degradation is a powerful therapeutic modality that uses heterobifunctional small-molecules to induce proximity between E3 ubiquitin ligases and target proteins to ubiquitinate and degrade specific proteins of interest. However, many proteins are ubiquitinated and degraded to drive disease pathology; in these cases targeted protein stabilization (TPS), rather than degradation, of the actively degraded target using a small-molecule would be therapeutically beneficial. Here, we present the Deubiquitinase-Targeting Chimera (DUBTAC) platform for TPS of specific proteins. Using chemoproteomic approaches, we discovered the covalent ligand EN523 that targets a non-catalytic allosteric cysteine C23 in the K48 ubiquitin-specific deubiquitinase OTUB1. We then developed a heterobifunctional DUBTAC consisting of our EN523 OTUB1 recruiter linked to lumacaftor, a drug used to treat cystic fibrosis that binds {Delta}F508-CFTR. We demonstrated proof-of-concept of TPS by showing that this DUBTAC robustly stabilized {Delta}F508-CFTR in human cystic fibrosis bronchial epithelial cells in an OTUB1-dependent manner. Our study underscores the utility of chemoproteomics-enabled covalent ligand discovery approaches to develop new induced proximity-based therapeutic modalities and introduces the DUBTAC platform for TPS. Editorial summaryWe have developed the Deubiquitinase Targeting Chimera (DUBTAC) platform for targeted protein stabilization. We have discovered a covalent recruiter against the deubiquitinase OTUB1 that we have linked to the mutant {Delta}F508-CFTR targeting cystic fibrosis drug Lumacaftor to stabilize mutant CFTR protein in cells.

biochemistry↗

bioRxiv2020

Screening a library of FDA-approved and bioactive compounds for antiviral activity against SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has emerged as a major global health threat. The COVID-19 pandemic has resulted in over 80 million cases and 1.7 million deaths to date while the number of cases continues to rise. With limited therapeutic options, the identification of safe and effective therapeutics is urgently needed. The repurposing of known clinical compounds holds the potential for rapid identification of drugs effective against SARS-CoV-2. Here we utilized a library of FDA-approved and well-studied preclinical and clinical compounds to screen for antivirals against SARS-CoV-2 in human pulmonary epithelial cells. We identified 13 compounds that exhibit potent antiviral activity across multiple orthogonal assays. Hits include known antivirals, compounds with anti-inflammatory activity, and compounds targeting host pathways such as kinases and proteases critical for SARS-CoV-2 replication. We identified seven compounds not previously reported to have activity against SARS-CoV-2, including B02, a human RAD51 inhibitor. We further demonstrated that B02 exhibits synergy with remdesivir, the only antiviral approved by the FDA to treat COVID-19, highlighting the potential for combination therapy. Taken together, our comparative compound screening strategy highlights the potential of drug repurposing screens to identify novel starting points for development of effective antiviral mono- or combination therapies to treat COVID-19.

microbiology↗

bioRxiv2020

A Strategy to Assess the Cellular Activity of E3 Ligases against Neo-Substrates using Electrophilic Probes

Targeted protein degradation is a rapidly developing therapeutic modality that promises lower dosing and enhanced selectivity as compared to traditional occupancy-driven inhibitors, and the potential to modulate historically intractable targets. While the well-characterized E3 ligases CRBN and VHL have been successfully redirected to degrade numerous proteins, there are approximately 600 predicted additional E3 family members that may offer improved activity, substrate selectivity, and/or tissue distribution; however, characterizing the potential applications of these many ligases for targeted protein degradation has proven challenging. Here, we report the development of an approach to evaluate the ability of recombinant E3 ligase components to support neo-substrate degradation. Bypassing the need for hit finding to identify specific E3 ligase binders, this approach makes use of simple chemistry for Covalent Functionalization Followed by E3 Electroporation into live cells (COFFEE). We demonstrate this method by electroporating recombinant VHL, covalently functionalized with JQ1 or dasatinib, to induce degradation of BRD4 or kinase targets, respectively. Furthermore, by applying COFFEE to SPSB2, a SOCS box and SPRY-domain E3 ligase that has not previously been redirected for targeted protein degradation, we validate this method as a powerful approach to define the activity of previously uncharacterized ubiquitin ligases against neo-substrates.

molecular biology↗