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1.
Cancer Discov ; 2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34544752

RESUMO

Cyclin-dependent kinases 4 and 6 (CDK4/6), represent a major therapeutic vulnerability for breast cancer. The kinases are clinically targeted via ATP competitive inhibitors (CDK4/6i); however, drug resistance commonly emerges over time. To understand CDK4/6i resistance, we surveyed over 1,300 breast cancers and identify several genetic alterations (e.g. FAT1, PTEN or ARID1A loss) converging on upregulation of CDK6. Mechanistically, we demonstrate CDK6 causes resistance by inducing and binding CDK inhibitor INK4 proteins (e.g. p18INK4C). In vitro binding and kinase assays together with physical modeling reveal that the p18INK4C/D-cyclin/CDK6 complex occludes CDK4/6i binding while only weakly suppressing ATP binding. Suppression of INK4 expression or its binding to CDK6 restores CDK4/6i sensitivity. To overcome this constraint, we developed bifunctional degraders conjugating palbociclib with E3 ligands. Two resulting lead compounds potently degraded CDK4/6, leading to substantial antitumor effects in vivo, demonstrating the promising therapeutic potential for retargeting CDK4/6 despite CDK4/6i resistance.

2.
Biochemistry ; 60(34): 2593-2609, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34411482

RESUMO

SHP2 is a protein tyrosine phosphatase that plays a critical role in the full activation of the Ras-MAPK pathway upon stimulation of receptor tyrosine kinases, which are frequently amplified or mutationally activated in human cancer. In addition, activating mutations in SHP2 result in developmental disorders and hematologic malignancies. Several allosteric inhibitors have been developed for SHP2 and are currently in clinical trials. Here, we report the development and evaluation of a SHP2 PROTAC created by conjugating RMC-4550 with pomalidomide using a PEG linker. This molecule is highly selective for SHP2, induces degradation of SHP2 in leukemic cells at submicromolar concentrations, inhibits MAPK signaling, and suppresses cancer cell growth. SHP2 PROTACs serve as an alternative strategy for targeting ERK-dependent cancers and are useful tools alongside allosteric inhibitors for dissecting the mechanisms by which SHP2 exerts its oncogenic activity.

3.
Mol Cell ; 81(16): 3323-3338.e14, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34352207

RESUMO

The emerging "epitranscriptomics" field is providing insights into the biological and pathological roles of different RNA modifications. The RNA methyltransferase METTL1 catalyzes N7-methylguanosine (m7G) modification of tRNAs. Here we find METTL1 is frequently amplified and overexpressed in cancers and is associated with poor patient survival. METTL1 depletion causes decreased abundance of m7G-modified tRNAs and altered cell cycle and inhibits oncogenicity. Conversely, METTL1 overexpression induces oncogenic cell transformation and cancer. Mechanistically, we find increased abundance of m7G-modified tRNAs, in particular Arg-TCT-4-1, and increased translation of mRNAs, including cell cycle regulators that are enriched in the corresponding AGA codon. Accordingly, Arg-TCT expression is elevated in many tumor types and is associated with patient survival, and strikingly, overexpression of this individual tRNA induces oncogenic transformation. Thus, METTL1-mediated tRNA modification drives oncogenic transformation through a remodeling of the mRNA "translatome" to increase expression of growth-promoting proteins and represents a promising anti-cancer target.


Assuntos
Carcinogênese/genética , Metiltransferases/genética , Neoplasias/genética , tRNA Metiltransferases/genética , Guanosina/análogos & derivados , Guanosina/genética , Humanos , Metilação , Neoplasias/patologia , Oncogenes/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , RNA de Transferência/genética
4.
Methods Mol Biol ; 2365: 247-263, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34432248

RESUMO

Assessment of small molecules that promote selective protein degradation (degraders) requires detailed characterization and measurement of protein levels in cells. Here we describe ratio-metric methods based on a dual fluorescent GFP/mCherry reporter system to quantify cellular protein levels. We further develop a kinetic framework for the analysis of such data. We describe two methods of generating the stable GFP-protein of interest (POI)/mCherry reporter cell lines, alternative readout methods by FACS and Laser Scanning Cytometry as well as the corresponding tools used for processing and analysis of such data. Finally, we show that the commonly used half-maximal degradation constant (DC50) or maximum degradation efficacy (Dmax) metrics are time-dependent and propose a time-invariant Michaelis-Menten-like analysis of degradation kinetics with analogous key parameters Km app and Vmax app.

5.
Cancer Cell ; 39(9): 1262-1278.e7, 2021 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-34329586

RESUMO

Fusion-transcription factors (fusion-TFs) represent a class of driver oncoproteins that are difficult to therapeutically target. Recently, protein degradation has emerged as a strategy to target these challenging oncoproteins. The mechanisms that regulate fusion-TF stability, however, are generally unknown. Using CRISPR-Cas9 screening, we discovered tripartite motif-containing 8 (TRIM8) as an E3 ubiquitin ligase that ubiquitinates and degrades EWS/FLI, a driver fusion-TF in Ewing sarcoma. Moreover, we identified TRIM8 as a selective dependency in Ewing sarcoma compared with >700 other cancer cell lines. Mechanistically, TRIM8 knockout led to an increase in EWS/FLI protein levels that was not tolerated. EWS/FLI acts as a neomorphic substrate for TRIM8, defining the selective nature of the dependency. Our results demonstrate that fusion-TF protein stability is tightly regulated and highlight fusion oncoprotein-specific regulators as selective therapeutic targets. This study provides a tractable strategy to therapeutically exploit oncogene overdose in Ewing sarcoma and potentially other fusion-TF-driven cancers.

6.
Mol Cell ; 81(17): 3468-3480.e7, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34314700

RESUMO

HECT ubiquitin ligases play essential roles in metazoan development and physiology. The HECT ligase HUWE1 is central to the cellular stress response by mediating degradation of key death or survival factors, including Mcl1, p53, DDIT4, and Myc. Although mutations in HUWE1 and related HECT ligases are widely implicated in human disease, our molecular understanding remains limited. Here we present a comprehensive investigation of full-length HUWE1, deepening our understanding of this class of enzymes. The N-terminal ∼3,900 amino acids of HUWE1 are indispensable for proper ligase function, and our cryo-EM structures of HUWE1 offer a complete molecular picture of this large HECT ubiquitin ligase. HUWE1 forms an alpha solenoid-shaped assembly with a central pore decorated with protein interaction modules. Structures of HUWE1 variants linked to neurodevelopmental disorders as well as of HUWE1 bound to a model substrate link the functions of this essential enzyme to its three-dimensional organization.

7.
J Med Chem ; 64(15): 11637-11650, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34279939

RESUMO

Chemical biology tools to modulate protein levels in cells are critical to decipher complex biology. Targeted protein degradation offers the potential for rapid and dose-dependent protein depletion through the use of protein fusion tags toward which protein degraders have been established. Here, we present a newly developed protein degradation tag BRD4BD1L94V along with the corresponding cereblon (CRBN)-based heterobifunctional degrader based on a "bump-and-hole" approach. The resulting compound XY-06-007 shows a half-degradation concentration (DC50, 6 h) of 10 nM against BRD4BD1L94V with no degradation of off-targets, as assessed by whole proteome mass spectrometry, and demonstrates suitable pharmacokinetics for in vivo studies. We demonstrate that BRD4BD1L94V can be combined with the dTAG approach to achieve simultaneous degrader-mediated depletion of their respective protein fusions. This orthogonal system complements currently available protein degradation tags and enables investigation into the consequences resulting from rapid degradation of previously undruggable disease codependencies.

8.
Cell Chem Biol ; 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34314730

RESUMO

Targeted protein degradation refers to the use of small molecules that recruit a ubiquitin ligase to a target protein for ubiquitination and subsequent proteasome-dependent degradation. While degraders have been developed for many targets, key questions regarding degrader development and the consequences of acute pharmacological degradation remain, specifically for targets that exist in obligate multi-protein complexes. Here, we synthesize a pan-histone deacetylase (HDAC) degrader library for the chemo-proteomic exploration of acute degradation of a key class of chromatin-modifying enzymes. Using chemo-proteomics, we not only map the degradability of the zinc-dependent HDAC family identifying leads for targeting HDACs 1-8 and 10 but also explore important aspects of degrading epigenetic enzymes. We discover cell line-driven target specificity and that HDAC degradation often results in collateral loss of HDAC-containing repressive complexes. These findings potentially offer a new mechanism toward controlling chromatin structure, and our resource will facilitate accelerated degrader design and development for HDACs.

9.
Nat Chem Biol ; 17(6): 711-717, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34035522

RESUMO

The zinc-finger transcription factor Helios is critical for maintaining the identity, anergic phenotype and suppressive activity of regulatory T (Treg) cells. While it is an attractive target to enhance the efficacy of currently approved immunotherapies, no existing approaches can directly modulate Helios activity or abundance. Here, we report the structure-guided development of small molecules that recruit the E3 ubiquitin ligase substrate receptor cereblon to Helios, thereby promoting its degradation. Pharmacological Helios degradation destabilized the anergic phenotype and reduced the suppressive activity of Treg cells, establishing a route towards Helios-targeting therapeutics. More generally, this study provides a framework for the development of small-molecule degraders for previously unligandable targets by reprogramming E3 ligase substrate specificity.


Assuntos
Proteínas de Ligação a DNA/efeitos dos fármacos , Fator de Transcrição Ikaros/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Fatores de Transcrição/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Humanos , Fator de Transcrição Ikaros/genética , Células Jurkat , Camundongos , Modelos Moleculares , Estrutura Molecular , Mutação/genética , Bibliotecas de Moléculas Pequenas , Especificidade por Substrato , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/metabolismo
10.
Angew Chem Int Ed Engl ; 60(29): 15905-15911, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-33915015

RESUMO

Aberrant activation of FGFR signaling occurs in many cancers, and ATP-competitive FGFR inhibitors have received regulatory approval. Despite demonstrating clinical efficacy, these inhibitors exhibit dose-limiting toxicity, potentially due to a lack of selectivity amongst the FGFR family and are poorly tolerated. Here, we report the discovery and characterization of DGY-09-192, a bivalent degrader that couples the pan-FGFR inhibitor BGJ398 to a CRL2VHL E3 ligase recruiting ligand, which preferentially induces FGFR1&2 degradation while largely sparing FGFR3&4. DGY-09-192 exhibited two-digit nanomolar DC50 s for both wildtype FGFR2 and several FGFR2-fusions, resulting in degradation-dependent antiproliferative activity in representative gastric cancer and cholangiocarcinoma cells. Importantly, DGY-09-192 induced degradation of a clinically relevant FGFR2 fusion protein in a xenograft model. Taken together, we demonstrate that DGY-09-192 has potential as a prototype FGFR degrader.


Assuntos
Descoberta de Drogas , Proteólise/efeitos dos fármacos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Linhagem Celular Tumoral , Humanos
11.
Eur J Med Chem ; 219: 113435, 2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-33892272

RESUMO

The eukaryotic translation initiation factor 4E (eIF4E) is the master regulator of cap-dependent protein synthesis. Overexpression of eIF4E is implicated in diseases such as cancer, where dysregulation of oncogenic protein translation is frequently observed. eIF4E has been an attractive target for cancer treatment. Here we report a high-resolution X-ray crystal structure of eIF4E in complex with a novel inhibitor (i4EG-BiP) that targets an internal binding site, in contrast to the previously described inhibitor, 4EGI-1, which binds to the surface. We demonstrate that i4EG-BiP is able to displace the scaffold protein eIF4G and inhibit the proliferation of cancer cells. We provide insights into how i4EG-BiP is able to inhibit cap-dependent translation by increasing the eIF4E-4E-BP1 interaction while diminishing the interaction of eIF4E with eIF4G. Leveraging structural details, we designed proteolysis targeted chimeras (PROTACs) derived from 4EGI-1 and i4EG-BiP and characterized these on biochemical and cellular levels. We were able to design PROTACs capable of binding eIF4E and successfully engaging Cereblon, which targets proteins for proteolysis. However, these initial PROTACs did not successfully stimulate degradation of eIF4E, possibly due to competitive effects from 4E-BP1 binding. Our results highlight challenges of targeted proteasomal degradation of eIF4E that must be addressed by future efforts.


Assuntos
Compostos de Bifenilo/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Sítios de Ligação , Compostos de Bifenilo/química , Compostos de Bifenilo/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Fator de Iniciação 4E em Eucariotos/antagonistas & inibidores , Fator de Iniciação 4E em Eucariotos/genética , Humanos , Cinética , Simulação de Acoplamento Molecular , Pró-Fármacos/síntese química , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteômica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
12.
Angew Chem Int Ed Engl ; 60(25): 13783-13787, 2021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-33768661

RESUMO

Therapeutically relevant proteins such as GPCRs, antibodies and kinases face clear limitations in NMR studies due to the challenges in site-specific isotope labeling and deuteration in eukaryotic expression systems. Here we describe an efficient and simple method to observe the methyl groups of leucine residues in proteins expressed in bacterial, eukaryotic or cell-free expression systems without modification of the expression protocol. The method relies on simple stereo-selective 13 C-labeling and deuteration of leucine that alleviates the need for additional deuteration of the protein. The spectroscopic benefits of "local" deuteration are examined in detail through Forbidden Coherence Transfer (FCT) experiments and simulations. The utility of this labeling method is demonstrated in the cell-free synthesis of bacteriorhodopsin and in the insect-cell expression of the RRM2 domain of human RBM39.


Assuntos
Eucariotos/química , Ressonância Magnética Nuclear Biomolecular , Receptores Acoplados a Proteínas G/química , Humanos , Estrutura Molecular
13.
Nat Med ; 27(3): 454-462, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33589825

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic continues to spread relentlessly, associated with a high frequency of respiratory failure and mortality. Children experience largely asymptomatic disease, with rare reports of multisystem inflammatory syndrome in children (MIS-C). Identifying immune mechanisms that result in these disparate clinical phenotypes in children could provide critical insights into coronavirus disease 2019 (COVID-19) pathogenesis. Using systems serology, in this study we observed in 25 children with acute mild COVID-19 a functional phagocyte and complement-activating IgG response to SARS-CoV-2, similar to the acute responses generated in adults with mild disease. Conversely, IgA and neutrophil responses were significantly expanded in adults with severe disease. Moreover, weeks after the resolution of SARS-CoV-2 infection, children who develop MIS-C maintained highly inflammatory monocyte-activating SARS-CoV-2 IgG antibodies, distinguishable from acute disease in children but with antibody levels similar to those in convalescent adults. Collectively, these data provide unique insights into the potential mechanisms of IgG and IgA that might underlie differential disease severity as well as unexpected complications in children infected with SARS-CoV-2.


Assuntos
Anticorpos Antivirais/sangue , COVID-19/epidemiologia , SARS-CoV-2/imunologia , Adolescente , Adulto , Idade de Início , Idoso , Anticorpos Neutralizantes/análise , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/análise , Infecções Assintomáticas , COVID-19/sangue , COVID-19/patologia , Portador Sadio/sangue , Portador Sadio/epidemiologia , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Imunidade/fisiologia , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Pandemias , Índice de Gravidade de Doença , Síndrome de Resposta Inflamatória Sistêmica/sangue , Síndrome de Resposta Inflamatória Sistêmica/epidemiologia , Adulto Jovem
14.
Sci Adv ; 7(6)2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33547076

RESUMO

Most intracellular proteins lack hydrophobic pockets suitable for altering their function with drug-like small molecules. Recent studies indicate that some undruggable proteins can be targeted by compounds that can degrade them. For example, thalidomide-like drugs (IMiDs) degrade the critical multiple myeloma transcription factors IKZF1 and IKZF3 by recruiting them to the cereblon E3 ubiquitin ligase. Current loss of signal ("down") assays for identifying degraders often exhibit poor signal-to-noise ratios, narrow dynamic ranges, and false positives from compounds that nonspecifically suppress transcription or translation. Here, we describe a gain of signal ("up") assay for degraders. In arrayed chemical screens, we identified novel IMiD-like IKZF1 degraders and Spautin-1, which, unlike the IMiDs, degrades IKZF1 in a cereblon-independent manner. In a pooled CRISPR-Cas9-based screen, we found that CDK2 regulates the abundance of the ASCL1 oncogenic transcription factor. This methodology should facilitate the identification of drugs that directly or indirectly degrade undruggable proteins.

15.
Cell Rep ; 34(1): 108532, 2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33406420

RESUMO

Heterobifunctional proteolysis-targeting chimeric compounds leverage the activity of E3 ligases to induce degradation of target oncoproteins and exhibit potent preclinical antitumor activity. To dissect the mechanisms regulating tumor cell sensitivity to different classes of pharmacological "degraders" of oncoproteins, we performed genome-scale CRISPR-Cas9-based gene editing studies. We observed that myeloma cell resistance to degraders of different targets (BET bromodomain proteins, CDK9) and operating through CRBN (degronimids) or VHL is primarily mediated by prevention of, rather than adaptation to, breakdown of the target oncoprotein; and this involves loss of function of the cognate E3 ligase or interactors/regulators of the respective cullin-RING ligase (CRL) complex. The substantial gene-level differences for resistance mechanisms to CRBN- versus VHL-based degraders explains mechanistically the lack of cross-resistance with sequential administration of these two degrader classes. Development of degraders leveraging more diverse E3 ligases/CRLs may facilitate sequential/alternating versus combined uses of these agents toward potentially delaying or preventing resistance.

16.
Cell Chem Biol ; 28(1): 78-87.e3, 2021 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-33007217

RESUMO

Deubiquitinating enzymes (DUBs) catalyze the removal of ubiquitin, thereby reversing the activity of E3 ubiquitin ligases and are central to the control of protein abundance and function. Despite the growing interest in DUBs as therapeutic targets, cellular functions for DUBs remain largely unknown and technical challenges often preclude the identification of DUB substrates in a comprehensive manner. Here, we demonstrate that treatment with potent DUB inhibitors coupled to mass spectrometry-based proteomics can identify DUB substrates at a proteome-wide scale. We applied this approach to USP7, a DUB widely investigated as a therapeutic target and identified many known substrates and additional targets. We demonstrate that USP7 substrates are enriched for DNA repair enzymes and E3 ubiquitin ligases. This work provides not only a comprehensive annotation of USP7 substrates, but a general protocol widely applicable to other DUBs, which is critical for translational development of DUB targeted agents.

17.
Cell ; 183(6): 1714-1731.e10, 2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33275901

RESUMO

Targeted protein degradation (TPD) refers to the use of small molecules to induce ubiquitin-dependent degradation of proteins. TPD is of interest in drug development, as it can address previously inaccessible targets. However, degrader discovery and optimization remains an inefficient process due to a lack of understanding of the relative importance of the key molecular events required to induce target degradation. Here, we use chemo-proteomics to annotate the degradable kinome. Our expansive dataset provides chemical leads for ∼200 kinases and demonstrates that the current practice of starting from the highest potency binder is an ineffective method for discovering active compounds. We develop multitargeted degraders to answer fundamental questions about the ubiquitin proteasome system, uncovering that kinase degradation is p97 dependent. This work will not only fuel kinase degrader discovery, but also provides a blueprint for evaluating targeted degradation across entire gene families to accelerate understanding of TPD beyond the kinome.


Assuntos
Proteínas Quinases/metabolismo , Proteólise , Proteoma/metabolismo , Adulto , Linhagem Celular , Bases de Dados de Proteínas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Quinases/genética , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adulto Jovem
18.
Nature ; 588(7836): 164-168, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33208943

RESUMO

Effective and sustained inhibition of non-enzymatic oncogenic driver proteins is a major pharmacological challenge. The clinical success of thalidomide analogues demonstrates the therapeutic efficacy of drug-induced degradation of transcription factors and other cancer targets1-3, but a substantial subset of proteins are resistant to targeted degradation using existing approaches4,5. Here we report an alternative mechanism of targeted protein degradation, in which a small molecule induces the highly specific, reversible polymerization of a target protein, followed by its sequestration into cellular foci and subsequent degradation. BI-3802 is a small molecule that binds to the Broad-complex, Tramtrack and Bric-à-brac (BTB) domain of the oncogenic transcription factor B cell lymphoma 6 (BCL6) and leads to the proteasomal degradation of BCL66. We use cryo-electron microscopy to reveal how the solvent-exposed moiety of a BCL6-binding molecule contributes to a composite ligand-protein surface that engages BCL6 homodimers to form a supramolecular structure. Drug-induced formation of BCL6 filaments facilitates ubiquitination by the SIAH1 E3 ubiquitin ligase. Our findings demonstrate that a small molecule such as BI-3802 can induce polymerization coupled to highly specific protein degradation, which in the case of BCL6 leads to increased pharmacological activity compared to the effects induced by other BCL6 inhibitors. These findings open new avenues for the development of therapeutic agents and synthetic biology.


Assuntos
Polimerização/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-6/química , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Microscopia Crioeletrônica , Humanos , Técnicas In Vitro , Ligantes , Modelos Moleculares , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/ultraestrutura , Solventes , Biologia Sintética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos
19.
ACS Chem Biol ; 15(10): 2722-2730, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-32865967

RESUMO

Cereblon (CRBN) is an E3 ligase adapter protein that can be reprogrammed by imide-class compounds such as thalidomide, lenalidomide, and pomalidomide to induce the degradation of neo-substrate proteins. In order to identify additional small molecule CRBN modulators, we implemented a focused combinatorial library approach where we fused an imide-based CRBN-binding pharmacophore to a heterocyclic scaffold, which could be further elaborated. We screened the library for CRBN-dependent antiproliferative activity in the multiple myeloma cell line MM1.S and identified five hit compounds. Quantitative chemical proteomics of hit compounds revealed that they induced selective degradation of GSPT1, a translation termination factor that is currently being explored as a therapeutic target for the treatment of acute myeloid leukemia. Molecular docking studies with CRBN and GSPT1 followed by analogue synthesis identified a possible hydrogen bond interaction with the central pyrimidine ring as a molecular determinant of hit compounds' selectivity. This study demonstrates that a focused combinatorial library design, phenotypic screening, and chemical proteomics can provide a suitable workflow to efficiently identify novel CRBN modulators.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Talidomida/análogos & derivados , Talidomida/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Fatores de Terminação de Peptídeos/química , Ligação Proteica , Bibliotecas de Moléculas Pequenas/metabolismo , Talidomida/metabolismo , Ubiquitina-Proteína Ligases/química
20.
medRxiv ; 2020 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-32909004

RESUMO

The human beta coronavirus SARS-CoV-2, causative virus of COVID-19, has infected more than 15 million people globally and continues to spread. Widespread, population level testing to detect active and past infections is critical to curb the COVID-19 pandemic. Antibody (serological) testing is the only option for detecting past infections outside the narrow window accessible to nucleic acid-based tests. However, currently available serological assays commonly lack scalability. Here, we describe the development of a rapid homogenous serological assay for the detection of antibodies to SARS-CoV-2 in patient plasma. We show that the fluorescence-based assay accurately detects seroconversion in COVID-19 patients from less than 1 microliter of plasma. Using a cohort of samples from COVID-19 infected or healthy individuals, we demonstrate detection with 100% sensitivity and specificity. This assay addresses an important need for a robust, low barrier to implementation, and scalable serological assay with complementary strengths to currently available serological platforms.

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