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1.
Nat Chem Biol ; 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34782742

RESUMO

Nuclear receptor-binding SET domain-containing 2 (NSD2) is the primary enzyme responsible for the dimethylation of lysine 36 of histone 3 (H3K36), a mark associated with active gene transcription and intergenic DNA methylation. In addition to a methyltransferase domain, NSD2 harbors two proline-tryptophan-tryptophan-proline (PWWP) domains and five plant homeodomains (PHDs) believed to serve as chromatin reading modules. Here, we report a chemical probe targeting the N-terminal PWWP (PWWP1) domain of NSD2. UNC6934 occupies the canonical H3K36me2-binding pocket of PWWP1, antagonizes PWWP1 interaction with nucleosomal H3K36me2 and selectively engages endogenous NSD2 in cells. UNC6934 induces accumulation of endogenous NSD2 in the nucleolus, phenocopying the localization defects of NSD2 protein isoforms lacking PWWP1 that result from translocations prevalent in multiple myeloma (MM). Mutations of other NSD2 chromatin reader domains also increase NSD2 nucleolar localization and enhance the effect of UNC6934. This chemical probe and the accompanying negative control UNC7145 will be useful tools in defining NSD2 biology.

2.
SLAS Discov ; 26(9): 1200-1211, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34192965

RESUMO

The COVID-19 pandemic has clearly brought the healthcare systems worldwide to a breaking point, along with devastating socioeconomic consequences. The SARS-CoV-2 virus, which causes the disease, uses RNA capping to evade the human immune system. Nonstructural protein (nsp) 14 is one of the 16 nsps in SARS-CoV-2 and catalyzes the methylation of the viral RNA at N7-guanosine in the cap formation process. To discover small-molecule inhibitors of nsp14 methyltransferase (MTase) activity, we developed and employed a radiometric MTase assay to screen a library of 161 in-house synthesized S-adenosylmethionine (SAM) competitive MTase inhibitors and SAM analogs. Among six identified screening hits, SS148 inhibited nsp14 MTase activity with an IC50 value of 70 ± 6 nM and was selective against 20 human protein lysine MTases, indicating significant differences in SAM binding sites. Interestingly, DS0464 with an IC50 value of 1.1 ± 0.2 µM showed a bisubstrate competitive inhibitor mechanism of action. DS0464 was also selective against 28 out of 33 RNA, DNA, and protein MTases. The structure-activity relationship provided by these compounds should guide the optimization of selective bisubstrate nsp14 inhibitors and may provide a path toward a novel class of antivirals against COVID-19, and possibly other coronaviruses.


Assuntos
COVID-19/genética , Exorribonucleases/genética , Ligação Proteica/genética , SARS-CoV-2/genética , Proteínas não Estruturais Virais/genética , Antivirais/farmacologia , Sítios de Ligação/genética , COVID-19/virologia , Humanos , Metilação , Pandemias , RNA Viral/genética , SARS-CoV-2/patogenicidade , Replicação Viral/genética
3.
SLAS Discov ; 26(8): 947-960, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34154424

RESUMO

SMYD3 (SET and MYND domain-containing protein 3) is a protein lysine methyltransferase that was initially described as an H3K4 methyltransferase involved in transcriptional regulation. SMYD3 has been reported to methylate and regulate several nonhistone proteins relevant to cancer, including mitogen-activated protein kinase kinase kinase 2 (MAP3K2), vascular endothelial growth factor receptor 1 (VEGFR1), and the human epidermal growth factor receptor 2 (HER2). In addition, overexpression of SMYD3 has been linked to poor prognosis in certain cancers, suggesting SMYD3 as a potential oncogene and attractive cancer drug target. Here we report the discovery of a novel SMYD3 inhibitor. We performed a thermal shift assay (TSA)-based high-throughput screening (HTS) with 410,000 compounds and identified a novel benzodiazepine-based SMYD3 inhibitor series. Crystal structures revealed that this series binds to the substrate binding site and occupies the hydrophobic lysine binding pocket via an unprecedented hydrogen bonding pattern. Biochemical assays showed substrate competitive behavior. Following optimization and extensive biophysical validation with surface plasmon resonance (SPR) analysis and isothermal titration calorimetry (ITC), we identified BAY-6035, which shows nanomolar potency and selectivity against kinases and other PKMTs. Furthermore, BAY-6035 specifically inhibits methylation of MAP3K2 by SMYD3 in a cellular mechanistic assay with an IC50 <100 nM. Moreover, we describe a congeneric negative control to BAY-6035. In summary, BAY-6035 is a novel selective and potent SMYD3 inhibitor probe that will foster the exploration of the biological role of SMYD3 in diseased and nondiseased tissues.

4.
J Med Chem ; 64(3): 1584-1592, 2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33522809

RESUMO

Increased activity of the lysine methyltransferase NSD2 driven by translocation and activating mutations is associated with multiple myeloma and acute lymphoblastic leukemia, but no NSD2-targeting chemical probe has been reported to date. Here, we present the first antagonists that block the protein-protein interaction between the N-terminal PWWP domain of NSD2 and H3K36me2. Using virtual screening and experimental validation, we identified the small-molecule antagonist 3f, which binds to the NSD2-PWWP1 domain with a Kd of 3.4 µM and abrogates histone H3K36me2 binding to the PWWP1 domain in cells. This study establishes an alternative approach to targeting NSD2 and provides a small-molecule antagonist that can be further optimized into a chemical probe to better understand the cellular function of this protein.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Proteínas Repressoras/antagonistas & inibidores , Simulação por Computador , Cristalografia por Raios X , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Histona-Lisina N-Metiltransferase/efeitos dos fármacos , Humanos , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Domínios Proteicos , Proteínas Repressoras/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade
5.
J Med Chem ; 64(7): 3697-3706, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33591753

RESUMO

Protein arginine methyltransferase 6 (PRMT6) catalyzes monomethylation and asymmetric dimethylation of arginine residues in various proteins, plays important roles in biological processes, and is associated with multiple cancers. To date, a highly selective PRMT6 inhibitor has not been reported. Here we report the discovery and characterization of a first-in-class, highly selective allosteric inhibitor of PRMT6, (R)-2 (SGC6870). (R)-2 is a potent PRMT6 inhibitor (IC50 = 77 ± 6 nM) with outstanding selectivity for PRMT6 over a broad panel of other methyltransferases and nonepigenetic targets. Notably, the crystal structure of the PRMT6-(R)-2 complex and kinetic studies revealed (R)-2 binds a unique, induced allosteric pocket. Additionally, (R)-2 engages PRMT6 and potently inhibits its methyltransferase activity in cells. Moreover, (R)-2's enantiomer, (S)-2 (SGC6870N), is inactive against PRMT6 and can be utilized as a negative control. Collectively, (R)-2 is a well-characterized PRMT6 chemical probe and a valuable tool for further investigating PRMT6 functions in health and disease.


Assuntos
Benzodiazepinonas/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Regulação Alostérica , Sítio Alostérico , Benzodiazepinonas/síntese química , Benzodiazepinonas/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Células HEK293 , Humanos , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteína-Arginina N-Metiltransferases/metabolismo , Estereoisomerismo
6.
bioRxiv ; 2021 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-33619486

RESUMO

The COVID-19 pandemic has clearly brought the healthcare systems world-wide to a breaking point along with devastating socioeconomic consequences. The SARS-CoV-2 virus which causes the disease uses RNA capping to evade the human immune system. Non-structural protein (nsp) 14 is one of the 16 nsps in SARS-CoV-2 and catalyzes the methylation of the viral RNA at N7-guanosine in the cap formation process. To discover small molecule inhibitors of nsp14 methyltransferase (MT) activity, we developed and employed a radiometric MT assay to screen a library of 161 in house synthesized S-adenosylmethionine (SAM) competitive methyltransferase inhibitors and SAM analogs. Among seven identified screening hits, SS148 inhibited nsp14 MT activity with an IC 50 value of 70 ± 6 nM and was selective against 20 human protein lysine methyltransferases indicating significant differences in SAM binding sites. Interestingly, DS0464 with IC 50 value of 1.1 ± 0.2 µM showed a bi-substrate competitive inhibitor mechanism of action. Modeling the binding of this compound to nsp14 suggests that the terminal phenyl group extends into the RNA binding site. DS0464 was also selective against 28 out of 33 RNA, DNA, and protein methyltransferases. The structure-activity relationship provided by these compounds should guide the optimization of selective bi-substrate nsp14 inhibitors and may provide a path towards a novel class of antivirals against COVID-19, and possibly other coronaviruses.

7.
Biochimie ; 183: 100-107, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33476699

RESUMO

The folate and methionine cycles, constituting one-carbon metabolism, are critical pathways for cell survival. Intersecting these two cycles, 5,10-methylenetetrahydrofolate reductase (MTHFR) directs one-carbon units from the folate to methionine cycle, to be exclusively used for methionine and S-adenosylmethionine (AdoMet) synthesis. MTHFR deficiency and upregulation result in diverse disease states, rendering it an attractive drug target. The activity of MTHFR is inhibited by the binding of AdoMet to an allosteric regulatory domain distal to the enzyme's active site, which we have previously identified to constitute a novel fold with a druggable pocket. Here, we screened 162 AdoMet mimetics using differential scanning fluorimetry, and identified 4 compounds that stabilized this regulatory domain. Three compounds were sinefungin analogues, closely related to AdoMet and S-adenosylhomocysteine (AdoHcy). The strongest thermal stabilisation was provided by (S)-SKI-72, a potent inhibitor originally developed for protein arginine methyltransferase 4 (PRMT4). Using surface plasmon resonance, we confirmed that (S)-SKI-72 binds MTHFR via its allosteric domain with nanomolar affinity. Assay of MTHFR activity in the presence of (S)-SKI-72 demonstrates inhibition of purified enzyme with sub-micromolar potency and endogenous MTHFR from HEK293 cell lysate in the low micromolar range, both of which are lower than AdoMet. Nevertheless, unlike AdoMet, (S)-SKI-72 is unable to completely abolish MTHFR activity, even at very high concentrations. Combining binding assays, kinetic characterization and compound docking, this work indicates the regulatory domain of MTHFR can be targeted by small molecules and presents (S)-SKI-72 as an excellent candidate for development of MTHFR inhibitors.


Assuntos
Inibidores Enzimáticos/química , Metilenotetra-Hidrofolato Redutase (NADPH2)/antagonistas & inibidores , Metilenotetra-Hidrofolato Redutase (NADPH2)/química , S-Adenosilmetionina/química , Regulação Alostérica , Humanos , Domínios Proteicos
9.
Phys Rev Lett ; 125(9): 090401, 2020 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-32915619

RESUMO

Alice and Bob each have half of a pair of entangled qubits. Bob measures his half and then passes his qubit to a second Bob who measures again and so on. The goal is to maximize the number of Bobs that can have an expected violation of the Clauser-Horne-Shimony-Holt (CHSH) Bell inequality with the single Alice. This scenario was introduced in [R. Silva et al., Phys. Rev. Lett. 114, 250401 (2015)PRLTAO0031-900710.1103/PhysRevLett.114.250401] where the authors mentioned evidence that when the Bobs act independently and with unbiased inputs then at most two of them can expect to violate the CHSH inequality with Alice. Here we show that, contrary to this evidence, arbitrarily many independent Bobs can have an expected CHSH violation with the single Alice. Our proof is constructive and our measurement strategies can be generalized to work with a larger class of two-qubit states that includes all pure entangled two-qubit states. Since violation of a Bell inequality is necessary for device-independent tasks, our work represents a step towards an eventual understanding of the limitations on how much device-independent randomness can be robustly generated from a single pair of qubits.

10.
J Med Chem ; 63(10): 5477-5487, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32367723

RESUMO

Protein arginine methyltransferase 6 (PRMT6) plays important roles in several biological processes associated with multiple cancers. Well-characterized potent, selective, and cell-active PRMT6 inhibitors are invaluable tools for testing biological and therapeutic hypotheses. Although there are several known reversible PRMT6 inhibitors, covalent PRMT6 inhibitors have not been reported. Based on a cocrystal structure of PRMT6-MS023 (a type I PRMT inhibitor), we discovered the first potent and cell-active irreversible PRMT6 inhibitor, 4 (MS117). The covalent binding mode of compound 4 to PRMT6 was confirmed by mass spectrometry and kinetic studies and by a cocrystal structure. Compound 4 did not covalently modify other closely related PRMTs, potently inhibited PRMT6 in cells, and was selective for PRMT6 over other methyltransferases. We also developed two structurally similar control compounds, 5 (MS167) and 7 (MS168). We provide these valuable chemical tools to the scientific community for further studying PRMT6 physiological and pathophysiological functions.


Assuntos
Descoberta de Drogas/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/metabolismo , Células HEK293 , Humanos , Células MCF-7 , Proteínas Nucleares/metabolismo , Estrutura Secundária de Proteína , Proteína-Arginina N-Metiltransferases/metabolismo
11.
Nat Commun ; 11(1): 2396, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32409666

RESUMO

Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increasingly being recognized for their potential as drug targets. Here we report the discovery of a potent, selective, and cell-active chemical probe for PRMT7. SGC3027 is a cell permeable prodrug, which in cells is converted to SGC8158, a potent, SAM-competitive PRMT7 inhibitor. Inhibition or knockout of cellular PRMT7 results in drastically reduced levels of arginine monomethylated HSP70 family stress-associated proteins. Structural and biochemical analyses reveal that PRMT7-driven in vitro methylation of HSP70 at R469 requires an ATP-bound, open conformation of HSP70. In cells, SGC3027 inhibits methylation of both constitutive and inducible forms of HSP70, and leads to decreased tolerance for perturbations of proteostasis including heat shock and proteasome inhibitors. These results demonstrate a role for PRMT7 and arginine methylation in stress response.


Assuntos
Arginina/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Estresse Fisiológico , Animais , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Metilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9
13.
Nat Commun ; 10(1): 5759, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848333

RESUMO

PRDM9 is a PR domain containing protein which trimethylates histone 3 on lysine 4 and 36. Its normal expression is restricted to germ cells and attenuation of its activity results in altered meiotic gene transcription, impairment of double-stranded breaks and pairing between homologous chromosomes. There is growing evidence for a role of aberrant expression of PRDM9 in oncogenesis and genome instability. Here we report the discovery of MRK-740, a potent (IC50: 80 ± 16 nM), selective and cell-active PRDM9 inhibitor (Chemical Probe). MRK-740 binds in the substrate-binding pocket, with unusually extensive interactions with the cofactor S-adenosylmethionine (SAM), conferring SAM-dependent substrate-competitive inhibition. In cells, MRK-740 specifically and directly inhibits H3K4 methylation at endogenous PRDM9 target loci, whereas the closely related inactive control compound, MRK-740-NC, does not. The discovery of MRK-740 as a chemical probe for the PRDM subfamily of methyltransferases highlights the potential for exploiting SAM in targeting SAM-dependent methyltransferases.


Assuntos
Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Sondas Moleculares/farmacologia , Cristalografia por Raios X , Metilação de DNA/efeitos dos fármacos , Inibidores Enzimáticos/química , Células HEK293 , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/ultraestrutura , Histonas/metabolismo , Humanos , Concentração Inibidora 50 , Simulação de Dinâmica Molecular , Sondas Moleculares/química , Domínios Proteicos , S-Adenosilmetionina/metabolismo
14.
Elife ; 82019 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-31657716

RESUMO

CARM1 is a cancer-relevant protein arginine methyltransferase that regulates many aspects of transcription. Its pharmacological inhibition is a promising anti-cancer strategy. Here SKI-73 (6a in this work) is presented as a CARM1 chemical probe with pro-drug properties. SKI-73 (6a) can rapidly penetrate cell membranes and then be processed into active inhibitors, which are retained intracellularly with 10-fold enrichment for several days. These compounds were characterized for their potency, selectivity, modes of action, and on-target engagement. SKI-73 (6a) recapitulates the effect of CARM1 knockout against breast cancer cell invasion. Single-cell RNA-seq analysis revealed that the SKI-73(6a)-associated reduction of invasiveness acts by altering epigenetic plasticity and suppressing the invasion-prone subpopulation. Interestingly, SKI-73 (6a) and CARM1 knockout alter the epigenetic plasticity with remarkable difference, suggesting distinct modes of action for small-molecule and genetic perturbations. We therefore discovered a CARM1-addiction mechanism of cancer metastasis and developed a chemical probe to target this process.


Assuntos
Neoplasias da Mama/genética , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Epigenômica/métodos , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Algoritmos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Feminino , Humanos , Cinética , Células MCF-7 , Modelos Químicos , Estrutura Molecular , Ligação Proteica , Proteína-Arginina N-Metiltransferases/metabolismo
15.
Sci Adv ; 5(8): eaav6410, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31489364

RESUMO

Global climate is critically sensitive to physical and biogeochemical dynamics in the subpolar Southern Ocean, since it is here that deep, carbon-rich layers of the world ocean outcrop and exchange carbon with the atmosphere. Here, we present evidence that the conventional framework for the subpolar Southern Ocean carbon cycle, which attributes a dominant role to the vertical overturning circulation and shelf-sea processes, fundamentally misrepresents the drivers of regional carbon uptake. Observations in the Weddell Gyre-a key representative region of the subpolar Southern Ocean-show that the rate of carbon uptake is set by an interplay between the Gyre's horizontal circulation and the remineralization at mid-depths of organic carbon sourced from biological production in the central gyre. These results demonstrate that reframing the carbon cycle of the subpolar Southern Ocean is an essential step to better define its role in past and future climate change.

16.
J Med Chem ; 62(17): 7669-7683, 2019 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-31415173

RESUMO

The first chemical probe to primarily occupy the co-factor binding site of a Su(var)3-9, enhancer of a zeste, trithorax (SET) domain containing protein lysine methyltransferase (PKMT) is reported. Protein methyltransferases require S-adenosylmethionine (SAM) as a co-factor (methyl donor) for enzymatic activity. However, SAM itself represents a poor medicinal chemistry starting point for a selective, cell-active inhibitor given its extreme physicochemical properties and its role in multiple cellular processes. A previously untested medicinal chemistry strategy of deliberate file enrichment around molecules bearing the hallmarks of SAM, but with improved lead-like properties from the outset, yielded viable hits against SET and MYND domain-containing protein 2 (SMYD2) that were shown to bind in the co-factor site. These leads were optimized to identify a highly biochemically potent, PKMT-selective, and cell-active chemical probe. While substrate-based inhibitors of PKMTs are known, this represents a novel, co-factor-derived strategy for the inhibition of SMYD2 which may also prove applicable to lysine methyltransferase family members previously thought of as intractable.


Assuntos
Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , S-Adenosilmetionina/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Sítios de Ligação/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Histona-Lisina N-Metiltransferase/isolamento & purificação , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , S-Adenosilmetionina/química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade
17.
JFMS Open Rep ; 5(2): 2055116919861248, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31308958

RESUMO

Case summary: A 3.5-year-old domestic shorthair cat presented with a 6 month history of weight loss and polyphagia. Clinical examination revealed a markedly reduced body condition score (2/9) and a quiet demeanour. Laboratory abnormalities comprised a mild non-regenerative anaemia, stress leukogram, hypoproteinaemia due to hypoalbuminaemia, azotaemia, hypokalaemia, total hypocalcaemia and sub-maximally concentrated urine (specific gravity 1.020). Abdominal ultrasonography revealed marked thickening of the gastric mucosa within the fundus, body and pylorus; the most dorsal portion of the fundus was spared. The thickened mucosa contained multiple small, anechoic cyst-like structures. The gastric submucosa, muscularis and serosa appeared normal. Histopathology, performed on a full-thickness gastric biopsy, revealed mucosal hypertrophy and markedly dilated gastric glands in areas; not all gastric glands were affected, with some appearing normal or atrophic. Focal interstitial fibrosis was present in some areas. The findings of hypoproteinaemia, gastric ultrasonographic changes and histopathology results share several similarities to those reported with Ménétrier disease. Relevance and novel information: Ménétrier disease is a rare condition of the stomach in humans. A similar condition, giant hypertrophic gastritis (or Ménétrier-like disease), has also been described rarely in dogs. To our knowledge, Ménétrier-like disease has not been previously described cats. This case shares features of Ménétrier-like disease, raising the suspicion of a similar aetiopathogenesis.

18.
Bioorg Med Chem ; 27(17): 3866-3878, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31327677

RESUMO

SET domain bifurcated protein 1 (SETDB1) is a human histone-lysine methyltransferase which is amplified in human cancers and was shown to be crucial in the growth of non-small and small cell lung carcinoma. In addition to its catalytic domain, SETDB1 harbors a unique tandem tudor domain which recognizes histone sequences containing both methylated and acetylated lysines, and likely contributes to its localization on chromatin. Using X-ray crystallography and NMR spectroscopy fragment screening approaches, we have identified the first small molecule fragment hits that bind to histone peptide binding groove of the Tandem Tudor Domain (TTD) of SETDB1. Herein, we describe the binding modes of these fragments and analogues and the biophysical characterization of key compounds. These confirmed small molecule fragments will inform the development of potent antagonists of SETDB1 interaction with histones.


Assuntos
Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Histona-Lisina N-Metiltransferase/isolamento & purificação , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/antagonistas & inibidores , Histonas/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Domínio Tudor/efeitos dos fármacos
19.
Nat Chem Biol ; 15(8): 822-829, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285596

RESUMO

Here, we report the fragment-based discovery of BI-9321, a potent, selective and cellular active antagonist of the NSD3-PWWP1 domain. The human NSD3 protein is encoded by the WHSC1L1 gene located in the 8p11-p12 amplicon, frequently amplified in breast and squamous lung cancer. Recently, it was demonstrated that the PWWP1 domain of NSD3 is required for the viability of acute myeloid leukemia cells. To further elucidate the relevance of NSD3 in cancer biology, we developed a chemical probe, BI-9321, targeting the methyl-lysine binding site of the PWWP1 domain with sub-micromolar in vitro activity and cellular target engagement at 1 µM. As a single agent, BI-9321 downregulates Myc messenger RNA expression and reduces proliferation in MOLM-13 cells. This first-in-class chemical probe BI-9321, together with the negative control BI-9466, will greatly facilitate the elucidation of the underexplored biological function of PWWP domains.


Assuntos
Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Sistemas CRISPR-Cas , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Domínios Proteicos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo
20.
Nat Commun ; 10(1): 2517, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31175292

RESUMO

Hyper-activated STAT5B variants are high value oncology targets for pharmacologic intervention. STAT5BN642H, a frequently-occurring oncogenic driver mutation, promotes aggressive T-cell leukemia/lymphoma in patient carriers, although the molecular origins remain unclear. Herein, we emphasize the aggressive nature of STAT5BN642H in driving T-cell neoplasia upon hematopoietic expression in transgenic mice, revealing evidence of multiple T-cell subset organ infiltration. Notably, we demonstrate STAT5BN642H-driven transformation of γδ T-cells in in vivo syngeneic transplant models, comparable to STAT5BN642H patient γδ T-cell entities. Importantly, we present human STAT5B and STAT5BN642H crystal structures, which propose alternative mutation-mediated SH2 domain conformations. Our biophysical data suggests STAT5BN642H can adopt a hyper-activated and hyper-inactivated state with resistance to dephosphorylation. MD simulations support sustained interchain cross-domain interactions in STAT5BN642H, conferring kinetic stability to the mutant anti-parallel dimer. This study provides a molecular explanation for the STAT5BN642H activating potential, and insights into pre-clinical models for targeted intervention of hyper-activated STAT5B.


Assuntos
Linfócitos Intraepiteliais , Leucemia de Células T/genética , Linfoma de Células T/genética , Mutação , Fator de Transcrição STAT5/genética , Animais , Neoplasias Hematológicas/genética , Humanos , Camundongos , Camundongos Transgênicos , Simulação de Acoplamento Molecular , Domínios de Homologia de src
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