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1.
Mol Cancer Res ; 18(10): 1512-1521, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32611550

RESUMO

O-GlcNAc transferase (OGT) is a nutrient-sensitive glycosyltransferase that is overexpressed in prostate cancer, the most common cancer in males. We recently developed a specific and potent inhibitor targeting this enzyme, and here, we report a synthetic lethality screen using this compound. Our screen identified pan-cyclin-dependent kinase (CDK) inhibitor AT7519 as lethal in combination with OGT inhibition. Follow-up chemical and genetic approaches identified CDK9 as the major target for synthetic lethality with OGT inhibition in prostate cancer cells. OGT expression is regulated through retention of the fourth intron in the gene and CDK9 inhibition blunted this regulatory mechanism. CDK9 phosphorylates carboxy-terminal domain (CTD) of RNA Polymerase II to promote transcription elongation. We show that OGT inhibition augments effects of CDK9 inhibitors on CTD phosphorylation and general transcription. Finally, the combined inhibition of both OGT and CDK9 blocked growth of organoids derived from patients with metastatic prostate cancer, but had minimal effects on normal prostate spheroids. We report a novel synthetic lethal interaction between inhibitors of OGT and CDK9 that specifically kills prostate cancer cells, but not normal cells. Our study highlights the potential of combining OGT inhibitors with other treatments to exploit cancer-specific vulnerabilities. IMPLICATIONS: The primary contribution of OGT to cell proliferation is unknown, and in this study, we used a compound screen to indicate that OGT and CDK9 collaborate to sustain a cancer cell-specific pro-proliferative program. A better understanding of how OGT and CDK9 cross-talk will refine our understanding of this novel synthetic lethal interaction.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Quinase 9 Dependente de Ciclina/metabolismo , Inibidores Enzimáticos/farmacologia , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Sinergismo Farmacológico , Inibidores Enzimáticos/administração & dosagem , Humanos , Masculino , Terapia de Alvo Molecular , N-Acetilglucosaminiltransferases/metabolismo , Piperidinas/farmacologia , Neoplasias da Próstata/genética , Inibidores de Proteínas Quinases/administração & dosagem , Pirazóis/farmacologia
2.
Nucleic Acids Res ; 48(10): 5656-5669, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32329777

RESUMO

Intron detention in precursor RNAs serves to regulate expression of a substantial fraction of genes in eukaryotic genomes. How detained intron (DI) splicing is controlled is poorly understood. Here, we show that a ubiquitous post-translational modification called O-GlcNAc, which is thought to integrate signaling pathways as nutrient conditions fluctuate, controls detained intron splicing. Using specific inhibitors of the enzyme that installs O-GlcNAc (O-GlcNAc transferase, or OGT) and the enzyme that removes O-GlcNAc (O-GlcNAcase, or OGA), we first show that O-GlcNAc regulates splicing of the highly conserved detained introns in OGT and OGA to control mRNA abundance in order to buffer O-GlcNAc changes. We show that OGT and OGA represent two distinct paradigms for how DI splicing can control gene expression. We also show that when DI splicing of the O-GlcNAc-cycling genes fails to restore O-GlcNAc homeostasis, there is a global change in detained intron levels. Strikingly, almost all detained introns are spliced more efficiently when O-GlcNAc levels are low, yet other alternative splicing pathways change minimally. Our results demonstrate that O-GlcNAc controls detained intron splicing to tune system-wide gene expression, providing a means to couple nutrient conditions to the cell's transcriptional regime.


Assuntos
Acetilglucosamina/metabolismo , Glicosídeo Hidrolases/genética , Íntrons , N-Acetilglucosaminiltransferases/genética , Splicing de RNA , Linhagem Celular , Glicosídeo Hidrolases/metabolismo , Células HEK293 , Humanos , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/metabolismo , Fosforilação , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/metabolismo , RNA-Seq
3.
J Am Chem Soc ; 140(42): 13542-13545, 2018 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-30285435

RESUMO

Reversible glycosylation of nuclear and cytoplasmic proteins is an important regulatory mechanism across metazoans. One enzyme, O-linked N-acetylglucosamine transferase (OGT), is responsible for all nucleocytoplasmic glycosylation and there is a well-known need for potent, cell-permeable inhibitors to interrogate OGT function. Here we report the structure-based evolution of OGT inhibitors culminating in compounds with low nanomolar inhibitory potency and on-target cellular activity. In addition to disclosing useful OGT inhibitors, the structures we report provide insight into how to inhibit glycosyltransferases, a family of enzymes that has been notoriously refractory to inhibitor development.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Células HCT116 , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , N-Acetilglucosaminiltransferases/química , N-Acetilglucosaminiltransferases/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
4.
J Am Chem Soc ; 139(31): 10597-10600, 2017 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-28727445

RESUMO

Antibiotic-resistant strains of Staphylococcus aureus pose a major threat to human health and there is an ongoing need for new antibiotics to treat resistant infections. In a high throughput screen (HTS) of 230 000 small molecules designed to identify bioactive wall teichoic acid (WTA) inhibitors, we identified one hit, which was expanded through chemical synthesis into a small panel of potent compounds. We showed that these compounds target TarG, the transmembrane component of the two-component ATP-binding cassette (ABC) transporter TarGH, which exports WTA precursors to the cell surface for attachment to peptidoglycan. We purified, for the first time, a WTA transporter and have reconstituted ATPase activity in proteoliposomes. We showed that this new compound series inhibits TarH-catalyzed ATP hydrolysis even though the binding site maps to TarG near the opposite side of the membrane. These are the first ABC transporter inhibitors shown to block ATPase activity by binding to the transmembrane domain. The compounds have potential as therapeutic agents to treat S. aureus infections, and purification of the transmembrane transporter will enable further development.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Ácidos Teicoicos/farmacologia , Adenosina Trifosfatases/antagonistas & inibidores , Sítios de Ligação , Parede Celular/química , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Modelos Biológicos , Estrutura Molecular , Peptidoglicano/química , Peptidoglicano/metabolismo , Ligação Proteica/efeitos dos fármacos
5.
Oncotarget ; 7(11): 12464-76, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26824323

RESUMO

Metabolic networks are highly connected and complex, but a single enzyme, O-GlcNAc transferase (OGT) can sense the availability of metabolites and also modify target proteins. We show that inhibition of OGT activity inhibits the proliferation of prostate cancer cells, leads to sustained loss of c-MYC and suppresses the expression of CDK1, elevated expression of which predicts prostate cancer recurrence (p=0.00179). Metabolic profiling revealed decreased glucose consumption and lactate production after OGT inhibition. This decreased glycolytic activity specifically sensitized prostate cancer cells, but not cells representing normal prostate epithelium, to inhibitors of oxidative phosphorylation (rotenone and metformin). Intra-cellular alanine was depleted upon OGT inhibitor treatment. OGT inhibitor increased the expression and activity of alanine aminotransferase (GPT2), an enzyme that can be targeted with a clinically approved drug, cycloserine. Simultaneous inhibition of OGT and GPT2 inhibited cell viability and growth rate, and additionally activated a cell death response. These combinatorial effects were predominantly seen in prostate cancer cells, but not in a cell-line derived from normal prostate epithelium. Combinatorial treatments were confirmed with two inhibitors against both OGT and GPT2. Taken together, here we report the reprogramming of energy metabolism upon inhibition of OGT activity, and identify synergistically lethal combinations that are prostate cancer cell specific.


Assuntos
Inibidores Enzimáticos/farmacologia , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Glicosilação , Humanos , Masculino , N-Acetilglucosaminiltransferases/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo
6.
Nat Chem Biol ; 12(1): 40-5, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26619249

RESUMO

The majority of bacterial proteins are dispensable for growth in the laboratory but nevertheless have important physiological roles. There are no systematic approaches to identify cell-permeable small-molecule inhibitors of these proteins. We demonstrate a strategy to identify such inhibitors that exploits synthetic lethal relationships both for small-molecule discovery and for target identification. Applying this strategy in Staphylococcus aureus, we have identified a compound that inhibits DltB, a component of the teichoic acid D-alanylation machinery that has been implicated in virulence. This D-alanylation inhibitor sensitizes S. aureus to aminoglycosides and cationic peptides and is lethal in combination with a wall teichoic acid inhibitor. We conclude that DltB is a druggable target in the D-alanylation pathway. More broadly, the work described demonstrates a systematic method to identify biologically active inhibitors of major bacterial processes that can be adapted to numerous organisms.


Assuntos
Amsacrina/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Staphylococcus aureus/efeitos dos fármacos , Aminoglicosídeos/farmacologia , Amsacrina/química , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Mutação , Bibliotecas de Moléculas Pequenas/farmacologia , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Ácidos Teicoicos/metabolismo
7.
Tetrahedron ; 70(27-28): 4250-4256, 2014 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-24914247

RESUMO

For the first time, nickel-catalyzed silyl-Heck reactions are reported. Using simple phosphine-supported nickel catalysts, direct activation of silyl triflates has been achieved. These results contrast earlier palladium-catalyzed systems, which require iodide additives to activate silyl-triflates. These nickel-based catalysts exhibit good functional group tolerance in the preparation of vinyl silanes, and unlike earlier systems, allows for the incorporation of trialkylsilanes larger than Me3Si.

8.
J Am Chem Soc ; 135(36): 13330-3, 2013 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-23984876

RESUMO

Vinyl silyl ethers and disiloxanes can now be prepared from aryl-substituted alkenes and related substrates using a silyl-Heck reaction. The reaction employs a commercially available catalyst system and mild conditions. This work represents a highly practical means of accessing diverse classes of vinyl silyl ether substrates in an efficient and direct manner with complete regiomeric and geometric selectivity.


Assuntos
Éteres/síntese química , Silanos/síntese química , Compostos de Vinila/síntese química , Alcenos/química , Éteres/química , Estrutura Molecular , Silanos/química , Compostos de Vinila/química
9.
Synlett ; 24(17): 2177-2182, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-29104371

RESUMO

Few methods exist to directly install silyl functionality onto olefins. This Synpacts highlights the state of the art of the silyl-Heck reaction and our recently developed conditions for preparing allyl and vinyl silanes from terminal alkenes using this method.

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