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1.
Nature ; 626(7997): 194-206, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38096902

RESUMO

The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.


Assuntos
Endonucleases , Elementos Nucleotídeos Longos e Dispersos , DNA Polimerase Dirigida por RNA , Transcrição Reversa , Humanos , Microscopia Crioeletrônica , Endonucleases/química , Endonucleases/genética , Endonucleases/metabolismo , Elementos Nucleotídeos Longos e Dispersos/genética , RNA/genética , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Cristalografia por Raios X , DNA/biossíntese , DNA/genética , Imunidade Inata , Interferons/biossíntese
2.
Bioorg Med Chem Lett ; 28(13): 2324-2327, 2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-29801997

RESUMO

To identify a potent and selective nucleoside inhibitor of dengue virus RNA-dependent RNA polymerase, a series of 2'- and/or 4'-ribose sugar modified uridine nucleoside phosphoramidate prodrugs and their corresponding triphosphates were synthesized and evaluated. Replacement of 2'-OH with 2'-F led to be a poor substrate for both dengue virus and human mitochondrial RNA polymerases. Instead of 2'-fluorination, the introduction of fluorine at the ribose 4'-position was found not to affect the inhibition of the dengue virus polymerase with a reduction in uptake by mitochondrial RNA polymerase. 2'-C-ethynyl-4'-F-uridine phosphoramidate prodrug displayed potent anti-dengue virus activity in the primary human peripheral blood mononuclear cell-based assay with no significant cytotoxicity in human hepatocellular liver carcinoma cell lines and no mitochondrial toxicity in the cell-based assay using human prostate cancer cell lines.


Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Pró-Fármacos/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Uridina Monofosfato/análogos & derivados , Uridina Monofosfato/farmacologia , Antivirais/química , Antivirais/toxicidade , Vírus da Dengue/enzimologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/toxicidade , Células Hep G2 , Humanos , Leucócitos Mononucleares/virologia , Estrutura Molecular , Sistema Fagocitário Mononuclear/virologia , Pró-Fármacos/química , Pró-Fármacos/toxicidade , Relação Estrutura-Atividade
3.
Bioorg Med Chem Lett ; 22(12): 4127-32, 2012 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-22578461
4.
Bioorg Med Chem Lett ; 22(8): 2705-7, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22446091

RESUMO

A series of 1'-substituted analogs of 4-aza-7,9-dideazaadenosine C-nucleoside were prepared and evaluated for the potential as antiviral agents. These compounds showed a broad range of inhibitory activity against various RNA viruses. In particular, the whole cell potency against HCV when R=CN was attributed to inhibition of HCV NS5B polymerase and intracellular concentration of the corresponding nucleoside triphosphate.


Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Nucleosídeos/síntese química , Nucleosídeos/farmacologia , Adenosina/síntese química , Adenosina/química , Adenosina/farmacologia , Antivirais/química , Compostos Aza/síntese química , Compostos Aza/química , Compostos Aza/farmacologia , Humanos , Estrutura Molecular , Nucleosídeos/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/efeitos dos fármacos
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