RESUMO
Many viruses have evolved structured RNA elements that can influence transcript abundance and translational efficiency, and help evade host immune factors by hijacking cellular machinery during replication. Here, we evaluated the functional impact of sub-genomic flaviviral RNAs (sfRNAs) known to stall exoribonuclease activity, by incorporating these elements into recombinant adeno-associated viral (AAV) genome cassettes. Specifically, sfRNAs from Dengue, Zika, Japanese Encephalitis, Yellow Fever, Murray Valley Encephalitis, and West Nile viruses increased transcript stability and transgene expression compared to a conventional woodchuck hepatitis virus element (WPRE). Further dissection of engineered transcripts revealed that sfRNA elements (i) require incorporation in cis within the 3' untranslated region (UTR) of AAV genomes, (ii) require minimal dumbbell structures to exert the observed effects, and (iii) can stabilize AAV transcripts independent of 5'-3' exoribonuclease 1 (XRN1)-mediated decay. Additionally, preliminary in vivo assessment of AAV vectors bearing sfRNA elements in mice achieved increased transcript abundance and expression in cardiac tissue. Leveraging the functional versatility of engineered viral RNA elements may help improve the potency of AAV vector-based gene therapies. IMPORTANCE: Viral RNA elements can hijack host cell machinery to control stability of transcripts and consequently, infection. Studies that help better understand such viral elements can provide insights into antiviral strategies and also potentially leverage these features for therapeutic applications. In this study, by incorporating structured flaviviral RNA elements into recombinant adeno-associated viral (AAV) vector genomes, we show improved AAV transcript stability and transgene expression can be achieved, with implications for gene transfer.
Assuntos
Dependovirus , Vetores Genéticos , RNA Viral , Dependovirus/genética , Animais , RNA Viral/genética , RNA Viral/metabolismo , Vetores Genéticos/genética , Camundongos , Humanos , Estabilidade de RNA , Flaviviridae/genética , Transgenes , Células HEK293 , Genoma Viral , Regiões 3' não Traduzidas/genética , Exorribonucleases/metabolismo , Exorribonucleases/genéticaRESUMO
We report the synthesis of 2,6-disubstituted pyrazines as potent cell active CSNK2A inhibitors. 4'-Carboxyphenyl was found to be the optimal 2-pyrazine substituent for CSNK2A activity, with little tolerance for additional modification. At the 6-position, modifications of the 6-isopropylaminoindazole substituent were explored to improve selectivity over PIM3 while maintaining potent CSNK2A inhibition. The 6-isopropoxyindole analogue 6c was identified as a nanomolar CSNK2A inhibitor with 30-fold selectivity over PIM3 in cells. Replacement of the 6-isopropoxyindole by isosteric ortho-methoxy anilines, such as 7c, generated analogues with selectivity for CSNK2A over PIM3 and improved the kinome-wide selectivity. The optimized 2,6-disubstituted pyrazines showed inhibition of viral replication consistent with their CSNK2A activity.
Assuntos
Benzoatos , Pirazinas , Relação Estrutura-Atividade , Pirazinas/farmacologia , Antivirais/farmacologiaRESUMO
Human cytomegalovirus (HCMV) requires the robust expression of two immediate early proteins, IE1 and IE2, immediately upon infection to suppress the antiviral response and promote viral gene expression. While transcriptional control of IE1 and IE2 has been extensively studied, the role of post-transcriptional regulation of IE1 and IE2 expression is relatively unexplored. We previously found that the shared major immediate early 5' untranslated region (MIE 5' UTR) of the mature IE1 and IE2 transcripts plays a critical role in facilitating the translation of the IE1 and IE2 mRNAs. As RNA secondary structure in 5' UTRs can regulate mRNA translation efficiency, we used selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) to identify RNA structures in the shared MIE 5' UTR. We found that the MIE 5' UTR contains three stable stem loop structures. Using a series of recombinant viruses to investigate the role of each stem loop in IE1 and IE2 protein synthesis, we found that the stem loop closest to the 5' end of the MIE 5' UTR (SL1) is both necessary and sufficient for efficient IE1 and IE2 mRNA translation and HCMV replication. The positive effect of SL1 on mRNA translation and virus replication was dependent on its location within the 5' UTR. Surprisingly, a synthetic stem loop with the same free energy as SL1 in its native location also supported wild type levels of IE1 and IE2 mRNA translation and virus replication, suggesting that the presence of RNA structure at a specific location in the 5' UTR, rather than the primary sequence of the RNA, is critical for efficient IE1 and IE2 protein synthesis. These data reveal a novel post-transcriptional regulatory mechanism controlling IE1 and IE2 expression and reinforce the critical role of RNA structure in regulating HCMV protein synthesis and replication.IMPORTANCEThese results reveal a new aspect of immediate early gene regulation controlled by non-coding RNA structures in viral mRNAs. Previous studies have largely focused on understanding viral gene expression at the level of transcriptional control. Our results show that a complete understanding of the control of viral gene expression must include an understanding of viral mRNA translation, which is driven in part by RNA structure(s) in the 5' UTR of viral mRNAs. Our results illustrate the importance of these additional layers of regulation by defining specific 5' UTR RNA structures regulating immediate early gene expression in the context of infection and identify important features of RNA structure that govern viral mRNA translation efficiency. These results may therefore broadly impact current thinking on how viral gene expression is regulated for human cytomegalovirus and other DNA viruses.
Assuntos
Citomegalovirus , Proteínas Imediatamente Precoces , Humanos , Regiões 5' não Traduzidas , Citomegalovirus/fisiologia , Proteínas Virais/metabolismo , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Replicação Viral , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
A series of 5-benzylamine-substituted pyrimido[4,5-c]quinoline derivatives of the CSNK2A chemical probe SGC-CK2-2 were synthesized with the goal of improving kinase inhibitor cellular potency and antiviral phenotypic activity while maintaining aqueous solubility. Among the range of analogs, those bearing electron-withdrawing (4c and 4g) or donating (4f) substituents on the benzyl ring as well as introduction of non-aromatic groups such as the cyclohexylmethyl (4t) were shown to maintain CSNK2A activity. The CSNK2A activity was also retained with N-methylation of SGC-CK2-2, but α-methyl substitution of the benzyl substituent led to a 10-fold reduction in potency. CSNK2A inhibition potency was restored with indene-based compound 4af, with activity residing in the S-enantiomer (4ag). Analogs with the highest CSNK2A potency showed good activity for inhibition of Mouse Hepatitis Virus (MHV) replication. Conformational analysis indicated that analogs with the best CSNK2A inhibition (4t, 4ac, and 4af) exhibited smaller differences between their ground state conformation and their predicted binding pose. Analogs with reduced activity (4ad, 4ae, and 4ai) required more substantial conformational changes from their ground state within the CSNK2A protein pocket.
RESUMO
Mammalian target of rapamycin (mTOR) is a key regulator of metabolism in the mammalian cell. Here, we show the essential role for mTOR signaling in the immune response to bacterial infection. Inhibition of mTOR during infection with Staphylococcus aureus revealed that mTOR signaling is required for bactericidal free radical production by phagocytes. Mechanistically, mTOR supported glucose transporter GLUT1 expression, potentially through hypoxia-inducible factor 1α, upon phagocyte activation. Cytokine and chemokine signaling, inducible nitric oxide synthase, and p65 nuclear translocation were present at similar levels during mTOR suppression, suggesting an NF-κB-independent role for mTOR signaling in the immune response during bacterial infection. We propose that mTOR signaling primarily mediates the metabolic requirements necessary for phagocyte bactericidal free radical production. This study has important implications for the metabolic requirements of innate immune cells during bacterial infection as well as the clinical use of mTOR inhibitors.IMPORTANCESirolimus, everolimus, temsirolimus, and similar are a class of pharmaceutics commonly used in the clinical treatment of cancer and the anti-rejection of transplanted organs. Each of these agents suppresses the activity of the mammalian target of rapamycin (mTOR), a master regulator of metabolism in human cells. Activation of mTOR is also involved in the immune response to bacterial infection, and treatments that inhibit mTOR are associated with increased susceptibility to bacterial infections in the skin and soft tissue. Infections caused by Staphylococcus aureus are among the most common and severe. Our study shows that this susceptibility to S. aureus infection during mTOR suppression is due to an impaired function of phagocytic immune cells responsible for controlling bacterial infections. Specifically, we observed that mTOR activity is required for phagocytes to produce antimicrobial free radicals. These results have important implications for immune responses during clinical treatments and in disease states where mTOR is suppressed.
Assuntos
Transportador de Glucose Tipo 1 , Fagócitos , Transdução de Sinais , Infecções Estafilocócicas , Staphylococcus aureus , Serina-Treonina Quinases TOR , Staphylococcus aureus/imunologia , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Fagócitos/imunologia , Fagócitos/metabolismo , Fagócitos/microbiologia , Humanos , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 1/genética , Animais , Radicais Livres/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
We previously reported that deletion of a 44-nucleotide element in the 3' untranslated region (UTR) of the Chikungunya virus (CHIKV) genome enhances the virulence of CHIKV infection in mice. Here, we find that while this 44-nucleotide deletion enhances CHIKV fitness in murine embryonic fibroblasts in a manner independent of the type I interferon response, the same mutation decreases viral fitness in C6/36 mosquito cells. Further, the fitness advantage conferred by the UTR deletion in mammalian cells is maintained in vivo in a mouse model of CHIKV dissemination. Finally, SHAPE-MaP analysis of the CHIKV 3' UTR revealed this 44-nucleotide element forms a distinctive two-stem-loop structure that is ablated in the mutant 3' UTR without altering additional 3' UTR RNA secondary structures.
Assuntos
Regiões 3' não Traduzidas , Febre de Chikungunya , Vírus Chikungunya , Replicação Viral , Vírus Chikungunya/genética , Vírus Chikungunya/fisiologia , Animais , Camundongos , Febre de Chikungunya/virologia , RNA Viral/genética , Virulência , Linhagem Celular , Fibroblastos/virologia , Aptidão Genética , Humanos , Deleção de Sequência , Conformação de Ácido Nucleico , Modelos Animais de DoençasRESUMO
HCMV genes UL135 and UL138 play opposing roles regulating latency and reactivation in CD34+ human progenitor cells (HPCs). Using the THP-1 cell line model for latency and reactivation, we designed an RNA sequencing study to compare the transcriptional profile of HCMV infection in the presence and absence of these genes. The loss of UL138 results in elevated levels of viral gene expression and increased differentiation of cell populations that support HCMV gene expression and genome synthesis. The loss of UL135 results in diminished viral gene expression during an initial burst that occurs as latency is established and no expression of eleven viral genes from the ULb' region even following stimulation for differentiation and reactivation. Transcriptional network analysis revealed host transcription factors with potential to regulate the ULb' genes in coordination with pUL135. These results reveal roles for UL135 and UL138 in regulation of viral gene expression and potentially hematopoietic differentiation.
RESUMO
Despite their widespread impact on human health there are no approved drugs for combating alphavirus infections. The heterocyclic ß-aminomethyl vinyl sulfone RA-0002034 (1a) is a potent irreversible covalent inhibitor of the alphavirus nsP2 cysteine protease with broad spectrum antiviral activity. Analogs of 1a that varied each of three regions of the molecule were synthesized to establish structure-activity relationships for inhibition of Chikungunya (CHIKV) nsP2 protease and viral replication. The covalent warhead was highly sensitive to modifications of the sulfone or vinyl substituents. However, numerous alterations to the core 5-membered heterocycle and its aryl substituent were well tolerated and several analogs were identified that enhanced CHIKV nsP2 binding. For example, the 4-cyanopyrazole analog 8d exhibited a kinact /Ki ratio >10,000 M-1s-1. 3-Arylisoxazole was identified an isosteric replacement for the 5-membered heterocycle, which circumvented the intramolecular cyclization that complicated the synthesis of pyrazole-based inhibitors like 1a. The accumulated structure-activity data was used to build a ligand-based model of the enzyme active site, which can be used to guide the design of covalent nsP2 protease inhibitors as potential therapeutics against alphaviruses.
RESUMO
The pyrazolo[1,5-a]pyrimidine scaffold is a promising scaffold to develop potent and selective CSNK2 inhibitors with antiviral activity against ß-coronaviruses. Herein, we describe the discovery of a 1,2,4-triazole group to substitute a key amide group for CSNK2 binding present in many potent pyrazolo[1,5-a]pyrimidine inhibitors. Crystallographic evidence demonstrates that the 1,2,4-triazole replaces the amide in forming key hydrogen bonds with Lys68 and a water molecule buried in the ATP-binding pocket. This isosteric replacement improves potency and metabolic stability at a cost of solubility. Optimization for potency, solubility, and metabolic stability led to the discovery of the potent and selective CSNK2 inhibitor 53. Despite excellent in vitro metabolic stability, rapid decline in plasma concentration of 53 in vivo was observed and may be attributed to lung accumulation, although in vivo pharmacological effect was not observed. Further optimization of this novel chemotype may validate CSNK2 as an antiviral target in vivo.
Assuntos
Antivirais , Caseína Quinase II , Pirimidinas , Triazóis , Replicação Viral , Triazóis/farmacologia , Triazóis/química , Triazóis/síntese química , Pirimidinas/farmacologia , Pirimidinas/química , Pirimidinas/síntese química , Antivirais/farmacologia , Antivirais/química , Antivirais/síntese química , Animais , Humanos , Replicação Viral/efeitos dos fármacos , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/metabolismo , Pirazóis/farmacologia , Pirazóis/química , Pirazóis/síntese química , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Amidas/química , Amidas/farmacologia , Amidas/síntese química , Relação Estrutura-Atividade , Camundongos , Ratos , SARS-CoV-2/efeitos dos fármacos , Descoberta de Drogas , MasculinoRESUMO
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has been responsible for numerous large-scale outbreaks in the last twenty years. Currently, there are no FDA-approved therapeutics for any alphavirus infection. CHIKV non-structural protein 2 (nsP2), which contains a cysteine protease domain, is essential for viral replication, making it an attractive target for a drug discovery campaign. Here, we optimized a CHIKV nsP2 protease (nsP2pro) biochemical assay for the screening of a 6,120-compound cysteine-directed covalent fragment library. Using a 50% inhibition threshold, we identified 153 hits (2.5% hit rate). In dose-response follow up, RA-0002034, a covalent fragment that contains a vinyl sulfone warhead, inhibited CHIKV nsP2pro with an IC 50 of 58 ± 17 nM, and further analysis with time-dependent inhibition studies yielded a k inact /K I of 6.4 x 10 3 M -1 s -1 . LC-MS/MS analysis determined that RA-0002034 covalently modified the catalytic cysteine in a site-specific manner. Additionally, RA-0002034 showed no significant off-target reactivity against a panel of cysteine proteases. In addition to the potent biochemical inhibition of CHIKV nsP2pro activity and exceptional selectivity, RA-0002034 was tested in cellular models of alphavirus infection and effectively inhibited viral replication of both CHIKV and related alphaviruses. This study highlights the discovery and characterization of the chemical probe RA-0002034 as a promising hit compound from covalent fragment-based screening for development toward a CHIKV or pan-alphavirus therapeutic. Significance Statement: Chikungunya virus is one of the most prominent and widespread alphaviruses and has caused explosive outbreaks of arthritic disease. Currently, there are no FDA-approved drugs to treat disease caused by chikungunya virus or any other alphavirus-caused infection. Here, we report the discovery of a covalent small molecule inhibitor of chikungunya virus nsP2 protease activity and viral replication of four diverse alphaviruses. This finding highlights the utility of covalent fragment screening for inhibitor discovery and represents a starting point towards the development of alphavirus therapeutics targeting nsP2 protease.
RESUMO
We report the synthesis of 2,6-disubstituted pyrazines as potent cell active CSNK2A inhibitors. 4'-Carboxyphenyl was found to be the optimal 2-pyrazine substituent for CSNK2A activity, with little tolerance for additional modification. At the 6-position, modifications of the 6-isopropylaminoindazole substituent were explored to improve selectivity over PIM3 while maintaining potent CSNK2A inhibition. The 6-isopropoxyindole analogue 6c was identified as a nanomolar CSNK2A inhibitor with 30-fold selectivity over PIM3 in cells. Replacement of the 6-isopropoxyindole by isosteric ortho-methoxy anilines, such as 7c, generated analogues with selectivity for CSNK2A over PIM3 and improved the kinome-wide selectivity. The optimized 2,6-disubstituted pyrazines showed inhibition of viral replication consistent with their CSNK2A activity.