Suppressing fatty acid synthase by type I interferon and chemical inhibitors as a broad spectrum anti-viral strategy against SARS-CoV-2

SARS-CoV-2 is an emerging viral pathogen and a major global public health challenge since December of 2019, with limited effective treatments throughout the pandemic. As part of the innate immune response to viral infection, type I interferons (IFN-I) trigger a signaling cascade that culminates in the activation of hundreds of genes, known as interferon stimulated genes (ISGs), that collectively foster an antiviral state. We report here the identification of a group of type I interferon suppressed genes, including fatty acid synthase (FASN), which are involved in lipid metabolism. Overexpression of FASN or the addition of its downstream product, palmitate, increased viral infection while knockout or knockdown of FASN reduced infection. More importantly, pharmacological inhibitors of FASN effectively blocked infections with a broad range of viruses, including SARS-CoV-2 and its variants of concern. Thus, our studies not only suggest that downregulation of metabolic genes may present an antiviral strategy by type I interferon, but they also introduce the potential for FASN inhibitors to have a therapeutic application in combating emerging infectious diseases such as COVID-19.

Development of Rhodotorula mucilaginosa strain via random mutagenesis for improved lipid production

Aims@#Oleaginous yeasts are widely used for the production of biodiesel feedstocks because of their high lipid content. This research was aimed to conduct random mutagenesis of Rhodotorula mucilaginosa using ethyl methane sulfonate (EMS) and identify the mutants with improved lipid production. @*Methodology and results@#A total of twenty-two mutant isolates prescreened with cerulenin were produced and further characterized via M13 PCR fingerprinting to determine their polymorphism and genetic distances. Eight strains, namely M1, M2, M3, M4, M7, M10, M11 and M18, were chosen based on their genetic distances from the parental strain for biomass production. Six mutants (M1, M2, M3, M4, M7 and M18) showing the highest dry cell weights were further selected for evaluation of lipid production in a laboratory-scale bioreactor using glucose as a carbon source. Results indicated that parental strain exhibited lipid content of 1.83 g/L, while strains M1, M2, M3, M7 and M18 generated 2.37 g/L, 2.27 g/L, 2.27 g/L, 3.10 g/L and 3.83 g/L of intracellular lipid, respectively. These five mutants were identified to have significant increase in lipid production compared to the parental strain. @*Conclusion, significance and impact of study@#This study demonstrated enhanced lipid production in R. mucilaginosa by random mutagenesis. New generated strains had higher lipid productivity compared to parental strain and application of these strains in industry may reduce the overall cost of biodiesel production.

Estudos da resposta estringente de Bacillus subtilis e busca por pequenas moléculas moduladoras de RelA / Studies of the stringent response of Bacillus subtilis and search for small molecules capable of modulating RelA activity

Seja no meio ambiente, dentro de um hospedeiro ou em outro habitat, bactérias estarão frequentemente enfrentando condições adversas, como exposição a compostos antibacterianos ou carência nutricional. Em situações como essas, as bactérias são capazes de ativar a chamada resposta estringente, modulada pelo alarmônio (p)ppGpp. O acúmulo de (p)ppGpp promove a inibição da transcrição de rRNAs e tRNAs e a supressão do processo de tradução, e a ativação de operons de biossíntese de aminoácidos. Sabe-se também hoje que a resposta estringente está relacionada a outras importantes carências nutricionais em Escherichia coli, como a falta de ácidos graxos, porém não se sabe se o mesmo ocorre em Bacillus subtilis ou em outras Grampositivas. (p)ppGpp atua também direta e indiretamente em vários outros processos celulares, como motilidade, resistência a antibióticos, virulência e persistência, indicando que (p)ppGpp é um regulador central que integra informação metabólica e respostas adaptativas. O presente trabalho buscou estudar a correlação da resposta estringente de B. subtilis com a carência de ácidos graxos e a busca por pequenas moléculas capazes de modular RelA (a principal proteína envolvida na síntese de (p)ppGpp) e impedir o acúmulo de (p)ppGpp. Para a indução da carência de ácidos graxos, foram utilizadas duas estratégias; uso da droga Cerulenina (inibidor de FabF) e mutantes condicionais no gene FabF. Observou-se que mutantes incapazes de ativar a resposta estringente (cepa ppGpp(0) ou RelAD264G) apresentaram grande perda de viabilidade celular durante a carência de ácidos graxos, ao passo que a cepa selvagem manteve sua viabilidade celular. A causa da morte se deu majoritariamente devido ao colapso do potencial de membrana. Apesar de não termos observado aumento de (p)ppGpp nas células selvagens durante a carência de ácidos graxos, observou-se uma redução da razão GTP/ATP, ao passo que na cepa ppGpp(0), a razão GTP/ATP aumentou, devido ao acúmulo de GTP. O uso da droga decoinina, capaz de reduzir os níveis intracelulares de GTP, resgatou parcialmente a viabilidade da cepa e impediu a perda do potencial de membrana, indicando que os níveis de GTP são importantes durante a carência de ácidos graxos em B. subtilis. Para a triagem de pequenas moléculas inibidoras do acúmulo de (p)ppGpp, foi utilizada uma biblioteca de 2320 diferentes compostos químicos, e buscou-se drogas capazes de reverter o fenótipo de crescimento lento de cepas de B. subtilis que acumulam (p)ppGpp (via mutação pontual; mutante RelAH77A e via tratamento com o indutor hidroxamato de arginina) em meio rico. A primeira etapa selecionou 40 moléculas capazes de resgatar o crescimento de células tratadas com arginina-hidroxamato, porém apenas uma, salicilanilida, foi capaz de também resgatar o crescimento da cepa RelAH77A. Todavia, apesar de ser capaz de acelerar o crescimento de B. subtilis esse efeito é limitado. Diversos análogos de salicilanilida foram testados, porém não apresentaram efeito superior a salicilanilida para a reversão do fenótipo de crescimento lento de B. subtilis. Em adição, a droga não foi capaz de aumentar a sensibilidade dos organismos a diversos antibióticos testados, e aparentemente é incapaz de alterar os níveis internos de (p)ppGpp, porém é capaz de causar alterações nos níveis de ATP. Logo, acredita-se que o efeito observado para o crescimento das células seja devido a efeitos indiretos, possivelmente envolvendo alteração de outros nucleotídeos fosforilados

In the environment, inside a host or other habitat, bacteria will always face adverse conditions, as for example exposure to antimicrobials or starvation. In situations like those, bacteria activate the stringent response, modulated by the alarmone (p)ppGpp. (p)ppGpp accumulation promotes inhibition of rRNA and tRNA transcription and suppression of translational process, at the same time that it activates several amino acid biosynthesis operons. It is known also that the stringent response it is related to other starvation stress in Escherichia coli, like lack of fatty acids, but there is no knowledge if the same occurs for Bacillus subtilis or other gram-positive bacteria. ppGpp acts directly and indirectly affecting several other cellular process, as motility, resistance to antibiotics, virulence and persistence, indicating that (p)ppGpp is a central regulator that integrates metabolic information and adaptive responses. This work aimed to study the correlation between the stringent response in B. subtilis with fatty acid starvation, and search for small moleculas capable of modulating RelA (the main enzyme responsible for ppGpp synthesis) and stop (p)ppGpp production. For fatty acid starvation induction, two strategies were used; use of the drug Cerulenin (inhibitor of the FabF protein) and conditional mutants of the FabF gene. We observed that mutants incapable of activating the stringent response (strains ppGpp(0) ou RelAD264G) presented great loss of viability during fatty acid starvation, whereas the wild-type strain keeps its viability. The main cause of death is due membrane rupture in some cells, but mainly due to membrane potential collapse. Although we did not observed increase of (p)ppGpp in wild-type strains during fatty acid starvation, we observed reduction in GTP/ATP ratios, a hallmark of (p)ppGpp production in gram-positive bacteria. In the strain ppGpp(0) GTP/ATP ratio increased, mainly due to GTP increase. Using the drug decoyinine, capable of reducing GTP levels, partially recued viability and protects cells of losing its membrane potential, indicating that GTP levels plays an important role during fatty acid starvation in B. subtilis. For the screening of small molecules capable of inhibit (p)ppGpp production, a library of 2320 different chemical compounds were used, and we looked for drugs capable of reverting the slow growth phenotype of B. subtilis strains with (p)ppGpp accumulation (using a mutant RelAH77A; and using a stringent response inductor, arginine hidroxamate). The first step selected for 40 molecules capable of rescuing the growth of cells treated with arginine hidroxamate, but only one drug, salicilanilyde could also rescue the growth of the strain RelAH77A. Although capable of rescuing growth of B. subtilis that accumulates (p)ppGpp, this rescue is limited. Several analogues of salicilanilyde were tested, but none were stronger than salicilanilyde itself in rescuing growth of slow growing strains of B. subtilis. In addition, the drug was not capable of increasing antibiotic sensibility and it is incapable of changing intracellular (p)ppGpp levels, but it does shifts ATP levels. Therefore, we believe that the observed effects of salicilanilyde is due indirect action, probably involving other phosphorylated nucleotides, rather than modifying (p)ppGpp levels

Effect of cerulenin combined with gemcitabine on proliferation and apoptosis in human pancreatic cell line BxPC3 / 中华胰腺病杂志

Objective To investigate the effect and mechanism of action in pancreatic cancer BxPC3 cell treated by cerulenin in combination with gemcitabine.Methods BxPC3 cells were cultivated with 10 μg/ml cerulenin,20 μmol/L gemcitabine or 10 μg/ml cerulenin + 20 μmol/L gemcitabine for 48 h,and cells without treatment were control.Cell proliferation was detected by CCK-8 assay,and early apoptosis was detected by AnnexinV/PI double staining method.The expression of Bcl-2 mRNA and Bax mRNA were detected by RT-PCR,and the protein level of Bcl-2,Bax were detected by Western Blot.Results The inhibition rate of BxPC3 cells were 0,(51.28 ± 1.84) ％,(53.59 ± 1.62) ％,(84.57 ± 1.01) ％ in control,cerulenin group,gemcitabine group,combination group; and the rate of early apoptosis was (0.83 ± 0.31) ％,(31.37 ± 1.04) ％,(38.33 ± 0.75) ％,(69.43 ± 0.83) ％,and the expression of Bcl-2 mRNA was 0.67 ± 0.01,0.44 ±0.01,0.36 ±0.08,0.27 ±0.07,and the expression of Bax mRNA was 0.14 ±0.01,0.31 ± 0.02,0.32 ± 0.03,0.91 ± 0.06 ; while the ratio of Bcl-2 mRNA/Bax mRNA was 4.78 ± 0.13,1.39 ± 0.04,1.15 ± 0.02,0.30 ± 0.02 ; the expression of Bcl-2 protein was 1.24 ± 0.04,0.51 ± 0.02,0.42 ± 0.02,0.13 ±0.01 ; and the expression of Bax protein was 0.20 ± 0.05,0.47 ± 0.01,0.54 ± 0.01,1.21 ± 0.03 ; while the ratio of Bcl-2/Bax was 6.00 ± 0.11,1.11 ± 0.01,0.77 ± 0.03,0.10 ± 0.06.And the difference among the groups was statistically significant (P ＜ 0.01).Conclusions Cerulenin combined with gemcitabin has synergistic effect on the inhibition of BxPC cells proliferation.Its mechanisms may be up-regulation of Bax,down-regulation of Bcl-2,and promoting apoptosis of cells through the decrease of Bcl-2/Bax ratio.

Cerulenin induces apoptosis of the cells in oral squamous cell carcinoma / 实用口腔医学杂志

Subjective: To observe apoptosis of the cells in oral sq uamous cell carcinoma(SCC) induced by cerulenin. Methods: SCC TCA-83 cells and fresh tissue of SCC of tongue from 5 patients were expos ed to cerulenin (10 mg/ml) for 24 hours, then the genosome DNA of the cells was extracted and electrophoresed; the fresh tissue of SCC was assessed by TUNEL labeling. Results: DNA gel electrophoresis showed typical apoptic DNA ladders from TCA-83 cells. The rate (%) of TUNEL-positive cells in cerulenin treated tissue of SCC was 29.0?2.6～40.6?16.2,that in the control 2 . 0?1.7～14.7?0.6 (P

The change in metabolism of fatty acids in breast cancer cells and its significance / 肠外与肠内营养

Objectives:To observe the changes in metabolism of fatty acids in breast cancer cells and the effects of inhibiting fatty acid synthase(FAS) on the growth of breast cancer cells. Methods:By RT PCR,the expression of FAS mRNA in breast cancer cell line was examined.The growth inhibition of breast cancer cells by specific FAS inhibitor, cerulenin, was determined by MTT assay.Flow cytometric analysis was used to study the changes of cell cycle. Results:FAS mRNA expression in MCF 7 cells was high.We found that cerulenin caused dose and time dependent inhibition of growth of MCF 7 cells.The growth inhibition of MCF 7 after 48 hours exposure of cerulenin at 2.5,5,10 and 20 mg/L was ( 43.47 ?4.58)%,(62.92?2.68)%,(81.93?0.91)% and (67.7?12.27)%( P