Identification of inhibitors of the E. coli cyclopropane fatty acid synthase from the screening of a chemical library: In vitro and in vivo studies

CLPB (Caseinolytic peptidase B) protein is a conformational mimetic of α-MSH, an anorectic hormone. Previous in vivo studies have already shown the potential effect of CLPB protein on food intake and on the production of peptide YY (PYY) by injection of E. coli wild type (WT) or E. coli ΔClpB. However, until now, no study has shown its direct effect on food intake. Furthermore, this protein can fragment naturally. Therefore, the aim of this study was (i) to evaluate the in vitro effects of CLPB fragments on PYY production; and (ii) to test the in vivo effects of a CLPB fragment sharing molecular mimicry with α-MSH (CLPB25) compared to natural fragments of the CLPB protein (CLPB96). To do that, a primary culture of intestinal mucosal cells from male Sprague–Dawley rats was incubated with proteins extracted from E. coli WT and ΔCLPB after fragmentation with trypsin or after a heat treatment of the CLPB protein. PYY secretion was measured by ELISA. CLPB fragments were analyzed by Western Blot using anti-α-MSH antibodies. In vivo effects of the CLPB protein on food intake were evaluated by intraperitoneal injections in male C57Bl/6 and ob/ob mice using the BioDAQ® system. The natural CLPB96 fragmentation increased PYY production in vitro and significantly decreased cumulative food intake from 2 h in C57Bl/6 and ob/ob mice on the contrary to CLPB25. Therefore, the anorexigenic effect of CLPB is likely the consequence of enhanced PYY secretion.

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Fatty acid is a potential agent for bone tissue induction: In vitro and in vivo approach

Experimental Biology and Medicine ◽

10.1177/1535370217731104 ◽

2017 ◽

Vol 242 (18) ◽

pp. 1765-1771 ◽

Cited By ~ 6

Author(s):

Guinea BC Cardoso ◽

Erivelto Chacon ◽

Priscila GL Chacon ◽

Pedro Bordeaux-Rego ◽

Adriana SS Duarte ◽

...

Keyword(s):

Stem Cells ◽

Oleic Acid ◽

Fatty Acid ◽

Bone Tissue ◽

In Vivo Studies ◽

Surrounding Tissue ◽

Bone Induction ◽

Acid Addition

Our hypothesis was to investigate the fatty acid potential as a bone induction factor. In vitro and in vivo studies were performed to evaluate this approach. Oleic acid was used in a 0.5 wt.% concentration. Polycaprolactone was used as the polymeric matrix by combining solvent-casting and particulate-leaching techniques, with a final porosity of 70 wt.%, investigated by SEM images. Contact angle measurements were produced to investigate the influence of oleic acid on polycaprolactone chains. Cell culture was performed using adipocyte-derived stem cells to evaluate biocompatibility and bioactivity properties. In addition, in vivo studies were performed to evaluate the induction potential of oleic acid addition. Adipocyte-derived stem cells were used to provide differentiation after 21 days of culture. Likewise, information were obtained with in vivo data and cellular invagination was observed on both scaffolds (polycaprolactone and polycaprolactone /oleic acid); interestingly, the scaffold with oleic acid addition demonstrated that cellular migrations are not related to the surrounding tissue, indicating bioactive potential. Our hypothesis is that fatty acid may be used as a potential induction factor for bone tissue engineering. The study’s findings indicate oleic acid as a possible agent for bone induction, according to data on cell differentiation, proliferation, and migration. Impact statement The biomaterial combined in this study on bone regeneration is innovative and shows promising results in the treatment of bone lesions. Polycaprolactone (PCL) and oleic acid have been studied separately. In this research, we combined biomaterials to assess the stimulus and the speed of bone healing.

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Targeting de novo lipogenesis and the Lands cycle induces ferroptosis in KRAS-mutant lung cancer

10.1101/2021.03.18.434804 ◽

2021 ◽

Author(s):

Caterina Bartolacci ◽

Cristina Andreani ◽

Goncalo Dias do Vale ◽

Stefano Berto ◽

Margherita Melegari ◽

...

Keyword(s):

Lung Cancer ◽

Fatty Acid ◽

Metabolic Networks ◽

Fatty Acid Synthase ◽

De Novo ◽

Genetically Engineered ◽

De Novo Lipogenesis ◽

Main Process

Mutant KRAS (KM) is the most common oncogene in lung cancer (LC). KM regulates several metabolic networks, but their role in tumorigenesis is still not sufficiently characterized to be exploited in cancer therapy. To identify metabolic networks specifically deregulated in KMLC, we characterized the lipidome of genetically engineered LC mice, cell lines, patient derived xenografts and primary human samples. We also determined that KMLC, but not EGFR-mutant (EGFR-MUT) LC, is enriched in triacylglycerides (TAG) and phosphatidylcholines (PC). We also found that KM upregulates fatty acid synthase (FASN), a rate-limiting enzyme in fatty acid (FA) synthesis promoting the synthesis of palmitate and PC. We determined that FASN is specifically required for the viability of KMLC, but not of LC harboring EGFR-MUT or wild type KRAS. Functional experiments revealed that FASN inhibition leads to ferroptosis, a reactive oxygen species (ROS)-and iron-dependent cell death. Consistently, lipidomic analysis demonstrated that FASN inhibition in KMLC leads to accumulation of PC with polyunsaturated FA (PUFA) chains, which are the substrate of ferroptosis. Integrating lipidomic, transcriptome and functional analyses, we demonstrated that FASN provides saturated (SFA) and monounsaturated FA (MUFA) that feed the Lands cycle, the main process remodeling oxidized phospholipids (PL), such as PC. Accordingly, either inhibition of FASN or suppression of the Lands cycle enzymes PLA2 and LPCAT3, promotes the intracellular accumulation of lipid peroxides and ferroptosis in KMLC both in vitro and in vivo. Our work supports a model whereby the high oxidative stress caused by KM dictates a dependency on newly synthesized FA to repair oxidated phospholipids, establishing a targetable vulnerability. These results connect KM oncogenic signaling, FASN induction and ferroptosis, indicating that FASN inhibitors already in clinical trial in KMLC patients (NCT03808558) may be rapidly deployed as therapy for KMLC.

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Crocetin Extracted from Saffron Shows Antitumor Effects in Models of Human Glioblastoma

International Journal of Molecular Sciences ◽

10.3390/ijms21020423 ◽

2020 ◽

Vol 21 (2) ◽

pp. 423 ◽

Cited By ~ 3

Author(s):

Alessandro Colapietro ◽

Andrea Mancini ◽

Flora Vitale ◽

Stefano Martellucci ◽

Adriano Angelucci ◽

...

Keyword(s):

Wound Repair ◽

Fatty Acid Synthase ◽

Disease Free Survival ◽

In Vivo Studies ◽

Comparable Efficacy ◽

Antitumor Effects ◽

Radio Therapy ◽

U87 Cells

Over recent years, many authors discussed the effects of different natural compounds on glioblastoma (GBM). Due to its capacity to impair survival and progression of different cancer types, saffron extract (SE), named crocetin (CCT), is particularly noteworthy. In this work, we elucidated the antitumor properties of crocetin in glioma in vivo and in vitro models for the first time. The in vitro results showed that the four tumor cell lines observed in this study (U251, U87, U138, and U373), which were treated with increasing doses of crocetin, showed antiproliferative and pro-differentiative effects as demonstrated by a significant reduction in the number of viable cells, deep changes in cell morphology, and the modulation of mesenchymal and neuronal markers. Indeed, crocetin decreased the expression of Cluster of Differentiation CD44, CD90, CXCR4, and OCT3/4 mesenchymal markers, but increased the expression of βIII-Tubulin and neurofilaments (NFH) neuronal linage-related markers. Epigenetic mechanisms may modulate these changes, since Histone Deacetylase, HDAC1 and HDAC3 were downmodulated in U251 and U87 cells, whereas HDAC1 expression was downmodulated in U138 and U373 cells. Western blotting analyses of Fatty Acid Synthase, FASN, and CD44 resulted in effective inhibition of these markers after CCT treatment, which was associated with important activation of the apoptosis program and reduced glioma cell movement and wound repair. The in vivo studies aligned with the results obtained in vitro. Indeed, crocetin was demonstrated to inhibit the growth of U251 and U87 cells that were subcutaneously injected into animal models. In particular, the Tumor To Progression or TTP values and Kaplan–Meier curves indicated that crocetin had more major effects than radiotherapy alone, but similar effects to temozolomide (TMZ). An intra-brain cell inoculation of a small number of luciferase-transfected U251 cells provided a model that was able to recapitulate recurrence after surgical tumor removal. The results obtained from the orthotopic intra-brain model indicated that CCT treatment increased the disease-free survival (DFS) and overall survival (OS) rates, inducing a delay in appearance of a detectable bioluminescent lesion. CCT showed greater efficacy than Radio Therapy (RT) but comparable efficacy to temozolomide in xenograft models. Therefore, we aimed to continue the study of crocetin’s effects in glioma disease, focusing our attention on the radiosensitizing properties of the natural compound and highlighting the ways in which this was realized.

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Once-daily gentamicin therapy for experimental Escherichia coli meningitis.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.1.49 ◽

1997 ◽

Vol 41 (1) ◽

pp. 49-53 ◽

Cited By ~ 23

Author(s):

A Ahmed ◽

M M París ◽

M Trujillo ◽

S M Hickey ◽

L Wubbel ◽

...

Keyword(s):

In Vivo Studies ◽

Bacterial Killing ◽

Once Daily ◽

E Coli ◽

Aminoglycoside Therapy ◽

Gentamicin Concentration ◽

Gentamicin Therapy ◽

The Impact

In vitro and in vivo studies have demonstrated that the bacteriologic efficacy of once-daily aminoglycoside therapy is equivalent to that achieved with conventional multiple daily dosing. The impact of once-daily dosing for meningitis has not been studied. Using the well-characterized rabbit meningitis model, we compared two regimens of the same daily dosage of gentamicin given either once or in three divided doses for 24 or 72 h. The initial 1 h mean cerebrospinal fluid (CSF) gentamicin concentration for animals receiving a single dose (2.9 +/- 1.7 micrograms/ml) was threefold higher than that for the animals receiving multiple doses. The rate of bacterial killing in the first 8 h of treatment was significantly greater for the animals with higher concentrations in their CSF (-0.21 +/- 0.19 versus -0.03 +/- 0.22 log10 CFU/ml/h), suggesting concentration-dependent killing. By 24h, the mean reduction in bacterial titers was similar for the two regimens. In animals treated for 72 h, no differences in bactericidal activity was noted for 24, 48, or 72 h. Gentamicin at two different dosages was administered intracisternally to a separate set of animals to achieve considerably higher CSF gentamicin concentrations. In these animals, the rate of bacterial clearance in the first 8 h (0.52 +/- 0.15 and 0.58 +/- 0.15 log10 CFU/ml/h for the lower and higher dosages, respectively) was significantly greater than that in animals treated intravenously. In conclusion, there is evidence of concentration-dependent killing with gentamicin early in treatment for experimental E. coli meningitis, and once-daily dosing therapy appears to be at least as effective as multiple-dose therapy in reducing bacterial counts in CSF.

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Fatty Acid Synthesis Is Essential for Survival of Cryptococcus neoformans and a Potential Fungicidal Target

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00442-07 ◽

2007 ◽

Vol 51 (10) ◽

pp. 3537-3545 ◽

Cited By ~ 28

Author(s):

Methee Chayakulkeeree ◽

Thomas H. Rude ◽

Dena L. Toffaletti ◽

John R. Perfect

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Cryptococcus Neoformans ◽

Fatty Acid Synthase ◽

Acid Synthesis ◽

Yeast Cells ◽

Synthase Inhibitor ◽

The Brain

ABSTRACT Fatty acid synthase in the yeast Cryptococcus neoformans is composed of two subunits encoded by FAS1 and FAS2 genes. We inserted a copper-regulated promoter (P CTR4-2 ) to regulate FAS1 and FAS2 expression in Cryptococcus neoformans (strains P CTR4-2 /FAS1 and P CTR4-2 /FAS2, respectively). Both mutants showed growth rates similar to those of the wild type in a low-copper medium in which FAS1 and FAS2 were expressed, but even in the presence of exogenous fatty acids, strains were suppressed in growth under high-copper conditions. The treatment of C. neoformans with fluconazole was shown to have an increased inhibitory activity and even became fungicidal when either FAS1 or FAS2 expression was suppressed. Furthermore, a subinhibitory dose of fluconazole showed anticryptococcal activity in vitro in the presence of cerulenin, a fatty acid synthase inhibitor. In a murine model of pulmonary cryptococcosis, a tissue census of yeast cells in P CTR4-2 /FAS2 strain at day 7 of infection was significantly lower than that in mice treated with tetrathiomolybdate, a copper chelator (P < 0.05), and a yeast census of P CTR4-2 /FAS1 strain at day 14 of infection in the brain was lower in the presence of more copper. In fact, no positive cultures from the brain were detected in mice (with or without tetrathiomolybdate treatment) infected with the P CTR4-2 /FAS2 strain, which implies that this mutant did not reach the brain in mice. We conclude that both FAS1 and FAS2 in C. neoformans are essential for in vitro and in vivo growth in conditions with and without exogenous fatty acids and that FAS1 and FAS2 can potentially be fungicidal targets for C. neoformans with a potential for synergistic behavior with azoles.

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Synthesis and Evaluation of Analogues of S-Adenosyl-L-methionine, as Inhibitors of the E. coli Cyclopropane Fatty Acid Synthase.

ChemInform ◽

10.1002/chin.200433224 ◽

2004 ◽

Vol 35 (33) ◽

Author(s):

Christine Guerard ◽

Maud Breard ◽

Fabienne Courtois ◽

Thierry Drujon ◽

Olivier Ploux

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthase ◽

Cyclopropane Fatty Acid ◽

E Coli

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In vitro antibiotic sensitivity and therapeutic efficacy of experimental salmonellosis, colibacillosis and pasteurellosis in broiler chickens

Bangladesh Journal of Veterinary Medicine ◽

10.3329/bjvm.v2i2.2538 ◽

1970 ◽

Vol 2 (2) ◽

pp. 99-102 ◽

Cited By ~ 6

Author(s):

MA Rahman ◽

MA Samad ◽

MB Rahman ◽

SML Kabir

Keyword(s):

Broiler Chickens ◽

Pasteurella Multocida ◽

Antibiotic Sensitivity ◽

Morbidity And Mortality ◽

In Vivo Studies ◽

Penicillin G ◽

E Coli ◽

Therapeutic Trials

Avian salmonellosis (AS), avian colibacillosis (AC) and avian pasteurellosis (AP) have been recognized as important bacterial diseases in poultry associated with morbidity and mortality in Bangladesh. The causative agents of these three diseases were isolated (5 isolates / disease) from dead chickens submitted for diagnosis at the BRAC Poultry Disease Diagnostic Centre, Gazipur during the period from January to December 2002. Five isolates of each of the Salmonella pullorum, Escherichia coli and Pasteurella multocida were evaluated against eight antibiotic containing disc which included ciprofloxacin, gentamicin, ampicillin, chloramphenicol, erythromycin, tetracycline, cephradine and penicillin G. Erythromycin in S. pullorum and Ciprofloxacin both in the E. coli and P. multocida were found highest sensitive, gentamicin, chloramphenicol, cephradine were found moderately sensitive to S. pullorum, gentamicin, tetracycline, erythromycin and ampicillin were found moderately sensitive to E. coli, and gentamicin ampicillin, cephradine and penicillin G were moderately sensitive to P. multocida. Therapeutic trials against experimentally produced S. pullorum, E. coli and P. multocida infection in three groups of broiler chickens showed that cephradine against S. pullorum and ciprofloxacin against both in E. coli and P. multocida were found highly effective both in vitro and in vivo studies, therefore, cephradine against salmonellosis and ciprofloxacin against colibacillosis and pasteurellosis are effective drugs of choice which could be used to control morbidity and mortality in poultry caused by these diseases.Key words: antibiotic sensitivity; salmonellosis; colibacillosis; pasteurellosis, broiler chickensdoi: 10.3329/bjvm.v2i2.2538Bangl. J. Vet. Med. (2004). 2 (2): 99-102

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Antimycobacterial action of thiolactomycin: an inhibitor of fatty acid and mycolic acid synthesis.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.12.2813 ◽

1996 ◽

Vol 40 (12) ◽

pp. 2813-2819 ◽

Cited By ~ 105

Author(s):

R A Slayden ◽

R E Lee ◽

J W Armour ◽

A M Cooper ◽

I M Orme ◽

...

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthase ◽

Acyl Carrier Protein ◽

Mycolic Acid ◽

Carrier Protein ◽

Type I ◽

Dependent Manner ◽

Fas Ii

Thiolactomycin (TLM) possesses in vivo antimycobacterial activity against the saprophytic strain Mycobacterium smegmatis mc2155 and the virulent strain M. tuberculosis Erdman, resulting in complete inhibition of growth on solid media at 75 and 25 micrograms/ml, respectively. Use of an in vitro murine macrophage model also demonstrated the killing of viable intracellular M. tuberculosis in a dose-dependent manner. Through the use of in vivo [1,2-14C]acetate labeling of M. smegmatis, TLM was shown to inhibit the synthesis of both fatty acids and mycolic acids. However, synthesis of the shorter-chain alpha'-mycolates of M. smegmatis was not inhibited by TLM, whereas synthesis of the characteristic longer-chain alpha-mycolates and epoxymycolates was almost completely inhibited at 75 micrograms/ml. The use of M. smegmatis cell extracts demonstrated that TLM specifically inhibited the mycobacterial acyl carrier protein-dependent type II fatty acid synthase (FAS-II) but not the multifunctional type I fatty acid synthase (FAS-I). In addition, selective inhibition of long-chain mycolate synthesis by TLM was demonstrated in a dose-response manner in purified, cell wall-containing extracts of M. smegmatis cells. The in vivo and in vitro data and knowledge of the mechanism of TLM resistance in Escherichia coli suggest that two distinct TLM targets exist in mycobacteria, the beta-ketoacyl-acyl carrier protein synthases involved in FAS-II and the elongation steps leading to the synthesis of the alpha-mycolates and oxygenated mycolates. The efficacy of TLM against M. smegmatis and M. tuberculosis provides the prospects of identifying fatty acid and mycolic acid biosynthetic genes and revealing a novel range of chemotherapeutic agents directed against M. tuberculosis.

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Pivotal Role of Fatty Acid Synthase in c-MYC Driven Hepatocarcinogenesis

International Journal of Molecular Sciences ◽

10.3390/ijms21228467 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8467

Author(s):

Jiaoyuan Jia ◽

Li Che ◽

Antonio Cigliano ◽

Xue Wang ◽

Graziella Peitta ◽

...

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthase ◽

De Novo ◽

Liver Tumors ◽

Cellular Growth ◽

De Novo Lipogenesis ◽

Liver Malignancy ◽

Genetic Ablation

Hepatocellular carcinoma (HCC) is a deadly form of liver malignancy with limited treatment options. Amplification and/or overexpression of c-MYC is one of the most frequent genetic events in human HCC. The mammalian target of Rapamycin Complex 1 (mTORC1) is a major functional axis regulating various aspects of cellular growth and metabolism. Recently, we demonstrated that mTORC1 is necessary for c-Myc driven hepatocarcinogenesis as well as for HCC cell growth in vitro. Among the pivotal downstream effectors of mTORC1, upregulation of Fatty Acid Synthase (FASN) and its mediated de novo lipogenesis is a hallmark of human HCC. Here, we investigated the importance of FASN on c-Myc-dependent hepatocarcinogenesis using in vitro and in vivo approaches. In mouse and human HCC cells, we found that FASN suppression by either gene silencing or soluble inhibitors more effectively suppressed proliferation and induced apoptosis in the presence of high c-MYC expression. In c-Myc/Myeloid cell leukemia 1 (MCL1) mouse liver tumor lesions, FASN expression was markedly upregulated. Most importantly, genetic ablation of Fasn profoundly delayed (without abolishing) c-Myc/MCL1 induced HCC formation. Liver tumors developing in c-Myc/MCL1 mice depleted of Fasn showed a reduction in proliferation and an increase in apoptosis when compared with corresponding lesions from c-Myc/MCL1 mice with an intact Fasn gene. In human HCC samples, a significant correlation between the levels of c-MYC transcriptional activity and the expression of FASN mRNA was detected. Altogether, our study indicates that FASN is an important effector downstream of mTORC1 in c-MYC induced HCC. Targeting FASN may be helpful for the treatment of human HCC, at least in the tumor subset displaying c-MYC amplification or activation.

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